Objective: Polyvalvularmyxomatous degeneration is a rare clinical condition. A 51-year-old male patient presented at our centre with all four heart valves with myxomatous degeneration and severe mitral and aortic regurgitation due to leaflet prolapse. The patient referred five further family members with valvular heart disease at different stages of presentation. The aim of this study was to investigate the genetic basis of this familial polyvalvularmyxomatous degeneration which was associated with mild dysmorphic facial anomalies and short stature. Design: A detailed family history was recorded. Nine members of the family, affected or not by valvular heart disease, were studied clinically, echocardiographically and by detailed genetic analyses. Results: Six of the nine family members had echocardiographic features of different degrees of degenerative heart valve disease. In addition, the affected subjects shared similar mild dysmorphic facial anomalies and short stature. Exome sequencing identified a rare heterozygous single nucleotide deletion in the TAB2 gene in all affected family members, which was absent in the unaffected members. Conclusions: A variant in the TAB2 gene is proposed as the cause of syndromic congenital heart disease, displaying congenital myxomatous degenerative heart valve disease, mild dysmorphic fascial anomalies and short stature in this family.

Patients diagnosed with T-cell leukemias and T-cell lymphomas (TCLs) still have a poor prognosis and an inadequate response to current therapies, highlighting the need for targeted treatments. We have analyzed the potential therapeutic value of the farnesyltransferase inhibitor, tipifarnib, in 25 TCL cell lines through the identification of genomic and/or immunohistochemical markers of tipifarnib sensitivity. More than half of the cell lines (60%) were considered to be sensitive. Tipifarnib reduced cell viability in these T-cell leukemia and TCL cell lines, induced apoptosis and modified the cell cycle. A mutational study showed TP53 , NOTCH1 and DNMT3 to be mutated in 84.6%, 69.2% and 30.0% of sensitive cell lines, and in 62.5%, 0% and 0% of resistant cell lines, respectively. An immunohistochemistry study showed that p-ERK and RelB were associated as potential biomarkers of tipifarnib sensitivity and resistance, respectively. Data from RNA-seq show that tipifarnib at IC 50 after 72 h downregulated a great variety of pathways, including those controlling cell cycle, metabolism, and ribosomal and mitochondrial activity. This study establishes tipifarnib as a potential therapeutic option in T-cell leukemia and TCL. The mutational state of NOTCH1 , p-ERK and RelB could serve as potential biomarkers of tipifarnib sensitivity and resistance.

Background The Manila clam (Ruditapes philippinarum) is a worldwide cultured bivalve species with important commercial value. Diseases affecting this species can result in large economic losses. Because knowledge of the molecular mechanisms of the immune response in bivalves, especially clams, is scarce and fragmentary, we sequenced RNA from immune-stimulated R. philippinarum hemocytes by 454-pyrosequencing to identify genes involved in their immune defense against infectious diseases. Methodology and Principal Findings High-throughput deep sequencing of R. philippinarum using 454 pyrosequencing technology yielded 974,976 high-quality reads with an average read length of 250 bp. The reads were assembled into 51,265 contigs and the 44.7% of the translated nucleotide sequences into protein were annotated successfully. The 35 most frequently found contigs included a large number of immune-related genes, and a more detailed analysis showed the presence of putative members of several immune pathways and processes like the apoptosis, the toll like signaling pathway and the complement cascade. We have found sequences from molecules never described in bivalves before, especially in the complement pathway where almost all the components are present. Conclusions This study represents the first transcriptome analysis using 454-pyrosequencing conducted on R. philippinarum focused on its immune system. Our results will provide a rich source of data to discover and identify new genes, which will serve as a basis for microarray construction and the study of gene expression as well as for the identification of genetic markers. The discovery of new immune sequences was very productive and resulted in a large variety of contigs that may play a role in the defense mechanisms of Ruditapes philippinarum.

In the past 5 years “Next-generation” Sequencing (NGS) technologies have transformed genomics by delivering fast, inexpensive and accurate genome information changing the way we think about scientific approaches in basic, applied and clinical research. The inexpensive production of large volumes of sequence data is the main advantage over the automated Sanger method, making this new technology useful for many applications. In this chapter, a brief technical review of NGS technologies is given, along with the keys to NGS success and a broad range of applications for NGS technologies. Handbook of instrumental techniques from CCiTUB “Next-generation” Sequencing (NGS) 1 BT.7

Gilthead seabream ( Sparus aurata ) is a teleost belonging to the family Sparidae with a high economical relevance in the Mediterranean countries. Although genomic tools have been developed in this species in order to investigate its physiology at the molecular level and consequently its culture, genomic information on post-embryonic development is still scarce. In this study, we have investigated the transcriptome of a marine teleost during the larval stage (from hatching to 60 days after hatching) by the use of 454 pyrosequencing technology. We obtained a total of 68,289 assembled contigs, representing putative transcripts, belonging to 54,606 different clusters. Comparison against all S. aurata expressed sequenced tags (ESTs) from the NCBI database revealed that up to 34,722 contigs, belonging to about 61% of gene clusters, are sequences previously not described. Contigs were annotated through an iterative Blast pipeline by comparison against databases such as NCBI RefSeq from Danio rerio , SwissProt or NCBI teleost ESTs. Our results indicate that we have enriched the number of annotated sequences for this species by more than 50% compared with previously existing databases for the gilthead seabream. Gene Ontology analysis of these novel sequences revealed that there is a statistically significant number of transcripts with key roles in larval development, differentiation, morphology, and growth. Finally, all information has been made available online through user-friendly interfaces such as GBrowse and a Blast server with a graphical frontend.

Background: Turbot (Scophthalmus maximus L.) is an important aquacultural resource both in Europe and Asia. However, there is little information on gene sequences available in public databases. Currently, one of the main problems affecting the culture of this flatfish is mortality due to several pathogens, especially viral diseases which are not treatable. In order to identify new genes involved in immune defense, we conducted 454-pyrosequencing of the turbot transcriptome after different immune stimulations.Methodology/Principal Findings: Turbot were injected with viral stimuli to increase the expression level of immune-related genes. High-throughput deep sequencing using 454-pyrosequencing technology yielded 915,256 high-quality reads. These sequences were assembled into 55,404 contigs that were subjected to annotation steps. Intriguingly, 55.16% of the deduced protein was not significantly similar to any sequences in the databases used for the annotation and only 0.85% of the BLASTx top-hits matched S. maximus protein sequences. This relatively low level of annotation is possibly due to the limited information for this specie and other flatfish in the database. These results suggest the identification of a large number of new genes in turbot and in fish in general. A more detailed analysis showed the presence of putative members of several innate and specific immune pathways.Conclusions/Significance: To our knowledge, this study is the first transcriptome analysis using 454-pyrosequencing for turbot. Previously, there were only 12,471 EST and less of 1,500 nucleotide sequences for S. maximus in NCBI database. Our results provide a rich source of data (55,404 contigs and 181,845 singletons) for discovering and identifying new genes, which will serve as a basis for microarray construction, gene expression characterization and for identification of genetic markers to be used in several applications. Immune stimulation in turbot was very effective, obtaining an enormous variety of sequences belonging to genes involved in the defense mechanisms.

PURPOSETo explore modifications in signal mechanisms involving CD11b and leukocyte adhesion in patients under haemodialysis (HD).METHODSSamples were obtained from uremic patients at baseline, 15 and 120 min of HD from both arterial and venous lines. CD11b expression was studied by flow cytometry. To study signalling mechanisms, CD11b was immunoprecipitated using a specific antibody. Immunoprecipitates were resolved by 8% SDS-PAGE to measure phosphorylation in immunoblots. Leukocyte adhesion was measured after blood perfusion using endothelial cells (EC) as adhesive substrate. Parallel studies were performed with blood from healthy donors.RESULTSThe percentage of CD11b+ cells increased during HD with a cellulose membrane in the venous line at 15 and 120 min (6.2+/-2.9% and 11.0+/-7.1%) and in the arterial line at 120 min (11.5+/-8.5 vs. 3.1+/-1.0% in control P < 0.05). After 120 min HD, CD11b phosphorylation decreased in leukocytes from both arterial (72.6+/-2.9) and venous lines (51.8+/-6.5) vs. basal samples (119.5+/-15.5 P < 0.005). Control leukocytes showed enhanced adhesion to uremic EC compared with control EC (3.0+/-0.3 vs. 2.3+/-1.0 leukocytes x100 EC(-1) P < 0.05). Uremic leukocyte adhesion was enhanced after HD compared with basal samples 4.2+/-0.2 leukocytes/100 EC in the arterial and 4.4+/-0.3 in the venous line; after 120 min vs 2.3+/-1.0 (P < 0.005).CONCLUSIONLeukocyte activation during HD through a cellulose membrane occurs with decreases in CD11b phosphorylation. Activation also induces increases in CD11b expression associated with enhanced leukocyte adhesion to uremic endothelial cells.

BACKGROUND Deficient hemostasis and accelerated atherosclerosis coexist in patients with chronic kidney disease. Endothelial dysfunction may be involved in the high incidence of atherothrombotic events in these patients. We established an in vitro model of endothelial dysfunction by exposing endothelial cells to uremic media and applied a proteomic approach to characterize endothelial cell dysfunction in uremia. STUDY DESIGN Cross-sectional study. SETTING AND PARTICIPANTS Serum samples from 8 patients with chronic kidney disease on hemodialysis treatment were collected. PREDICTOR Exposure of cultured endothelial cells to normal and uremic serum. OUTCOME AND MEASUREMENTS: Proteins from lysed cells were characterized by isoelectric point and molecular weight by using 2-dimensional gel electrophoresis. Spots were visualized by means of silver staining and identified by using mass spectrometry. RESULTS Identification of the most prominent proteins showed molecules related to inflammation (high mobility group box 1, aldose reductase, and proteasome components) and oxidative stress (superoxide dismutase and glutathione peroxidase), both associated with chronic kidney disease. These changes may be caused by activation of the nuclear factor-kappaB transcription factor. Changes in expression of cytoskeletal proteins (destrin and vimentin) also were detected. LIMITATIONS In vitro study. CONCLUSION Proteomic techniques proved to be a powerful tool to investigate endothelial dysfunction in uremia. A more exhaustive analysis will provide answers and potential therapeutic targets in the near future.

Introduction: Platelet activation leads to signal transduction mechanisms, in which phosphotyrosine proteins play a relevant role.Material and methods: Platelet suspensions were independently activated by collagen and thrombin in the absence and in the presence of two tyrosine kinase inhibitors, tyrphostin 47 and genistein. Samples were processed to visualize morphological changes by electron microscopy, to evaluate changes in cytoskeletal assembly, to analyze modifications in the expression of activation dependent antigens, and the procoagulant activity at the surface level by flow cytometry. Additional experiments applying flow conditions were performed to assess the effect of inhibiting tyrosine phosphorylation on primary platelet adhesion and fibrin formation.Results: Inhibition of tyrosine phosphorylation blocked shape change and cytoskeletal assembly induced by collagen, and inhibited, though partially, those effects due to thrombin. Both activating agents induced the expression of the intraplatelet antigens CD62P and CD63 at the surface, although only collagen promoted expression of anionic phospholipids. Both tyrphostin 47 and genistein prevented those effects. The extent of platelet adhesion on both collagen-coated and subendothelial surfaces was significantly diminished by the presence of the tyrosine kinase inhibitors assayed. Fibrin formation was also significantly reduced.Conclusions: Platelet shape change and secretion during platelet activation depends on tyrosine phosphorylation. In addition, primary adhesion of platelets induces signaling through tyrosine kinases to achieve full spreading, and results in the exposure of a procoagulant surface on platelets. (c) 2007 Elsevier Ltd. All rights reserved.

Background: Although the bleeding tendency in hemophilia A can be explained by a defect in coagulation, a possible platelet dysfunction has not been formally investigated. Methods: We studied collagen and TF-induced hemostasis using the blood of patients with severe hemophilia A in an in vitro set-up. Blood samples from healthy donors (n=11) or from patients (n=9) were anticoagulated with low molecular weight heparin. Perfusion studies at moderate shear rates (600s), were performed using damaged vascular segments; whereas PFA-100® with specially devised cartridges of collagen alone (COL) or collagen-tissue factor (COL-TF) were used for high shear rates (5000s ). We also tested the ability of rFVIIa to improve hemostasis. Results: Addition of rFVIIa (equivalent to 380 μg/Kg) to blood samples from healthy individuals induced an increase of fibrin formation (53.8±12.1% vs. 37.7±8.2%). In experiments performed with blood from hemophiliacs, fibrin deposition was almost absent (< 5%), but increased to 48.1±9.2% with rFVIIa. Experiments at high shear rate showed a prolonged closure times in samples from hemophilic patients using COL cartridges (255±22s vs.191±16s in healthy donors; p<0.05). Presence of TF in the apertures caused a 20% shortening in closure times in both cases. Addition of rFVIIa to hemophilic blood samples induced a further statistically significant reduction of closure times in COL-TF cartridges (p<0.05). This reduction was not observed in healthy individuals. Conclusions: Under our experimental settings, hemophilic patients showed a platelet dysfunction at high shear rate. rFVIIa, in the presence of TF, contributed to a partial correction of this effect. Studies with healthy donors, indirectly suggest no additional prothrombotic profile for rFVIIa under very high shear rate conditions. Potentially, PFA-100 could be used as a system for monitoring FVIIa in hemophilic patients. INTRODUCTION Normal hemostasis is the result of an adequate balance of platelet function interplaying with activators and inhibitors of the coagulation system. Predominance of activating factors can lead to a prothrombotic state, whereas lack or impairment of the coagulation factors can result in severe bleeding. After vessel injury, highly reactive proteins, such as collagen and Tissue Factor, are exposed to flowing blood. The TF/FVIIa complex activates the coagulation protease cascade, which leads to fibrin deposition and platelet activation though thrombin generation [1;2]. Absences of factor VIII or factor IX (hemophilia A and B respectively) are the coagulopathies that course with the most severe bleeding episodes. Interestingly, the hemorrhagic symptoms seem to be related to alteration in coagulation mechanisms since platelet functions are thought to be normal. Recombinant factor VIIa (rFVIIa) was developed for the management and prevention of bleeding episodes in patients with FVII deficiency [3], as well as in patients with hemophilia A and B who have developed inhibitors [4;5]. Additionally, in vivo and in vitro studies have evidenced that rFVIIa improves hemostasis in several coagulopathies and thrombopathies favoring the development of an hemostatic response at sites where TF is exposed [6-13]. Several studies have investigated the mechanism of action of rFVIIa in patients with hemophilia in detail [14;15]. Recent studies suggest that, in these patients, hemostatic action of rFVIIa is the result of an increase of coagulation mechanisms and a decrease of fibrinolysis due to the activation of an inhibitor of fibrinolysis such as ThrombinActivatable Fibrinolysis Inhibitor (TAFI) [16]. Previous in vitro studies from our group and others consistently found that rFVIIa can exert procoagulant effects in blood from patients with severe thrombocytopenia, major glycoprotein deficiencies [11;17;18], patients with impaired coagulation system as hemophiliacs and patients treated with oral anticoagulation [19] and cirrhotic patients [10] through an enhancement of thrombin

Aims: We evaluated modifications in formation of heterotypic platelet-leukocyte aggregation induced by dialysis through cellulosic or synthetic membranes and evaluated the effects of such procedures promoting adhesive interactions between leukocytes and normal endothelial cells (ECs). Methods: Samples were obtained from arterial and venous lines at baseline, after 15 and 120 min of hemodialysis. Heterotypic aggregation was assessed using flow-cytometric techniques. Experiments to determine leukocyte adhesion to ECs were performed in parallel plate perfusion chambers at 450 s–1. Results: Patients dialyzed with a cellulosic membrane showed a significantly higher baseline granulocyte heterotypic aggregation (median 22.5%, range 8.6–32%) versus healthy subjects (median 10%, range 3.2–14.6%; p < 0.05). Granulocyte heterotypic aggregation values remained increased throughout the hemodialysis session not only in the arterial line (median 18 and 24.5%, range 7–30 and 8.7–36% at 15 and 2 h, respectively) but also in the venous line (median 20 and 25%, range 8.6–32 and 11.5–35% at 15 min and 2 h, respectively). Basal lymphocytes heterotypic aggregation values observed in uremic patients were 6% (0.1–7.1%) versus 1.0% (0.5–2.8%) in the control group (p < 0.05). The increase remained during the hemodialysis session both in the arterial line (median 5 and 4%, range 0.2–14 and 0.5–7.1 % at 15 min and 2 h, respectively) and in the venous line (median 7 and 7%, range 1.4–14 and 0.5–10.6% at 15 min and 2 h). In contrast, patients dialyzed with a synthetic membrane showed a decreased basal granulocyte heterotypic aggregation compared to healthy subjects (median 3.5 vs. 10%, range 2.8–7 vs. 3.2–14.6%, respectively; p < 0.05). For lymphocytes, basal heterotypic aggregation values were 0.2% (range 0.1–0.5%) in dialyzed patients vs. 0.98% (range 0.5–2.8%) in healthy subjects (p < 0.05), without changes throughout the dialysis session. Changes in leukocyte adhesion during hemodialysis did not reach statistical significance with either hemodialysis membrane. Our studies confirm a differential activation of platelets and leukocytes depending on the nature of the dialysis membranes. However, activation of circulating cellular elements by hemodialysis procedures did not enhance cross-talk interactions between leukocytes and unaltered ECs.

Summary We investigated the interactions of vesicles containing human tissue factor (TF) with platelets and evaluated responses induced by rFVIIa using standard aggregometry, ultrastructural and flow-cytometry techniques. Washed platelets were exposed to a preparation of placental human TF (pTF) or to a relipidated formulation of recombinant humanTF (rTF). Under stirring conditions, pTF induced reversible aggregation with platelets returning to their resting state after 5 minutes. This reversible response to pTF was partially inhibited by antibodies against CD62-P, but not by antithrombin agents, and was not observed with rTF. Sequential ultrastructural studies revealed uptake of both TF preparations by platelets involving traffic of vesicles through channels of the open canalicular system (OCS). Immunocytochemical studies on cryosections identified TF in the OCS, and occasionally in the alpha-granules of the platelets. These processes were faster with pTF than with rTF, but bothTF preparations accumulated in platelets at the end of incubation periods. Flow cytometry studies revealed the presence of other cellular antigens (CD62-P, CD14 and CD45) associated to the pTF. Addition of rFVIIa to washed platelets exposed to pTF or rTF, caused a thrombin dependent irreversible platelet aggregation. Our studies demonstrate that platelets possess mechanisms to capture and incorporate TF-rich vesicles. These processes are accelerated by the presence of other cellular antigens in the vesicles. Our findings may explain the hemostatic action of rFVIIa in severely hemodiluted patients, but are also relevant for the understanding of potential implications of TF-associated to platelets in the propagation of thrombus.

The role of platelet glycoprotein Ib as a thrombin receptor has been often a subject of controversy. We have investigated the role of the thrombin receptors, GPIb and protease-activated receptor (PAR)-1. Tyrosine phosphorylation in whole platelet lysates and in cytoskeletal extracts was evaluated after activation with thrombin and with the thrombin receptor-activating peptide (TRAP). Different experimental approaches were applied including: (i) congenital deficiency of platelet GPIb (Bernard Soulier syndrome, BSS), (ii) antibody to GPIb (AP1), (iii) selective protease cleavage (metalloprotease), and (iv) antibody to (PAR)-1. After activation of control platelets with thrombin or TRAP, multiple proteins became tyrosine phosphorylated in platelet lysates and some of them associated with the cytoskeletal fraction. These effects were absent in BSS platelets. Presence of AP1 or metalloprotease treatment showed an inhibitory effect when platelets were activated with a low concentration of thrombin or TRAP. Blockade of PAR-1 with a specific antibody, SPAN 12, inhibited platelet response to both agonists. This study reinforces the hypothesis that GPIb is the high-affinity receptor for thrombin. The signaling mechanisms occurring through tyrosine phosphorylation of proteins triggered by thrombin seem to be dependent on intact GPIb. Moreover, our results indicate that both receptors, GPIb and PAR-1, are necessary to achieve a full platelet response to thrombin.

We demonstrate that exposure of cultured human endothelial cells to rHuEPO resulted in a dose-dependent increase in the tyrosine kinase activity, with phosphorylation of JAK-2 followed by rapid phosphorylation of STAT-5. Simultaneously, rHuEPO induced long-lasting phosphorylation of MAPK p42/44. Activation of this signaling pathways was directly associated with an increase in the thrombogenic properties of the extracellular matrix generated by these cells, when they were exposed to flowing blood. The enhancement in the reactivity of the resulting extracellular matrix towards platelets was associated with a higher expression of tissue factor. All these effects were blocked by an antibody to the EPO receptor and by specific inhibitors of tyrosine phosphorylation. The observed action of rHuEPO on endothelial cells seemed to be specifically triggered by the subsequent events that follow receptor binding, and occurred even at pharmacological concentrations of the cytokine. Our results indicate that rHuEPO has a direct action on the endothelium, increasing the reactivity of the underlying extracellular matrix towards platelets, effect that may be attributed to an increase in the expression of TF.

Recombinant human erythropoietin (rHuEPO) improves platelet function and signaling through tyrosine phosphorylation in uremic platelets in response to thrombin. Novel erythropoiesis-stimulating protein (NESP) has been recently introduced for the treatment of anemia in uremic patients with the advantage of requiring less frequent dosing. We analyzed the effects of NESP on intraplatelet signaling to thrombin, and compared these with the effects of rHuEPO. Results indicate that NESP is equivalent to rHuEPO with respect to its effects on platelet function.