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Analysis of a new strain of Euphorbia mosaic virus with distinct replication specificity unveils a lineage of begomoviruses with short Rep sequences in the DNA-B intergenic region
Get PDF"Background: Euphorbia mosaic virus (EuMV) is a member of the SLCV clade, a lineage of New World begomoviruses that display distinctive features in their replication-associated protein (Rep) and virion-strand replication origin. The first entirely characterized EuMV isolate is native from Yucatan Peninsula, Mexico; subsequently, EuMV was detected in weeds and pepper plants from another region of Mexico, and partial DNA-A sequences revealed significant differences in their putative replication specificity determinants with respect to EuMV-YP. This study was aimed to investigate the replication compatibility between two EuMV isolates from the same country. Results: A new isolate of EuMV was obtained from pepper plants collected at Jalisco, Mexico. Full-length clones of both genomic components of EuMV-Jal were biolistically inoculated into plants of three different species, which developed symptoms indistinguishable from those induced by EuMV-YP. Pseudorecombination experiments with EuMV-Jal and EuMV-YP genomic components demonstrated that these viruses do not form infectious reassortants in Nicotiana benthamiana, presumably because of Rep-iteron incompatibility. Sequence analysis of the EuMV-Jal DNA-B intergenic region (IR) led to the unexpected discovery of a 35-nt-long sequence that is identical to a segment of the rep gene in the cognate viral DNA-A. Similar short rep sequences ranging from 35- to 51-nt in length were identified in all EuMV isolates and in three distinct viruses from South America related to EuMV. These short rep sequences in the DNA-B IR are positioned downstream to a similar to 160-nt non-coding domain highly similar to the CP promoter of begomoviruses belonging to the SLCV clade. Conclusions: EuMV strains are not compatible in replication, indicating that this begomovirus species probably is not a replicating lineage in nature. The genomic analysis of EuMV-Jal led to the discovery of a subgroup of SLCV clade viruses that contain in the non-coding region of their DNA-B component, short rep gene sequences located downstream to a CP-promoter-like domain. This assemblage of DNA-A-related sequences within the DNA-B IR is reminiscent of polyomavirus microRNAs and could be involved in the posttranscriptional regulation of the cognate viral rep gene, an intriguing possibility that should be experimentally explored.
Replicative and integrative plasmids for production of human interferon gamma in Bacillus subtilis
Get PDF"Integrative and replicative plasmids for the expression driven by the P43 promoter and secretion of recombinant proteins in Bacillus subtilis were constructed. The plasmids named pInt and pRep respectively were tested for the production of recombinant human interferon gamma (rhIFN-γ). A synthetic hIFN-γ gene employing the optimized B. subtilis codon usage was fused with the Bacillus licheniformis α-amylase signal peptide (sp-amyL) encoding sequence. The integrative construct produced 2.5 ± 0.2 mg l−1 and the replicative system produced 20.3 ± 0.8 mg l−1 of total recombinant rhIFN-γ. The results showed that secretion of hIFN-γ was the bottleneck for the overexpression of mature rhIFN-γ by B. subtilis.
Análisis de los complejos multiproteínicos de Bifidobacterium longum por Electroforesisi Blue Native de doble dimensión, efecto del sustrato.
Get PDFTesis (Maestría en Ciencias en Biología Molecular)"Bifidobacterium longum es una de las especies bacterianas nativas de la flora intestinal, conocida como probiótica; es decir , que proporciona beneficios a la salud del huésped. En el intestino los probióticos consumen preferentemente azúcares no digeribles por el huésped como los fructanos que se encuentran en las plantas y son conocidos como prebióticos. El estudio del complexoma (todos los complejos de proteínas de la célula), ayuda a tener una mejor comprensión y una visión global de la función celular. La doble-dimensión Blue Native (BN)/SDSPAGE fue inicialmente reportada para analizar complejos de proteínas de membrana. En el presente trabajo hemos aplicado la técnica BN/SDS-PAGE para caracterizar los complejos citoplásmicos en B. longum, cepa ATCC 15707 y sus cambios cuando se tiene como fuente de carbono glucosa o fructanos. Para ello se optimizó la electroforesis BN y el usó de tricina en lugar de glicina mejoró la separación. Fue posible identificar 53 proteínas agrupadas en 8 complejos, además de identificar las proteínas expresadas diferencialmente en las diferentes fuentes de carbono. Las proteínas componentes de los complejos fueron identificadas por espectrometría de masas.""Bifidobacterium longum is one of the bacterial species native of the intestinal flora, it is known as a probiotic, i.e. bacteria that provide health benefits to the host. In the intestine, the probiotics consume indigestible sugars preferentially by the host as fructans found in plants and are known as prebiotics. The study of the complexoma interest (all protein complexes in the cell), is helps in the understanding and to have a overview of cellular function. The two-dimensional Blue Native BN/SDS-PAGE was initially reported for analysis of membrane protein complexes. In this study we assessed the study of cytoplasmic protein that form complexes in B. longum ATCC 15707 in the presences of fructans as a carbon source by this method. BN electrophoresis was improved by using tricine instead of glycine. It was possible to identify 53 proteins grouped into eight complexes, and identify differentially expressed proteins in different carbon sources. The protein components of the complex were identified by mass spectrometry.
Spectral parameter power series representation for Hill´s discriminant
Get PDF"We establish a series representation of the Hill discriminant based on the spectral parameter power series (SPPS) recently introduced by V. Kravchenko. We also show the invariance of the Hill discriminant under a Darboux transformation and employing the Mathieu case the feasibility of this type of series for numeri-cal calculations of the eigenspectrum.
Producción de antígenos inmunogénicos en plantas: un enfoque para mejorar la vacuna contra la tos ferina
No full textTesis (Maestría en Ciencias en Biología Molecular)"La tos ferina es una enfermedad infecciosa de distribución mundial en la que se ha observado un aumento en la incidencia de la enfermedad en adolescentes y niños menores de 6 meses de edad. Además se ha establecido que la inmunidad adquirida con la vacuna se pierde alrededor de los siete años posteriores a la última aplicación, lo que aumenta el riesgo de contagio a partir de los diez años de edad. En la actualidad existen las vacunas acelulares contra tos ferina, pero su alto precio limita su aplicación. Por lo anterior, el presente trabajo se enfoca a expresar epítopos de Bordatella pertussis relevantes en plantas como un primer paso en el desarrollo de una vacuna comestible contra la tosferina, que produzca una respuesta inmune más duradera y/o permita el suministro de dosis de refuerzos vía oral, la cual es una vía más aceptada por el público. Se desarrollaron construcciones que codifican para epítopos relevantes de la toxina pertúsica, la hemaglutinina filamentosa y la pertactina, además, cuentan con espaciadores ricos en prolina y cola de histidinas, esta última para su purificación. Las secuencias fueron optimizadas para su expresión estable tanto en citoplasma de lechuga vía Agrobacterium tumefaciens, como en cloroplastos de tabaco vía biobalística. Se obtuvieron plantas adultas T0 transplastómicas, a las que se verificó la integración del transgén por PCR, así como la presencia del transcrito específico del tamaño esperado por RT-PCR, y se confirmó la concentración de proteína de manera indirecta medio de un ensayo inmunoenzimático con anticuerpos específicos contra la cola de histidinas. En la transformación nuclear de lechuga, se logró regenerar callos y brotes resistentes al agente de selección con la construcción utilizada. El material obtenido será analizado in vivo para ver demostrar su inmunogenicidad, para posteriormente realizar retos en ratones y demostrar su inmunoprotección. Este es el primer trabajo de la expresión de un polipéptido donde se han incluido 3 diferentes epítopos de regiones antigénicas de B. pertussis, el cual se ha expresado en plantas.
Net ecosystem exchange of carbon and water vapor among contrasting land-uses types in the semiarid short-grass steppe in Central México.
Get PDFTesis (Maestría en Ciencias Ambientales)"Ecosistemas áridos como los pastizales cubren el 47% de la superficie terrestre y almacenan el 15% del carbono (C) global del suelo. El cambio de uso de suelo (LUC) es la principal actividad humana que controla el intercambio de C, agua y energía entre el continuo suelo-atmósfera. Se requiere incrementar nuestro conocimiento sobre el impacto del LUC sobre el intercambio y almacenamiento de C por los ecosistemas para desarrollar herramientas de predicción y estrategias de mitigación del cambio ambiental global. En este estudio se determinó el intercambio neto de CO2 (NEE) y vapor de agua (ET) a escala diurna y estacional en cinco sitios de pastizal mediano abierto en los Llanos de Ojuelos, Jalisco: 1) una exclusión de ganado de 28 años (Exclusión), 2) un sitio con dominancia de especies de gramíneas generalmente subordinadas (Cambio de especies), 3) un sitio sobrepastoreado (sobrepastoreo), 4) un sitio sobrepastoreado invadido por arbustivas perennes y herbáceas exóticas (Invasión de arbustos), y 5) un sitio con agricultura de temporal (Agricultura). Se midió el índice de área foliar (LAI), la temperatura del aire (T), la densidad de flujo fotónico fotosintético (PPFD) y contenido volumétrico de agua en el suelo (SWC) como controles bióticos y abióticos de NEE y ET. Se midió el NEE y ET durante ocho fechas de octubre de 2008 a julio de 2009 usando el método del domo geodésico. Se establecieron 6 parcelas por sitio, sobre las que se hicieron 4 mediciones al día (08:00, 12:00, 16:00 y 20:00 h). Se usó un análisis de medidas repetidas (ANOVA, α=0.05) para comparar los flujos entre sitios. Se realizaron análisis de regresión lineares y no lineares entre los flujos y sus controles. No fue observado un patrón claro del NEE a través del gradiente de disturbio: el sitio Cambio de especies mostró mayores tasas de NEE diurnas y nocturnas con un balance positivo de captura de CO2 de 0.781 μmol m-2 s-1; el sitio sobrepastoreo mostró bajas tasas de intercambio, pero aún con captura de CO2 (0.236 μmol m-2 s-1); en contraste, la Exclusión mostró pérdida de CO2 (0.719 μmol m-2 s-1). Los sitios Cambio de especies y Exclusión mostraron las más altas tasas de ET (1.353 y 1.221 mmol m-2 s-1, respectivamente).""Dryland ecosystems such as grasslands cover up to 47% of terrestrial land and store around 15% of the global soil carbon. Land-use change (LUC) is the main human activity that controls the carbon, water and energy fluxes in the soilatmosphere continuum. International research agendas request to increase our understanding on the impact of LUC on the capacity of ecosystems to capture and store carbon to improve forecasting tools and mitigation strategies. In the region “Llanos de Ojuelos”, Jalisco, this study measured at diurnal and seasonal scales, net ecosystem exchange (NEE) and evapotranspiration (ET) of the short-grass steppe. Five different sites were included; 1) a 28 year exclosure (Exclosure), 2) a site dominated by commonly subordinated grasses (Species-shift), 3) an overgrazed site (Overgrazed), 4) an overgrazed site with shrub encroachment and exotic species invasion (Shrub encroachment) and 5) a site converted to rainfed oat crop (Oat crop). Biotic (leaf area index, LAI) and abiotic drivers (air temperature T, photosynthetic photon flux density PPFD, and volumetric soil water content SWC) were measured. NEE and ET were recorded on eight dates from October 2008 to July 2009 using the geodesic dome method. Six plots per site were established and gas exchange was measured on four times during the day (08:00, 12:00, 16:00 and 20:00 h). A repeated measures ANOVA (α=0.05) was performed to compare NEE and ET among sites. Linear and non-linear regression analyses between fluxes and drivers were performed. Results showed not clear trends for NEE following the gradient of disturbance; still, species-shift exhibited the highest diurnal and nocturnal NEE rates with a positive CO2 balance of 0.781 μmol m-2 s-2. The overgrazed site showed significantly lower rates but still a positive CO2 balance (0.236 μmol m-2 s-1). In contrast, the Exclosure site showed in this period a net CO2 efflux of 0.719 μmol m-2 s-1. Regarding ET rates, the Species shift and the Exclosure sites observed the largest rates (1.353 and 1.221 mmol m-2 s-1, respectively) that was attributable to a significant larger leaf area index (LAI). Diel time patterns of NEE and ET were driven by PPFD (R2=0.95 y 0.93, respectively) during the wet months (October, June and July); while in the dry and warm months (March-May) the main driver was air temperature.
Subtelomeric silencing of the MTL3 locus of Candida glabrata requires yKu70, yKu80, and Rif1 proteins
No full text"Candida glabrata is a haploid opportunistic fungal pathogen that is phylogenetically related to Saccharomyces cerevisiae. Even though C. glabrata has no known sexual cycle, it contains, like S. cerevisiae, three mating type-like loci (MTL) called MTL1, MTL2, and MTL3, as well as most of the genes required for mating, meiosis, and sporulation. MTL1 is localized at an internal position on chromosome B and is thought to be the locus corresponding to the MAT locus in S. cerevisiae. MTL2 and MTL3 are localized close to two telomeres on different chromosomes (29.4 kb from Chr E-L and 10.5 kb from Chr B-L, respectively). By using URA3 reporter gene insertions at the three MTL loci, we found that in contrast to the case for S. cerevisiae, only MTL3 is subject to transcriptional silencing while MTL2 is transcriptionally active, and this is in agreement with previously reported data. We found that the silencing of MTL3 is nucleated primarily at the left telomere of chromosome B and spreads over 12 kb to MTL3, rather than nucleating at flanking, closely positioned cis-acting silencers, like those flanking HMR and HML of S. cerevisiae. Interestingly, the silencing of MTL3 absolutely requires the yKu70, yKu80, and Rif1 proteins, in sharp contrast to the silencing of the HM loci of S. cerevisiae. In addition, we found that several cell type-specific genes are expressed in C. glabrata regardless of the presence, or even absence, of mating type information at any of the MTL loci.
Diseño de herramientas moleculares para la caracterización de las proteínas Sir3 y Sir4 en Candida glabrata.
Get PDFTesis (Maestría en Ciencias en Biología Molecular)"En los últimos años, Candida glabrata ha surgido como patógeno oportunista, especialmente en individuos inmunocomprometidos. Una característica que presenta esta levadura es que se adhiere a células epiteliales. Esta adherencia está mediada por proteínas de pared celular codificadas por una familia de más de 20 genes parálogos (genes EPA). La mayoría de estos genes se encuentran localizados en regiones subteloméricas, donde están sujetos al silenciamiento transcripcional dependiente de la cromatina. Este silenciamiento lo lleva acabo el complejo Sir (Sir2, Sir3 y Sir4), Rap1, Ku70, Ku80 y Rif1. Mutaciones en cualquiera de los genes que codifican para la maquinaria del silenciamiento subtelomérico, vuelve a las células hiperadherentes y resistentes a estrés oxidativo. Esto sugiere que la adherencia y la resistencia a estrés oxidativo, estarían coregulados en este hongo patógeno por un mismo sistema de regulación global, como lo es el del silenciamiento subtelomérico. Adicionalmente ello sugiere que este sistema de regulación global pudiera detectar señales del medio ambiente y responder a éstas, reprogramando la expresión de genes subteloméricos específicos. Hay evidencia que muestra que la maquinaria del silenciamiento subtelomérico puede estar detectando señales extracelulares. En C. glabrata, la proteína Sir2 (una desacetilasa de histonas dependiente de NAD+) detecta los niveles de ácido nicotínico (el acido nicotínico es precursor de NAD+) extracelular. Al disminuir los niveles de ácido nicotínico, disminuyen también los niveles de NAD+ y Sir2 pierde su actividad, esto permite la relajación de la cromatina y la transcripción de algunos genes EPA subteloméricos. En S. cerevisiae, Sir3 se fosforila en respuesta a estrés nutricional y osmótico. No obstante, si el complejo Sir pierde su actividad, ocurre de manera consecuente una desrepresión específica de los genes silenciados. Con base en estos antecedentes nos propusimos determinar si Sir3 y Sir4, pudieran también ser blancos de señales ambientales. Para caracterizar a éstas proteínas diseñamos una serie de herramientas moleculares, que nos permitirán etiquetar a las proteínas Sir3 y Sir4 con diferentes epítopes, y determinar si estas proteínas Sir son blancos de señales extracelulares.""In recent years, Candida glabrata has emerged as an opportunistic fungal pathogen, especially in individuals with weakened immune systems. C. glabrata is able to adhere to cultured epithelial cells and this adherence is mediated by the Epa cell wall proteins. These cell wall proteins are encoded by a family of around 20 paralogue genes, which are localized mainly in subtelomeric regions. Subtelomeric regions are subject to chromatin-based transcriptional silencing. The proteins involved in subtelomeric silencing are the Sir complex (Sir2, Sir3 and Sir4), Rap1, Ku70, Ku80 and Rif1. Mutations in any of the genes that encode for subtelomeric silencing machinery, render the yeast hyperadherente and resistant to oxidative stress. Adherence and resistance to oxidative stress might be coregulated in this fungal pathogen by a single global regulatory system, subtelomeric silencing. Subtelomeric silencing might be able to sense and respond to environmental signals, thus reprogramming the expression of specific subtelomeric genes. There is evidence that demostrates that the silencing machinery could bethe target of extracelullar signals. In C. glabrata, Sir2 (a NAD+ dependent histone deacetylase) detects the levels of nicotinic acid (NA, NAD+ precursor) as an extracellular signal. Reduced levels of NA, causes reduced levels of NAD+, thus Sir2 loses its activity, allowing the relaxation of the chromatin, and transcription of subtelomeric genes like the EPA genes, which then become hyperadherent. In S. cerevisiae, Sir3 is phosphorylated in response to nutritional and osmotic stress, which causes loss of activity of the Sir complex, and consequently a specific derepression of silenced genes. Based on these findings, we sought to determine whether Sir3p and Sir4, might also be targets of environmental cues. To further understand the role of Sir3 and Sir4, we designed a series of vectors to epitope-tagged these proteins. We envisage that such specific antibodies, would allow us to determine if Sir3 and/or Sir4 are in fact detecting environmental signals.
Grahp and automata in arterial vascular tree of the kidney
Get PDF"The renal vascular development is not known and occurs through two mechanisms that sometimes overlap: vasculogenesis and angiogenesis. Here we only consider growth through angiogenesis, i.e., the arterial vascular tree of the kidney. There are two types of vascular angiogenesis in development: sprouting and splitting an- giogenesis. We study these processes through mathematical tools. The graphs and automatas allow modeling the vascular growth, can generated tree structures by incorporating the physiological laws of the arterial branching. That is, the graph prescribes topology of the vascular tree and the automaton can include the rule of dynamics in the phenomena of vascularization.
Ultrasensitive quantitation of human papillomavirus type 16 E6 oncogene sequences by nested real time PCR
Get PDF"Background We have developed an ultrasensitive method based on conventional PCR preamplification followed by nested amplification through real time PCR (qPCR) in the presence of the DNA intercalating agent EvaGreen. Results Amplification mixtures calibrated with a known number of pHV101 copies carrying a 645 base pair (bp)-long insert of the human papillomavirus type 16 (HPV16) E6 oncogene were used to generate the E6-1 amplicon of 645 bp by conventional PCR and then the E6-2 amplicon of 237 bp by nested qPCR. Direct and nested qPCR mixtures for E6-2 amplification corresponding to 2.5 × 102-2.5 × 106 initial pHV101 copies had threshold cycle (Ct) values in the ranges of 18.7-29.0 and 10.0-25.0, respectively. The Ct of qPCR mixtures prepared with 1/50 volumes of preamplified mixtures containing 50 ng of DNA of the SiHa cell line (derived from an invasive cervical cancer with one HPV16 genome per cell) was 19.9. Thermal fluorescence extinction profiles of E6-2 amplicons generated from pHV101 and SiHa DNA were identical, with a peak at 85.5°C. Conclusions Our method based on conventional preamplification for 15 cycles increased 10,750 times the sensitivity of nested qPCR for the quantitation of the E6 viral oncogene and confirmed that the SiHa cell line contains one E6-HPV16 copy per cell.