The development of a protocol for the analysis of genetic expression through «differential display», as a means to reducing the number of false positives

Entre los métodos empleados para los análisis de la expresión de genes, el método de "differentialdisplay" ha sido ampliamente utilizado y, a pesar del uso extendido de los "microarrays", es aún unmétodo válido para el análisis con muestras cuyo transcriptoma es desconocido. Con el objeto de reducirel elevado número de falsos positivos que genera esta técnica, hemos optimizado el protocolo parareducir la posibilidad de generar falsos positivos. En primer lugar, hemos marcado radiactivamente elcebador oligo-dT con lo que los fragmentos de DNA identificados son extremos 3'-UTR de RNAm. Pormuestra hemos realizado dos transcripciones inversas y dos reacciones de PCR en cada una de ellas. Paraseleccionar un fragmento de DNA, debía estar diferencialmente expresado en las 4 reacciones de PCR.Por último, todos los fragmentos fueron clonados y secuenciados por triplicado. Estas modificacionesal protocolo nos ha permitido identificar 5 genes expresados diferencialmente entre células epitelialesde intestino en estado proliferativo y diferenciado.The analysis of genetic expression, the differential display (DD) method has been widely used, but inspite of the extensive use of the «microarrays» method, it is still to be considered as a valid methodfor the analysis of samples whose transcriptone is not known. In this work, an attempt has been madeto reduce the high number of false positives generated by this technique by optimising method protocol.As a preliminary step, we radioactively marked the oligo dT primer with which the fragments ofidentified DNA were extreme 3'-UTR of mRNA. For each sample two inverse transcriptions and twoPCR reactions were performed. Only fragments of DNA that are expressed differentially in all 4 PCRreactions should be selected. Finally, all of the fragments were cloned and sequenced in triplicate. Theseprotocol modifications have allowed us to identify 5 differentially expressed genes, in intestinal epithelialcells in both proliferative and differentiated states

The importance of comparative phylogeography in diagnosing introduced species: a lesson from the seal salamander, Desmognathus monticola

<p>Abstract</p> <p>Background</p> <p>In most regions of the world human influences on the distribution of flora and fauna predate complete biotic surveys. In some cases this challenges our ability to discriminate native from introduced species. This distinction is particularly critical for isolated populations, because relicts of native species may need to be conserved, whereas introduced species may require immediate eradication. Recently an isolated population of seal salamanders, <it>Desmognathus monticola</it>, was discovered on the Ozark Plateau, ~700 km west of its broad continuous distribution in the Appalachian Mountains of eastern North America. Using Nested Clade Analysis (NCA) we test whether the Ozark isolate results from population fragmentation (a natural relict) or long distance dispersal (a human-mediated introduction).</p> <p>Results</p> <p>Despite its broad distribution in the Appalachian Mountains, the primary haplotype diversity of <it>D. monticola </it>is restricted to less than 2.5% of the distribution in the extreme southern Appalachians, where genetic diversity is high for other co-distributed species. By intensively sampling this genetically diverse region we located haplotypes identical to the Ozark isolate. Nested Clade Analysis supports the hypothesis that the Ozark population was introduced, but it was necessary to include haplotypes that are less than or equal to 0.733% divergent from the Ozark population in order to arrive at this conclusion. These critical haplotypes only occur in < 1.2% of the native distribution and NCA excluding them suggest that the Ozark population is a natural relict.</p> <p>Conclusion</p> <p>Our analyses suggest that the isolated population of <it>D. monticola </it>from the Ozarks is not native to the region and may need to be extirpated rather than conserved, particularly because of its potential negative impacts on endemic Ozark stream salamander communities. Diagnosing a species as introduced may require locating nearly identical haplotypes in the known native distribution, which may be a major undertaking. Our study demonstrates the importance of considering comparative phylogeographic information for locating critical haplotypes when distinguishing native from introduced species.</p

Alimentación de las ranas pardas, Rana gr. temporaria, en el circo de Piedrafita, (Pirineos, España)

Feeding of high Pyrenean mountain brown frogs were studied in the pre-wintering period. The main prey is beetles (44'97o), two-winged flies (12'9%) and bees, wasps, ants, etc (11'4%). For the whole of the frogs, the most frequent prey size is the small (smaller than 10 mm). Big frogs tend to consume bigger prey in not ,o. many quantity, while small frogs consume more quantity of preys but smaller in size. From prey way of life, frogs eat mainly terrestrial and aerial preys, although capture of aquatic prey was observed (11'5%) and these preys were eaten inside the water.Se ha estudiado la dieta de las ranas pardas en alta montaña pirenaica durante el periodo pre-invernal. Las ranas consumen principalmente coleópteros (44'9%), dípteros (12'9%) e himenópteros (11'4%). Para el conjunto de todas las ranas estudiadas, las presas más consumidas son las de pequeño tamaño (menores de 10 mm). Las ranas de mayor talla tienden a consumir presas más grandes y en poca cantidad, mientras que las ranas pequeñas consumen más presas aunque de pequeño tamaño. En cuanto a forma de vida las presas más consumidas son terrestres y aéreas, aunque también se ha comprobado la captura de presas acuáticas (11'5%) que son consumidas por las ranas dentro del agua

Benzoxazinoids in wheat allelopathy – From discovery to application for sustainable weed management

Financiado para publicación en acceso aberto: Universidade de Vigo/CISUGAllelopathic activity of wheat (Triticum aestivum L.) has previously been associated with the production of phenolic acids and flavonoids (PAF), benzoxazinones (BXZs) and phenoxazinones (PXZs). The biosynthesis of BXZs is closely regulated during cereal growth, with accumulation highest in young tissues with variation associated with genotype and environmental conditions. This review is focused on BXZ metabolites and their impact on germination, seedling growth and physiological, biochemical, transcriptional and proteome traits of surrounding plants and weeds. The major pathways employed by plants for benzoxazinoid detoxification involve hydroxylation and glucosylation and polymerisation of intermediates in these pathways. Allelochemicals from various wheat genotypes have been shown to inhibit the growth of selected weed species, including Bromus japonicus, Chenopodium album, Portulaca oleracea, Avena fatua and Lolium rigidum. Wheat allelopathy is potentially exploited from the standpoint of crop mulches, incorporation of crop residues, tissue disruption, intercropping with allelopathic cultivars and application of aqueous wheat extracts. BXZs have been shown to suppress the growth and development of certain agricultural pests, including insects, fungal pathogens, and weeds. Many native plants, fungi and insect herbivores inherently possess varying tolerance levels towards BXZs. However, other BXZ- susceptible species are adversely impacted by elevated BXZ levels in crop plants. Thus, considerations for the selection and breeding of wheat genotypes possessing enhanced defensive ability via elevated BXZ contents are discussed. Here, these objectives are reconsidered with a focus on co-evolutionary aspects and their potential impacts on biodiversity in the agroecosystems under study. For future breeding efforts to be successful, it is important to take such potential adverse environmental impacts into account, in combination with an increased focus on enhancing beneficial allelopathic effects within agricultural systems

Australian Sphingidae – DNA Barcodes Challenge Current Species Boundaries and Distributions

© 2014 Rougerie et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The attached file is the published version of the article

RNASeq analysis reveals biological processes governing the clinical behaviour of endometrioid and serous endometrial cancers

BACKGROUND: Endometrial carcinoma comprises a group of tumors with distinct histologic and molecular features, and clinical behavior. Here we sought to define the biological processes that govern the clinical behavior of endometrial cancers. METHODS: Sixteen prototype genes representative of different biological processes that would likely play a role in endometrial and other hormone-driven cancers were defined. RNA-sequencing gene expression data from 323 endometrial cancers from The Cancer Genome Atlas were used to determine the transcription module of each prototype gene. The expression of prototype genes and modules and their association with outcome was assessed in univariate and multivariate survival analyses. The association of MSH6 expression with outcome was validated in an independent cohort of 243 primary endometrial cancers using immunohistochemistry. RESULTS: We observed that the clinical behavior of endometrial carcinomas as a group was associated with hormone receptor signaling, PI3K pathway signaling and DNA mismatch repair processes. When analyzed separately, in endometrioid carcinomas, hormone receptor, PI3K and DNA mismatch repair modules were significantly associated with outcome in univariate analysis, whereas the clinical behavior of serous cancers was likely governed by apoptosis and Wnt signaling. Multivariate survival analysis revealed that MSH6 expression was associated with outcome of endometrial cancer patients independently from traditional prognostic clinicopathologic parameters, which was confirmed in an independent cohort at the protein level. CONCLUSION: Endometrioid and serous endometrial cancers are underpinned by distinct molecular pathways. MSH6 expression levels may be associated with outcome in endometrial cancers as a group

Clinical Characteristics and Long-term outcomes of patients undergoing combined heart-kidney transplantation: a single-center experience

[Abstract] Background. The purpose of the study was to describe clinical characteristics and long-term survival of patients undergoing combined heart-kidney transplant in a single center. Methods. We conducted a retrospective analysis of 22 consecutive patients who underwent combined heart-kidney transplant at our institution between 1995 and 2013. Long-term outcomes were analyzed by means of the Kaplan-Meier method. Results. Four patients underwent re-do transplant (2 cardiac re-transplants, 1 kidney re-transplant, and 1 combined heart-kidney re-transplant). Most frequent underlying cardiac conditions were coronary artery disease (54%), dilated cardiomyopathy (23%), and chronic rejection of a previous heart graft (18%). Known causes of chronic renal dysfunction were nephroangioesclerosis (23%), drug-related toxicity (14%), and Wegener granulomatosis (5%). Non-specified chronic renal dysfunction was present in 50% patients. In-hospital postoperative mortality rate was 5 of 22 (23%). Causes of early death were directly related to kidney transplant surgery in 4 of 5 (80%) patients. Among the remaining 17 patients who surmounted the postoperative period, long-term survival rates 1 year, 5 years, and 10 years after HKT were 88%, 82%, and 65%, respectively. Over a mean follow-up of 6.7 ± 6.4 years, cumulative incidences of cytomegalovirus infection, coronary allograft vasculopathy, malignancy, and acute cardiac graft rejection were 41%, 6%, 24%, and 41%, respectively. There was no episode of acute renal graft rejection. At the end of follow-up, all survivors (n = 11) were in functional New York Heart Association class I. Mean creatinine serum level was 1.68 mg/dL. Conclusions. In our experience, combined heart-kidney transplant is a feasible therapeutic option that yielded favorable long-term outcomes, with a low cumulative incidence of cardiac graft dysfunction. These results were obtained at the expense of a significant risk of early postoperative mortality, which was mainly related to complications of kidney transplant surgery