New Tetrachromic VOF Stain (Type III-G.S) for Normal and Pathological Fish Tissues

10 páginas, 3 figuras, 1 tabla. Este trabajo esta dedicado a Sra. María Armenta.A new VOF Type III-G.S stain was applied to histological sections of different organs and tissues of healthy and pathological larvae, juvenile and adult fish species (Solea senegalensis; Sparus aurata; Diplodus sargo; Pagrus auriga; Argyrosomus regius and Halobatrachus didactylus). In comparison to the original Gutiérrez VOF stain, more acid dyes of contrasting colours and polychromatic/metachromatic properties were incorporated as essential constituents of the tetrachromic VOF stain. This facilitates the selective staining of different basic tissues and improves the morphological analysis of histochemical approaches of the cell components. The VOF Type III -6.5 stain is composed of a mixture of several dyes of varying size and molecular weight (Orange G<acid Fuchsin<Light green<Methyl Blue<Fast Green), which are used simultaneously, and it enables the individual tissues to be selectively differentiated and stained. Muscle fibers, collagen, reticulin and elastin fibers, erythrocytes, cartilage, bone, mucous cells, oocytes and larvae were selectively stained and differentiated. Dyes with small size and molecular weight (i.e Orange G), penetrate all tissue structures rapidly, but are only tightly retained in densely textured tissues (i.e erythrocytes). Methyl Blue is an interesting triarylmethane dye (large size and molecular weight), which is incorporated in this new VOF tetrachrome stain, and acquires histochemical significance when used at acid pH (2.8) because collagen and reticulin fibers, as well basophilic and metachromatic substances (strongly ionized sulphated glycoconjugates) can be identified. Muscle tissues show an evident green colour (Fast Green or Light Green affinities), even those isolated and/or diffuse muscle fibers present in the digestive submucosa layer. Connective tissues showed a specific and strong blue colour (Methyl Blue affinity) or mixed blue-red staining (Methyl Blue and Acid Fucshin affinities). Very noticeable is the staining of the mucous cells, as well as the hyaline capsule of the viral lymphocystic cells, which were stained blue-purple (carboxylated and/or strongly ionized sulphated groups). Cartilaginous tissues showed a blue or purple (Methyl Blue affinity) staining, and a specific red colour (Acid Fucshin affinity) was evident during calcification or in bone structures (i.e skeleton, fins, gills, teeth).This work (Spanish MCYT/AGL2003-03558).Peer reviewe

Histochemical aspects of the yolk-sac and digestive tract of larvae of the Senegal sole, Solea senegalensis (Kaup, 1858)

8 páginas, 3 figuras, 1 tabla.Histochemical distribution of glycoproteins, carbohydrates and proteins rich in different aminoacids were studied using histological and histochemical procedures, in Senegal sole, Solea senegalensis (Kaup, 1858) larvae from hatching until day 15. Glycogen, proteins and glycoproteins were detected in the yolk-sac of the larvae at hatching and during the yolk-resorption. The epithelia1 digestive system (brush border, enterocytes and goblet cells) contained neutral and acid mucins (carboxylated andlor sulphated). Glycogen was observed in the cytoplasm of the digestive absortive cells (enterocytes) and in the liver (hepatocytes) on day 3-4 posthatching. Protein reactions, and specially those that showed proteins rich in arginine, tyrosine and tryptophan, were very intense in the zymogen granules of the pancreatic cells. Oesophageal and intestinal goblet cells contained glucose N-acetyl and sialic acid residues, but the mucin content of these mucous cells did not show affinity towards Con-A, suggesting the absence of glycoproteins with Mannose andlor glucose residues. WGA showed a very intense positivity in the microvilli of the digestive epithelium of the larvae and positive granules for both lectins, specially for Con-A, were detected in the cytoplasm of the anterior intestinal enterocytes.This work was supported by the DGICYT and ClCM of Spain (Projects PB93-0756 and AGF94-0756, 1994-1997).Peer reviewe

Contrasting outcomes of Vibrio harveyi pathogenicity in gilthead seabream, Sparus aurata and European seabass, Dicentrachus labrax

Vibrio harveyi has been reported as the dominant heterotrophic bacterial species in western Mediterranean coastal areas during warm seasons, and is recognized as an economically significant pathogen for the aquaculture industry. The present work aimed to evaluate the pathogenicity of a V. harveyi strain isolated from ascitic fluid collected from cultured gilthead seabream and then used in a challenge experiment involving the two most important fish species in Mediterranean aquaculture: gilthead seabream, Sparus aurata and European seabass, Dicentrarchus labrax. The ascitic fluid from diseased juvenile seabreams, previously vaccinated against Photobacterium damselae and Vibrio anguillarum, was extracted and bacteria cultivated for isolation and characterization. Additionally, different tissues were sampled for histological evaluation and description. Significant histopathological responses were observed in hepatic and mucosal tissues. One of the strains isolated from ascitic fluid, IRTA 17-43, was selected for a bacterial challenge. Additionally, the attenuation of virulence through sequential passage of the strain on solid media was also assessed. In parallel, a co-habitation trial was performed in order to evaluate the possible transfer of the bacteria between injected and healthy individuals. Pathogenicity trials in gilthead seabream resulted in only 25% mortality when injected with 107 CFU mL−1, whereas, for European seabass, a mortality of 95% was recorded, with clear signs of vibriosis. When passed sequentially on solid media, the strain IRTA-17-43 showed a decrease of 35% in cumulative mortality for European seabass. No apparent transmission of the pathogen occurred during the co-habitation trial for both species. In conclusion, although few external signs of V. harveyi are observed in vaccinated carriers, internal effects of the infection were clear and severe. Although no horizontal transfer of infection was observed, the risk of occurrence between carriers and immunosuppressed individuals or between different species should be considered. This further validates that the establishment of a good health management system within fish farms is of major importance in order to avoid the onset of disease outbreaks.info:eu-repo/semantics/acceptedVersio

Potential risk of Artemia sp as a transmission vector of lymphocystis disease virus (LCDV)

4 páginas, 1 figura. XI Congreso Nacional de Acuicultura (Vigo, 24-28 septiembre 2007). Ed. Antonio Cerviño Eiroa, Alejandro Guerra Díaz y Carmen Pérez Acosta.[EN] Cysts and naupliis of Artemia sp. were analysed. Specific nested-PCR revealed lymphocystis disease virus (LCDV) genome. Infective viral particle has been observed by CPE development in inoculated cell cultures. Viral genome was found by in situ hybridization in nauplii and adults of natural infected artemia, as in nauplii of bath challenged artemia. No morphological damages have been observed. Artemia is a bio-accumulator of fish pathogens, with a possible role as environmental reservoir of fish pathogens. These results have shown the risk of artemia as a source of viral pathogens to fish larvae.[ES] En el presente trabajo se han realizado estudios virológicos en distintos lotes de quistes comerciales de artemias utilizados como alimento de larvas de peces marinos en piscifactorías. Mediante nested-PCR se ha detectado genoma del virus de linfocistis (LCDV) en homogeneizados de quistes y nauplios de artemia. La existencia de partículas víricas infectivas en estos homogeneizados se ha demostrado mediante la aparición de efectos citopáticos en cultivos celulares. Los ensayos de hibridación in situ han demostrado la existencia del LCDV en artemias infectadas naturalmente, así como en artemias inoculadas mediante baño, sin observarse alteraciones morfológicas. Las artemias actúan por tanto como bioacumuladores, pudiendo desempeñar un papel importante como reservorios ambientales de patógenos de peces. Estos resultados ponen de manifiesto el riesgo potencial de las artemias como fuente de patógenos víricos en estadios larvarios.Irene Cano es contratada postdoctoral en el ICMAN.CSIC dentro del Fondo social europeo- I3P-CSIC, en el marco del proyecto AGL2006-17777-C03-02/ACU (IP: Carmen Sarasquete).Peer reviewe

Comparative gene expression of gonadotropins (FSH and LH) and peptide levels of gonadotropin-releasing hormones (GnRHs) in the pituitary of wild and cultured Senegalese sole (Solea senegalensis) broodstocks

12 páginas, 8 figuras, 2 tablas.The Senegalese sole (Solea senegalensis) is a valuable flatfish for aquaculture, but it presents important reproductive problems in captivity. Spawning is achieved by wild-caught breeders but cultured broodstocks fail to spawn spontaneously and, when they do, eggs are unfertilized. To gain knowledge on the physiological basis underlying this reproductive dysfunction, this study aimed at analyzing comparative hormone levels between wild and cultured broodstocks at the spawning season. The Senegalese sole gonadotropin (GTH) subunits, FSHβ, LHβ and GPα, were cloned and qualitative (in situ hybridization) and quantitative (real-time PCR) assays developed to analyze pituitary GTH gene expression. In females, FSHβ and GPα mRNA levels were higher in wild than in cultured broodstocks, whereas in males all three subunits were highest in cultured. By ELISA, three GnRH forms were detected in the pituitary, displaying a relative abundance of GnRH2 > GnRH1 > GnRH3. All GnRHs were slightly more abundant in wild than cultured females, whereas no differences were observed in males. Plasma levels of vitellogenin and sex steroids were also analyzed. Results showed endocrine differences between wild and cultured broodstocks at the spawning period, which could be related to the endocrine failure of the reproductive axis in cultured breeders.This research was funded by the Spanish Ministry of Education and Science (MEC) (AGL2006-13777-C01), the Ministry of Agriculture, Fisheries and Food (MAPA) (JACUMAR, II National Plan for the Cultivation of Sole) and the Regional Government of Galicia (PGIDIT06RMA004E). J.M. Guzmán received a FPI fellowship from the MEC. J.B. Ortiz-Delgado was supported by the “Ramón y Cajal” program (MEC, Spain).Peer reviewe

Restoration of microRNA-214 expression reduces growth of myeloma cells through positive regulation of P53 and inhibition of DNA replication

This is an open-access paper.-- et al.MicroRNA have been demonstrated to be deregulated in multiple myeloma. We have previously reported that miR-214 is down-regulated in multiple myeloma compared to in normal plasma cells. The functional role of miR- 214 in myeloma pathogenesis was explored by transfecting myeloma cell lines with synthetic microRNA followed by gene expression profiling. Putative miR-214 targets were validated by luciferase reporter assay. Ectopic expression of miR-214 reduced cell growth and induced apoptosis of myeloma cells. In order to identify the potential direct target genes of miR-214 which could be involved in the biological pathways regulated by this microRNA, gene expression profiling of the H929 myeloma cell line transfected with precursor miR-214 was carried out. Functional analysis revealed significant enrichment for DNA replication, cell cycle phase and DNA binding. miR- 214 directly down-regulated the expression of PSMD10, which encodes the oncoprotein gankyrin, and ASF1B, a histone chaperone required for DNA replication, by binding to their 3'-untranslated regions. In addition, gankyrin inhibition induced an increase of P53mRNA levels and subsequent up-regulation of CDKN1A (p21Waf1/Cip1) and BAX transcripts, which are direct transcriptional targets of p53. In conclusion, MiR-214 functions as a tumor suppressor in myeloma by positive regulation of p53 and inhibition of DNA replication.This work was partially supported by the Spanish FIS (PI080568 and PS0901897), the >Gerencia Regional de Salud, Junta de Castilla y León> (GRS202/A08 and GRS 702/A/11), and the Spanish Myeloma Network Program (RD06/0020/0006). MES is supported by the Ministerio de Sanidad y Consumo (CA08/00212).Peer Reviewe

High-resolution copy number analysis of paired normal-tumor samples from diffuse large B cell lymphoma

Copy number analysis can be useful for assessing prognosis in diffuse large B cell lymphoma (DLBCL). We analyzed copy number data from tumor samples of 60 patients diagnosed with DLBCL de novo and their matched normal samples. We detected 63 recurrent copy number alterations (CNAs), including 33 gains, 30 losses, and nine recurrent acquired copy number neutral loss of heterozygosity (CNN-LOH). Interestingly, 20 % of cases acquired CNN-LOH of 6p21 locus, which involves the HLA region. In normal cells, there were no CNAs but we observed CNN-LOH involving some key lymphoma regions such as 6p21 and 9p24.1 (5 %) and 17p13.1 (2.5 %) in DLBCL patients. Furthermore, a model with some specific CNA was able to predict the subtype of DLBCL, 1p36.32 and 10q23.31 losses being restricted to germinal center B cell-like (GCB) DLBCL. In contrast, 8p23.3 losses and 11q24.3 gains were strongly associated with the non-GCB subtype. A poor prognosis was associated with biallelic inactivation of TP53 or 18p11.32 losses, while prognosis was better in cases carrying 11q24.3 gains. In summary, CNA abnormalities identify specific DLBCL groups, and we describe CNN-LOH in germline cells from DLBCL patients that are associated with genes that probably play a key role in DLBCL development.This work was supported by research funding from the Health Council of Castilla y León (GRS265/A/08), the Health Research Program (PS09/01382), and the Red Temática de Investigación Cooperativa en Cáncer (RTICC) grant RD12/0036 (groups 0069, 0029, 0036, 0058, and 0060) included in the National Plan I+D+I supported by the Instituto Carlos III and the Fondo Europeo de Desarrollo Regional (FEDER), the Spanish Ministry of Economy and Competitiveness, and the European Regional Development Fund (ERDF) 'Una manera de hacer Europa' (Innocampus; CEI-2010-1-0010). ES was supported by CM10/00078-Río Hortega, an ISCIII contract, FEHH grant 2013–2014 and JR14/00025-Juan Rodés, an ISCIII contract. IS was supported by the Subprograma Juan de la Cierva (JCI-2011-10232) and a Miguel Servet contract (CP13/00159).Peer Reviewe

Medicinal Plant Leaf Extract From Sage and Lemon Verbena Promotes Intestinal Immunity and Barrier Function in Gilthead Seabream (Sparus aurata)

The inclusion of a medicinal plant leaf extract (MPLE) from sage (Salvia officinalis) and lemon verbena (Lippia citriodora), rich in verbascoside and triterpenic compounds like ursolic acid, was evaluated in gilthead seabream (Sparus aurata) fed a low fishmeal-based diet (48% crude protein, 17% crude fat, 21.7 MJ kg-1, 7% fishmeal, 15% fish oil) for 92 days. In particular, the study focused on the effect of these phytogenic compounds on the gut condition by analyzing the transcriptomic profiling (microarray analysis) and histological structure of the intestinal mucosa, as well as the histochemical properties of mucins stored in goblet cells. A total number of 506 differentially expressed genes (285 up- and 221 down-regulated) were found when comparing the transcriptomic profiling of the intestine from fish fed the control and MPLE diets. The gut transcripteractome revealed an expression profile that favored biological mechanisms associated to the 1) immune system, particularly involving T cell activation and differentiation, 2) gut integrity (i.e., adherens and tight junctions) and cellular proliferation, and 3) cellular proteolytic pathways. The histological analysis showed that the MPLE dietary supplementation promoted an increase in the number of intestinal goblet cells and modified the composition of mucins’ glycoproteins stored in goblet cells, with an increase in the staining intensity of neutral mucins, as well as in mucins rich in carboxylated and weakly sulfated glycoconjugates, particularly those rich in sialic acid residues. The integration of transcriptomic and histological results showed that the evaluated MPLE from sage and lemon verbena is responsible for the maintenance of intestinal health, supporting gut homeostasis and increasing the integrity of the intestinal epithelium, which suggests that this phytogenic may be considered as a promising sustainable functional additive for aquafeeds.info:eu-repo/semantics/publishedVersio

Transcriptome analysis reveals molecular profiles associated with evolving steps of monoclonal gammopathies

This is an open-access paper.-- et al.A multistep model has been proposed of disease progression starting in monoclonal gammopathy of undetermined significance continuing through multiple myeloma, sometimes with an intermediate entity called smoldering myeloma, and ending in extramedullary disease. To gain further insights into the role of the transcriptome deregulation in the transition from a normal plasma cell to a clonal plasma cell, and from an indolent clonal plasma cell to a malignant plasma cell, we performed gene expression profiling in 20 patients with monoclonal gammopathy of undetermined significance, 33 with high-risk smoldering myeloma and 41 with multiple myeloma. The analysis showed that 126 genes were differentially expressed in monoclonal gammopathy of undetermined significance, smoldering myeloma and multiple myeloma as compared to normal plasma cell. Interestingly, 17 and 9 out of the 126 significant differentially expressed genes were small nucleolar RNA molecules and zinc finger proteins. Several proapoptotic genes (AKT1 and AKT2) were down-regulated and antiapoptotic genes (APAF1 and BCL2L1) were up-regulated in multiple myeloma, both symptomatic and asymptomatic, compared to monoclonal gammopathy of undetermined significance. When we looked for those genes progressively modulated through the evolving stages of monoclonal gammopathies, eight snoRNA showed a progressive increase while APAF1, VCAN and MEGF9 exhibited a progressive downregulation. In conclusion, our data show that although monoclonal gammopathy of undetermined significance, smoldering myeloma and multiple myeloma are not clearly distinguishable groups according to their gene expression profiling, several signaling pathways and genes were significantly deregulated at different steps of the transformation process.This study was partially supported by Spanish FIS (PI080568, PS09/01450 and PS0901897), “Gerencia Regional de Salud, Junta de Castilla y León” (GRS 702/A/11) grant, and the Spanish Myeloma Network Program (RD06/0020/0006, RD12/0036/0058 and RD12/0036/0046).Peer Reviewe

Phenotypic, genomic and functional characterization reveals no differences between CD138++ and CD138low subpopulations in multiple myeloma cell lines

This is an open-access article distributed under the terms of the Creative Commons Attribution License.Despite recent advances in the treatment of multiple myeloma (MM), it remains an incurable disease potentially due to the presence of resistant myeloma cancer stem cells (MM-CSC). Although the presence of clonogenic cells in MM was described three decades ago, the phenotype of MM-CSC is still controversial, especially with respect to the expression of syndecan-1 (CD138). Here, we demonstrate the presence of two subpopulations - CD138++ (95-99%) and CD138low (1-5%) - in eight MM cell lines. To find out possible stem-cell-like features, we have phenotypically, genomic and functionally characterized the two subpopulations. Our results show that the minor CD138low subpopulation is morphologically identical to the CD138++ fraction and does not represent a more immature B-cell compartment (with lack of CD19, CD20 and CD27 expression). Moreover, both subpopulations have similar gene expression and genomic profiles. Importantly, both CD138++ and CD138low subpopulations have similar sensitivity to bortezomib, melphalan and doxorubicin. Finally, serial engraftment in CB17-SCID mice shows that CD138++ as well as CD138low cells have self-renewal potential and they are phenotypically interconvertible. Overall, our results differ from previously published data in MM cell lines which attribute a B-cell phenotype to MM-CSC. Future characterization of clonal plasma cell subpopulations in MM patients' samples will guarantee the discovery of more reliable markers able to discriminate true clonogenic myeloma cells.This work was supported by the Cooperative Research Thematic Network (RTICs; RD06/0020/0006), the “Junta de Castilla y León. Consejería de Sanidad” (GRS 391/B/09), the “Ministerio de Ciencia e Innovación” (PS09/01897), the “Fundación Memoria D. Samuel Solórzano Barruso” (FS/2-2010) and Asociación Española Contra el Cáncer (AECC)(GCB120981SAN).Peer Reviewe