Taxonomy, phylogeny and molecular epidemiology of Echinococcus multilocularis: From fundamental knowledge to health ecology.

Alveolar echinococcosis, caused by the tapeworm Echinococcus multilocularis, is one of the most severe parasitic diseases in humans and represents one of the 17 neglected diseases prioritised by the World Health Organisation (WHO) in 2012. Considering the major medical and veterinary importance of this parasite, the phylogeny of the genus Echinococcus is of considerable importance; yet, despite numerous efforts with both mitochondrial and nuclear data, it has remained unresolved. The genus is clearly complex, and this is one of the reasons for the incomplete understanding of its taxonomy. Although taxonomic studies have recognised E. multilocularis as a separate entity from the Echinococcus granulosus complex and other members of the genus, it would be premature to draw firm conclusions about the taxonomy of the genus before the phylogeny of the whole genus is fully resolved. The recent sequencing of E. multilocularis and E. granulosus genomes opens new possibilities for performing in-depth phylogenetic analyses. In addition, whole genome data provide the possibility of inferring phylogenies based on a large number of functional genes, i.e. genes that trace the evolutionary history of adaptation in E. multilocularis and other members of the genus. Moreover, genomic data open new avenues for studying the molecular epidemiology of E. multilocularis: genotyping studies with larger panels of genetic markers allow the genetic diversity and spatial dynamics of parasites to be evaluated with greater precision. There is an urgent need for international coordination of genotyping of E. multilocularis isolates from animals and human patients. This could be fundamental for a better understanding of the transmission of alveolar echinococcosis and for designing efficient healthcare strategies.

Identification and characterization of a molecular marker in epithelial ovarian cancer

Carcinomas that arise from the ovarian surface epithelium represent a great challenge in gynecologic oncology. Although the prognosis of ovarian cancer is influenced by many factors capable of predicting clinical outcome, including tumor stage, pathological grade, and amount of residual disease following primary surgery, the biological aspects of ovarian cancer are not completely understood, thus implying that there may be other predictive indicators that could be used independently or in conjunction with these factors to provide a clearer clinical picture. The identification of additional markers with biological relevance is desirable. To identify disease-associated peptides, a phage display random peptide library was used to screen immunoglobulins derived from a patient with ovarian cancer. One peptide was markedly enriched following three rounds of affinity selection. The presence of autoantibodies against the peptide was examined in a panel of ovarian cancer patients. Stage IV patients exhibited a high percentage of positive reactivity (59%). This was in contrast to stage III patients, who only displayed 7% positive reactivity. Antibodies against the peptide were affinity purified, and heat-shock protein 90 (Hsp90) was identified as the corresponding autoantigen. The expression profile of the identified antigen was determined. Hsp90 was expressed in all sections examined regardless of degree of anaplasia. This thesis shows that utilizing the humoral response to ovarian cancer can be used to identify a tumor antigen in ovarian cancer. The data show that certain antigens may be expressed in ovarian tumors independent of the disease stage or grade, whereas circulating antibodies against such epitopes are only found in a subset of patients. ^

Identifying and Targeting Molecular Deregulations in Uterine Leiomyosarcoma: A Role for mTOR Inhibition-Based Combination Therapies

Uterine leiomyosarcoma (ULMS) is an aggressive malignancy characterized by marked chemoresistance, frequent relapses, and poor outcome. Despite efforts to improve survival over the past several decades, only minimal advances have been made. Hence, there is an urgent and unmet need for better understanding of the molecular deregulations that underlay ULMS and development of more effective therapeutic strategies. This work identified several common deregulations in a large (n=208) tissue microarray of ULMS compared to GI smooth muscle, myometrium, and leiomyoma controls. Our results suggest that significant loss of smooth muscle and gynecological differentiation markers is common in ULMS, a finding that could help render improved ULMS diagnosis, especially for advanced disease. Similarly to reports in other malignancies, we found that several cancer-related proteins were differentially expressed; these could be useful together as biomarkers for ULMS. Notably, we identified significant upregulation and overexpression of the mTOR pathway in ULMS, examined the possible contribution of tyrosine kinase receptor deregulation promoting mTOR activation, and unraveled a role for pS6RP and p4EBP1 as molecular disease prognosticators. The significance of mTOR activation in ULMS and its potential as a therapeutic target were further investigated. Rapamycin abrogated ULMS cell growth and cell cycle progression in vitro but induced only sight growth delay in vivo. Given that effective mTOR therapies likely require combination mTOR blockade with inhibition of other targets, coupled with recent observations suggesting that Aurora A kinase (Aurk A) deregulations commonly occur in ULMS, the preclinical impact of dually targeting both pathways was evaluated. Combined therapy with rapamycin (an mTORC1 inhibitor) and MLN8237 (an investigational Aurk A inhibitor) profoundly and synergistically abrogated ULMS growth in vitro. Interestingly, the superior effects were noted only when MLN8237 was pre-administered. This novel therapeutic combination and scheduling regimen resulted in marked tumor growth inhibition in vivo. Together, these data support further exploration of dual mTOR and Aurk A blockade for the treatment of human ULMS.

Molecular Mechanisms of Vascular Disease in Patients with Rare Variants in MYH11

Thoracic aortic aneurysms and dissections (TAAD) are the primary disease affecting the thoracic ascending aorta, with an incidence rate of 10.4/100,000. Although about 20% of patients carry a mutation in a single gene that causes their disease, the remaining 80% of patients may also have genetic factors that increase their risk for developing TAAD. Many of the genes that predispose to TAAD encode proteins involved in smooth muscle cell (SMC) contraction and the disease-causing mutations are predicted to disrupt contractile function. SMCs are the predominant cell type in the ascending aortic wall. Mutations in MYH11, encoding the smooth muscle specific myosin heavy chain, are a rare cause of inherited TAAD. However, rare but recurrent non-synonymous variants in MYH11 are present in the general population but do not cause inherited TAAD. The goal of this study was to assess the potential role of these rare variants in vascular diseases. Two distinct variants were selected: the most commonly seen rare variant, MYH11 R247C, and a duplication of the chromosomal region spanning the MYH11 locus at 16p13.1. Genetic analyses indicated that both of these variants were significantly enriched in patients with TAAD compared with controls. A knock-in mouse model of the Myh11 R247C rare variant was generated, and these mice survive and reproduce normally. They have no structural abnormalities of the aorta or signs of aortic disease, but do have decreased aortic contractility. Myh11R247C/R247C mice also have increased proliferative response to vascular injury in vivo and increased proliferation of SMCs in vitro. Myh11R247C/R247C SMCs have decreased contractile gene and protein expression and are dedifferentiated. In fibroblasts, myosin force generation is required for maturation of focal adhesions, and enhancers of RhoA activity replace enhancers of Rac1 activity as maturation occurs. Consistent with these previous findings, focal adhesions are smaller in Myh11R247C/R247C SMCs, and there is decreased RhoA activation. A RhoA activator (CN03) rescues the dedifferentiated phenotype of Myh11R247C/R247C SMCs. Myh11R247C/R247C mice were bred with an existing murine model of aneurysm formation, the Acta2-/- mouse. Over time, mice carrying the R247C allele in conjunction with heterozygous or homozygous loss of Acta2 had significantly increased aortic diameter, and a more rapid accumulation of pathologic markers. These results suggest that the Myh11 R247C rare variant acts as a modifier gene increasing the risk for and severity of TAAD in mice. In patients with 16p13.1 duplications, aortic MYH11 expression is increased, but there is no corresponding increase in smooth muscle myosin heavy chain protein. Using SMCs that overexpress Myh11, we identified alterations in SMC phenotype leading to excessive protein turnover. All contractile proteins, not just myosin, are affected, and the proteins are turned over by autophagic degradation. Surprisingly, these cells are also more contractile compared with wild-type SMCs. The results described in this dissertation firmly establish that rare variants in MYH11 significantly affect the phenotype of SMCs. Further, the data suggests that these rare variants do increase the risk of TAAD via pathways involving altered SMC phenotype and contraction. Therefore, this study validates that these rare genetic variants alter vascular SMCs and provides model systems to explore the contribution of rare variants to disease.

Caracterización molecular de variedades de vid ( Vitis vinifera L.) de calidad enológica por marcadores microsatelitales

Siete variedades de vid empleadas en Argentina para la elaboración de vinos de alta gama fueron caracterizadas molecularmente a través del empleo de microsatélites. Seis de los 8 pares de cebadores usados amplificaron patrones de bandas reproducibles. El número de alelos detectados por locus varió entre 5 y 10, con un total de 42 alelos, registrándose desde 1 a 7 alelos únicos. El número de genotipos microsatélites encontrados para cada locus osciló entre 3 y 7, con un total de 28. Las variedades Tempranillo y Chenin mostraron 6 y 5 genotipos «únicos», respectivamente, con los 6 loci analizados. La heterocigosidad observada varió entre 42,9 y 100 %, mientras que la heterocigosidad esperada osciló entre 64,3 y 87,8 %. El locus más informativo fue VrZAG79 (con un contenido de información polimórfica de 84,9 % y un número de alelos efectivos de 8,17) mientras que el menos informativo fue VrZAG62. La probabilidad acumulada de obtener genotipos idénticos fue de 7,68 x 10-05 indicando que, mediante el uso combinado de estos 6 cebadores microsatélites se pueden discriminar todas las variedades ensayadas, con alto grado de certeza. El análisis de agrupamiento por el método UPGMA separó claramente las variedades francesas (Merlot, Pinot Noir y Cabernet Sauvignon) y Syrah de los cepajes Tempranillo y Bonarda. Esta técnica podría ofrecerse como servicio a viveristas y productores vitícolas interesados en garantizar la identidad genética de sus materiales comercializados.

Genetic diversity among alfalfa genotypes (Medicago sativa L.) of non-dormant cultivars using SSR markers and agronomic traits

The aim of this study was to assess genetic diversity among 40 alfalfa (Medicago sativa L.) genotypes of different non-dormant (FD=8) cultivars. Biomass yield, regrowth speed and reaction to spring black stem, lepto leaf spot, and rust were evaluated. Analyses of variances were performed using a mixed model to examine the agronomic variation among individuals. A principal component analysis on standardized agronomic data was performed. Agronomic data were also used to calculate Gower's distance and UPGMA algorithm. For the molecular analysis, six SSR markers were evaluated and 84 alleles were identified. The genetic distance was estimated using standard Nei's distance. Average standard genetic diversity was 0.843, indicating a high degree of variability among genotypes. Finally, a generalized procrustes analysis was performed to calculate the correlation between molecular and agronomic distance, indicating a 65.4% of consensus. This value is likely related to the low number of individuals included in the study, which might have underestimated the real phenotypic variability among genotypes. Despite the low number of individuals and SSR markers analyzed, this study provides a baseline for future diversity studies to identify genetically distant alfalfa individuals or cultivars.

Percentage of cultured cell-populations that stained positively and/or negatively for apoptotic markers cleaved caspase-3 and cleaved PARP, following DNA damage treatments induced by doxorubicin and TNF-alpha co-treatments

The present dataset contains the source data for Figure 2B of Tentner et al. (2012). The data shows the percentage of cultured cell-populations that stained positively and/or negatively for apoptotic markers cleaved caspase-3 and cleaved PARP, following DNA damage treatments induced by various doses of doxorubicin (0, 2 and 10 µmole/L) in the presence (100 ng/mL) or absence (0 ng/mL) of TNF-alpha co-treatment. For the six treatment conditions investigated, cell counts were made by flow cytometry at times 6, 12, 24, and 48 h following treatment; CULTURE DETAILS: U2OS cells were obtained from ATCC were maintained at 21% oxygen and 5% CO2 in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum, penicillin, streptomycin, 2mM L-glutamine, and used within 15-20 passages. The first thymidine block was released by washing the plates three times with PBS, and incubating them in fresh thymidine-free media for 12 h. A second thymidine block was then performed by re-addition of thymidine to 2.5 mM followed by incubation for an additional 18 h. Media was aspirated, plates were washed 3 with PBS, and replaced with fresh media in the presence or absence of 10 mM aphidicolin; ANALYSIS DETAILS: See supplementary journal publication; RESULT: The authors of the supplementary journal publication conclude that TNF enhances dose-dependent cell death following doxorubicin-induced DNA damage with minimal affect on dose-dependent cell-cycle arrest.

Effectiveness of AFLPs and retrotransposon-based markers for the identification of portuguese grapevine cultivars and clones

Grapevine germplasm, including 38 of the main Portuguese cultivars and three foreign cultivars, Pinot Noir, Pinot Blanc and Chasselas, used as a reference, and 37 true-to-type clones from the Alvarinho, Arinto, Loureiro, Moscatel Galego Branco, Trajadura and Vinhão cultivars were studied using AFLP and three retrotransposon-based molecular techniques, IRAP, REMAP and SSAP. To study the retrotransposon-based polymorphisms, 18 primers based on the LTR sequences of Tvv1, Gret1 and Vine-1 were used. In the analysis of 41 cultivars, 517 IRAP, REMAP, AFLP and SSAP fragments were obtained, 83% of which were polymorphic. For IRAP, only the Tvv1Fa primer amplified DNA fragments. In the REMAP analysis, the Tvv1Fa-Ms14 primer combination only produced polymorphic bands, and the Vine-1 primers produced mainly ISSR fragments. The highest number of polymorphic fragments was found for AFLP. Both AFLP and SSAP showed a greater capacity for identifying clones, resulting in 15 and 9 clones identified, respectively. Together, all of the techniques allowed for the identification of 54% of the studied clones, which is an important step in solving one of the challenges that viticulture currently faces.

Biodiversidad de frijol (Phaseolus vulgaris L.) en Honduras. Caracterización agromorfológica, molecular y ecogeográfica

En el presente trabajo se ha llevado a cabo un estudio de la biodiversidad del frijol común (Phaseolus vulgaris L.) en Honduras, que es el segundo de los cultivos de granos básicos en importancia. Dicho estudio se ha realizado mediante una caracterización agromorfológica, molecular y ecogeográfica en una selección de 300 accesiones conservadas en el banco de germoplasma ubicado en la Escuela Agrícola Panamericana (EAP) El Zamorano, y que se colectaron en 13 departamentos del país durante el periodo de 1990 a 1994. Estas accesiones fueron colectadas cuatro años antes del acontecimiento del huracán Mitch, el cual a su paso afectó al 96% del área total cultivable en su momento, lo cual nos hace considerar que la biodiversidad de razas locales (landraces) de frijol común existentes in situ fueron severamente afectadas. Los trabajos dirigidos a analizar la biodiversidad de razas locales de frijol común en Honduras son escasos, y este trabajo se constituye como el primero que incluye una amplia muestra a ser estudiada a través de una caracterización en tres aspectos complementarios (agromorfológico, molecular y ecogeográfico). Se evaluaron 32 caracteres agromorfológicos, 12 cuantitativos y 20 cualitativos, en distintas partes de la planta. Se establecieron las correlaciones entre los caracteres agromorfológicos y se elaboró un dendrograma con los mismos, en el que se formaron ocho grupos, en parte relacionados principalmente con los colores y tamaños de la semilla. Mediante el análisis de componentes principales se estudiaron los caracteres de más peso en cada uno de los tres primeros componentes. Asimismo, se estudiaron las correlaciones entre caracteres, siendo las más altas la longitud y anchura de la hoja, días a madurez y a cosecha y longitud y peso de semilla. Por otra parte, el mapa de diversidad agromorfológica mostró la existencia de tres zonas con mayor diversidad: en el oeste (en los departamentos de Santa Bárbara, Lempira y Copán), en el centro-norte (en los departamentos de Francisco Morazán, Yoro y Atlántida) y en el sur (en el departamento de El Paraíso y al sur de Francisco Morazán). Para la caracterización molecular partimos de 12 marcadores de tipo microsatélite, evaluados en 54 accesiones, que fueron elegidas por constituir grupos que compartían un mismo nombre local. Finalmente, se seleccionaron los cuatro microsatélites (BM53, GATS91, BM211 y PV-AT007) que resultaron ser más polimórficos e informativos para el análisis de las 300 accesiones, con los que se detectaron un total de 119 alelos (21 de ellos únicos o privados de accesión) y 256 patrones alélicos diferentes. Para estudiar la estructura y relaciones genéticas en las 300 accesiones se incluyeron en el análisis tres controles o accesiones de referencia, pertenecientes dos de ellas al acervo genético Andino y una al Mesoamericano. En el dendrograma se obtuvieron 25 grupos de accesiones con idénticas combinaciones de alelos. Al comparar este dendrograma con el de caracteres agromorfológicos se observaron diversos grupos con marcada similitud en ambos. Un total de 118 accesiones resultaron ser homogéneas y homocigóticas, a la vez que representativas del grupo de 300 accesiones, por lo que se analizaron con más detalle. El análisis de la estructura genética definió la formación de dos grupos, supuestamente relacionados con los acervos genéticos Andino (48) y Mesoamericano (61), y un reducido número de accesiones (9) que podrían tener un origen híbrido, debido a la existencia de un cierto grado de introgresión entre ambos acervos. La diferenciación genética entre ambos grupos fue del 13,3%. Asimismo, 66 de los 82 alelos detectados fueron privados de grupo, 30 del supuesto grupo Andino y 36 del Mesoamericano. Con relación al mapa de diversidad molecular, presentó una distribución bastante similar al de la diversidad agromorfológica, detectándose también las zonas de mayor diversidad genética en el oeste (en los departamentos de Lempira y Santa Bárbara), en el centro-norte (en los departamentos de Yoro y Atlántida) y en el sur (en el departamento de El Paraíso y al sur de Francisco Morazán). Para la caracterización ecogeográfica se seleccionaron variables de tipo bioclimático (2), geofísico (2) y edáfico (8), y mediante el método de agrupamiento de partición alrededor de los medoides, la combinación de los grupos con cada uno de los tres tipos de variables definió un total de 32 categorías ecogeográficas en el país, detectándose accesiones en 16 de ellas. La distribución de las accesiones previsiblemente esté relacionada con la existencia de condiciones más favorables al cultivo de frijol. En el mapa de diversidad ecogeográfica, nuevamente, se observaron varias zonas con alta diversidad tanto en el oeste, como en el centro-norte y en el sur del país. Como consecuencia del estudio realizado, se concluyó la existencia de una marcada biodiversidad en el material analizado, desde el punto de vista tanto agromorfológico como molecular. Por lo que resulta de gran importancia plantear la conservación de este patrimonio genético tanto ex situ, en bancos de germoplasma, como on farm, en las propias explotaciones de los agricultores del país, siempre que sea posible. ABSTRACT In the present work we have carried out a study of the biodiversity of the common bean (Phaseolus vulgaris L) in Honduras, which is the second of the basic grain crops in importance. This study was conducted through agro-morphological, molecular and ecogeographical characterization of a selection of 300 accessions conserved in the genebank located in the ‘Escuela Agrícola Panamericana (EAP) El Zamorano’ that were collected in 13 departments of the country during the 1990 to 1994 period. These accessions were collected four years before the occurrence of Mitch hurricane, which affected 96% of the total cultivable area at the time, which makes us to consider that the biodiversity of local landraces of common bean existing in situ were severely affected. The work aimed to analyze the biodiversity of local races of common bean in Honduras are scarce, and this work constitutes the first to include a large sample to be studied through a characterization on three complementary aspects (agromorphological, molecular and ecogeographical). Thirty two agromorphological characters, 12 quantitative and 20 qualitative, in various parts of the plant were evaluated. Correlations between agromorphological characters were established and a dendrogram with them was constructed, in which eight groups were formed, in part mainly related to the colors and sizes of the seeds. By principal component analysis the characters with more weight in each of the first three components were studied. Also, correlations between characters were studied, the highest of them being length and leaf width, days to maturity and harvest, and seed length and weight. Moreover, the map of agromorphological diversity showed the existence of three areas with more diversity: the west (departments of Santa Barbara, Copan and Lempira), the center-north (departments of Francisco Morazán, Yoro and Atlántida) and the south (department of El Paraiso and south of Francisco Morazán). For molecular characterization we started with 12 microsatellite markers, evaluated in 54 accessions, which were chosen because they formed groups that shared the same local name. Finally, four microsatellites (BM53, GATS91, BM211 and PV-AT007) were selected for the analysis of 300 accessions, since they were the most polymorphic and informative. They gave a total of 119 alleles (21 of them unique or private for the accession) and 256 different allelic patterns. To study the structure and genetic relationships in the 300 accessions, three controls or accessions of reference were included in the analysis: two of them belonging to the Andean gene pool and one to the Mesoamerican. In the dendrogram, 25 accession groups with identical allele combinations were obtained. Comparing this dendrogram to the obtained with agromorphological characters, several groups with marked similarity in both were observed. A total of 118 accessions were homozygous and homogeneous, while representing the group of 300 accessions, therefore they were analyzed in more detail. The analysis of the genetic structure defined the formation of two groups, supposedly related to the Andean (48) and the Mesoamerican (61) gene pools, and a small number of accessions (9) which may have a hybrid origin, due to the existence of some degree of introgression between both gene pools. Genetic differentiation between both groups was 13.3%. Also, 66 of the 82 detected alleles were private or unique for the group, 30 of the supposed Andean group and 36 of the Mesoamerican. With relation to the map of molecular diversity, it showed a quite similar distribution to the agromorphological, also detecting the areas of greatest genetic diversity in the west (departments of Lempira and Santa Bárbara), in the center-north (departments Atlántida and Yoro) and in the south (departments of El Paraíso and south of Francisco Morazán). For the ecogeographical characterization, bioclimatic (2), geophysical (2) and edaphic (8) variables were selected, and by the method of clustering partition around the medoids, the combination of the groups to each of the three types of variables defined a total of 32 ecogeographical categories in the country, having accessions in 16 of them. The distribution of accessions is likely related to the existence of more favorable conditions for the cultivation of beans. The map of ecogeographical diversity, again, several areas with high diversity both in the west and in the center-north and in the south of the country were observed. As a result of study, the existence of marked biodiversity in the analyzed material was concluded, both from the agromorphological and from the molecular point of view. Consequently it is very important to propose the conservation of this genetic heritage both ex situ, in genebanks, as on farm, in the holdings of the farmers of the country, whenever possible.

Molecular tools to improve chestnut management: El Bierzo as a case study

The European chestnut (Castanea sativa Mill.) is a multipurpose species that has been widely cultivated around the Mediterranean basin since ancient times. New varieties were brought to the Iberian Peninsula during the Roman Empire, which coexist since then with native populations that survived the last glaciation. The relevance of chestnut cultivation has being steadily growing since the Middle Ages, until the rural decline of the past century put a stop to this trend. Forest fires and diseases were also major factors. Chestnut cultivation is gaining momentum again due to its economic (wood, fruits) and ecologic relevance, and represents currently an important asset in many rural areas of Europe. In this Thesis we apply different molecular tools to help improve current management strategies. For this study we have chosen El Bierzo (Castile and Leon, NW Spain), which has a centenary tradition of chestnut cultivation and management, and also presents several unique features from a genetic perspective (next paragraph). Moreover, its nuts are widely appreciated in Spain and abroad for their organoleptic properties. We have focused our experimental work on two major problems faced by breeders and the industry: the lack of a fine-grained genetic characterization and the need for new strategies to control blight disease. To characterize with sufficient detail the genetic diversity and structure of El Bierzo orchards, we analyzed DNA from 169 trees grafted for nut production covering the entire region. We also analyzed 62 nuts from all traditional varieties. El Bierzo constitutes an outstanding scenario to study chestnut genetics and the influence of human management because: (i) it is located at one extreme of the distribution area; (ii) it is a major glacial refuge for the native species; (iii) it has a long tradition of human management (since Roman times, at least); and (iv) its geographical setting ensures an unusual degree of genetic isolation. Thirteen microsatellite markers provided enough informativeness and discrimination power to genotype at the individual level. Together with an unexpected level of genetic variability, we found evidence of genetic structure, with three major gene pools giving rise to the current population. High levels of genetic differentiation between groups supported this organization. Interestingly, genetic structure does not match with spatial boundaries, suggesting that the exchange of material and cultivation practices have strongly influenced natural gene flow. The microsatellite markers selected for this study were also used to classify a set of 62 samples belonging to all traditional varieties. We identified several cases of synonymies and homonymies, evidencing the need to substitute traditional classification systems with new tools for genetic profiling. Management and conservation strategies should also benefit from these tools. The avenue of high-throughput sequencing technologies, combined with the development of bioinformatics tools, have paved the way to study transcriptomes without the need for a reference genome. We took advantage of RNA sequencing and de novo assembly tools to determine the transcriptional landscape of chestnut in response to blight disease. In addition, we have selected a set of candidate genes with high potential for developing resistant varieties via genetic engineering. Our results evidenced a deep transcriptional reprogramming upon fungal infection. The plant hormones ET and JA appear to orchestrate the defensive response. Interestingly, our results also suggest a role for auxins in modulating such response. Many transcription factors were identified in this work that interact with promoters of genes involved in disease resistance. Among these genes, we have conducted a functional characterization of a two major thaumatin-like proteins (TLP) that belongs to the PR5 family. Two genes encoding chestnut cotyledon TLPs have been previously characterized, termed CsTL1 and CsTL2. We substantiate here their protective role against blight disease for the first time, including in silico, in vitro and in vivo evidence. The synergy between TLPs and other antifungal proteins, particularly endo-p-1,3-glucanases, bolsters their interest for future control strategies based on biotechnological approaches.

Unique molecular surface features of in vivo tolerized T cells

Differential expression of surface markers can frequently be used to distinguish functional subsets of T cells, yet a surface phenotype unique to T cells induced into an anergic state has not been described. Here, we report that CD4 T cells rendered anergic in vivo by superantigen can be identified by loss of the 6C10 T cell marker. Inoculation of Vβ8.1 T cell antigen receptor (TCR) transgenic mice with a Vβ8.1-reactive minor lymphocyte-stimulating superantigen (Mls-1a) induces tolerance to Mls-1a by clonal anergy. CD4 lymph node T cells from Mls-1a inoculated transgenic mice enriched for the 6C10− phenotype neither proliferate nor produce interleukin-2 upon TCR engagement, whereas 6C10+ CD4 T cells retain responsiveness. Analysis of T cell memory markers demonstrate that 6C10− T cells remain 3G11hi but express heterogeneous levels of CD45RB, CD62L, CD44, and the CD69 early activation marker, suggesting that T cells at various degrees of activation can be functionally anergic. These studies demonstrate that anergic T cells can be purified based on 6C10 expression permitting examination of issues concerning biochemical and biological features specific to T cell anergy.

Molecular immune responses of the mosquito Anopheles gambiae to bacteria and malaria parasites

Immune responses of the malaria vector mosquito Anopheles gambiae were monitored systematically by the induced expression of five RNA markers after infection challenge. One newly isolated marker encodes a homologue of the moth Gram-negative bacteria-binding protein (GNBP), and another corresponds to a serine protease-like molecule. Additional previously described markers that respond to immune challenge encode the antimicrobial peptide defensin, a putative galactose lectin, and a putative serine protease. Specificity of the immune responses was indicated by differing temporal patterns of induction of specific markers in bacteria-challenged larvae and adults, and by variations in the effectiveness of different microorganisms and their components for marker induction in an immune-responsive cell line. The markers exhibit spatially distinct patterns of expression in the adult female mosquito. Two of them are highly expressed in different regions of the midgut, one in the anterior and the other in the posterior midgut. Marker induction indicates a significant role of the midgut in insect innate immunity. Immune responses to the penetration of the midgut epithelium by a malaria parasite occur both within the midgut itself and elsewhere in the body, suggesting an immune-related signaling process.

Molecular cytogenetic dissection of human chromosomes 3 and 21 evolution

Chromosome painting in placental mammalians illustrates that genome evolution is marked by chromosomal synteny conservation and that the association of chromosomes 3 and 21 may be the largest widely conserved syntenic block known for mammals. We studied intrachromosomal rearrangements of the syntenic block 3/21 by using probes derived from chromosomal subregions with a resolution of up to 10–15 Mbp. We demonstrate that the rearrangements visualized by chromosome painting, mostly translocations, are only a fraction of the actual chromosomal changes that have occurred during evolution. The ancestral segment order for both primates and carnivores is still found in some species in both orders. From the ancestral primate/carnivore condition an inversion is needed to derive the pig homolog, and a fission of chromosome 21 and a pericentric inversion is needed to derive the Bornean orangutan condition. Two overlapping inversions in the chromosome 3 homolog then would lead to the chromosome form found in humans and African apes. This reconstruction of the origin of human chromosome 3 contrasts with the generally accepted scenario derived from chromosome banding in which it was proposed that only one pericentric inversion was needed. From the ancestral form for Old World primates (now found in the Bornean orangutan) a pericentric inversion and centromere shift leads to the chromosome ancestral for all Old World monkeys. Intrachromosomal rearrangements, as shown here, make up a set of potentially plentiful and informative markers that can be used for phylogenetic reconstruction and a more refined comparative mapping of the genome.

Molecular and genealogical evidence for a founder effect in Fanconi anemia families of the Afrikaner population of South Africa

Fanconi anemia (FA) is a rare, genetically heterogeneous autosomal recessive disorder associated with progressive aplastic anemia, congenital abnormalities, and cancer. FA has a very high incidence in the Afrikaner population of South Africa, possibly due to a founder effect. Previously we observed allelic association between polymorphic markers flanking the FA group A gene (FANCA) and disease chromosomes in Afrikaners. We genotyped 26 FA families with microsatellite and single nucleotide polymorphic markers and detected five FANCA haplotypes. Mutation scanning of the FANCA gene revealed association of these haplotypes with four different mutations. The most common was an intragenic deletion of exons 12–31, accounting for 60% of FA chromosomes in 46 unrelated Afrikaner FA patients, while two other mutations accounted for an additional 20%. Screening for these mutations in the European populations ancestral to the Afrikaners detected one patient from the Western Ruhr region of Germany who was heterozygous for the major deletion. The mutation was associated with the same unique FANCA haplotype as in Afrikaner patients. Genealogical investigation of 12 Afrikaner families with FA revealed that all were descended from a French Huguenot couple who arrived at the Cape on June 5, 1688, whereas mutation analysis showed that the carriers of the major mutation were descendants of this same couple. The molecular and genealogical evidence is consistent with transmission of the major mutation to Western Germany and the Cape near the end of the 17th century, confirming the existence of a founder effect for FA in South Africa.

Patterns of kinship in groups of free-living sperm whales (Physeter macrocephalus) revealed by multiple molecular genetic analyses.

Mature female sperm whales (Physeter macrocephalus) live in socially cohesive groups of 10-30, which include immature animals of both sexes, and within which there is communal care of the young. We examined kinship in such groups using analyses of microsatellite DNA, mitochondrial DNA sequence, and sex-linked markers on samples of sloughed skin collected noninvasively from animals in three groups off the coast of Ecuador. Social groups were defined through photographic identification of individuals. Each group contained about 26 members, mostly female (79%). Relatedness was greater within groups, as compared to between groups. Particular mitochondrial haplotypes were characteristic of groups, but all groups contained more than one haplotype. The data are generally consistent with each group being comprised of several matrillines from which males disperse at about the age of 6 years. There are indications of paternal relatedness among grouped individuals with different mitochondrial haplotypes, suggesting long-term associations between different matrilines.