Caracterización de Cryphonectria hypovirus (CHV) mediante patrones RFLP en cepas hipovirulentas de Cryphonectria parasitica (Murrill) M.E. Barr de Cataluña

La detecció de soques hipovirulentes a Catalunya ha fet necessari l'estudi genètic de Cryphonectria hypovirus. Mitjançant mostrejos, es van obtenir les soques hipovirulentes i es va comprovar el seu caràcter hipovirulent mitjançant l'extracció del dsRNA. Mitjançant RT-PCR i RFLP es va estudiar la diversitat genètica de les poblacions de Cryphonectria hypovirus. En aquest estudi s'ha observat que la hipovirulència a Catalunya és encara un fenomen localitzat. D'altra banda, s'ha obervat certa diversitat genètica del hipovirus, fet que suggereix que C. hypovirus va ser introduït a Catalunya reiterades vegades. Aquestes reiterades introduccions de C. hypovirus també s'han observat a nivell parcel•lari. La majoria de les mostres pertanyen al subtipus CHV1-I, la qual cosa significa que és d'esperar un increment de la presència del hipovirus a les masses, en ser aquest subtipus el més adequat per dispersar en el medi. S'han detectat altres soques amb patrons RFLP encara no descrits, que podrien pertànyer a altres subtipus. Per tant, s'hauria de fer un estudi genètic més detallat d'aquestes mostres. D'altra banda, hi ha certa diversitat de GCVs associada a les soques amb l'hipovirus. Aquest fet podria suggerir que els GCVs presents a Catalunya tenen una bona capacitat per transmetre el virus o bé seria una altra evidència que C. hypovirus ha estat introduït reiteradament a Catalunya. Finalment s'ha detectat una zona especialment interessant amb diversitat de hipovirus i amb hipovirus encara no descrits, així com soques que podrien pertànyer a CHV1-E. Això suggereix que a la zona podria haver encara més diversitat del hipovirus i per tant podria ser interessant realitzar-hi nous mostreigs.

Avances en el estudio del "torrao" o cribado del tomate

El "torrao" es una enfermedad presente en nuestro país desde 2001, que sigue presentándose en cada campaña de tomate con mayor o menor incidencia según el año. Las plantas afectadas muestran necrosis en la parte basal de los foliolos que puede evolucionar a cribado, manchas longitudinales en los peciolos y manchas necróticas en fruto, que terminan por rajarlo. El presente trabajo es la continuación del publicado en el número 32 de esta revista titulado "Necrosis del tomate: "torrao" o cribado" y surge de los resultados obtenidos tras la reciente publicación de la identificación y caracterización del nuevo virus "Tomato torrado virus" (ToTV) como agente implicado en la enfermedad conocida como "torrao". En este estudio se seleccionaron 94 muestras procedentes de prospecciones realizadas en invernaderos de Murcia durante los años 2003 a 2006. La aplicación RT-PCR e hibridación molecular para la detección de ToTV ha permitido detectar la presencia de esta nueva virosis en 87 de las muestras analizadas. En 83 de ellas, se encontró la presencia conjunta de este nuevo virus con el Pepino mosaic virus (PepMV), mayoritariamente con el aislado tipo Chileno 2 (Accesión number: DQ00095). Se plantean nuevos estudios para determinar la implicación de ambos virus, ToTV y PepMV, en el desarrollo del síndrome conocido como "torrao" del tomate.

Necrosis del tomate: "torrao "o "cribado"

Desde la primavera de 2001, viene presentándose en España una nueva enfermedad conocida con el nombre de "torrao" o "cribado". Los síntomas que habitualmente presentan las plantas afectadas son una necrosis en la parte basal del foliolo que evoluciona a cribado, en los peciolos aparecen manchas longitudinales en ocasiones endurecidas que llegan a curvar los foliolos, y los frutos manifiestan manchas necróticas, deformaciones que finalmente lo rajan, quedando comercialmente inviables. Muestreos realizados desde su aparición han determinado la mayor incidencia de la enfermedad en la zona de Murcia, seguido de Canarias y en menor proporción Almería, y Alicante. Los resultados de los análisis realizados a las 369 muestras recogidas determinan que el 67% de las muestras analizadas eran positivas a Pepino mosaic virus (PepMV). En los ensayos de transmisión, únicamente mediante el injerto, se consiguió reproducir los síntomas de la enfermedad en dos casos, en el resto las plantas inoculadas e injertadas únicamente mostraban síntomas típicos de PepMV y los análisis realizados confirmaron este aspecto. A la vista de los resultados obtenidos, se diseñó un nuevo método de diagnóstico que ha permitido la caracterización del 89% de las muestras analizadas como aislado Chileno 2 de PepMV, recientemente publicado en el Gen Bank (Accesión number: DQ000985). De acuerdo con lo expuesto podría tratarse de uno de los agentes implicados en el desarrollo del síndrome junto con otros factores aún por determinar

A novel source for miR-21 expression through the alternative polyadenylation of VMP1 gene transcripts

miR-21 is the most commonly over-expressed microRNA (miRNA) in cancer and a proven oncogene. Hsa-miR-21 is located on chromosome 17q23.2, immediately downstream of the vacuole membrane protein-1 (VMP1) gene, also known as TMEM49. VMP1 transcripts initiate ∼130 kb upstream of miR-21, are spliced, and polyadenylated only a few hundred base pairs upstream of the miR-21 hairpin. On the other hand, primary miR-21 transcripts (pri-miR-21) originate within the last introns of VMP1, but bypass VMP1 polyadenylation signals to include the miR-21 hairpin. Here, we report that VMP1 transcripts can also bypass these polyadenylation signals to include miR-21, thus providing a novel and independently regulated source of miR-21, termed VMP1–miR-21. Northern blotting, gene-specific RT-PCR, RNA pull-down and DNA branching assays support that VMP1–miR-21 is expressed at significant levels in a number of cancer cell lines and that it is processed by the Microprocessor complex to produce mature miR-21. VMP1 and pri-miR-21 are induced by common stimuli, such as phorbol-12-myristate-13-acetate (PMA) and androgens, but show differential responses to some stimuli such as epigenetic modifying agents. Collectively, these results indicate that miR-21 is a unique miRNA capable of being regulated by alternative polyadenylation and two independent gene promoters.

The importance of sonographer experience and machine quality with regards to the role of musculoskeletal ultrasound in routine care of rheumatoid arthritis patients.

OBJECTIVES: Regarding recent progress, musculoskeletal ultrasound (US) will probably soon be integrated in standard care of patient with rheumatoid arthritis (RA). However, in daily care, quality of US machines and level of experience of sonographers are varied. We conducted a study to assess reproducibility and feasibility of an US scoring for RA, including US devices of different quality and rheumatologist with various levels of expertise in US as it would be in daily care. METHODS: The Swiss Sonography in Arthritis and Rheumatism (SONAR) group has developed a semi-quantitative score using OMERACT criteria for synovitis and erosion in RA. The score was taught to 108 rheumatologists trained in US. One year after the last workshop, 19 rheumatologists participated in the study. Scans were performed on 6 US machines ranging from low to high quality, each with a different patient. Weighted kappa was calculated for each pair of readers. RESULTS: Overall, the agreement was fair to moderate. Quality of device, experience of the sonographers and practice of the score before the study improved substantially the agreement. Agreement assessed on higher quality machine, among sonographers with good experience in US increased to substantial (median kappa for B-mode and Doppler: 0.64 and 0.41 for erosion). CONCLUSIONS: This study demonstrated feasibility and reproducibility of the Swiss US SONAR score for RA. Our results confirmed importance of the quality of US machine and the training of sonographers for the implementation of US scoring in the routine daily care of RA.

Two different epidemiological scenarios of border disease in the populations of Pyrenean chamois (Rupicapra p. pyrenaica) after the first disease outbreaks

Since 2001 several outbreaks of a new disease associated with Border disease virus (BDV) infection have caused important declines in Pyrenean chamois (Rupicapra pyrenaica) populations in the Pyrenees. The goal of this study was to analyze the post-outbreak BDV epidemiology in the first two areas affected by disease with the aim to establish if the infection has become endemic. We also investigated if BDV infected wild and domestic ruminants sharing habitat with chamois. Unexpectedly, we found different epidemiological scenarios in each population. Since the disease outbreaks, some chamois populations recuperated quickly, while others did not recover as expected. In chamois from the first areas, prevalence was high (73.47%) and constant throughout the whole study period and did not differ between chamois born before and after the BDV outbreak; in all, BDV was detected by RT-PCR in six chamois. In the other areas, prevalence was lower (52.79%) and decreased during the study period; as well, prevalence was significantly lower in chamois born after the disease outbreak. No BDV were detected in this population. A comparative virus neutralisation test performed with four BDV strains and one Bovine viral diarrhoea virus (BVDV) strain showed that all the chamois had BDV-specific antibodies. Pestivirus antibodies were detected in all the rest of analyzed species, with low prevalence values in wild ruminants and moderate values in domestic ruminants. No viruses were detected in these species. These results confirm the hypothesis that outbreaks of BDV infection only affect the Pyrenean chamois, although other wild ruminants can occasionally be infected. In conclusion, two different scenarios have appeared since the first border disease outbreaks in Pyrenean chamois: on the one hand frequent BDV circulation with possible negative impact on population dynamics in some areas and on the other, lack of virus circulation and quick recovery of the chamois population.

Caracterização de genes diferencialmente expressos na interação entre maçã 'Fuji' e Botryosphaeria dothidea

O objetivo deste estudo foi investigar a interação entre o fungo Botryosphaeria dothidea e maçãs cv. Fuji por meio da técnica de Differential Display RT-PCR. O cDNA de frutos infectados e não infectados pelo fungo foi amplificado com uma combinação de 15 oligonucleotídeos iniciadores. Foram isolados 400 fragmentos de cDNA diferencialmente expressos, dos quais 120 foram sequenciados e comparados com sequências disponíveis no GenBank, por meio do programa BLASTX. As sequências obtidas foram similares à metalotioninas, profilina alergênica, proteína de resistência e fosfatase.

Comportamento de híbridos de citros em relação à infecção natural pelo Citrus tristeza virus e à presença de sintomas de descamamento eruptivo

O Programa de Melhoramento Genético de Citros da Embrapa Mandioca e Fruticultura vem gerando híbridos para utilização como porta-enxertos, que necessitam ser avaliados em relação ao comportamento frente à infecção natural por isolados locais de Citrus tristeza virus (CTV) e à presença de sintomas de descamamento eruptivo (BahiaBarkScaling disease - BBS). Este trabalho apresenta resultados da avaliação do comportamento de 141 híbridos (sob a forma de pés-francos ou enxertados) estabelecidos na área experimental da Embrapa Mandioca e Fruticultura, no Recôncavo Sul da Bahia. Foram avaliadas a presença e a severidade de sintomas de caneluras e descamamento por meio de escala de notas. Para detectar a presença do CTV, foi utilizado o método sorológico de ELISA indireto e RT-PCR. Os híbridos avaliados foram classificados como imunes, tolerantes e intolerantes ao CTV. A maioria dos híbridos que apresentaram sintomas de BBS tem uma tangerineira como parental.

COMPARISON OF RNA EXTRACTION METHODS FOR Passiflora edulis SIMS LEAVES

ABSTRACT Functional genomic analyses require intact RNA; however, Passiflora edulis leaves are rich in secondary metabolites that interfere with RNA extraction primarily by promoting oxidative processes and by precipitating with nucleic acids. This study aimed to analyse three RNA extraction methods, Concert™ Plant RNA Reagent (Invitrogen, Carlsbad, CA, USA), TRIzol® Reagent (Invitrogen) and TRIzol® Reagent (Invitrogen)/ice -commercial products specifically designed to extract RNA, and to determine which method is the most effective for extracting RNA from the leaves of passion fruit plants. In contrast to the RNA extracted using the other 2 methods, the RNA extracted using TRIzol® Reagent (Invitrogen) did not have acceptable A260/A280 and A260/A230 ratios and did not have ideal concentrations. Agarose gel electrophoresis showed a strong DNA band for all of the Concert™ method extractions but not for the TRIzol® and TRIzol®/ice methods. The TRIzol® method resulted in smears during electrophoresis. Due to its low levels of DNA contamination, ideal A260/A280 and A260/A230 ratios and superior sample integrity, RNA from the TRIzol®/ice method was used for reverse transcription-polymerase chain reaction (RT-PCR), and the resulting amplicons were highly similar. We conclude that TRIzol®/ice is the preferred method for RNA extraction for P. edulis leaves.

Mini invasive skeletal muscle biopsy technique with a tri-axial end cut needle.

OBJECTIVE: Skeletal Muscle Biopsy is a minor surgical procedure for the diagnosis of different neuromuscular pathological conditions and has recently gained popularity also in the research field of age-related muscular modifications and sarcopenia. Few studies focused on the application of mini-invasive muscular biopsy in both normal and pathological conditions. The aim of our study was to describe a mini invasive ultrasound-guided skeletal muscular biopsy technique in complete spinal cord injured (SCI) patients and healthy controls with a tri-axial end-cut needle. PATIENTS AND METHODS: Skeletal muscle biopsies were collected from 6 chronic SCI patients and 3 healthy controls vastus lateralis muscle with a tri-axial end cut needle (Biopince© - Angiotech). Muscle samples were stained for ATPase to determine fibers composition, moreover, gene expression of cyclooxygenase-1 (COX-1) and prostaglandin E2 receptor has been analyzed by Real Time RT-PCR. RESULTS: All the procedures were perfomed easily without failures and complications. Control tissue was macroscopically thicker than SCI one. Control specimen displayed an equal distribution of type I and type II fibers, while SCI sample displayed a prevalence of type II fibers SCI specimen displayed a significant reduction in COX-1 gene expression. This mini-invasive approach was easy, accurate and with low complication rate in performing skeletal muscle biopsy in both SCI patients and controls. CONCLUSIONS: This technique could be useful in conditions in which the overall quantity of specimen required is small like for molecular biology analysis. For histological diagnostic purposes and/or conditions in which the original tissue is already pathologically modified, this technique should be integrated with more invasive techniques.

Low-fat dairy products and blood pressure: follow-up of 2290 older persons at high cardiovascular risk participating in the PREDIMED study

High blood pressure (BP) has been ranked as the most important risk factor worldwide regarding attributable deaths. Dietary habits are major determinants of BP. Among them, frequent intake of low-fat dairy products may protect against hypertension. Our aim was to assess the relationship between low-fat dairy product intake and BP levels and their changes after 12 month follow-up in a cohort of asymptomatic older persons at high cardiovascular risk recruited into a large-scale trial assessing the effects of Mediterranean diets on cardiovascular outcomes. Data from 2290 participants, including 1845 with hypertension, were available for analyses. Dairy products were not a specific part of the intervention; thus, data were analysed as an observational cohort. Dietary information was collected with validated semi-quantitative FFQ and trained personnel measured BP. To assess BP changes, we undertook cross-sectional analyses at baseline and at the end of follow-up and longitudinal analyses. A statistically significant inverse association between low-fat dairy product intake and systolic BP was observed for the 12-month longitudinal analysis. In the longitudinal analysis, the adjusted systolic and diastolic BP were significantly lower in the highest quintile of low-fat dairy product intake ( 2 4·2 (95% CI 2 6·9, 2 1·4) and 2 1·8 (95% CI 2 3·2, 2 0·4) mmHg respectively), whereas the point estimates for the difference in diastolic BP indicated a modest non-significant inverse association. Intake of low-fat dairy products was inversely associated with BP in an older population at high cardiovascular risk, suggesting a possible protective effect against hypertension.

CD4 expression decrease by antisense oligonucleotides. Inhibition of rat T CD4+ cell reactivity

In previous studies, we have demonstrated the inhibition of CD4 expression in rat lymphocytes treated with phorbol myristate acetate (PMA) by antisense oligonucleotides (AS-ODNs) directed against the AUG start region of the cd4 gene. The aim of the present study was to inhibit CD4 expression in lymphocytes without promoting CD4 synthesis and to determine the effect of this inhibition on CD4+ T cell function. Four 21-mer ODNs against the rat cd4 gene (AS-CD4-1 to AS-CD4-4) were used. Surface CD4 expression was measured by immunofluorescence staining and flow cytometry, and mRNA CD4 expression was measured by RT-PCR. T CD4+ cell function was determined by specific and unspecific proliferative response of rat-primed lymphocytes. After 24 hours of incubation, AS-CD4-2 and AS-CD4-4 reduced lymphocyte surface CD4 expression by 40%. This effect remained for 72 hours and was not observed on other surface molecules, such as CD3, CD5, or CD8. CD4 mRNA expression was reduced up to 40% at 24 hours with AS-CD4-2 and AS-CD4-4. After 48 hours treatment, CD4 mRNA decreased up to 27% and 29% for AS-CD4-2 and AS-CD4-4, respectively. AS-CD4-2 and AS-CD4-4 inhibited T CD4+ cell proliferative response upon antigen-specific and unspecific stimuli. Therefore, AS-ODNs against CD4 molecules inhibited surface and mRNA CD4 expression, under physiologic turnover and, consequently, modulate T CD4+ cell reactivity.

CD4 expression decrease by antisense oligonucleotides. Inhibition of rat T CD4+ cell reactivity

In previous studies, we have demonstrated the inhibition of CD4 expression in rat lymphocytes treated with phorbol myristate acetate (PMA) by antisense oligonucleotides (AS-ODNs) directed against the AUG start region of the cd4 gene. The aim of the present study was to inhibit CD4 expression in lymphocytes without promoting CD4 synthesis and to determine the effect of this inhibition on CD4+ T cell function. Four 21-mer ODNs against the rat cd4 gene (AS-CD4-1 to AS-CD4-4) were used. Surface CD4 expression was measured by immunofluorescence staining and flow cytometry, and mRNA CD4 expression was measured by RT-PCR. T CD4+ cell function was determined by specific and unspecific proliferative response of rat-primed lymphocytes. After 24 hours of incubation, AS-CD4-2 and AS-CD4-4 reduced lymphocyte surface CD4 expression by 40%. This effect remained for 72 hours and was not observed on other surface molecules, such as CD3, CD5, or CD8. CD4 mRNA expression was reduced up to 40% at 24 hours with AS-CD4-2 and AS-CD4-4. After 48 hours treatment, CD4 mRNA decreased up to 27% and 29% for AS-CD4-2 and AS-CD4-4, respectively. AS-CD4-2 and AS-CD4-4 inhibited T CD4+ cell proliferative response upon antigen-specific and unspecific stimuli. Therefore, AS-ODNs against CD4 molecules inhibited surface and mRNA CD4 expression, under physiologic turnover and, consequently, modulate T CD4+ cell reactivity.

Pandemic influença A (H1N1) infection in pregnant and nonpregnant women in Spain (2009-2010)

The present study aimed to compare the main features of infection with pandemic influenza A virus in pregnant and nonpregnant women admitted to hospitals in Spain during the first waves of the 2009-2010 influenza pandemic. This was a prospective (November 2009 to June 2010), multicenter observational study. All cases were women of reproductive age who had not been vaccinated against seasonal or pandemic influenza A. Influenza infection was confirmed by reverse transcription-polymerase chain reaction (RT-PCR). The sociodemographic and clinical data of all cases were reviewed. A total of 219 inpatients, including 49 pregnant women and 170 nonpregnant women, were enrolled in the study upon admission to participating hospitals. The most substantially different symptoms between the groups were respiratory distress and unilobar consolidation, both of which were more frequent among nonpregnant women. Antibiotics and systemic corticosteroids were more frequently used in nonpregnant women; however, there were no differences in the rates of treatment with antivirals. Our findings indicated that the compared with nonpregnant women, pregnant women in this study did not have significantly different symptoms and were not at increased risk of complications from pandemic influenza virus infection.

Optimisation d'une approche pour l'analyse de l'expression de gènes au niveau unicellulaire chez des sous-populations spécialisées de lymphocytes T CD4+ humaines

Chez les patients cancéreux, les cellules malignes sont souvent reconnues et détruites par les cellules T cytotoxiques du patient. C'est pourquoi, depuis plusieurs années, des recherches visent à produire des vaccins sensibilisant les cellules de l'immunité adaptative, afin de prévenir certains cancers. Bien que les vaccins ciblant les cellules T CD8+ (cytotoxiques) ont une efficacité in-vitro élevée, un vaccin pouvant cibler les cellules T CD8+ et CD4+ aurait une plus grande efficacité (1-3). En effet, les cellules T helper (CD4+) favorisent la production et la maintenance des cellules T CD8+ mémoires à longue durée de vie. Il existe un grand nombre de sous-types de cellules T CD4+ et leur action envers les cellules cancéreuses est différente. Par exemple, les lymphocytes Treg ont une activité pro-tumorale importante (4) et les lymphocytes Th1 ont une activité anti-tumorale (5). Cependant, le taux naturel des différents sous-types de cellules T CD4+ spécifiques aux antigènes tumoraux est variable. De plus, une certaine flexibilité des différents sous-types de cellules T CD4+ a été récemment démontrée (6). Celle-ci pourrait être ciblée par des protocoles de vaccination avec des antigènes tumoraux administrés conjointement à des adjuvants définis. Pour cela, il faut approfondir les connaissances sur le rôle des cellules T CD4+ spécifiques aux antigènes dans l'immunité anti-tumorale et connaître précisément la proportion des sous-types de cellules T CD4+ activées avant et après la vaccination. L'analyse des cellules T, par la cytométrie de flux, est très souvent limité par le besoin d'un nombre très élevé de cellules pour l'analyse de l'expression protéique. Or dans l'analyse des cellules T CD4+ spécifiques aux antigènes tumoraux cette technique n'est souvent pas applicable, car ces cellules sont présentes en très faible quantité dans le sang et dans les tissus tumoraux. C'est pourquoi, une approche basée sur l'analyse de la cellule T individuelle a été mise en place afin d'étudier l'expression du profil génétique des cellules T CD8+ et CD4+. (7,8) Méthode : Ce nouveau protocole (« single cell ») a été élaboré à partir d'une modification du protocole PCR-RT, qui permet la détection spécifique de l'ADN complémentaire (ADNc) après la transcription globale de l'ARN messager (ARNm) exprimé par une cellule T individuelle. Dans ce travail, nous optimisons cette nouvelle technique d'analyse pour les cellules T CD4+, en sélectionnant les meilleures amorces. Tout d'abord, des clones à profils fonctionnels connus sont générés par cytométrie de flux à partir de cellules T CD4+ d'un donneur sain. Pour cette étape d'optimisation des amorces, la spécificité des cellules T CD4+ n'est pas prise en considération. Il est, donc, possible d'étudier et de trier ces clones par cytométrie de flux. Ensuite, grâce au protocole « single cell », nous testons par PCR les amorces des différents facteurs spécifiques de chaque sous-type des T CD4+ sur des aliquotes issus d'une cellule provenant des clones générés. Nous sélectionnons les amorces dont la sensibilité, la spécificité ainsi que les valeurs prédictives positives et négatives des tests sont les meilleures. (9) Conclusion : Durant ce travail nous avons généré de l'ADNc de cellules T individuelles et sélectionné douze paires d'amorces pour l'identification des sous-types de cellules T CD4+ par la technique d'analyse PCR « single cell ». Les facteurs spécifiques aux cellules Th2 : IL-4, IL-5, IL-13, CRTh2, GATA3 ; les facteurs spécifiques aux cellules Th1 : TNFα, IL-2 ; les facteurs spécifiques aux cellules Treg : FOXP3, IL-2RA ; les facteurs spécifiques aux cellules Th17 : RORC, CCR6 et un facteur spécifique aux cellules naïves : CCR7. Ces amorces peuvent être utilisées dans le futur en combinaison avec des cellules antigènes-spécifiques triées par marquage des multimères pMHCII. Cette méthode permettra de comprendre le rôle ainsi que l'amplitude et la diversité fonctionnelle de la réponse de la cellule T CD4+ antigène-spécifique dans les cancers et dans d'autres maladies. Cela afin d'affiner les recherches en immunothérapie oncologique. (8)