Tactile Stimulation in Adult Rats Modulates Dopaminergic Molecular Parameters in the Nucleus accumbens Preventing Amphetamine Relapse.

Amphetamine (AMPH) is a psychostimulant drug frequently related to addiction, which is characterized by functional and molecular changes in the brain reward system, favoring relapse development, and pharmacotherapies have shown low effectiveness. Considering the beneficial influences of tactile stimulation (TS) in different diseases that affect the central nervous system (CNS), here we evaluated if TS applied in adult rats could prevent or minimize the AMPH-relapse behavior also accessing molecular neuroadaptations in the nucleus accumbens (NAc). Following AMPH conditioning in the conditioned place preference (CPP) paradigm, male rats were submitted to TS (15-min session, 3 times a day, for 8 days) during the drug abstinence period, which were re-exposed to the drug in the CPP paradigm for additional 3 days for relapse observation and molecular assessment. Our findings showed that besides AMPH relapse, TS prevented the dopamine transporter (DAT), dopamine 1 receptor (D1R), tyrosine hydroxylase (TH), mu opioid receptor (MOR) increase, and AMPH-induced delta FosB (ΔFosB). Based on these outcomes, we propose TS as a useful tool to treat psychostimulant addiction, which is subsequent to clinical studies; it could be included in detoxification programs together with pharmacotherapies and psychological treatments already conventionally established.

Binge and Subchronic Exposure to Ketamine Promote Memory Impairments and Damages in the Hippocampus and Peripheral Tissues in Rats: Gallic Acid Protective Effects.

Ketamine (KET) is a dissociative anesthetic for restrict medical use with high potential for abuse and neurotoxicity which does not prevent its recreational use. Gallic acid (GA) is a natural free radical "scavenger." We evaluated the GA protective role regarding binge or subchronic (SbChro) KET-induced toxicity in adolescent rats. In the binge protocol, animals were treated with GA (one dose of 13.5 mg/kg, p.o. every 2 h, totaling 3 doses) 12 h after KET exposure (one dose of 10 mg/kg, i.p., every 3 h, totaling 5 doses). In the SbChro, animals were treated with GA (one dose of 13.5 mg/kg/day, p.o., for 3 days) 48 h following KET exposure (one dose of 10 mg/kg/day, i.p) for 10 days. Our findings show that binge-KET impaired memory, increased pro-BDNF and TrkB levels in the hippocampus, and increased lipid peroxidation (LP) in the kidney and hippocampus, while SbChro-KET impaired memory, increased pro-BDNF, and decreased both BDNF and TrkB levels in the hippocampus, and increased LP in the kidney, liver, and hippocampus. GA treatment reversed the subchronically KET-induced harmful influences better. Interestingly, only memory impairment observed in the SbChro-KET protocol was reversed by GA. Memory impairments showed a positive correlation with hippocampal BDNF levels and negative with LP levels in the same brain area. This last hippocampal damage (LP) showed a negative correlation with BDNF levels in the hippocampus, indicating an interesting and close causal connection. Our outcomes show that the deleterious effects of SbChro-KET exposure can be attenuated or abolished with GA administration, a natural antioxidant that could be considered in KET abuse treatment.

Physical exercise modifies behavioral and molecular parameters related to opioid addiction regardless of training time.

Addiction is a devastating worldwide disorder that requires effective and innovative therapies. Physical exercise could be useful in addiction treatment because it shares a common neural circuit with addictive drugs. Based on this, molecular adaptations consequent to time of exercise in opioid exposed animals were evaluated. Rats were designed as sedentary (SED) or exercised (EXE). This last group was separated to perform three different periods of swimming: short-term (S-EXE), medium-term (M-EXE) and long-term (L-EXE) for 14, 28 and 42 days, respectively. On the last exercising week, one-half of the animals from SED and all animals from S-, M- and l-EXE were concomitantly exposed to morphine-conditioned place preference (CPP) paradigm and y-maze task for behavioral assessments followed by molecular assays in both Nucleus accumbens (NAc) and hippocampus. Between SED groups, morphine conditioning showed drug-CPP and increased dopamine transporter (DAT), dopamine receptor type-1 (D1R), type-2 (D2R) and glucocorticoid receptor (GR) in both brain areas in relation to saline group. Besides the small morphine-CPP in relation to SED group, all periods decreased DAT, D1R, and GR immunoreactivity in NAc, DAT and D1R in hippocampus, while D2R in both brain areas and GR in hippocampus were primarily decreased by L-EXE. Our findings show that even a short-term exercise modifies behaviors related to drug withdrawal, changing DA targets and GR, which are closely linked to addiction. Therefore, our outcomes involving physical exercise are interesting to perform a possible clinical trial, thus expanding the knowledge about drug addiction.

Gallic acid prevents ketamine-induced oxidative damages in brain regions and liver of rats.

INTRODUCTION: Ketamine (KET) is an anesthetic agent widely used in human and veterinary medicine. According to studies, KET is associated to direct neutorotoxic damages due to its capacity to induce oxidative stress. Because of the free radical generation in the organism and its relation with diseases' development, there is a growing interest to study antioxidant molecules, such as gallic acid (GA), a natural phenolic compound. AIM: Evaluate the GA antioxidant potential for the prevention of oxidative damage in the brain and liver tissue of rats exposed to acute KET administration. MATERIAL AND METHODS: 32 Wistar male rats received GA (by gavage, 13.5 mg/kg) for three consecutive days, 24 h after the last GA dose, animals were anesthetized with KET (50 mg/kg, i.m.). All animals were euthanized by decapitation 60 min after KET administration. The liver, brain cortex and hippocampus were removed and homogenized for biochemical analysis. RESULTS: In brain cortex, KET increased reactive species (RS) generation, protein carbonyls (PC) levels and reduced non-protein thiols (NPSH) levels, while GA pre-treatment reduced PC and increased NPSH levels. KET increased PC and decreased NPSH levels in the hippocampus, and GA reduced PC and NPSH levels. In the liver, no difference was observed in the RS generation, while KET induced and increase of PC levels and decreased NPSH levels, while GA pre-treatment prevented it. CONCLUSION: GA administration can prevent oxidative damage caused by acute KET administration and minimize its noxious effects. Further studies are needed to evidence GA antioxidant properties regarding KET chronic use.

The role of extracellular matrix in mouse and human corneal neovascularization.

Corneal neo-vascularization (CNV) is a highly prevalent medical condition which impairs visual acuity. The role of specific proteins in modulating CNV has been extensively reported, although no studies have described the entire human proteome in CNV corneas. In this paper, we performed a proteomic analysis of vascularized vs healthy corneal stroma, in a CNV mouse model and in CNV-affected patients, with a specific focus on extracellular matrix (ECM) proteins. We identified and quantified 2315 murine proteins, 691 human proteins and validated 5 proteins which are differentially expressed in vascularized samples and conserved in mice and humans: tenascin-C and fibronectin-1 were upregulated, while decorin, lumican and collagen-VI were downregulated in CNV samples. Interestingly, among CNV patients, those affected with Acanthamoeba keratitis showed the highest levels of fibronectin-1 and tenascin-C, suggesting a specific role of these two proteins in Acanthamoeba driven corneal CNV. On a broader picture, our findings support the hypothesis that the corneal stroma in CNV samples is disorganized and less compact. We are confident that the dissection of the human corneal proteome may shed new light on the complex pathophysiology of human CNV, and finally lead to improved treatments.

Tactile Stimulation on Adulthood Modifies the HPA Axis, Neurotrophic Factors, and GFAP Signaling Reverting Depression-Like Behavior in Female Rats.

Depression is a common psychiatric disease which pharmacological treatment relieves symptoms, but still far from ideal. Tactile stimulation (TS) has shown beneficial influences in neuropsychiatric disorders, but the mechanism of action is not clear. Here, we evaluated the TS influence when applied on adult female rats previously exposed to a reserpine-induced depression-like animal model. Immediately after reserpine model (1 mg/kg/mL, 1×/day, for 3 days), female Wistar rats were submitted to TS (15 min, 3×/day, for 8 days) or not (unhandled). Imipramine (10 mg/kg/mL) was used as positive control. After behavioral assessments, animals were euthanized to collect plasma and prefrontal cortex (PFC). Behavioral observations in the forced swimming test, splash test, and sucrose preference confirmed the reserpine-induced depression-like behavior, which was reversed by TS. Our findings showed that reserpine increased plasma levels of adrenocorticotropic hormone and corticosterone, decreased brain-derived neurotrophic factor (BDNF) and tropomyosin receptor kinase B, and increased proBDNF immunoreactivity in the PFC, which were also reversed by TS. Moreover, TS reestablished glial fibrillary acidic protein and glucocorticoid receptor levels, decreased by reserpine in PFC, while glial cell line-derived neurotrophic factor was increased by TS per se. Our outcomes are showing that TS applied in adulthood exerts a beneficial influence in depression-like behaviors, modulating the HPA axis and regulating neurotrophic factors more effectively than imipramine. Based on this, our proposal is that TS, in the long term, could be considered a new therapeutic strategy for neuropsychiatric disorders improvement in adult life, which may represent an interesting contribution to conventional pharmacological treatment.

Montelukast reduces seizures in pentylenetetrazol-kindled mice.

Cysteinyl leukotrienes (CysLTs) have been implicated in seizures and kindling; however, the effect of CysLT receptor antagonists on seizure frequency in kindled animals and changes in CysLT receptor expression after pentylenetetrazol (PTZ)-induced kindling have not been investigated. In this study, we evaluated whether the CysLT1 inverse agonist montelukast, and a classical anticonvulsant, phenobarbital, were able to reduce seizures in PTZ-kindled mice and alter CysLT receptor expression. Montelukast (10 mg/kg, sc) and phenobarbital (20 mg/kg, sc) increased the latency to generalized seizures in kindled mice. Montelukast increased CysLT1 immunoreactivity only in non-kindled, PTZ-challenged mice. Interestingly, PTZ challenge decreased CysLT2 immunoreactivity only in kindled mice. CysLT1 antagonists appear to emerge as a promising adjunctive treatment for refractory seizures. Nevertheless, additional studies are necessary to evaluate the clinical implications of this research.

Ochratoxin A as possible factor trigging autism and its male prevalence via epigenetic mechanism.

The role of dysbiosis causing leaky gut with xenobiotic production and absorption is increasingly demonstrated in autism spectrum disorder (ASD) pathogenesis. Among xenobiotics, we focused on ochratoxin A (one of the major food contaminating mycotoxin), that in vitro and in vivo exerts a male-specific neurotoxicity probably via microRNA modulation of a specific target gene. Among possible targets, we focused on neuroligin4X. Interestingly, this gene carries some single nucleotide polymorphisms (SNPs) already correlated with the disease and with illegitimate microRNA binding sites and, being located on X-chromosome, could explain the male prevalence. In conclusion, we propose a possible gene-environment interaction triggering ASD explaining the epigenetic neurotoxic mechanism activated by ochratoxin A in genetically predisposed children. This mechanism offers a clue for male prevalence of the disease and may have an important impact on prevention and cure of ASD.

Montelukast reduces seizures in pentylenetetrazol-kindled mice

Cysteinyl leukotrienes (CysLTs) have been implicated in seizures and kindling; however, the effect of CysLT receptor antagonists on seizure frequency in kindled animals and changes in CysLT receptor expression after pentylenetetrazol (PTZ)-induced kindling have not been investigated. In this study, we evaluated whether the CysLT1 inverse agonist montelukast, and a classical anticonvulsant, phenobarbital, were able to reduce seizures in PTZ-kindled mice and alter CysLT receptor expression. Montelukast (10 mg/kg, sc) and phenobarbital (20 mg/kg, sc) increased the latency to generalized seizures in kindled mice. Montelukast increased CysLT1 immunoreactivity only in non-kindled, PTZ-challenged mice. Interestingly, PTZ challenge decreased CysLT2 immunoreactivity only in kindled mice. CysLT1 antagonists appear to emerge as a promising adjunctive treatment for refractory seizures. Nevertheless, additional studies are necessary to evaluate the clinical implications of this research.

Genome Sequence of Rhodococcus opacus Strain R7, a Biodegrader of Mono- and Polycyclic Aromatic Hydrocarbons.

Rhodococcus opacus strain R7 (CIP107348) degrades several mono- and polycyclic aromatic hydrocarbons. Here, we present the high-quality draft genome sequence of strain R7, consisting of 10,118,052 bp, with a G+C content of 67.0%, 9,602 protein-coding genes, and 62 RNAs genes.

Genome Sequence of Rhodococcus sp. Strain BCP1, a Biodegrader of Alkanes and Chlorinated Compounds.

Rhodococcus sp. strain BCP1 cometabolizes chlorinated compounds and mineralizes a broad range of alkanes, as it is highly tolerant to them. The high-quality draft genome sequence of Rhodococcus sp. strain BCP1, consisting of 6,231,823 bp, with a G+C content of 70.4%, 5,902 protein-coding genes, and 58 RNA genes, is presented here.

Myosin-binding protein C DNA variants in domestic cats (A31P, A74T, R820W) and their association with hypertrophic cardiomyopathy.

BACKGROUND: Two mutations in the MYBPC3 gene have been identified in Maine Coon (MCO) and Ragdoll (RD) cats with hypertrophic cardiomyopathy (HCM). OBJECTIVE: This study examined the frequency of these mutations and of the A74T polymorphism to describe their worldwide distribution and correlation with echocardiography. ANIMALS: 1855 cats representing 28 breeds and random-bred cats worldwide, of which 446 underwent echocardiographic examination. METHODS: This is a prospective cross-sectional study. Polymorphisms were genotyped by Illumina VeraCode GoldenGate or by direct sequencing. The disease status was defined by echocardiography according to established guidelines. Odds ratios for the joint probability of having HCM and the alleles were calculated by meta-analysis. Functional analysis was simulated. RESULTS: The MYBPC3 A31P and R820W were restricted to MCO and RD, respectively. Both purebred and random-bred cats had HCM and the incidence increased with age. The A74T polymorphism was not associated with any phenotype. HCM was most prevalent in MCO homozygote for the A31P mutation and the penetrance increased with age. The penetrance of the heterozygote genotype was lower (0.08) compared with the P/P genotype (0.58) in MCO. CONCLUSIONS AND CLINICAL IMPORTANCE: A31P mutation occurs frequently in MCO cats. The high incidence of HCM in homozygotes for the mutation supports the causal nature of the A31P mutation. Penetrance is incomplete for heterozygotes at A31P locus, at least at a young age. The A74T variant does not appear to be correlated with HCM.

IBDsite: a Galaxy-interacting, integrative database for supporting inflammatory bowel disease high throughput data analysis.

BACKGROUND: Inflammatory bowel diseases (IBD) refer to a group of inflammatory conditions concerning colon and small intestine, which cause socially uncomfortable symptoms and often are associated with an increased risk of colon cancer. IBD are complex disorders, which rely on genetic susceptibility, environmental factors, deregulation of the immune system, and host relationship with commensal microbiota. The complexity of these pathologies makes difficult to clearly understand the mechanisms of their onset. Therefore, the study of IBD must be faced exploiting an integrated and multilevel approach, ranging from genes, transcripts and proteins to pathways altered in affected tissues, and carefully considering their regulatory mechanisms, which may intervene in the pathology onset. It is also crucial to have a knowledge base about the symbiotic bacteria that are hosted in the human gut. To date, much data exist regarding IBD and human commensal bacteria, but this information is sparse in literature and no free resource provides a homogeneously and rationally integrated view of biomolecular data related to these pathologies. METHODS: Human genes altered in IBD have been collected from literature, paying particular interest for the immune system alterations prompted by the interaction with the gut microbiome. This process has been performed manually to assure the reliability of collected data. Heterogeneous metadata from different sources have been automatically formatted and integrated in order to enrich information about these altered genes. A user-friendly web interface has been created for easy access to structured data. Tools such as gene clustering coefficients, all-pairs shortest paths and pathway lengths calculation have been developed to provide data analysis support. Moreover, the implemented resource is compliant to the Galaxy framework, allowing the collected data to be exploited in the context of high throughput bioinformatics analysis. RESULTS: To fill the lack of a reference resource for 'omics' science analysis in the context of IBD, we developed the IBDsite (available at http://www.itb.cnr.it/ibd), a disease-oriented platform, which collects data related to biomolecular mechanisms involved in the IBD onset. The resource provides a section devoted to human genes identified as altered in IBD, which can be queried at different biomolecular levels and visualised in gene-centred report pages. Furthermore, the system presents information related to the gut microbiota involved in IBD affected patients. The IBDsite is compliant with all Galaxy installations (in particular, it can be accessed from our custom version of Galaxy, http://www.itb.cnr.it/galaxy), in order to facilitate high-throughput data integration and to enable evaluations of the genomic basis of these diseases, complementing the tools embedded in the IBDsite. CONCLUSIONS: Lots of sparse data exist concerning IBD studies, but no on-line resource homogeneously and rationally integrate and collect them. The IBDsite is an attempt to group available information regarding human genes and microbial aspects related to IBD, by means of a multilevel mining tool. Moreover, it constitutes a knowledge base to filter, annotate and understand new experimental data in order to formulate new scientific hypotheses, thanks to the possibility of integrating genomics aspects by employing the Galaxy framework. Discussed use-cases demonstrate that the developed system is useful to infer not trivial knowledge from the existing widespread data or from novel experiments.

Nanoinformatics: developing new computing applications for nanomedicine.

Nanoinformatics has recently emerged to address the need of computing applications at the nano level. In this regard, the authors have participated in various initiatives to identify its concepts, foundations and challenges. While nanomaterials open up the possibility for developing new devices in many industrial and scientific areas, they also offer breakthrough perspectives for the prevention, diagnosis and treatment of diseases. In this paper, we analyze the different aspects of nanoinformatics and suggest five research topics to help catalyze new research and development in the area, particularly focused on nanomedicine. We also encompass the use of informatics to further the biological and clinical applications of basic research in nanoscience and nanotechnology, and the related concept of an extended "nanotype" to coalesce information related to nanoparticles. We suggest how nanoinformatics could accelerate developments in nanomedicine, similarly to what happened with the Human Genome and other -omics projects, on issues like exchanging modeling and simulation methods and tools, linking toxicity information to clinical and personal databases or developing new approaches for scientific ontologies, among many others.

Modeling the cell cycle: from deterministic models to hybrid systems.

The cell cycle is a complex biological system frequently investigated from a mathematical perspective. In fact, over the past years a huge number of deterministic mathematical models describing the dynamics and the regulation of this process have been proposed. A crucial point concerning the cell cycle modeling is the combination of continuous and discrete dynamics in order to obtain results which are coherent with the biological context. To face with this problem we propose a novel approach to the mathematical modeling of biological processes based on the use of hybrid systems. This new methodology essentially consists in a model reduction (using the modified Prony's method) which allows to define the crucial features of the dynamical system. The final aim is to implement a corresponding hybrid system which preserves the properties of the starting deterministic model. Thus, we implemented a methodology which allows to describe the cellular system by combining continuous behavior with discrete events by using the hybrid automata technology. In this way we try to overcome some drawbacks of the deterministic approach, especially regarding the possibility to introduce new variables during simulation and the associated variation of parameters in a more efficient way than the continuous method can do. We applied this innovative methodology to the reconstruction of a simplified hybrid model concerning one of the crucial mammalian cell cycle control point. In particular, we investigated the role of the transcription factors E2F in the R-point transition. The resulting hybrid model preserve the properties of the deterministic one and it allows the identification of the parameter which controls the transition from the inactive (quiescent) to the active state (R-point transition) after the mitogenic stimulation. At the best of our knowledge no hybrid model for the R-point transition are available in literature.

Rol protector del 17ß-Estradiol y la testosterona en la apoptosis del músculo esquelético / Protective role of 17ß-Estradiol and testosterone in apoptosis of skeletal muscle

La sarcopenia, pérdida de masa y fuerza del músculo esqueléico, es una condición frecuente durante el envejecimiento. Conduce a incapacidad motora resultando en internación y mortalidad. Puesto que los niveles de estrógenos y/o testosterona disminuyen con la edad, la sarcopenia se ha asociado al déficit de estas hormonas. Aunque los mecanismos moleculares involucrados en esta patología no están totalmente dilucidados, existen evidencias indicando que la apoptosis es en parte responsable de la pérdida de miocitos en la adultez. Previamente demostramos que el 17ß-estradiol (E2) inhibe la apoptosis en la línea celular C2C12 de músculo esquelético a través de PI3K/Akt, MAPKs, HSP27 y receptores estrogénicos (ERs) con localización no clásica. Usando siRNAs específicos para silenciar las isoformas del ER, comprobamos que el E2 activa ERK involucrando a ERa, mientras que la activación de p38 MAPK es independiente de ERs. Confirmamos que el E2 puede inhibir la apoptosis a través de las MAPKs en cultivos primarios de músculo esquelético de ratón. Al igual que la E2, la testosterona bloquea la apoptosis. Las alteraciones morfológicas típicas de la apoptosis como fragmentación nuclear, desorganización del citoesqueleto, reorganización/disfunción mitocondrial y liberación de citocromo c, inducidos por H2O2 fueron suprimidas al preincubar las células con testosterona. Se requieren investigaciones adicionales para establecer un paralelismo entre los mecanismos de acción de ambas hormonas, que podrían estar implicados en patologías musculares asociadas a apoptosis. Los datos presentados en este estudio profundizan el conocimiento de las bases moleculares de la sarcopenia relacionada con estados de déficit de hormonas sexuales.

Medical informatics and bioinformatics: a bibliometric study.

This paper reports on an analysis of the bioinformatics and medical informatics literature with the objective to identify upcoming trends that are shared among both research fields to derive benefits from potential collaborative initiatives for their future. Our results present the main characteristics of the two fields and show that these domains are still relatively separated.

High performance GRID based implementation for genomics and protein analysis.

Starting from the genomic and proteomic sequence data, a complex computational infrastructure as been established with the objective to develop a GRID based system to to automate the analysis, prediction and annotation processes of genomic DNA. To support of this type of analysis, several algorithms as been used to recognize biological signals involved in the identification of genes and proteins. The system implemented can be use to analyse the content of the large number of genomic sequences. For this reason, the system realized is capable of using a computational architecture specifically designed for intensive computing based on GRID technologies developed throughout the BIOINFOGRID European project. We developed a GRID based workflow to correlate different kind of Bioinformatics data, going from the Genomics Nucleotide to the Protein Sequence. The first step in the workflow consists of submitting a nucleotide sequence that is elaborated by a specific software for gene prediction. In particular this tool performs a search in the nucleotide sequence to find out the key components of gene. The predicted gene is then translated in the corresponding protein sequence. Based on protein sequence is then possible to identify the domains that characterize the protein functionality using specific tools of domain prediction. Protein domains classification are very important in the analysis of the macromolecular functionality. To analyze a whole protein family from large genome of various organism means to elaborate a large amount of data that requires huge computational resources. To analyze all this data we suggest the use of a high performance platform based on grid technology. We have implemented our applications on a wide area grid platform for scientific applications [http://www.grid.it and http://grid-it.cnaf.infn.it] composed of about 1000 CPU's. The grid infrastructure consists in a collection of computing elements and storage elements that jointly concur to define a platform for high performance elaboration. In this study a grid based application is presented to compute the protein domain analysis in a distributed way. This approach has high performance because the protein domains are checked with different software in parallel in different grid sites.

Presence of estrogens and estrogen receptor-like proteins in Solanum glaucophyllum.

The synthesis of mammalian steroid hormones by plants has been reported. However, their physiological role in plants is controversial. The existence of receptor molecules as those of animal cells could provide clues into a possible steroid mechanism of action. Solanum glaucophyllum callus cultures were found to contain not only 17beta-estradiol and estrone but also abundant estrogen binding sites. These sites were specific for 17beta-estradiol ( approximately 550 fmol/mg protein) and could also be competed by the known estrogen receptor (ER) agonist diethylstilbestrol. Antibodies directed against specific sequences of the classical ER alpha isoform, labelled a approximately 67 kDa band which comigrated with the mammalian ER alpha antigen. ER alpha-like proteins were tested positive as estrogen binders in Ligand blot experiments using 17beta-estradiol macromolecular derivatives as ligands. Our results provide first evidences on the existence of estrogen binding proteins structurally related to the mammalian ER alpha subtype in a higher plant system.

Presence of ATG triplets in 5' untranslated regions of eukaryotic cDNAs correlates with a 'weak' context of the start codon.

MOTIVATION: The context of the start codon (typically, AUG) and the features of the 5' Untranslated Regions (5' UTRs) are important for understanding translation regulation in eukaryotic mRNAs and for accurate prediction of the coding region in genomic and cDNA sequences. The presence of AUG triplets in 5' UTRs (upstream AUGs) might effect the initiation rate and, in the context of gene prediction, could reduce the accuracy of the identification of the authentic start. To reveal potential connections between the presence of upstream AUGs and other features of 5' UTRs, such as their length and the start codon context, we undertook a systematic analysis of the available eukaryotic 5' UTR sequences. RESULTS: We show that a large fraction of 5' UTRs in the available cDNA sequences, 15-53% depending on the organism, contain upstream ATGs. A negative correlation was observed between the information content of the translation start signal and the length of the 5' UTR. Similarly, a negative correlation exists between the 'strength' of the start context and the number of upstream ATGs. Typically, cDNAs containing long 5' UTRs with multiple upstream ATGs have a 'weak' start context, and in contrast, cDNAs containing short 5' UTRs without ATGs have 'strong' starts. These counter-intuitive results may be interpreted in terms of upstream AUGs having an important role in the regulation of translation efficiency by ensuring low basal translation level via double negative control and creating the potential for additional regulatory mechanisms. One of such mechanisms, supported by experimental studies of some mRNAs, includes removal of the AUG-containing portion of the 5' UTR by alternative splicing. AVAILABILITY: An ATG_ EVALUATOR program is available upon request or at www.itba.mi.cnr.it/webgene. CONTACT: rogozin@ncbi.nlm.nih.gov, milanesi@itba.mi.cnr.it.