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Metastasis is the primary cause of death for most breast cancer (BC) patients who succumb to the disease. During the hematogenous dissemination, circulating tumor cells interact with different blood components. Thus, there are microenvironmental and systemic processes contributing to cancer regulation. We have recently published that red blood cells (RBCs) that accompany circulating tumor cells have prognostic value in metastatic BC patients. RBC alterations are related to several diseases. Although the principal known role is gas transport, it has been recently assigned additional functions as regulatory cells on circulation. Hence, to explore their potential contribution to tumor progression, we characterized the proteomic composition of RBCs from 53 BC patients from stages I to III and IV, compared with 33 cancer-free controls. In this work, we observed that RBCs from BC patients showed a different proteomic profile compared to cancer-free controls and between different tumor stages. The differential proteins were mainly related to extracellular components, proteasome, and metabolism. Embryonic hemoglobins, not expected in adults' RBCs, were detected in BC patients. Besides, lysosome-associated membrane glycoprotein 2 emerge as a new RBCs marker with diagnostic and prognostic potential for metastatic BC patients. Seemingly, RBCs are acquiring modifications in their proteomic composition that probably represents the systemic cancer disease, conditioned by the tumor microenvironment.
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Neoplasias de la Mama , Células Neoplásicas Circulantes , Adulto , Humanos , Femenino , Neoplasias de la Mama/metabolismo , Células Neoplásicas Circulantes/metabolismo , Proteómica , Eritrocitos/metabolismo , Hemoglobinas/metabolismo , Biomarcadores de Tumor/metabolismo , Microambiente TumoralRESUMEN
BACKGROUND: Understanding sex determination (SD) across taxa is a major challenge for evolutionary biology. The new genomic tools are paving the way to identify genomic features underlying SD in fish, a group frequently showing limited sex chromosome differentiation and high SD evolutionary turnover. Turbot (Scophthalmus maximus) is a commercially important flatfish with an undifferentiated ZW/ZZ SD system and remarkable sexual dimorphism. Here we describe a new long-read turbot genome assembly used to disentangle the genetic architecture of turbot SD by combining genomics and classical genetics approaches. RESULTS: The new turbot genome assembly consists of 145 contigs (N50 = 22.9 Mb), 27 of them representing >95% of its estimated genome size. A genome wide association study (GWAS) identified a ~ 6.8 Mb region on chromosome 12 associated with sex in 69.4% of the 36 families analyzed. The highest associated markers flanked sox2, the only gene in the region showing differential expression between sexes before gonad differentiation. A single SNP showed consistent differences between Z and W chromosomes. The analysis of a broad sample of families suggested the presence of additional genetic and/or environmental factors on turbot SD. CONCLUSIONS: The new chromosome-level turbot genome assembly, one of the most contiguous fish assemblies to date, facilitated the identification of sox2 as a consistent candidate gene putatively driving SD in this species. This chromosome SD system barely showed any signs of differentiation, and other factors beyond the main QTL seem to control SD in a certain proportion of families.
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Peces Planos , Estudio de Asociación del Genoma Completo , Factores de Transcripción SOXB1 , Animales , Mapeo Cromosómico , Cromosomas , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Peces Planos/genética , Genoma , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismoRESUMEN
The production of Manila clam (Ruditapes philippinarum) is seriously threatened by the protistan parasite Perkinsus olseni. We characterized and compared gene expression of Manila clam haemocytes in response to P. olseni in a time-course (10 h, 24 h, 8 d) controlled laboratory challenge (LC), representing the first step of infection, and in a more complex infection in the wild (WI), using a validated oligo-microarray containing 11,232 transcripts, mostly annotated. Several immune-genes involved in NIK/NF-kappaB signalling, Toll-like receptor signalling and apoptosis were activated at LC-10 h. However, down-regulation of genes encoding lysozyme, histones, cathepsins and heat shock proteins indicated signals of immunodepression, which persisted at LC-24 h, when only down-regulated genes were detected. A rebound of haemocyte activity occurred at LC-8 d as shown by up-regulation of genes involved in cytoskeleton organization and cell survival. The WI study showed a more complex picture, and several immune-relevant processes including cytoskeleton organization, cell survival, apoptosis, encapsulation, cell redox- and lipid-homeostasis were activated, illustrating the main mechanism of host response. Our results provide useful information, including potential biomarkers, to develop strategies for controlling Manila clam perkinsosis.
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Alveolados/fisiología , Bivalvos/genética , Bivalvos/inmunología , Hemocitos/inmunología , Inmunidad Innata/genética , Transcriptoma/inmunología , Animales , Apoptosis/genética , Hemocitos/parasitología , Interacciones Huésped-Parásitos/inmunología , Análisis de Secuencia por Matrices de Oligonucleótidos , Transducción de Señal/genéticaRESUMEN
The flat oyster, Ostrea edulis, is one of the main farmed oysters, not only in Europe but also in the United States and Canada. Bonamiosis due to the parasite Bonamia ostreae has been associated with high mortality episodes in this species. This parasite is an intracellular protozoan that infects haemocytes, the main cells involved in oyster defence. Due to the economical and ecological importance of flat oyster, genomic data are badly needed for genetic improvement of the species, but they are still very scarce. The objective of this study is to develop a sequence database, OedulisDB, with new genomic and transcriptomic resources, providing new data and convenient tools to improve our knowledge of the oyster's immune mechanisms. Transcriptomic and genomic sequences were obtained using 454 pyrosequencing and compiled into an O. edulis database, OedulisDB, consisting of two sets of 10,318 and 7159 unique sequences that represent the oyster's genome (WG) and de novo haemocyte transcriptome (HT), respectively. The flat oyster transcriptome was obtained from two strains (naïve and tolerant) challenged with B. ostreae, and from their corresponding non-challenged controls. Approximately 78.5% of 5619 HT unique sequences were successfully annotated by Blast search using public databases. A total of 984 sequences were identified as being related to immune response and several key immune genes were identified for the first time in flat oyster. Additionally, transcriptome information was used to design and validate the first oligo-microarray in flat oyster enriched with immune sequences from haemocytes. Our transcriptomic and genomic sequencing and subsequent annotation have largely increased the scarce resources available for this economically important species and have enabled us to develop an OedulisDB database and accompanying tools for gene expression analysis. This study represents the first attempt to characterize in depth the O. edulis haemocyte transcriptome in response to B. ostreae through massively sequencing and has aided to improve our knowledge of the immune mechanisms of flat oyster. The validated oligo-microarray and the establishment of a reference transcriptome will be useful for large-scale gene expression studies in this species.
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Bases de Datos Genéticas , Genoma , Haplosporidios/inmunología , Inmunidad Innata , Análisis de Secuencia por Matrices de Oligonucleótidos , Ostrea/genética , Ostrea/parasitología , Animales , Etiquetas de Secuencia Expresada , Hemocitos/inmunología , Hemocitos/metabolismo , Hemocitos/parasitología , Ostrea/inmunología , Análisis de Secuencia de ADN , Análisis de Secuencia de ARN , TranscriptomaRESUMEN
The protistan parasite Perkinsus olseni is a deadly causative agent of perkinsosis, a molluscan disease affecting Manila clam (Ruditapes philippinarum), having a significant impact on world mollusc production. Deciphering the underlying molecular mechanisms in R. philippinarum-P. olseni interaction is crucial for controlling this parasitosis. The present study investigated the transcriptional expression in the parasite trophozoite using RNA-seq. Control and treatment (in vitro challenged with Manila clam-plasma) P. olseni trophozoite RNA were extracted and sequenced on the Illumina HiSeq 2000 instrument using a 100-bp paired-end sequencing strategy. Paired reads (64.7 million) were de novo assembled using Trinity, and the resultant transcripts were further clustered using CAP3. The re-constructed P. olseni transcriptome contains 47,590 unique transcripts of which 23,505 were annotated to 9764 unique proteins. A large number of genes were associated with Gene Ontology terms such as stress and immune-response, cell homeostasis, antioxidation, cell communication, signal transduction, signalling and proteolysis. Among annotated transcripts, a preliminary gene expression analysis detected 679 up-regulated and 478 down-regulated genes, linked to virulence factors, anti-oxidants, adhesion and immune-response molecules. Genes of several metabolic pathways such as DOXP/MEP, FAS II or folate biosynthesis, which are potential therapeutic targets, were identified. This study is the first description of the P. olseni transcriptome, and provides a substantial genomic resource for studying the molecular mechanisms of the host-parasite interaction in perkinsosis. In this sense, it is also the first evaluation of the parasite gene expression after challenge with clam extracellular products.
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Alveolados/genética , Bivalvos/parasitología , Interacciones Huésped-Parásitos/genética , Transcriptoma/genética , Trofozoítos/fisiología , Alveolados/patogenicidad , Aminoacil-ARNt Sintetasas/metabolismo , Animales , Ácido Fólico/biosíntesis , Regulación de la Expresión Génica , Hemolinfa/química , Lípidos/biosíntesis , Lípidos/genética , Péptido Hidrolasas/metabolismo , Inhibidores de Proteasas/metabolismo , Pirimidinas/biosíntesis , ARN/química , ARN/aislamiento & purificación , Transducción de Señal/genética , Factores de Virulencia/fisiologíaRESUMEN
DNA profiling is a key tool for forensic analysis; however, current methods identify a suspect either by direct comparison or from DNA database searches. In cases with unidentified suspects, prediction of visible physical traits e.g. pigmentation or hair distribution of the DNA donors can provide important probative information. This study aimed to explore single nucleotide polymorphism (SNP) variants for their effect on hair colour prediction. A discovery panel of 63 SNPs consisting of already established hair colour markers from the HIrisPlex hair colour phenotyping assay as well as additional markers for which associations to human pigmentation traits were previously identified was used to develop multiplex assays based on SNaPshot single-base extension technology. A genotyping study was performed on a range of European populations (n = 605). Hair colour phenotyping was accomplished by matching donor's hair to a graded colour category system of reference shades and photography. Since multiple SNPs in combination contribute in varying degrees to hair colour predictability in Europeans, we aimed to compile a compact marker set that could provide a reliable hair colour inference from the fewest SNPs. The predictive approach developed uses a naïve Bayes classifier to provide hair colour assignment probabilities for the SNP profiles of the key SNPs and was embedded into the Snipper online SNP classifier ( http://mathgene.usc.es/snipper/ ). Results indicate that red, blond, brown and black hair colours are predictable with informative probabilities in a high proportion of cases. Our study resulted in the identification of 12 most strongly associated SNPs to hair pigmentation variation in six genes.
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Color del Cabello/genética , Polimorfismo de Nucleótido Simple , Población Blanca/genética , Europa (Continente) , Femenino , Marcadores Genéticos , Genotipo , Humanos , Funciones de Verosimilitud , Modelos Logísticos , Masculino , FenotipoRESUMEN
Circulating tumor cells (CTCs), proposed as major players in cancer dissemination, have demonstrated clinical prognostic significance in several cancer types. However, their predictive value remains unclear. Here we evaluated the clinical utility of six CTC markers (tissue specific and epithelial to mesenchymal transition transcripts) both as prognostic and predictive tools in metastatic colorectal cancer (mCRC) patients. CTCs were immunoisolated from blood in 50 mCRC patients at baseline and at 4 and 16 weeks after treatment onset. Expression levels of GAPDH, VIL1, CLU, TIMP1, LOXL3 and ZEB2 were determined by qualitative polymerase chain reaction and normalized to the unspecific cell isolation marker CD45. At baseline, median progression-free survival (PFS) and overall survival (OS) for patients with high CTC markers were 6.3 and 12.7 months, respectively, versus 12.7 and 24.2 for patients with low CTC markers (PFS; p = 0.0003; OS; p = 0.044). Concerning response to therapy, PFS and OS for patients with increased CTC markers along treatment were, respectively, 6.6 and 13.1 months, compared with 12.7 and 24.3 for patients presenting CTC markers reduction (PFS; p = 0.004; OS; p = 0.007). Of note, CTC markers identified therapy-refractory patients not detected by standard image techniques. Patients with increased CTC markers along treatment, but classified as responders by computed tomography, showed significantly shorter survival times (PFS: 7.8 vs. 13.2; OS: 14.4 vs. 24.4; months). In conclusion, we have generated a CTC marker panel for prognosis evaluation and the identification of patients benefiting or not from therapy in mCRC. Our methodology efficiently classified patients earlier than routine computed tomography and from a minimally invasive liquid biopsy.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Biomarcadores de Tumor/análisis , Neoplasias Colorrectales/sangre , Neoplasias Hepáticas/sangre , Células Neoplásicas Circulantes/patología , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales Humanizados/administración & dosificación , Bevacizumab , Camptotecina/administración & dosificación , Camptotecina/análogos & derivados , Capecitabina , Cetuximab , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/mortalidad , Neoplasias Colorrectales/patología , Desoxicitidina/administración & dosificación , Desoxicitidina/análogos & derivados , Transición Epitelial-Mesenquimal , Femenino , Fluorouracilo/administración & dosificación , Fluorouracilo/análogos & derivados , Estudios de Seguimiento , Humanos , Irinotecán , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/mortalidad , Neoplasias Hepáticas/secundario , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Compuestos Organoplatinos/administración & dosificación , Oxaliplatino , Pronóstico , Tasa de SupervivenciaRESUMEN
BACKGROUND: Genomic resources for plant and animal species that are under exploitation primarily for human consumption are increasingly important, among other things, for understanding physiological processes and for establishing adequate genetic selection programs. Current available techniques for high-throughput sequencing have been implemented in a number of species, including fish, to obtain a proper description of the transcriptome. The objective of this study was to generate a comprehensive transcriptomic database in turbot, a highly priced farmed fish species in Europe, with potential expansion to other areas of the world, for which there are unsolved production bottlenecks, to understand better reproductive- and immune-related functions. This information is essential to implement marker assisted selection programs useful for the turbot industry. RESULTS: Expressed sequence tags were generated by Sanger sequencing of cDNA libraries from different immune-related tissues after several parasitic challenges. The resulting database ("Turbot 2 database") was enlarged with sequences generated from a 454 sequencing run of brain-hypophysis-gonadal axis-derived RNA obtained from turbot at different development stages. The assembly of Sanger and 454 sequences generated 52,427 consensus sequences ("Turbot 3 database"), of which 23,661 were successfully annotated. A total of 1,410 sequences were confirmed to be related to reproduction and key genes involved in sex differentiation and maturation were identified for the first time in turbot (AR, AMH, SRY-related genes, CYP19A, ZPGs, STAR FSHR, etc.). Similarly, 2,241 sequences were related to the immune system and several novel key immune genes were identified (BCL, TRAF, NCK, CD28 and TOLLIP, among others). The number of genes of many relevant reproduction- and immune-related pathways present in the database was 50-90% of the total gene count of each pathway. In addition, 1,237 microsatellites and 7,362 single nucleotide polymorphisms (SNPs) were also compiled. Further, 2,976 putative natural antisense transcripts (NATs) including microRNAs were also identified. CONCLUSIONS: The combined sequencing strategies employed here significantly increased the turbot genomic resources available, including 34,400 novel sequences. The generated database contains a larger number of genes relevant for reproduction- and immune-associated studies, with an excellent coverage of most genes present in many relevant physiological pathways. This database also allowed the identification of many microsatellites and SNP markers that will be very useful for population and genome screening and a valuable aid in marker assisted selection programs.
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Cruzamiento/métodos , Enfermedades de los Peces/prevención & control , Peces Planos/genética , Peces Planos/fisiología , Genómica , Reproducción/genética , Análisis de Secuencia de ARN , Animales , Bases de Datos Genéticas , Peces Planos/inmunología , Marcadores Genéticos/genética , Humanos , Anotación de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Polimorfismo de Nucleótido Simple , ARN sin Sentido/genética , ARN Mensajero/genéticaRESUMEN
The accuracy in the diagnosis of metastatic colorectal cancer (mCRC) represents one of the challenges in the clinical management of patients. The detection of circulating tumour cells (CTC) is becoming a promising alternative to current detection techniques, as it focuses on one of the players of the metastatic disease and it should provide with more specific and sensitive detection rates. Here, we describe an improved method of detection of CTC from mCRC patients by combining immune-enrichment, optimal purification of RNA from very low cell numbers, and the selection of accurate PCR probes. As a result, we obtained a logistic model that combines GAPDH and VIL1 normalized to CD45 rendering powerful results in the detection of CTC from mCRC patients (AUROC value 0.8599). We further demonstrated the utility of this model at the clinical setting, as a reliable prognosis tool to determine progression-free survival in mCRC patients. Overall, we developed a strategy that ameliorates the specificity and sensitivity in the detection of CTC, resulting in a robust and promising logistic model for the clinical management of metastatic colorectal cancer patients.
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Biomarcadores de Tumor/análisis , Neoplasias Colorrectales/diagnóstico , Neoplasias Colorrectales/patología , Células Neoplásicas Circulantes/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Supervivencia sin Enfermedad , Células Epiteliales/metabolismo , Femenino , Humanos , Antígenos Comunes de Leucocito/genética , Antígenos Comunes de Leucocito/metabolismo , Modelos Logísticos , Masculino , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Persona de Mediana Edad , Pronóstico , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: The turbot (Scophthalmus maximus) is a relevant species in European aquaculture. The small turbot genome provides a source for genomics strategies to use in order to understand the genetic basis of productive traits, particularly those related to sex, growth and pathogen resistance. Genetic maps represent essential genomic screening tools allowing to localize quantitative trait loci (QTL) and to identify candidate genes through comparative mapping. This information is the backbone to develop marker-assisted selection (MAS) programs in aquaculture. Expressed sequenced tag (EST) resources have largely increased in turbot, thus supplying numerous type I markers suitable for extending the previous linkage map, which was mostly based on anonymous loci. The aim of this study was to construct a higher-resolution turbot genetic map using EST-linked markers, which will turn out to be useful for comparative mapping studies. RESULTS: A consensus gene-enriched genetic map of the turbot was constructed using 463 SNP and microsatellite markers in nine reference families. This map contains 438 markers, 180 EST-linked, clustered at 24 linkage groups. Linkage and comparative genomics evidences suggested additional linkage group fusions toward the consolidation of turbot map according to karyotype information. The linkage map showed a total length of 1402.7 cM with low average intermarker distance (3.7 cM; ~2 Mb). A global 1.6:1 female-to-male recombination frequency (RF) ratio was observed, although largely variable among linkage groups and chromosome regions. Comparative sequence analysis revealed large macrosyntenic patterns against model teleost genomes, significant hits decreasing from stickleback (54%) to zebrafish (20%). Comparative mapping supported particular chromosome rearrangements within Acanthopterygii and aided to assign unallocated markers to specific turbot linkage groups. CONCLUSIONS: The new gene-enriched high-resolution turbot map represents a useful genomic tool for QTL identification, positional cloning strategies, and future genome assembling. This map showed large synteny conservation against model teleost genomes. Comparative genomics and data mining from landmarks will provide straightforward access to candidate genes, which will be the basis for genetic breeding programs and evolutionary studies in this species.
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Mapeo Cromosómico/métodos , Etiquetas de Secuencia Expresada , Peces/genética , Peces Planos/genética , Animales , Femenino , Masculino , Repeticiones de Microsatélite , Sitios de Carácter Cuantitativo , SinteníaRESUMEN
The development of microhaplotype (MH) panels for massively parallel sequencing (MPS) platforms is gaining increasing relevance for forensic analysis. Here, we expand the applicability of a 102 autosomal and 11 X-chromosome panel of MHs, previously validated with both MiSeq and Ion S5 MPS platforms and designed for identification purposes. We have broadened reference population data for identification purposes, including data from 240 HGDP-CEPH individuals of native populations from North Africa, the Middle East, Oceania and America. Using the enhanced population data, the panel was evaluated as a marker set for bio-geographical ancestry (BGA) inference, providing a clear differentiation of the five main continental groups of Africa, Europe, East Asia, Native America, and Oceania. An informative degree of differentiation was also achieved for the population variation encompassing North Africa, Middle East, Europe, South Asia, and East Asia. In addition, we explored the potential for individual BGA inference from simple mixed DNA, by simulation of mixed profiles followed by deconvolution of mixture components.
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BACKGROUND: Current therapeutic options in the course of metastatic castration-resistant prostate cancers (mCRPC) reinforce the need for reliable tools to characterize the tumor in a dynamic way. Circulating tumor cells (CTCs) have emerged as a viable solution to the problem, whereby patients with a variety of solid tumors, including PC, often do not have recent tumor tissue available for analysis. The biomarker characterization in CTCs could provide insights into the current state of the disease and an overall picture of the intra-tumor heterogeneity. METHODS: in the present study, we applied a global gene expression characterization of the CTC population from mCRPC (n = 9), with the goal to better understand the biology of these cells and identify the relevant molecules favoring this tumor progression. RESULTS: This analysis allowed the identification of 50 genes specifically expressed in CTCs from patients. Six of these markers (HOXB13, QKI, MAOA, MOSPD1, SDK1, and FGD4), were validated in a cohort of 28 mCRPC, showing clinical interest for the management of these patients. Of note, the activity of this CTC signature was related to the regulation of MYC, a gene strongly implicated in the biology of mCRPC. CONCLUSIONS: Overall, our results represent new evidence on the great value of CTCs as a non-invasive biopsy to characterize PC.
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The Manila clam (Ruditapes philippinarum) is the bivalve species with the highest global production from both fisheries and aquaculture, but its production is seriously threatened by perkinsosis, a disease caused by the protozoan parasite Perkinsus olseni. To understand the molecular mechanisms underlying R. philippinarum-P. olseni interactions, we analysed the gene expression profiles of in vitro challenged clam hemocytes and P. olseni trophozoites, using two oligo-microarray platforms, one previously validated for R. philippinarum hemocytes and a new one developed and validated in this study for P. olseni. Manila clam hemocytes were in vitro challenged with trophozoites, zoospores, and extracellular products from P. olseni in vitro cultures, while P. olseni trophozoites were in vitro challenged with Manila clam plasma along the same time-series (1 h, 8 h, and 24 h). The hemocytes showed a fast activation of the innate immune response, particularly associated with hemocyte recruitment, in the three types of challenges. Nevertheless, different immune-related pathways were activated in response to the different parasite stages, suggesting specific recognition mechanisms. Furthermore, the analyses provided useful complementary data to previous in vivo challenges, and confirmed the potential of some proposed biomarkers. The combined analysis of gene expression in host and parasite identified several processes in both the clam and P. olseni, such as redox and glucose metabolism, protease activity, apoptosis and iron metabolism, whose modulation suggests cross-talk between parasite and host. This information might be critical to determine the outcome of the infection, thus highlighting potential therapeutic targets. Altogether, the results of this study aid understanding the response and interaction between R. philippinarum and P. olseni, and will contribute to developing effective control strategies for this threatening parasitosis.
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Alveolados , Bivalvos/parasitología , Alveolados/genética , Alveolados/metabolismo , Animales , Bivalvos/genética , Bivalvos/metabolismo , Células Sanguíneas/metabolismo , Interacciones Huésped-Parásitos/inmunología , Inmunidad Innata , Técnicas In Vitro/métodos , Parásitos/genética , Parásitos/metabolismo , Mariscos/parasitología , Transcriptoma , Trofozoítos/genética , Trofozoítos/metabolismoRESUMEN
BACKGROUND: The turbot (Scophthalmus maximus; Scophthalmidae; Pleuronectiformes) is a flatfish species of great relevance for marine aquaculture in Europe. In contrast to other cultured flatfish, very few genomic resources are available in this species. Aeromonas salmonicida and Philasterides dicentrarchi are two pathogens that affect turbot culture causing serious economic losses to the turbot industry. Little is known about the molecular mechanisms for disease resistance and host-pathogen interactions in this species. In this work, thousands of ESTs for functional genomic studies and potential markers linked to ESTs for mapping (microsatellites and single nucleotide polymorphisms (SNPs)) are provided. This information enabled us to obtain a preliminary view of regulated genes in response to these pathogens and it constitutes the basis for subsequent and more accurate microarray analysis. RESULTS: A total of 12584 cDNAs partially sequenced from three different cDNA libraries of turbot (Scophthalmus maximus) infected with Aeromonas salmonicida, Philasterides dicentrarchi and from healthy fish were analyzed. Three immune-relevant tissues (liver, spleen and head kidney) were sampled at several time points in the infection process for library construction. The sequences were processed into 9256 high-quality sequences, which constituted the source for the turbot EST database. Clustering and assembly of these sequences, revealed 3482 different putative transcripts, 1073 contigs and 2409 singletons. BLAST searches with public databases detected significant similarity (e-value < or = 1e-5) in 1766 (50.7%) sequences and 816 of them (23.4%) could be functionally annotated. Two hundred three of these genes (24.9%), encoding for defence/immune-related proteins, were mostly identified for the first time in turbot. Some ESTs showed significant differences in the number of transcripts when comparing the three libraries, suggesting regulation in response to these pathogens. A total of 191 microsatellites, with 104 having sufficient flanking sequences for primer design, and 1158 putative SNPs were identified from these EST resources in turbot. CONCLUSION: A collection of 9256 high-quality ESTs was generated representing 3482 unique turbot sequences. A large proportion of defence/immune-related genes were identified, many of them regulated in response to specific pathogens. Putative microsatellites and SNPs were identified. These genome resources constitute the basis to develop a microarray for functional genomics studies and marker validation for genetic linkage and QTL analysis in turbot.
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Etiquetas de Secuencia Expresada , Enfermedades de los Peces/inmunología , Peces Planos/inmunología , Regulación de la Expresión Génica/inmunología , Infecciones por Bacterias Gramnegativas/inmunología , Infecciones Protozoarias en Animales/inmunología , Aeromonas salmonicida , Animales , Biomarcadores , Biología Computacional , Perfilación de la Expresión Génica , Biblioteca de Genes , Datos de Secuencia Molecular , OligohimenóforosRESUMEN
DNA methylation is the most extensively studied epigenetic signature, with a large number of studies reporting age-correlated CpG sites in overlapping genes. However, most of these studies lack sample coverage of individuals under 18â¯years old and therefore little is known about the progression of DNA methylation patterns in children and adolescents. In the present study we aimed to select candidate age-correlated DNA methylation markers based on public datasets from Illumina BeadChip arrays and previous publications, then to explore the resulting markers in 209 blood samples from donors aged between 2 to 18â¯years old using the EpiTYPER® DNA methylation analysis system. Results from our analyses identified six genes highly correlated with age in the young, in particular the gene KCNAB3, which indicates its potential as a highly informative and specific age biomarker for childhood and adolescence. We outline a preliminary age prediction model based on quantile regression that uses data from the six CpG sites most strongly correlated with age ranges extended to include children and adolescents.
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Envejecimiento/genética , Metilación de ADN , Genética Forense/métodos , Marcadores Genéticos , Acetiltransferasas/genética , Adolescente , Amidohidrolasas/genética , Niño , Preescolar , Islas de CpG/genética , Proteína Quinasa Dependiente de GMP Cíclico Tipo II/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/genética , Proteína de Dominio de Muerte Asociada a Edar/genética , Elongasas de Ácidos Grasos , Humanos , Proteínas con Homeodominio LIM/genética , Proteínas Musculares/genética , Proteínas del Tejido Nervioso/genética , Reacción en Cadena de la Polimerasa , Canales de Potasio de la Superfamilia Shaker , Canales de Potasio Shaw/genética , Programas Informáticos , Factores de Transcripción/genéticaRESUMEN
INTRODUCTION: There is a critical need of new surrogate markers for improving the therapeutic selection and monitoring of metastatic prostate cancer patients. Nowadays clinical management of these patients is been driven by biochemical and clinical parameters without enough accuracy to allow a real personalized medicine. The present study was conducted to go insight the molecular profile of circulating tumor cells (CTCs) isolated from advanced metastatic castration-resistant prostate cancer (mCRPC) with the aim of identifying prognostic marker with potential utility for therapy selection and monitoring. MATERIALS AND METHODS: CTCs isolation was carried out in peripheral blood samples from 29 mCRPC patients that undergo systemic chemotherapy based on taxanes (docetaxel/cabazitaxel) and 19 healthy controls using in parallel CellSearch and an alternative EpCAM-based immunoisolation followed by RT-qPCR analysis to characterize the CTC population. A panel of 17 genes related with prostate biology, hormone regulation, stem properties, tumor aggressiveness and taxanes responsiveness was analysed to identify an expression signature characterizing the CTCs. RESULTS: Patients with ≥ 5 CTCs/7.5ml of peripheral blood at baseline and during the treatment showed lower progression free survival (PFS) and overall survival (OS). Changes of CTCs levels during the treatment were also associated with the patient's outcome. These results confirmed previous data obtained using CellSearch in mCRPC. In addition, we found a CTC profile mainly characterized by the expression of relevant genes for the hormone dependent regulation of PCa such as AR and CYP19 together with genes strongly implicated in PCa progression and resistance development such as BIRC5, TUB1A, GDF15, RAB7 and SPINK1. Our gene-expression profiling also permitted the identification of valuable prognostic biomarkers. Thus, high levels of AR, CYP19 and GDF15 were associated with poor PFS rates while AR, GDF15 and BIRC5 were also found as reliable predictors of OS. Besides, a logistic model using KLK3 and BIRC5 showed a high specificity and sensitivity compared to CellSearch to discriminate patients with a more aggressive evolution. CONCLUSIONS: The molecular characterization of CTCs from advanced mCRPC patients provided with a panel of specific biomarkers, including genes related to taxanes resistance, with a promising applicability as "liquid biopsy" for the management of these patients.
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Purpose: Endometrial cancer (EC) diagnosis relies on the observation of tumor cells in endometrial biopsies obtained by aspiration (i.e., uterine aspirates), but it is associated with 22% undiagnosed patients and up to 50% of incorrectly assigned EC histotype and grade. We aimed to identify biomarker signatures in the fluid fraction of these biopsies to overcome these limitations.Experimental Design: The levels of 52 proteins were measured in the fluid fraction of uterine aspirates from 116 patients by LC-PRM, the latest generation of targeted mass-spectrometry acquisition. A logistic regression model was used to assess the power of protein panels to differentiate between EC and non-EC patients and between EC histologic subtypes. The robustness of the panels was assessed by the "leave-one-out" cross-validation procedure performed within the same cohort of patients and an independent cohort of 38 patients.Results: The levels of 28 proteins were significantly higher in patients with EC (n = 69) compared with controls (n = 47). The combination of MMP9 and KPYM exhibited 94% sensitivity and 87% specificity for detecting EC cases. This panel perfectly complemented the standard diagnosis, achieving 100% of correct diagnosis in this dataset. Nine proteins were significantly increased in endometrioid EC (n = 49) compared with serous EC (n = 20). The combination of CTNB1, XPO2, and CAPG achieved 95% sensitivity and 96% specificity for the discrimination of these subtypes.Conclusions: We developed two uterine aspirate-based signatures to diagnose EC and classify tumors in the most prevalent histologic subtypes. This will improve diagnosis and assist in the prediction of the optimal surgical treatment. Clin Cancer Res; 23(21); 6458-67. ©2017 AACR.
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Biomarcadores de Tumor/genética , Neoplasias Endometriales/diagnóstico , Carioferinas/genética , Biopsia Líquida/métodos , Proteínas de Microfilamentos/genética , Proteínas Nucleares/genética , alfa Catenina/genética , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/metabolismo , Líquidos Corporales/metabolismo , Neoplasias Endometriales/genética , Neoplasias Endometriales/patología , Neoplasias Endometriales/cirugía , Endometrio/metabolismo , Endometrio/patología , Femenino , Humanos , Espectrometría de Masas , Persona de Mediana Edad , Pronóstico , Proteoma/genética , Neoplasias Uterinas/diagnóstico , Neoplasias Uterinas/genética , Neoplasias Uterinas/patología , Neoplasias Uterinas/cirugíaRESUMEN
We used a microarray approach to examine changes in gene expression in turbot peritoneal cells after injection of the fish with vaccines containing the ciliate parasite Philasterides dicentrarchi as antigen and one of the following adjuvants: chitosan-PVMMA microspheres, FreundÌs complete adjuvant, aluminium hydroxide gel or Matrix-Q (Isconova, Sweden). We identified 374 genes that were differentially expressed in all groups of fish. Forty-two genes related to tight junctions and focal adhesions and/or actin cytoskeleton were differentially expressed in free peritoneal cells. The profound changes in gene expression related to cell adherence and cytoskeleton may be associated with cell migration and also with the formation of cell-vaccine masses and their attachment to the peritoneal wall. Thirty-five genes related to apoptosis were differentially expressed. Although most of the proteins coded by these genes have a proapoptotic effect, others are antiapoptotic, indicating that both types of signals occur in peritoneal leukocytes of vaccinated fish. Interestingly, many of the genes related to lymphocytes and lymphocyte activity were downregulated in the groups injected with vaccine. We also observed decreased expression of genes related to antigen presentation, suggesting that macrophages (which were abundant in the peritoneal cavity after vaccination) did not express these during the early inflammatory response in the peritoneal cavity. Finally, several genes that participate in the inflammatory response were differentially expressed, and most participated in resolution of inflammation, indicating that an M2 macrophage response is generated in the peritoneal cavity of fish one day post vaccination.
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Adyuvantes Inmunológicos/farmacología , Infecciones por Cilióforos/veterinaria , Peces Planos/genética , Peces Planos/parasitología , Expresión Génica/efectos de los fármacos , Vacunas/farmacología , Animales , Ensayo de Inmunoadsorción Enzimática , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/prevención & control , Peces Planos/inmunología , Oligohimenóforos , Análisis de Secuencia por Matrices de Oligonucleótidos , Peritoneo/efectos de los fármacos , Reacción en Cadena de la PolimerasaRESUMEN
Knowledge on the molecular mechanisms underlying metastasis colonization in Non-Small Cell Lung Cancer (NSCLC) remains incomplete. A complete overview integrating driver mutations, primary tumour heterogeneity and overt metastasis lacks the dynamic contribution of disseminating metastatic cells due to the inaccessibility to the molecular profiling of Circulating Tumour Cells (CTCs). By combining immunoisolation and whole genome amplification, we performed a global gene expression analysis of EpCAM positive CTCs from advanced NSCLC patients. We identified an EpCAM+ CTC-specific expression profile in NSCLC patients mostly associated with cellular movement, cell adhesion and cell-to-cell signalling mediated by PI3K/AKT, ERK1/2 and NF-kB pathways. NOTCH1 emerged as a driver connecting active signalling pathways, with a reduced number of related candidate genes (NOTCH1, PTP4A3, LGALS3 and ITGB3) being further validated by RT-qPCR on an independent cohort of NSCLC patients. In addition, these markers demonstrated high prognostic value for Progression-Free Survival (PFS). In conclusion, molecular characterization of EpCAM+ CTCs from advanced NSCLC patients provided with highly specific biomarkers with potential applicability as a "liquid biopsy" for monitoring of NSCLC patients and confirmed NOTCH1 as a potential therapeutic target to block lung cancer dissemination.