Producción de plantas libres de virus y morfogénesis indirecta a partir del cultivo de meristemo de tres genotipos de quequisque (Xanthosoma saggittifolium (L) Schott

Se establecieron dos ensayos con el objetivo de determinar en que condición de cultivo se induce: 1) mayor cantidad de plantas libres del DMV diagnosticado con la prueba ELISA; 2) mayor cantidad de callos y plantas regeneradas a partir de callos en tres genotipos de quequisque. Ambos ensayos se iniciaron utilizando meristemos como explantes. Los ensayos se establecieron en esquema de diseño DCA bifactorial: a) genotipos de quequisque, Masaya (My), Nueva Guinea (NG) y Blanco (Bco); b) 5 variantes medios de cultivos. Se realizaron 3 pruebas ELISA a plantas de los tres genotipos que presentasen síntomas del DMV en el campo, a plantas del campo seleccionadas al azar y a plantas provenientes del cultivo de meristemos. En el ensayo de saneamiento del DMV se empleó en el medio MS suplido con combinaciones de AIA y 6-BAP, y para el ensayo de producción de callos se utilizaron combinaciones de 2,4-D y 6-BAP. La regeneración de plantas a partir de callos se logró en el medio MS sencillo. El desarrollo de los explantes en el primer ensayo se cuantificó utilizando escalas arbitrarias para el color y el crecimiento. En el segundo ensayo se utilizó una escala para medir el desarrollo. A los datos de cada variable se realizó un ANDEVA y la prueba de rangos múltiples Tukey, al α= 0.05, cuando hubo diferencias estadísticas entre tratamientos. Cien porciento de las muestras de hojas de plantas de los 3 genotipos que presentaban síntomas del DMV en el campo, resultaron positivas. Las plantas del campo de los genotipos My, NG y Bco elegidas al azar, registraron 88.32, 98.70 y el 90.00% de infección con el DMV; en cambio las plantas in vitro registraron 93.7, 100 y 100% libres de DMV en los mismos genotiposrespectivamente. A los 90 días de establecido el primer ensayo, el medio MS sin reguladores de crecimiento resultó estadísticamente superior con 84% de explantes creciendo y 8% plantas formadas. El medio MS + 1 mgl-16-BAP + 1 mgl-1 AIA registró los valores más discretos con 53% de explantes sin crecer y 41% plantas creciendo. El genotipo My fue superior en todos los medios con 90% de explantes creciendo y 7% plantas formadas. El genotipo Bco registró los más bajos valores con 58% de explantes sin crecer y 5% plantas formadas. Las variables color y crecimiento reportaron similar comportamiento. El medio de cultivo testigo fue estadísticamente superior con 64% de explantes color verde, 30% amarillo y 6% necróticos. El genotipo My sobresalió con 40% de explantes amarillos y 60% verdes. Los genotipos NG y Bco tuvieron comportamientos similares. En el ensayo de producción de callos, tres tratamientos resultaron estadísticamente superiores: el genotipo NG en el medio MS + 3 mgl-12,4-D (60% de explantes formando callos) y el genotipo Masaya en los medios MS+ 5 mg l-12,4-D y MS + 3 mg l-12,4-D (26.7 y 40% callos formados, respectivamente). No se registraron diferencias estadísticas entre los genotipos (16, 17 y 17% callos formados). El medio de cultivo MS + 3 mg l-12,4-D indujo la formación de 4% callos y 44% plantas. El medio de cultivo testigo (MS sin reguladores de crecimiento) resultó en 47% explantes sin crecer y ningún callo formado. Después de los 60 días del trasplante de los callos a un medio sin hormonas, los genotipos Masaya, Nueva Guinea y Blanco reportaron que el 94, 83 y 58 % regeneraron yemas respectivamente.

Comportamiento agronómico en vitroplantas sanas e infestadas con el virus del dasheen (DMV) en tres cultivares de quequisque (Xanthosomas spp.) en el CNIA-INTA, Managua

Con el objetivo de evaluar el comportamiento agronómico de vitroplantas de tres cultivaresde quequisque (Xanthosoma spp.) libres del virus DMV producidas a través del cultivo de meristemos, se estableció el ensayo en el CNIA-INTA (Centro Nacional de Investigación Agropecuaria–Instituto Nicaragüense de Tecnología Agropecuaria) en el departamento de Managua, entre los meses abril-diciembre del año 2004. Se evaluaron variables morfológicas: altura de la planta (cm), área foliar (cm²), diámetro del pseudotallo (cm) y número de hojas; de rendimiento: número de cormelos por planta, peso de cormelo (g), rendimiento (kg ha), diámetro de cormelo (cm) y largo de cormelo (cm) y la presencia del virus e insectos asociados al cultivo en los cultivares Masaya (MY), Nueva Guinea (NG) y Blanco (Bco). Se utilizó un diseño de arreglos en Parcelas Divididas conformado por tres bloques, en la parcela grande se ubicaron los cultivares y en las pequeñas la condición sanitaria (sana e infectada). Se realizó ANDEVA y la separación de medias de rangos múltiples de Tukey ( ∞ =0.05). Los cultivares y la condición sanitaria presentaron diferencias estadísticas entre ellas en las variables morfológicas, habiendo obtenido el cultivar NG los mayores promedios: altura de la planta con (64.23 cm ), área foliar con (1129 cm²), diámetro del pseudotallo con (4.68 cm) y número de hojas con (3.90 cm). El rendimiento presentó efecto significativo en las condiciones fitosanitarias, pero no en los cultivares e interacción. Se encontró tendencia al incremento del porcentaje de reinfección de plantas con presencia del DMV a través del tiempo, el cultivar Bco a los 192 dds reportó un máximo valor del (90 %), seguido de los cultivares MY (65 %) y NG (60 %). Las principales plagas asociadas a los cultivares de quequisque fueron del orden Diptera, Homoptera, Coleoptero, Heminoptero y Lepidoptero, considerándose al áfido Aphis gossypii del orden Homoptera como el posible vector del virus DMV.

Estudio preliminar de la relación mosca blanca virus-maleza en agroecosistema frijol común (Phaseolus vulgaris L) con riego en Nicaragua

En los diferentes agroecosistemas del frijol las malezas representan una de las amenazas más serias y constantes de pérdidas en el rendimiento por lo que se han sido estudiadas de el punto de vista de la competencia, pero estas puedes ser huéspedes alternos de insectos benéficos y dañinos y jugar otro papel de importancia. El frijol es atacado por diferentes patógenos virales siendo en su mayoría transmitidos por insectos entre los que se cuenta Mosca blanca para el caso del virus del mosaico dorado del frijol común Gámez (1971). Con el fin de estudiar la relación Mosca –Malezas virus se realizó un estudio en el centro de Granos Básicos San Cristóbal en el departamento de Managua entre los meses de Enero a Abril de 1987, en dicho centro se muestrearon tres áreas diferentes: cultivada (frijol común con riego), No cultivada (malezas). Ronda permanente (malezas). En las áreas se tomaron en cuenta diferente parámetros para determinar la composición florística, los huéspedes de mosca blanca, huéspedes de patógenos viral y preferencia de la mosca blanca entre malezas y frijol. Los resultados indican que el complejo de malezas predominantes las especies Abutilon crispum, Euphorbia heterpphylla, Baltimora recta y Sida sp. Se pueden considerar huéspedes de Mosca blanca. Existen preferencia de la mosca por especias de malezas como Abutilòn crispum, Eurphorbia heterophylla, chanmaesyce hisopifolìa más que por frijol y que especies como Abutilon crispum y Sida sp son huéspedes de patógenos virales. Estos resultados en conjunto sugieren una situación riesgosa para el futuro del frijol común con riego, ya que se presentan condiciones necesarias para la escorrentía de epifitorias virales en el campo encontramos el inóculo viral, el vector especies susceptibles y condiciones ambientales favorables que podrías llevar a una explosión de la enfermedad en términos mayores; hecho que no podemos asegurar sin estudios más detallados.

Evaluation of bioactive sphingolipids in 4-HPR-resistant leukemia cells

Background -- N-(4-hydroxyphenyl)retinamide (4-HPR, fenretinide) is a synthetic retinoid with potent pro-apoptotic activity against several types of cancer, but little is known regarding mechanisms leading to chemoresistance. Ceramide and, more recently, other sphingolipid species (e.g., dihydroceramide and dihydrosphingosine) have been implicated in 4-HPR-mediated tumor cell death. Because sphingolipid metabolism has been reported to be altered in drug-resistant tumor cells, we studied the implication of sphingolipids in acquired resistance to 4-HPR based on an acute lymphoblastic leukemia model. Methods -- CCRF-CEM cell lines resistant to 4-HPR were obtained by gradual selection. Endogenous sphingolipid profiles and in situ enzymatic activities were determined by LC/MS, and resistance to 4-HPR or to alternative treatments was measured using the XTT viability assay and annexin V-FITC/propidium iodide labeling. Results -- No major crossresistance was observed against other antitumoral compounds (i.e. paclitaxel, cisplatin, doxorubicin hydrochloride) or agents (i.e. ultra violet C, hydrogen peroxide) also described as sphingolipid modulators. CCRF-CEM cell lines resistant to 4-HPR exhibited a distinctive endogenous sphingolipid profile that correlated with inhibition of dihydroceramide desaturase. Cells maintained acquired resistance to 4-HPR after the removal of 4-HPR though the sphingolipid profile returned to control levels. On the other hand, combined treatment with sphingosine kinase inhibitors (unnatural (dihydro)sphingosines ((dh)Sph)) and glucosylceramide synthase inhibitor (PPMP) in the presence or absence of 4-HPR increased cellular (dh)Sph (but not ceramide) levels and were highly toxic for both parental and resistant cells. Conclusions -- In the leukemia model, acquired resistance to 4-HPR is selective and persists in the absence of sphingolipid profile alteration. Therapeutically, the data demonstrate that alternative sphingolipid-modulating antitumoral strategies are suitable for both 4-HPR-resistant and sensitive leukemia cells. Thus, whereas sphingolipids may not be critical for maintaining resistance to 4-HPR, manipulation of cytotoxic sphingolipids should be considered a viable approach for overcoming resistance.

Investigation of Interactions between Two Monoclonal Antibodies and SARS Virus with a Label-Free Protein Array

The investigation of interactions between two kinds of monoclonal antibodies and SARS virus with a label-free protein array technique were presented in this paper. The performance consists of three parts: a surface modification for ligand immobilization/surface, a protein array fabrication with an integrated microfluidic system for patterning, packaging and liquid handling, and a protein array reader of imaging ellipsometer. This revealed the technique could be used as an immunoassay for qualitative and quantitative detection as wen as kinetic analysis of biomolecule interaction.

New genetic markers for treatment personalization in pediatric acute lymphoblastic leukemia

La Leucemia Linfoblástica Aguda (LLA) es el cáncer pediátrico más común. Es un desorden de las células linfoblásticas, que son las precursoras de las células linfáticas, y se caracteriza por la acumulación en médula ósea y sangre de pequeñas células blásticas con poco citoplasma y cromatina dispersa. En las últimas décadas, se ha conseguido aumentar la supervivencia del 10% al 80% pero todavía hay un 20% de pacientes que no responden al tratamiento. Esta mejoría se ha conseguido mediante la implantación de terapias combinadas y la adecuación de la terapia a grupos de riesgo. Los pacientes se separan en tres grupos de riesgo, Riesgo Estándar (RE), Alto Riesgo (AR) y Muy Alto Riesgo (MAR), en base a marcadores pronósticos, entre los que se incluyen alteraciones citogenéticas. Sin embargo, a lo largo del tratamiento, nos encontramos con dos problemas:1) Por un lado, algunos de los pacientes incluidos en el grupo de RE y AR no responden bien al tratamiento y pasan AR y MAR respectivamente. Esto quiere decir que los grupos de riesgo no están bien definidos. Por lo tanto, sería de interés poder caracterizar los pacientes que realmente son RE y AR y aquéllos que desde un principio deberían haber sido considerados como de mayor riesgo.2) Por otro lado, un alto porcentaje de pacientes experimenta toxicidad, que puede llegar a ser muy grave en algunos casos, siendo necesario parar el tratamiento. Por este motivo, sería altamente beneficioso poder reconocer a los pacientes que van a ser más sensibles al tratamiento para, de ese modo, poder ajustar la dosis.Por todo esto, creemos que una mejor asignación de los pacientes de LLA a grupos de riesgo y la personalización de la dosis, mediante nuevos marcadores genéticos, permitiría mejorar la respuesta al tratamiento.En este estudio nos planteamos, por lo tanto, dos objetivos: 1) Llevar a cabo la identificación de nuevas alteraciones genéticas presentes en el tumor para una mejor caracterización del riesgo y 2) Realizar una caracterización genética del individuo que permita predecir la respuesta al tratamiento.

Inoculation of Triatoma Virus (Dicistroviridae: Cripavirus) elicits a non-infective immune response in mice

Background: Dicistroviridae is a new family of small, non-enveloped, +ssRNA viruses pathogenic to both beneficial arthropods and insect pests. Little is known about the dicistrovirus replication mechanism or gene function, and any knowledge on these subjects comes mainly from comparisons with mammalian viruses from the Picornaviridae family. Due to its peculiar genome organization and characteristics of the per os viral transmission route, dicistroviruses make good candidates for use as biopesticides. Triatoma virus (TrV) is a pathogen of Triatoma infestans (Hemiptera: Reduviidae), one of the main vectors of the human trypanosomiasis disease called Chagas disease. TrV was postulated as a potential control agent against Chagas' vectors. Although there is no evidence that TrV nor other dicistroviruses replicate in species outside the Insecta class, the innocuousness of these viruses in humans and animals needs to be ascertained. Methods: In this study, RT-PCR and ELISA were used to detect the infectivity of this virus in Mus musculus BALB/c mice. Results: In this study we have observed that there is no significant difference in the ratio IgG2a/IgG1 in sera from animals inoculated with TrV when compared with non-inoculated animals or mice inoculated only with non-infective TrV protein capsids. Conclusions: We conclude that, under our experimental conditions, TrV is unable to replicate inmice. This study constitutes the first test to evaluate the infectivity of a dicistrovirus in a vertebrate animal model.

Chiral gold(I) vs chiral silver complexes as catalysts for the enantioselective synthesis of the second generation GSK-hepatitis C virus inhibitor

The synthesis of a GSK 2(nd) generation inhibitor of the hepatitis C virus, by enantioselective 1,3-dipolar cycloaddition between a leucine derived iminoester and tert-butyl acrylate, was studied. The comparison between silver(I) and gold(I) catalysts in this reaction was established by working with chiral phosphoramidites or with chiral BINAP. The best reaction conditions were used for the total synthesis of the hepatitis C virus inhibitor by a four step procedure affording this product in 99% ee and in 63% overall yield. The origin of the enantioselectivity of the chiral gold(I) catalyst was justified according to DFT calculations, the stabilizing coulombic interaction between the nitrogen atom of the thiazole moiety and one of the gold atoms being crucial.

Novel Papillomaviruses in Free-Ranging Iberian Bats: No Virus-Host Co-evolution, No Strict Host Specificity, and Hints for Recombination

Papillomaviruses (PVs) are widespread pathogens. However, the extent of PV infections in bats remains largely unknown. This work represents the first comprehensive study of PVs in Iberian bats. We identified four novel PVs in the mucosa of free-ranging Eptesicus serotinus (EserPV1, EserPV2, and EserPV3) and Rhinolophus ferrumequinum (RferPV1) individuals and analyzed their phylogenetic relationships within the viral family. We further assessed their prevalence in different populations of E. serotinus and its close relative E. isabellinus. Although it is frequent to read that PVs co-evolve with their host, that PVs are highly species-specific, and that PVs do not usually recombine, our results suggest otherwise. First, strict virus-host co-evolution is rejected by the existence of five, distantly related bat PV lineages and by the lack of congruence between bats and bat PVs phylogenies. Second, the ability of EserPV2 and EserPV3 to infect two different bat species (E. serotinus and E. isabellinus) argues against strict host specificity. Finally, the description of a second noncoding region in the RferPV1 genome reinforces the view of an increased susceptibility to recombination in the E2-L2 genomic region. These findings prompt the question of whether the prevailing paradigms regarding PVs evolution should be reconsidered.

Detection of hepatitis B virus markers using a biosensor based on imaging ellipsometry

A biosensor based on imaging ellipsometry (BIE) has been developed and validated in 169 patients for detecting five markers of hepatitis B virus (HBV) infection. The methodology has been established to pave the way for clinical diagnosis, including ligand screening, determination of the sensitivity, set-up of cut-off values (CoVs) and comparison with other clinical methods. A matrix assay method was established for ligand screening. The CoVs of HBV markers were derived with the help of receiver operating characteristic curves. Enzyme-linked immunosorbent assay (ELISA) was the reference method. Ligands with high bioactivity were selected and sensitivities of 1 ng/mL and 1 IU/mL for hepatitis B surface antigen (HBsAg) and surface antibody (anti-HBs) were obtained respectively. The CoVs of HBsAg, anti-HBs, hepatitis B e antigen, hepatitis B e antibody and core antibody were as follows: 15%, 18%, 15%, 20% and 15%, respectively, which were the percentages over the values of corresponding ligand controls. BIE can simultaneously detect up to five markers within 1 h with results in acceptable agreement with ELISA, and thus shows a potential for diagnosing hepatitis B with high throughput.

Distinct intron DNA structures in simian virus 40 T-antigen and adenovirus 2 E1A genes

Distinct structures delineating the introns of Simian Virus 40 T-antigen and Adenovirus 2 E1A genes have been discovered. The structures, which are centered around the branch points of the genes inserted in supercoiled double-stranded plasmids, are specifically targeted through photoactivated strand cleavage by the metal complex tris(4,7-diphenyl-1,10-phenanthroline)rhodium(III). The DNA sites that are recognized lack sequence homology but are similar in demarcating functionally important sites on the RNA level. The single-stranded DNA fragments corresponding to the coding strands of the genes were also found to fold into a structure apparently identical to that in the supercoiled genes based on the recognition by the metal complex. Further investigation of different single-stranded DNA fragments with other structural probes, such as another metal complex bis(1,10-phenanthroline)(phenanthrenequinone diimine)rhodium(III), AMT (4'aminomethyl-4,5',8 trimethylpsoralen), restriction enzyme Mse I, and mung bean nuclease, showed that the structures require the sequ ences at both ends of the intron plus the flanking sequences but not the middle of the intron. The two ends form independent helices which interact with each other to form the global tertiary structures. Both of the intron structures share similarities to the structure of the Holliday junction, which is also known to be specifically targeted by the former metal complex. These structures may have arisen from early RNA intron structures and may have been used to facilitate the evolution of genes through exon shuffling by acting as target sites for recombinase enzymes.

Chemical studies of viral entry mechanisms: I. Hydrophobic protein-lipid interactions during sendai virus membrane fusion. II. Kinetics of bacteriophage λ DNA injection.

Viruses possess very specific methods of targeting and entering cells. These methods would be extremely useful if they could also be applied to drug delivery, but little is known about the molecular mechanisms of the viral entry process. In order to gain further insight into mechanisms of viral entry, chemical and spectroscopic studies in two systems were conducted, examining hydrophobic protein-lipid interactions during Sendai virus membrane fusion, and the kinetics of bacteriophage λ DNA injection.

Sendai virus glycoprotein interactions with target membranes during the early stages of fusion were examined using time-resolved hydrophobic photoaffinity labeling with the lipid-soluble carbene generator3-(trifluoromethyl)-3-(m-^(125 )I] iodophenyl)diazirine (TID). The probe was incorporated in target membranes prior to virus addition and photolysis. During Sendai virus fusion with liposomes composed of cardiolipin (CL) or phosphatidylserine (PS), the viral fusion (F) protein is preferentially labeled at early time points, supporting the hypothesis that hydrophobic interaction of the fusion peptide at the N-terminus of the F_1 subunit with the target membrane is an initiating event in fusion. Correlation of the hydrophobic interactions with independently monitored fusion kinetics further supports this conclusion. Separation of proteins after labeling shows that the F_1 subunit, containing the putative hydrophobic fusion sequence, is exclusively labeled, and that the F_2 subunit does not participate in fusion. Labeling shows temperature and pH dependence consistent with a need for protein conformational mobility and fusion at neutral pH. Higher amounts of labeling during fusion with CL vesicles than during virus-PS vesicle fusion reflects membrane packing regulation of peptide insertion into target membranes. Labeling of the viral hemagglutinin/neuraminidase (HN) at low pH indicates that HN-mediated fusion is triggered by hydrophobic interactions, after titration of acidic amino acids. HN labeling under nonfusogenic conditions reveals that viral binding may involve hydrophobic as well as electrostatic interactions. Controls for diffusional labeling exclude a major contribution from this source. Labeling during reconstituted Sendai virus envelope-liposome fusion shows that functional reconstitution involves protein retention of the ability to undergo hydrophobic interactions.

Examination of Sendai virus fusion with erythrocyte membranes indicates that hydrophobic interactions also trigger fusion between biological membranes, and that HN binding may involve hydrophobic interactions as well. Labeling of the erythrocyte membranes revealed close membrane association of spectrin, which may play a role in regulating membrane fusion. The data show that hydrophobic fusion protein interaction with both artificial and biological membranes is a triggering event in fusion. Correlation of these results with earlier studies of membrane hydration and fusion kinetics provides a more detailed view of the mechanism of fusion.

The kinetics of DNA injection by bacteriophage λ. into liposomes bearing reconstituted receptors were measured using fluorescence spectroscopy. LamB, the bacteriophage receptor, was extracted from bacteria and reconstituted into liposomes by detergent removal dialysis. The DNA binding fluorophore ethidium bromide was encapsulated in the liposomes during dialysis. Enhanced fluorescence of ethidium bromide upon binding to injected DNA was monitored, and showed that injection is a rapid, one-step process. The bimolecular rate law, determined by the method of initial rates, revealed that injection occurs several times faster than indicated by earlier studies employing indirect assays.

It is hoped that these studies will increase the understanding of the mechanisms of virus entry into cells, and to facilitate the development of virus-mimetic drug delivery strategies.