A Time Point Proteomic Analysis Reveals Protein Dynamics of Plasmodium Oocysts.

The oocyst is a sporogonic stage of Plasmodium development that takes place in the mosquito midgut in about 2 weeks. The cyst is protected by a capsule of unknown composition, and little is known about oocyst biology. We carried out a proteomic analysis of oocyst samples isolated at early, mid, and late time points of development. Four biological replicates for each time point were analyzed, and almost 600 oocyst-specific candidates were identified. The analysis revealed that, in young oocysts, there is a strong activity of protein and DNA synthesis, whereas in mature oocysts, proteins involved in oocyst and sporozoite development, gliding motility, and invasion are mostly abundant. Among the proteins identified at early stages, 17 candidates are specific to young oocysts. Thirty-four candidates are common to oocyst and the merosome stages (sporozoite proteins excluded), sharing common features as replication and egress. Western blot and immunofluorescence analyses of selected candidates confirm the expression profile obtained by proteomic analysis.

Screening of the activity of sixty essential oils against plasmodium early mosquito stages in vitro and machine learning analysis reveals new putative inhibitors of malaria parasites.

Malaria, an infectious disease with a tremendous impact on human health is caused by Plasmodium parasites, and transmitted by Anopheles mosquitoes. New approaches to control the disease involve transmission blocking strategies aiming to target the parasite in the mosquito. Here, we investigated the putative inhibitory activity of essential oils and their components on the early mosquito stages of the parasite. We employed an in vitro assay of gametocyte-to-ookinete development of the rodent model parasite Plasmodium berghei combined with high content screening. 60 essential oils with known composition were tested. The results revealed that fifteen EOs had inhibitory activity. Furthermore, a machine learning approach was used to identify the putative inhibitory components. Five of the most important chemical components indicated by the machine learning-based models were actually confirmed by the experimental approach. This combined approach was used for the first time to identify the potential transmission blocking activity of essential oils and single components at the zygote and ookinete stages.

A Plasma Circular RNA Profile Differentiates Subjects with Alzheimer's Disease and Mild Cognitive Impairment from Healthy Controls.

The most frequently used biomarkers to support the diagnosis of Alzheimer's Disease (AD) are Aß42, total-Tau, and phospho-tau protein levels in CSF. Moreover, magnetic resonance imaging is used to assess hippocampal atrophy, 18F-FDG PET to identify abnormal brain metabolism, and PET imaging for amyloid deposition. These tests are rather complex and invasive and not easily applicable to clinical practice. Circulating non-coding RNAs, which are inherently stable and easy to manage, have been reported as promising biomarkers for central nervous system conditions. Recently, circular RNAs (circRNAs) as a novel class of ncRNAs have gained attention. We carried out a pilot study on five participants with AD and five healthy controls (HC) investigating circRNAs by Arraystar Human Circular RNA Microarray V2.0. Among them, 26 circRNAs were differentially expressed (FC ≥ 1.5, p < 0.05) in participants with AD compared to HC. From a top 10 of differentially expressed circRNAs, a validation study was carried out on four up-regulated (hsa_circRNA_050263, hsa_circRNA_403959, hsa_circRNA_003022, hsa_circRNA_100837) and two down-regulated (hsa_circRNA_102049, hsa_circRNA_102619) circRNAs in a larger population. Moreover, five subjects with mild cognitive impairment (MCI) were investigated. The analysis confirmed the upregulation of hsa_circRNA_050263, hsa_circRNA_403959, and hsa_circRNA_003022 both in subjects with AD and in MCI compared to HCs. We also investigated all microRNAs potentially interacting with the studied circRNAs. The GO enrichment analysis shows they are involved in the development of the nervous system, and in the cellular response to nerve growth factor stimuli, protein phosphorylation, apoptotic processes, and inflammation pathways, all of which are processes related to the pathology of AD.

Identification and preliminary characterization of Plasmodium falciparum proteins secreted upon gamete formation.

Malaria long-term elimination depends on parasite transmission control. Plasmodium sexual stage maturation in the mosquito, including egress from the host erythrocyte, is one of the prime targets for transmission-blocking interventions. This work aims to identify candidate molecules potentially involved in gamete emergence from the host erythrocyte, as novel transmission blocking targets. We analyzed by quantitative mass spectrometry the proteins released/secreted by purified Plasmodium falciparum gametocytes upon induction of gametogenesis. The proteome obtained showed a good overlap (74%) with the one previously characterized in similar conditions from gametocytes of the rodent malaria parasite P. berghei. Four candidates were selected based on comparative analysis of their abundance values in released vs total gametocyte proteome. We also characterized the P. falciparum orthologue of the microgamete surface protein (MiGS), a marker of male gametocyte secretory vesicles in murine models of malaria. The findings of this study reveal that all the selected candidate proteins are expressed in both genders and localize to vesicle-like structures that respond to gametogenesis stimuli. This result, together with the fact that the selected proteins are released during gamete emergence in both Plasmodium species, makes them interesting candidates for future functional studies to investigate their potential role in the gametogenesis process.

Structural organization of erythrocyte membrane microdomains and their relation with malaria susceptibility.

Cholesterol-rich microdomains are membrane compartments characterized by specific lipid and protein composition. These dynamic assemblies are involved in several biological processes, including infection by intracellular pathogens. This work provides a comprehensive analysis of the composition of human erythrocyte membrane microdomains. Based on their floating properties, we also categorized the microdomain-associated proteins into clusters. Interestingly, erythrocyte microdomains include the vast majority of the proteins known to be involved in invasion by the malaria parasite Plasmodium falciparum. We show here that the Ecto-ADP-ribosyltransferase 4 (ART4) and Aquaporin 1 (AQP1), found within one specific cluster, containing the essential host determinant CD55, are recruited to the site of parasite entry and then internalized to the newly formed parasitophorous vacuole membrane. By generating null erythroid cell lines, we showed that one of these proteins, ART4, plays a role in P. falciparum invasion. We also found that genetic variants in both ART4 and AQP1 are associated with susceptibility to the disease in a malaria-endemic population.

A Comprehensive Gender-related Secretome of Plasmodium berghei Sexual Stages.

Plasmodium, the malaria parasite, undergoes a complex life cycle alternating between a vertebrate host and a mosquito vector of the genus Anopheles In red blood cells of the vertebrate host, Plasmodium multiplies asexually or differentiates into gamete precursors, the male and female gametocytes, responsible for parasite transmission. Sexual stage maturation occurs in the midgut of the mosquito vector, where male and female gametes egress from the host erythrocytes to fuse and form a zygote. Gamete egress entails the successive rupture of two membranes surrounding the parasite, the parasitophorous vacuole membrane and the erythrocyte plasma membrane. In this study, we used the rodent model parasite Plasmodium berghei to design a label-free quantitative proteomic approach aimed at identifying gender-related proteins differentially released/secreted by purified mature gametocytes when activated to form gametes. We compared the abundance of molecules secreted by wild type gametocytes of both genders with that of a transgenic line defective in male gamete maturation and egress. This enabled us to provide a comprehensive data set of egress-related molecules and their gender specificity. Using specific antibodies, we validated eleven candidate molecules, predicted as either gender-specific or common to both male and female gametocytes. All of them localize to punctuate, vesicle-like structures that relocate to cell periphery upon activation, but only three of them localize to the gametocyte-specific secretory vesicles named osmiophilic bodies. Our results confirm that the egress process involves a tightly coordinated secretory apparatus that includes different types of vesicles and may put the basis for functional studies aimed at designing novel transmission-blocking molecules.

Caveolin-1 Endows Order in Cholesterol-Rich Detergent Resistant Membranes.

Cholesterol-enriched functional portions of plasma membranes, such as caveolae and rafts, were isolated from lungs of wild-type (WT) and caveolin-1 knockout (Cav-1 KO) mice within detergent resistant membranes (DRMs). To gain insight into their molecular composition we performed proteomic and lipid analysis on WT and Cav-1 KO-DRMs that showed predicted variations of proteomic profiles and negligible differences in lipid composition, while Langmuir monolayer technique and small and wide-angle X-ray scattering (SAXS-WAXS) were here originally introduced to study DRMs biophysical association state. Langmuir analysis of Cav-1 containing DRMs displayed an isotherm with a clear-cut feature, suggesting the coexistence of the liquid-ordered (Lo) phase typical of the raft structure, namely "cholesterol-rich Lo phase," with a phase fully missing in Cav-1 KO that we named "caveolin-induced Lo phase." Furthermore, while the sole lipid component of both WT and KO-DRMs showed qualitatively similar isotherm configuration, the reinsertion of recombinant Cav-1 into WT-DRMs lipids restored the WT-DRM pattern. X-ray diffraction results confirmed that Cav-1 causes the formation of a "caveolin-induced Lo phase," as suggested by Langmuir experiments, allowing us to speculate about a possible structural model. These results show that the unique molecular link between Cav-1 and cholesterol can spur functional order in a lipid bilayer strictly derived from biological sources.

Acidic microenvironment plays a key role in human melanoma progression through a sustained exosome mediated transfer of clinically relevant metastatic molecules.

BACKGROUND: Microenvironment cues involved in melanoma progression are largely unknown. Melanoma is highly influenced in its aggressive phenotype by the changes it determinates in its microenvironment, such as pH decrease, in turn influencing cancer cell invasiveness, progression and tissue remodelling through an abundant secretion of exosomes, dictating cancer strategy to the whole host. A role of exosomes in driving melanoma progression under microenvironmental acidity was never described. METHODS: We studied four differently staged human melanoma lines, reflecting melanoma progression, under microenvironmental acidic pHs pressure ranging between pH 6.0-6.7. To estimate exosome secretion as a function of tumor stage and environmental pH, we applied a technique to generate native fluorescent exosomes characterized by vesicles integrity, size, density, markers expression, and quantifiable by direct FACS analysis. Functional roles of exosomes were tested in migration and invasion tests. Then we performed a comparative proteomic analysis of acid versus control exosomes to elucidate a specific signature involved in melanoma progression. RESULTS: We found that metastatic melanoma secretes a higher exosome amount than primary melanoma, and that acidic pH increases exosome secretion when melanoma is in an intermediate stage, i.e. metastatic non-invasive. We were thus able to show that acidic pH influences the intercellular cross-talk mediated by exosomes. In fact when exposed to exosomes produced in an acidic medium, pH naïve melanoma cells acquire migratory and invasive capacities likely due to transfer of metastatic exosomal proteins, favoring cell motility and angiogenesis. A Prognoscan-based meta-analysis study of proteins enriched in acidic exosomes, identified 11 genes (HRAS, GANAB, CFL2, HSP90B1, HSP90AB1, GSN, HSPA1L, NRAS, HSPA5, TIMP3, HYOU1), significantly correlating with poor prognosis, whose high expression was in part confirmed in bioptic samples of lymph node metastases. CONCLUSIONS: A crucial step of melanoma progression does occur at melanoma intermediate -stage, when extracellular acidic pH induces an abundant release and intra-tumoral uptake of exosomes. Such exosomes are endowed with pro-invasive molecules of clinical relevance, which may provide a signature of melanoma advancement.

Phylo_dCor: distance correlation as a novel metric for phylogenetic profiling.

BACKGROUND: Elaboration of powerful methods to predict functional and/or physical protein-protein interactions from genome sequence is one of the main tasks in the post-genomic era. Phylogenetic profiling allows the prediction of protein-protein interactions at a whole genome level in both Prokaryotes and Eukaryotes. For this reason it is considered one of the most promising methods. RESULTS: Here, we propose an improvement of phylogenetic profiling that enables handling of large genomic datasets and infer global protein-protein interactions. This method uses the distance correlation as a new measure of phylogenetic profile similarity. We constructed robust reference sets and developed Phylo-dCor, a parallelized version of the algorithm for calculating the distance correlation that makes it applicable to large genomic data. Using Saccharomyces cerevisiae and Escherichia coli genome datasets, we showed that Phylo-dCor outperforms phylogenetic profiling methods previously described based on the mutual information and Pearson's correlation as measures of profile similarity. CONCLUSIONS: In this work, we constructed and assessed robust reference sets and propose the distance correlation as a measure for comparing phylogenetic profiles. To make it applicable to large genomic data, we developed Phylo-dCor, a parallelized version of the algorithm for calculating the distance correlation. Two R scripts that can be run on a wide range of machines are available upon request.

An Integrated Approach to Explore Composition and Dynamics of Cholesterol-rich Membrane Microdomains in Sexual Stages of Malaria Parasite.

Membrane microdomains that include lipid rafts, are involved in key physiological and pathological processes and participate in the entry of endocellular pathogens. These assemblies, enriched in cholesterol and sphingolipids, form highly dynamic, liquid-ordered phases that can be separated from the bulk membranes thanks to their resistance to solubilization by nonionic detergents. To characterize complexity and dynamics of detergent-resistant membranes of sexual stages of the rodent malaria parasite Plasmodium berghei, here we propose an integrated study of raft components based on proteomics, lipid analysis and bioinformatics. This analysis revealed unexpected heterogeneity and unexplored pathways associated with these specialized assemblies. Protein-protein relationships and protein-lipid co-occurrence were described through multi-component networks. The proposed approach can be widely applied to virtually every cell type in different contexts and perturbations, under physiological and/or pathological conditions.

Proteomic analysis of detergent-resistant membrane microdomains in trophozoite blood stage of the human malaria parasite Plasmodium falciparum.

Intracellular pathogens contribute to a significant proportion of infectious diseases worldwide. The successful strategy of evading the immune system by hiding inside host cells is common to all the microorganism classes, which exploit membrane microdomains, enriched in cholesterol and sphingolipids, to invade and colonize the host cell. These assemblies, with distinct biochemical properties, can be isolated by means of flotation in sucrose density gradient centrifugation because they are insoluble in nonionic detergents at low temperature. We analyzed the protein and lipid contents of detergent-resistant membranes from erythrocytes infected by Plasmodium falciparum, the most deadly human malaria parasite. Proteins associated with membrane microdomains of trophic parasite blood stages (trophozoites) include an abundance of chaperones, molecules involved in vesicular trafficking, and enzymes implicated in host hemoglobin degradation. About 60% of the identified proteins contain a predicted localization signal suggesting a role of membrane microdomains in protein sorting/trafficking. To validate our proteomic data, we raised antibodies against six Plasmodium proteins not characterized previously. All the selected candidates were recovered in floating low-density fractions after density gradient centrifugation. The analyzed proteins localized either to internal organelles, such as the mitochondrion and the endoplasmic reticulum, or to exported membrane structures, the parasitophorous vacuole membrane and Maurer's clefts, implicated in targeting parasite proteins to the host erythrocyte cytosol or surface. The relative abundance of cholesterol and phospholipid species varies in gradient fractions containing detergent-resistant membranes, suggesting heterogeneity in the lipid composition of the isolated microdomain population. This study is the first report showing the presence of cholesterol-rich microdomains with distinct properties and subcellular localization in trophic stages of Plasmodium falciparum.

Global proteomic analysis of the oocyst/sporozoite of Toxoplasma gondii reveals commitment to a host-independent lifestyle.

BACKGROUND: Toxoplasmosis is caused by the apicomplexan parasite Toxoplasma gondii and can be acquired either congenitally or via the oral route. In the latter case, transmission is mediated by two distinct invasive stages, i.e., bradyzoites residing in tissue cysts or sporozoites contained in environmentally resistant oocysts shed by felids in their feces. The oocyst plays a central epidemiological role, yet this stage has been scarcely investigated at the molecular level and the knowledge of its expressed proteome is very limited. RESULTS: Using one-dimensional gel electrophoresis coupled to liquid chromatography-linked tandem mass spectrometry, we analysed total or fractionated protein extracts of partially sporulated T. gondii oocysts, producing a dataset of 1304 non reduntant proteins (~18% of the total predicted proteome), ~59% of which were classified according to the MIPS functional catalogue database. Notably, the comparison of the oocyst dataset with the extensively covered proteome of T. gondii tachyzoite, the invasive stage responsible for the clinical signs of toxoplasmosis, identified 154 putative oocyst/sporozoite-specific proteins, some of which were validated by Western blot. The analysis of this protein subset showed that, compared to tachyzoites, oocysts have a greater capability of de novo amino acid biosynthesis and are well equipped to fuel the Krebs cycle with the acetyl-CoA generated through fatty acid ß-oxidation and the degradation of branched amino acids. CONCLUSIONS: The study reported herein significantly expanded our knowledge of the proteome expressed by the oocyst/sporozoite of T. gondii, shedding light on a stage-specifc subset of proteins whose functional profile is consistent with the adaptation of T. gondii oocysts to the nutrient-poor and stressing extracellular environment.

Revisiting the Plasmodium falciparum RIFIN family: from comparative genomics to 3D-model prediction.

BACKGROUND: Subtelomeric RIFIN genes constitute the most abundant multigene family in Plasmodium falciparum. RIFIN products are targets for the human immune response and contribute to the antigenic variability of the parasite. They are transmembrane proteins grouped into two sub-families (RIF_A and RIF_B). Although recent data show that RIF_A and RIF_B have different sub-cellular localisations and possibly different functions, the same structural organisation has been proposed for members of the two sub-families. Despite recent advances, our knowledge of the regulation of RIFIN gene expression is still poor and the biological role of the protein products remain obscure. RESULTS: Comparative studies on RIFINs in three clones of P. falciparum (3D7, HB3 and Dd2) by Multidimensional scaling (MDS) showed that gene sequences evolve differently in the 5'upstream, coding, and 3'downstream regions, and suggested a possible role of highly conserved 3' downstream sequences. Despite the expected polymorphism, we found that the overall structure of RIFIN repertoires is conserved among clones suggesting a balance between genetic drift and homogenisation mechanisms which guarantees emergence of novel variants but preserves the functionality of genes. Protein sequences from a bona fide set of 3D7 RIFINs were submitted to predictors of secondary structure elements. In contrast with the previously proposed structural organisation, no signal peptide and only one transmembrane helix were predicted for the majority of RIF_As. Finally, we developed a strategy to obtain a reliable 3D-model for RIF_As. We generated 265 possible structures from 53 non-redundant sequences, from which clustering and quality assessments selected two models as the most representative for putative RIFIN protein structures. CONCLUSION: First, comparative analyses of RIFIN repertoires in different clones of P. falciparum provide insights on evolutionary mechanisms shaping the multigene family. Secondly, we found that members of the two sub-families RIF_As and RIF_Bs have different structural organization in accordance with recent experimental results. Finally, representative models for RIF_As have an "Armadillo-like" fold which is known to promote protein-protein interactions in diverse contexts.

A genomic glimpse of aminoacyl-tRNA synthetases in malaria parasite Plasmodium falciparum.

BACKGROUND: Plasmodium parasites are causative agents of malaria which affects >500 million people and claims approximately 2 million lives annually. The completion of Plasmodium genome sequencing and availability of PlasmoDB database has provided a platform for systematic study of parasite genome. Aminoacyl-tRNA synthetases (aaRSs) are pivotal enzymes for protein translation and other vital cellular processes. We report an extensive analysis of the Plasmodium falciparum genome to identify and classify aaRSs in this organism. RESULTS: Using various computational and bioinformatics tools, we have identified 37 aaRSs in P. falciparum. Our key observations are: (i) fraction of proteome dedicated to aaRSs in P. falciparum is very high compared to many other organisms; (ii) 23 out of 37 Pf-aaRS sequences contain signal peptides possibly directing them to different cellular organelles; (iii) expression profiles of Pf-aaRSs vary considerably at various life cycle stages of the parasite; (iv) several PfaaRSs posses very unusual domain architectures; (v) phylogenetic analyses reveal evolutionary relatedness of several parasite aaRSs to bacterial and plants aaRSs; (vi) three dimensional structural modelling has provided insights which could be exploited in inhibitor discovery against parasite aaRSs. CONCLUSION: We have identified 37 Pf-aaRSs based on our bioinformatics analysis. Our data reveal several unique attributes in this protein family. We have annotated all 37 Pf-aaRSs based on predicted localization, phylogenetics, domain architectures and their overall protein expression profiles. The sets of distinct features elaborated in this work will provide a platform for experimental dissection of this family of enzymes, possibly for the discovery of novel drugs against malaria.

Core promoters are predicted by their distinct physicochemical properties in the genome of Plasmodium falciparum.

Little is known about the structure and distinguishing features of core promoters in Plasmodium falciparum. In this work, we describe the first method to computationally identify core promoters in this AT-rich genome. This prediction algorithm uses solely DNA physicochemical properties as descriptors. Our results add to a growing body of evidence that a physicochemical code for eukaryotic genomes plays a crucial role in core promoter recognition.

Plasmodium lipid rafts contain proteins implicated in vesicular trafficking and signalling as well as members of the PIR superfamily, potentially implicated in host immune system interactions.

Plasmodium parasites, the causal agents of malaria, dramatically modify the infected erythrocyte by exporting parasite proteins into one or multiple erythrocyte compartments, the cytoplasm and the plasma membrane or beyond. Despite advances in defining signals and specific cellular compartments implicated in protein trafficking in Plasmodium-infected erythrocytes, the contribution of lipid-mediated sorting to this cellular process has been poorly investigated. In this study, we examined the proteome of cholesterol-rich membrane microdomains or lipid rafts, purified from erythrocytes infected by the rodent parasite Plasmodium berghei. Besides structural proteins associated with invasive forms, we detected chaperones, proteins implicated in vesicular trafficking, membrane fusion events and signalling. Interestingly, the raft proteome of mixed P. berghei blood stages included proteins encoded by members of a large family (bir) of putative variant antigens potentially implicated in host immune system interactions and targeted to the surface of the host erythrocytes. The generation of transgenic parasites expressing BIR/GFP fusions confirmed the dynamic association of members of this protein family with membrane microdomains. Our results indicated that lipid rafts in Plasmodium-infected erythrocytes might constitute a route to sort and fold parasite proteins directed to various host cell compartments including the cell surface.

A novel series of compositionally biased substitution matrices for comparing Plasmodium proteins.

BACKGROUND: The most common substitution matrices currently used (BLOSUM and PAM) are based on protein sequences with average amino acid distributions, thus they do not represent a fully accurate substitution model for proteins characterized by a biased amino acid composition. This problem has been addressed recently by adjusting existing matrices, however, to date, no empirical approach has been taken to build matrices which offer a substitution model for comparing proteins sharing an amino acid compositional bias. Here, we present a novel procedure to construct series of symmetrical substitution matrices to align proteins from similarly biased Plasmodium proteomes. RESULTS: We generated substitution matrices by selecting from the BLOCKS database those multiple alignments with a compositional bias similar to that of P. falciparum and P. yoelii proteins. A novel 'fuzzy' clustering method was adopted to group sequences within these alignments, showing that this method retains more complete information on the amino acid substitutions when compared to hierarchical clustering. We assessed the performance against the BLOSUM62 series and showed that the usage of our matrices results in an improvement in the performance of BLAST database searches, greatly reducing the number of false positive hits. We then demonstrated applications of the use of novel matrices to improve the annotation of homologs between the two Plasmodium species and to classify members of the P. falciparum RIFIN/STEVOR family. CONCLUSION: We confirmed that in the case of compositionally biased proteins, standard BLOSUM matrices are not suited for optimal alignments, and specific substitution matrices are required. In addition, we showed that the usage of these matrices leads to a reduction of false positive hits, facilitating the automatic annotation process.

The immunological selection of recombinant peptides from Cryptosporidium parvum reveals 14 proteins expressed at the sporozoite stage, 7 of which are conserved in other apicomplexa.

Cryptosporidium parvum is an apicomplexan parasite that infects various mammals, including humans, yet no specific treatment has been developed. C. parvum sporozoites are the initial invasive forms that infect the intestinal epithelial cells of the host. To identify novel proteins expressed at the sporozoite stage, we analyzed around 100 recombinant peptides from a C. parvum expression library with an anti-sporozoite serum. We selected 14 peptides recognized by the serum and identified the corresponding genes in the C. parvum genomic database. Twelve of the 14 genes had been previously annotated in the genome database, whereas 2 of them (the CpC2C and the CpMT1 genes) were newly identified. We established that 13 of the 14 genes are expressed in the sporozoites and that the only multi-exon gene (CpC2C) produces a detectable amount of unspliced mRNA. The search for conserved domains revealed various structural features of these proteins, including signal peptides, transmembrane domains, WD repeats, C2 domain, and Myosin tails. Interestingly, among the 14 proteins, we also identified a putative rhomboid (CpRom) which, similarly to those found in other apicomplexa, could be involved in the host-cell invasion process. The search for similar proteins, conducted on 13 proteins, showed that 4 of these proteins belong to widely conserved families, whereas 7 of them are of apicomplexan origin and only 2 are restricted to the Cryptosporidium genus.

A new parameter to study compositional properties of non-coding regions in eukaryotic genomes.

Genomes are characterized by global and local compositional properties that are interesting in an evolutionary perspective but also provide useful information for the identification of some functional elements. Following previous studies, in this work we investigated compositional properties of non-coding sequences in four eukaryotic genomes (C. elegans, D. melanogaster, M. musculus, H. sapiens). We developed a procedure based on Principal Component Analysis (PCA) and Linear Discriminant Analysis (LDA) to identify pentamers that are over-represented in introns (intron vocabulary) and to define a new parameter (LD) that reflects oligonucleotide composition of a given sequence. We analyzed genomic sequences and we found that all non-coding parts of a genome are characterized by similar LD values. Furthermore, we used the new parameter to analyze potentially regulatory regions. We extracted non-redundant sets of promoter sequences for D. melanogaster and H. sapiens and we studied their compositional (G+C content and LD parameter) and conformational (bendability propensity) properties. We found that regions immediately surrounding transcription start sites are distinguishable because of their %G+C, LD and bendability values.