Performance of a blood-based RNA signature for gemcitabine-based treatment in metastatic pancreatic adenocarcinoma.

Background: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer, and chemotherapy is a key treatment for advanced PDAC. Gemcitabine chemotherapy is still an important component of treatment; however, there is no routine biomarker to predict its efficacy. Predictive tests may help clinicians to decide on the best first-line chemotherapy. Methods: This study is a confirmatory study of a blood-based RNA signature, called the GemciTest. This test measures the expression levels of nine genes using real-time polymerase chain reaction (PCR) processes. Clinical validation was carried out, through a discovery and a validation phases, on 336 patients (mean 68.7 years; range, 37-88 years) for whom blood was collected from two prospective cohorts and two tumor biobanks. These cohorts included previously untreated advanced PDAC patients who received either a gemcitabine- or fluoropyrimidine-based regimen. Results: Gemcitabine-based treated patients with a positive GemciTest (22.9%) had a significantly longer progression-free survival (PFS) {5.3 vs. 2.8 months; hazard ratio (HR) =0.53 [95% confidence interval (CI): 0.31-0.92]; P=0.023} and overall survival (OS) [10.4 vs. 4.8 months; HR =0.49 (95% CI: 0.29-0.85); P=0.0091]. On the contrary, fluoropyrimidine-based treated patients showed no significant difference in PFS and OS using this blood signature. Conclusions: The GemciTest demonstrated that a blood-based RNA signature has the potential to aid in personalized therapy for PDAC, leading to better survival rates for patients receiving a gemcitabine-based first-line treatment.

Leishmania Parasites Differently Regulate Antioxidant Genes in Macrophages Derived From Resistant and Susceptible Mice.

Macrophage-Leishmania interactions are central to parasite growth and disease outcome. Macrophages have developed various strategies to fight invaders, including oxidative burst. While some microorganisms seem to survive and even thrive in an oxidative environment, others are susceptible and get killed. To counter oxidative stress, macrophages switch the expressions of cytoprotective and detoxifying enzymes, which are downstream targets of the nuclear factor erythroid 2-related factor 2 (Nrf2), to enhance cell survival. We have explored the transcription of NRF2 and of its target genes and compared the effect of the parasite on their transcription in bone marrow-derived macrophages (BMdMs) from Leishmania-resistant and Leishmania-susceptible mice. While heme oxygenase 1 (HO-1) transcription is independent of the genetic background, the transcription of glutathione reductase (Gsr) and of cysteine/glutamate exchange transporter (Slc7a11), involved in glutathione accumulation, was differentially regulated in BMdMs from both mouse strains. We also show that, except for HO-1, known to favor the survival of the parasite, the transcription of the selected genes, including Gsr, CD36, and catalase (CAT), was actively repressed, if not at all time points at least at the later ones, by the parasite, especially in Balb/c BMdMs. Consistent with these results, we found that the silencing of NRF2 in this study increases the survival and multiplication of the parasite.

Molecular Profiling of Spermatozoa Reveals Correlations between Morphology and Gene Expression: A Novel Biomarker Panel for Male Infertility.

Choosing spermatozoa with an optimum fertilizing potential is one of the major challenges in assisted reproductive technologies (ART). This selection is mainly based on semen parameters, but the addition of molecular approaches could allow a more functional evaluation. To this aim, we used sixteen fresh sperm samples from patients undergoing ART for male infertility and classified them in the high- and poor-quality groups, on the basis of their morphology at high magnification. Then, using a DNA sequencing method, we analyzed the spermatozoa methylome to identify genes that were differentially methylated. By Gene Ontology and protein-protein interaction network analyses, we defined candidate genes mainly implicated in cell motility, calcium reabsorption, and signaling pathways as well as transmembrane transport. RT-qPCR of high- and poor-quality sperm samples allowed showing that the expression of some genes, such as AURKA, HDAC4, CFAP46, SPATA18, CACNA1C, CACNA1H, CARHSP1, CCDC60, DNAH2, and CDC88B, have different expression levels according to sperm morphology. In conclusion, the present study shows a strong correlation between morphology and gene expression in the spermatozoa and provides a biomarker panel for sperm analysis during ART and a new tool to explore male infertility.

Could a Multi-Marker and Machine Learning Approach Help Stratify Patients with Heart Failure?

Half of the patients with heart failure (HF) have preserved ejection fraction (HFpEF). To date, there are no specific markers to distinguish this subgroup. The main objective of this work was to stratify HF patients using current biochemical markers coupled with clinical data. The cohort study included HFpEF (n = 24) and heart failure with reduced ejection fraction (HFrEF) (n = 34) patients as usually considered in clinical practice based on cardiac imaging (EF ≥ 50% for HFpEF; EF < 50% for HFrEF). Routine blood tests consisted of measuring biomarkers of renal and heart functions, inflammation, and iron metabolism. A multi-test approach and analysis of peripheral blood samples aimed to establish a computerized Machine Learning strategy to provide a blood signature to distinguish HFpEF and HFrEF. Based on logistic regression, demographic characteristics and clinical biomarkers showed no statistical significance to differentiate the HFpEF and HFrEF patient subgroups. Hence a multivariate factorial discriminant analysis, performed blindly using the data set, allowed us to stratify the two HF groups. Consequently, a Machine Learning (ML) strategy was developed using the same variables in a genetic algorithm approach. ML provided very encouraging explorative results when considering the small size of the samples applied. The accuracy and the sensitivity were high for both validation and test groups (69% and 100%, 64% and 75%, respectively). Sensitivity was 100% for the validation and 75% for the test group, whereas specificity was 44% and 55% for the validation and test groups because of the small number of samples. Lastly, the precision was acceptable, with 58% in the validation and 60% in the test group. Combining biochemical and clinical markers is an excellent entry to develop a computer classification tool to diagnose HFpEF. This translational approach is a springboard for improving new personalized treatment methods and identifying "high-yield" populations for clinical trials.

Predictive Values of Blood-Based RNA Signatures for the Gemcitabine Response in Advanced Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is expected to be the second cause of cancer death by 2022. For nearly 80% of patients, diagnosis occurs at an advanced, nonsurgical stage, making such patients incurable. Gemcitabine is still an important component in PDAC treatment and is most often used as a backbone to test new targeted therapies and there is, to date, no routine biomarker to predict its efficacy. Samples from a phase III randomized trial were used to develop through a large approach based on blood-based liquid biopsy, transcriptome profiling, and machine learning, a nine gene predictive signature for gemcitabine sensitivity. Patients with a positive test (41.6%) had a significantly longer progression free survival (PFS) (3.8 months vs. 1.9 months p = 0.03) and a longer overall survival (OS) (14.5 months vs. 5.1, p < 0.0001). In multivariate analyses, this signature was independently associated with PFS (HR = 0.5 (0.28-0.9) p = 0.025) and OS (HR = 0.39 (0.21-0.7) p = 0.002).

Transcriptomic study in women with trisomy 21 identifies a possible role of the GTPases of the immunity-associated proteins (GIMAP) in the protection of breast cancer.

BACKGROUND: People with trisomy 21 (T21) are predisposed to developing hematological tumors, but have significantly lower-than-expected age-adjusted incidence rates of having a solid tumor. MATERIAL AND METHODS: To identify novel genetic factors implicated in the lower breast cancer (BC) frequency observed in women with T21 than in the general population, we compared the transcriptome pattern of women with a homogeneous T21, aged more than 30 years, with or without BC, and tumoral BC tissue of control women with a normal karyotype from the study of Varley et al. (2014). RESULTS: Differential analysis of gene expression between the 15 women in the T21 without BC group and BC patients in the other groups (two women with T21 and fifteen control women, respectively) revealed 154 differentially expressed genes, of which 63 were found to have similar expression profile (up- or downregulated). Of those 63 genes, four were in the same family, namely GIMAP4, GIMAP6, GIMAP7 and GIMAP8, and were strongly upregulated in the T21 without BC group compared to the other groups. A significant decrease in mRNA levels of these genes in BC tissues compared to non-tumor breast tissues was also noted. CONCLUSION: We found that the expression of some GIMAPs is significantly higher in women with T21 without BC than in patients with sporadic BC. Our findings support the hypothesis that GIMAPs may play a tumor-suppressive role against BC, and open the possibility that they may also have the same role for other solid tumors in T21 patients. The search for new prognostic factors and hopefully new therapeutic or preventive strategies against BC are discussed.

Parental diuron-exposure alters offspring transcriptome and fitness in Pacific oyster Crassostrea gigas.

One of the primary challenges in ecotoxicology is to contribute to the assessment of the ecological status of ecosystems. In this study, we used Pacific oyster Crassostrea gigas to explore the effects of a parental exposure to diuron, a herbicide frequently detected in marine coastal environments. The present toxicogenomic study provides evidence that exposure of oyster genitors to diuron during gametogenesis results in changes in offspring, namely, transcriptomic profile alterations, increased global DNA methylation levels and reduced growth and survival within the first year of life. Importantly, we highlighted the limitations to identify particular genes or gene expression signatures that could serve as biomarkers for parental herbicide-exposure and further for multigenerational and transgenerational effects of specific chemical stressors. By analyzing samples from two independent experiments, we demonstrated that, due to complex confounding effects with both tested solvent vehicles, diuron non-specifically affected the offspring transcriptome. These original results question the potential development of predictive genomic tools for detecting specific indirect impacts of contaminants in environmental risk assessments. However, our results indicate that chronic environmental exposure to diuron over several generations may have significant long term impacts on oyster populations with adverse health outcomes.

Bronchial Epithelial Cells from Asthmatic Patients Display Less Functional HLA-G Isoform Expression.

Not all asthmatic patients adequately respond to current available treatments, such as inhaled corticosteroids or omalizumab®. New treatments will aim to target the bronchial epithelium-immune response interaction using different pathways. HLA-G is involved in immunomodulation and may promote epithelial cell differentiation and proliferation. HLA-G protein has several isoforms generated by alternative splicing that might have differential functionalities. HLA-G protein expression and genetic polymorphisms have been reported to be associated with asthma. Our hypothesis is that bronchial epithelium from asthmatic patients displays less functional HLA-G isoforms. HLA-G transcriptional isoforms were quantified by real-time PCR in human bronchial epithelium cells (HBEC) grown in air-liquid interface culture obtained from five healthy controls (HC), seven patients with mild asthma (MA), and seven patients with severe asthma (SA). They were re-differentiated, and IL-13 exposure was used as a proxy for a pro-inflammatory cytokine. HLA-G protein expression was assessed by western blot analysis. HLA-G allele was typed by direct sequencing. Our results showed that both MA and SA display less functional HLA-G isoforms than HC (p < 0.05); in vitro HBEC re-differentiation from SA displays a particular isoform expression profile compared to MA and HC (p = 0.03); HLA-G*01:06 frequency in MA and SA was significantly higher than in the healthy population (p = 0.03 and p < 0.001, respectively); and IL-13 exposure had no impact on HLA-G expression. Our results support that an impaired expression of HLA-G isoforms in asthmatic patients could contribute to the loss of inflammation control and epithelium structural remodeling. Therefore, HLA-G might be an interesting alternative target for asthmatic patients not adequately responding to current drugs.

Bacterial community characterization of water and intestine of the shrimp Litopenaeus stylirostris in a biofloc system.

BACKGROUND: Biofloc technology (BFT), a rearing method with little or no water exchange, is gaining popularity in aquaculture. In the water column, such systems develop conglomerates of microbes, algae and protozoa, together with detritus and dead organic particles. The intensive microbial community presents in these systems can be used as a pond water quality treatment system, and the microbial protein can serve as a feed additive. The current problem with BFT is the difficulty of controlling its bacterial community composition for both optimal water quality and optimal shrimp health. The main objective of the present study was to investigate microbial diversity of samples obtained from different culture environments (Biofloc technology and clear seawater) as well as from the intestines of shrimp reared in both environments through high-throughput sequencing technology. RESULTS: Analyses of the bacterial community identified in water from BFT and "clear seawater" (CW) systems (control) containing the shrimp Litopenaeus stylirostris revealed large differences in the frequency distribution of operational taxonomic units (OTUs). Four out of the five most dominant bacterial communities were different in both culture methods. Bacteria found in great abundance in BFT have two principal characteristics: the need for an organic substrate or nitrogen sources to grow and the capacity to attach to surfaces and co-aggregate. A correlation was found between bacteria groups and physicochemical and biological parameters measured in rearing tanks. Moreover, rearing-water bacterial communities influenced the microbiota of shrimp. Indeed, the biofloc environment modified the shrimp intestine microbiota, as the low level (27 %) of similarity between intestinal bacterial communities from the two treatments. CONCLUSION: This study provides the first information describing the complex biofloc microbial community, which can help to understand the environment-microbiota-host relationship in this rearing system.

Lipid Droplet Formation, Their Localization and Dynamics during Leishmania major Macrophage Infection.

Leishmania, the causative agent of vector-borne diseases, known as leishmaniases, is an obligate intracellular parasite within mammalian hosts. The outcome of infection depends largely on the activation status of macrophages, the first line of mammalian defense and the major target cells for parasite replication. Understanding the strategies developed by the parasite to circumvent macrophage defense mechanisms and to survive within those cells help defining novel therapeutic approaches for leishmaniasis. We previously showed the formation of lipid droplets (LDs) in L. major infected macrophages. Here, we provide novel insights on the origin of the formed LDs by determining their cellular distribution and to what extent these high-energy sources are directed to the proximity of Leishmania parasites. We show that the ability of L. major to trigger macrophage LD accumulation is independent of parasite viability and uptake and can also be observed in non-infected cells through paracrine stimuli suggesting that LD formation is from cellular origin. The accumulation of LDs is demonstrated using confocal microscopy and live-cell imagin in parasite-free cytoplasmic region of the host cell, but also promptly recruited to the proximity of Leishmania parasites. Indeed LDs are observed inside parasitophorous vacuole and in parasite cytoplasm suggesting that Leishmania parasites besides producing their own LDs, may take advantage of these high energy sources. Otherwise, these LDs may help cells defending against parasitic infection. These metabolic changes, rising as common features during the last years, occur in host cells infected by a large number of pathogens and seem to play an important role in pathogenesis. Understanding how Leishmania parasites and different pathogens exploit this LD accumulation will help us define the common mechanism used by these different pathogens to manipulate and/or take advantage of this high-energy source.

Evidence of zoonotic Poxviridae coinfections in clinically diagnosed papillomas using a newly developed mini-array test.

Our study describes a newly developed mini-array test for the rapid detection of poxviruses in animals and humans. The method is based on detection that combines target nucleic acid amplification by polymerase chain reaction and specific hybridization, using enzyme-linked antibodies, allowing identification of zoonotic orthopoxviruses and parapoxviruses in animal and human biological samples. With 100% specificity, the test rules out the possibility of cross-reactions with viral agents causing look-alike diseases. The assay was employed in the field to investigate the causes of several outbreaks of a malignant proliferative skin disease that affected domestic ruminants in Sicily during 2011-2014. Due to specific aspects of the lesions, the animals were clinically diagnosed with papillomatosis. The mini-array test allowed the identification of coinfections caused by more than 1 viral species belonging to the Parapoxvirus and Orthopoxvirus genera, either in goats or in cattle. Our study suggests that the so-called "papillomatosis" can be the result of multiple infections with epitheliotropic viruses, including zoonotic poxviruses that cannot be properly identified with classical diagnostic techniques.

Comparative analysis of resistant and susceptible macrophage gene expression response to Leishmania major parasite.

BACKGROUND: Leishmania are obligated intracellular pathogens that replicate almost exclusively in macrophages. The outcome of infection depends largely on parasite pathogenicity and virulence but also on the activation status and genetic background of macrophages. Animal models are essential for a better understanding of pathogenesis of different microbes including Leishmania. RESULTS: Here we compared the transcriptional signatures of resistant (C57BL/6) and susceptible (BALB/c) mouse bone marrow-derived macrophages in response to Leishmania major (L. major) promastigotes infection.Microarray results were first analyzed for significant pathways using the Kyoto Encylopedia of Genes and Genomes (KEGG) database. The analysis revealed that a large set of the shared genes is involved in the immune response and that difference in the expression level of some chemokines and chemokine receptors could partially explain differences in resistance. We next focused on up-regulated genes unique to either BALB/c or C57BL/6 derived macrophages and identified, using KEGG database, signal transduction pathways among the most relevant pathways unique to both susceptible and resistant derived macrophages. Indeed, genes unique to C57BL/6 BMdMs were associated with target of rapamycin (mTOR) signaling pathway while a range of genes unique to BALB/c BMdMs, belong to p53 signaling pathway. We next investigated whether, in a given mice strain derived macrophages, the different up-regulated unique genes could be coordinately regulated. Using GeneMapp Cytoscape, we showed that the induced genes unique to BALB/c or C57BL/6 BMdMs are interconnected. Finally, we examined whether the induced pathways unique to BALB/c derived macrophages interfere with the ones unique to C57BL/6 derived macrophages. Protein-protein interaction analysis using String database highlights the existence of a cross-talk between p53 and mTOR signaling pathways respectively specific to susceptible and resistant BMdMs. CONCLUSIONS: Taken together our results suggest that strains specific pathogenesis may be due to a difference in the magnitude of the same pathways and/or to differentially expressed pathways in the two mouse strains derived macrophages. We identify signal transduction pathways among the most relevant pathways modulated by L. major infection, unique to BALB/c and C57BL/6 BMdM and postulate that the interplay between these potentially interconnected pathways could direct the macrophage response toward a given phenotype.

The intellectual disability of trisomy 21: differences in gene expression in a case series of patients with lower and higher IQ.

Trisomy 21 (T21), or Down syndrome (DS), is the most frequent and recognizable cause of intellectual disabilities. The level of disability, as evaluated by the intelligence quotient (IQ) test, varies considerably between patients independent of other factors. To determine the genetic or molecular basis of this difference, a high throughput transcriptomic analysis was performed on twenty T21 patients with high and low IQ, and 10 healthy controls using Digital Gene Expression. More than 90 millions of tags were sequenced in the three libraries. A total of 80 genes of potential interest were selected for the qPCR experiment validation, and three housekeeping genes were used for normalizing purposes. HLA DQA1 and HLA DRB1 were significantly downregulated among the patients with a low IQ, the values found in the healthy controls being intermediate between those noted in the IQ+ and IQ- T21 patients. Interestingly, the intergenic region between these genes contains a binding sequence for the CCCTC-binding factor, or CTCF, and cohesin (a multisubunit complex), both of which are essential for expression of HLA DQA1 and HLA DRB1 and numerous other genes. Our results might lead to the discovery of genes, or genetic markers, that are directly involved in several phenotypes of DS and, eventually, to the identification of potential targets for therapeutic interventions.

Different secretory repertoires control the biomineralization processes of prism and nacre deposition of the pearl oyster shell.

Mollusca evolutionary success can be attributed partly to their efficiency to sustain and protect their soft body with an external biomineralized structure, the shell. Current knowledge of the protein set responsible for the formation of the shell microstructural polymorphism and unique properties remains largely patchy. In Pinctada margaritifera and Pinctada maxima, we identified 80 shell matrix proteins, among which 66 are entirely unique. This is the only description of the whole "biomineralization toolkit" of the matrices that, at least in part, is thought to regulate the formation of the prismatic and nacreous shell layers in the pearl oysters. We unambiguously demonstrate that prisms and nacre are assembled from very different protein repertoires. This suggests that these layers do not derive from each other.

Of mice and men: divergence of gene expression patterns in kidney.

Since the development of methods for homologous gene recombination, mouse models have played a central role in research in renal pathophysiology. However, many published and unpublished results show that mice with genetic changes mimicking human pathogenic mutations do not display the human phenotype. These functional differences may stem from differences in gene expression between mouse and human kidneys. However, large scale comparison of gene expression networks revealed conservation of gene expression among a large panel of human and mouse tissues including kidneys. Because renal functions result from the spatial integration of elementary processes originating in the glomerulus and the successive segments constituting the nephron, we hypothesized that differences in gene expression profiles along the human and mouse nephron might account for different behaviors. Analysis of SAGE libraries generated from the glomerulus and seven anatomically defined nephron segments from human and mouse kidneys allowed us to identify 4644 pairs of gene orthologs expressed in either one or both species. Quantitative analysis shows that many transcripts are present at different levels in the two species. It also shows poor conservation of gene expression profiles, with less than 10% of the 4644 gene orthologs displaying a higher conservation of expression profiles than the neutral expectation (p<0.05). Accordingly, hierarchical clustering reveals a higher degree of conservation of gene expression patterns between functionally unrelated kidney structures within a given species than between cognate structures from the two species. Similar findings were obtained for sub-groups of genes with either kidney-specific or housekeeping functions. Conservation of gene expression at the scale of the whole organ and divergence at the level of its constituting sub-structures likely account for the fact that although kidneys assume the same global function in the two species, many mouse "models" of human pathologies do not display the expected phenotype.

Transcriptomic signature of Leishmania infected mice macrophages: a metabolic point of view.

We analyzed the transcriptional signatures of mouse bone marrow-derived macrophages at different times after infection with promastigotes of the protozoan parasite Leishmania major. Ingenuity Pathway Analysis revealed that the macrophage metabolic pathways including carbohydrate and lipid metabolisms were among the most altered pathways at later time points of infection. Indeed, L. major promastiogtes induced increased mRNA levels of the glucose transporter and almost all of the genes associated with glycolysis and lactate dehydrogenase, suggesting a shift to anaerobic glycolysis. On the other hand, L. major promastigotes enhanced the expression of scavenger receptors involved in the uptake of Low-Density Lipoprotein (LDL), inhibited the expression of genes coding for proteins regulating cholesterol efflux, and induced the synthesis of triacylglycerides. These data suggested that Leishmania infection disturbs cholesterol and triglycerides homeostasis and may lead to cholesterol accumulation and foam cell formation. Using Filipin and Bodipy staining, we showed cholesterol and triglycerides accumulation in infected macrophages. Moreover, Bodipy-positive lipid droplets accumulated in close proximity to parasitophorous vacuoles, suggesting that intracellular L. major may take advantage of these organelles as high-energy substrate sources. While the effect of infection on cholesterol accumulation and lipid droplet formation was independent on parasite development, our data indicate that anaerobic glycolysis is actively induced by L. major during the establishment of infection.

A hemocyte gene expression signature correlated with predictive capacity of oysters to survive Vibrio infections.

BACKGROUND: The complex balance between environmental and host factors is an important determinant of susceptibility to infection. Disturbances of this equilibrium may result in multifactorial diseases as illustrated by the summer mortality syndrome, a worldwide and complex phenomenon that affects the oysters, Crassostrea gigas. The summer mortality syndrome reveals a physiological intolerance making this oyster species susceptible to diseases. Exploration of genetic basis governing the oyster resistance or susceptibility to infections is thus a major goal for understanding field mortality events. In this context, we used high-throughput genomic approaches to identify genetic traits that may characterize inherent survival capacities in C. gigas. RESULTS: Using digital gene expression (DGE), we analyzed the transcriptomes of hemocytes (immunocompetent cells) of oysters able or not able to survive infections by Vibrio species shown to be involved in summer mortalities. Hemocytes were nonlethally collected from oysters before Vibrio experimental infection, and two DGE libraries were generated from individuals that survived or did not survive. Exploration of DGE data and microfluidic qPCR analyses at individual level showed an extraordinary polymorphism in gene expressions, but also a set of hemocyte-expressed genes whose basal mRNA levels discriminate oyster capacity to survive infections by the pathogenic V. splendidus LGP32. Finally, we identified a signature of 14 genes that predicted oyster survival capacity. Their expressions are likely driven by distinct transcriptional regulation processes associated or not associated to gene copy number variation (CNV). CONCLUSIONS: We provide here for the first time in oyster a gene expression survival signature that represents a useful tool for understanding mortality events and for assessing genetic traits of interest for disease resistance selection programs.

Large-scale discovery of conopeptides and conoproteins in the injectable venom of a fish-hunting cone snail using a combined proteomic and transcriptomic approach.

Predatory marine snails of the genus Conus use venom containing a complex mixture of bioactive peptides to subdue their prey. Here we report on a comprehensive analysis of the protein content of injectable venom from Conus consors, an indo-pacific fish-hunting cone snail. By matching MS/MS data against an extensive set of venom gland transcriptomic mRNA sequences, we identified 105 components out of ~400 molecular masses detected in the venom. Among them, we described new conotoxins belonging to the A, M- and O1-superfamilies as well as a novel superfamily of disulphide free conopeptides. A high proportion of the deduced sequences (36%) corresponded to propeptide regions of the A- and M-superfamilies, raising the question of their putative role in injectable venom. Enzymatic digestion of higher molecular mass components allowed the identification of new conkunitzins (~7 kDa) and two proteins in the 25 and 50 kDa molecular mass ranges respectively characterised as actinoporin-like and hyaluronidase-like protein. These results provide the most exhaustive and accurate proteomic overview of an injectable cone snail venom to date, and delineate the major protein families present in the delivered venom. This study demonstrates the feasibility of this analytical approach and paves the way for transcriptomics-assisted strategies in drug discovery.

New prognostic markers, determined using gene expression analyses, reveal two distinct subtypes of chronic myelomonocytic leukaemia patients.

Chronic myelomonocytic leukaemia (CMML) is a heterogeneous haematopoietic disorder characterized by myeloproliferative or myelodysplastic features. At present, the pathogenesis of this malignancy is not completely understood. In this study, we sought to analyse gene expression profiles of CMML in order to characterize new molecular outcome predictors. A learning set of 32 untreated CMML patients at diagnosis was available for TaqMan low-density array gene expression analysis. From 93 selected genes related to cancer and cell cycle, we built a five-gene prognostic index after multiplicity correction. Using this index, we characterized two categories of patients with distinct overall survival (94% vs. 19% for good and poor overall survival, respectively; P = 0·007) and we successfully validated its strength on an independent cohort of 21 CMML patients with Affymetrix gene expression data. We found no specific patterns of association with traditional prognostic stratification parameters in the learning cohort. However, the poor survival group strongly correlated with high-risk treated patients and transformation to acute myeloid leukaemia. We report here a new multigene prognostic index for CMML, independent of the gene expression measurement method, which could be used as a powerful tool to predict clinical outcome and help physicians to evaluate criteria for treatments.

Recruitment of glycosyl hydrolase proteins in a cone snail venomous arsenal: further insights into biomolecular features of Conus venoms.

Cone snail venoms are considered an untapped reservoir of extremely diverse peptides, named conopeptides, displaying a wide array of pharmacological activities. We report here for the first time, the presence of high molecular weight compounds that participate in the envenomation cocktail used by these marine snails. Using a combination of proteomic and transcriptomic approaches, we identified glycosyl hydrolase proteins, of the hyaluronidase type (Hyal), from the dissected and injectable venoms ("injectable venom" stands for the venom variety obtained by milking of the snails. This is in contrast to the "dissected venom", which was obtained from dissected snails by extraction of the venom glands) of a fish-hunting cone snail, Conus consors (Pionoconus clade). The major Hyal isoform, Conohyal-Cn1, is expressed as a mixture of numerous glycosylated proteins in the 50 kDa molecular mass range, as observed in 2D gel and mass spectrometry analyses. Further proteomic analysis and venom duct mRNA sequencing allowed full sequence determination. Additionally, unambiguous segment location of at least three glycosylation sites could be determined, with glycans corresponding to multiple hexose (Hex) and N-acetylhexosamine (HexNAc) moieties. With respect to other known Hyals, Conohyal-Cn1 clearly belongs to the hydrolase-type of Hyals, with strictly conserved consensus catalytic donor and positioning residues. Potent biological activity of the native Conohyals could be confirmed in degrading hyaluronic acid. A similar Hyal sequence was also found in the venom duct transcriptome of C. adamsonii (Textilia clade), implying a possible widespread recruitment of this enzyme family in fish-hunting cone snail venoms. These results provide the first detailed Hyal sequence characterized from a cone snail venom, and to a larger extent in the Mollusca phylum, thus extending our knowledge on this protein family and its evolutionary selection in marine snail venoms.