Quantification of reachable attractors in asynchronous discrete dynamics

Motivation: Models of discrete concurrent systems often lead to huge and complex state transition graphs that represent their dynamics. This makes difficult to analyse dynamical properties. In particular, for logical models of biological regulatory networks, it is of real interest to study attractors and their reachability from specific initial conditions, i.e. to assess the potential asymptotical behaviours of the system. Beyond the identification of the reachable attractors, we propose to quantify this reachability. Results: Relying on the structure of the state transition graph, we estimate the probability of each attractor reachable from a given initial condition or from a portion of the state space. First, we present a quasi-exact solution with an original algorithm called Firefront, based on the exhaustive exploration of the reachable state space. Then, we introduce an adapted version of Monte Carlo simulation algorithm, termed Avatar, better suited to larger models. Firefront and Avatar methods are validated and compared to other related approaches, using as test cases logical models of synthetic and biological networks. Availability: Both algorithms are implemented as Perl scripts that can be freely downloaded from http://compbio.igc.gulbenkian.pt/nmd/node/59 along with Supplementary Material.Comment: 19 pages, 2 figures, 2 algorithms and 2 table

Co-designed FreeRTOS deployed on FPGA

Most embedded systems are bound to real-time constraints. Two of the critical metrics presented in these systems are determinism and latency. Due to growing in complexity of embedded applications, real time operating systems (RTOS) are needed, not only to hide the increasingly complex hardware, but also to provide services to the system’s running tasks. Unfortunately, this new layer on an embedded system puts more pressure on the aforementioned metrics. One of the ways to cope with this problem is to offload RTOS run-time services to the hardware layer. This paper presents a hybrid hardware/software implementation of this technique upon the well known FreeRTOS, improving system’s latency and predictability, by migrating critical runtime services to hardware. The developed hardware accelerators were synthesized on a field-programmable gate array (FPGA), exploiting the point-to-point bus Fast Simplex Link (FSL) to interconnect to the Xilinx’s Microbaze soft-core processor.This work has been supported by FCT - Foundation for Science and Technology within the Project Scope: PEst-OE/EEI/UI0319/2014

Presence of Gardnerella vaginalis in healthy portuguese women: a pilot study

Bacterial vaginosis (BV) has an important position worldwide, as the leading vaginal disorder in women, and affects 30-50% of African women and 10-20% of White women of reproductive age. This condition although not mortal causes great discomfort and may lead to other complications such as pre-term labour or increase susceptibility for HIV infection. During BV occur a decrease of Lactobacillus spp. present in the vaginal epithelium and an increase in the number of anaerobic microorganisms like Gardnerella vaginalis, Pretovella spp., Mobilincus spp.; Mycoplasma hominis and Atopobium vaginae. Gardnerella vaginalis is also responsible for the formation of a biofilm in the vaginal epithelium in sick women’s. However, the direct correlation between the pathology and the causing agent (or agents) has not been clearly established. Currently there is only one article in PubMed (Guerreiro et. al, 1998) referring to the prevalence of bacterial vaginosis in Portugal and we aim to extend the research in this field specifically to the portuguese population. As part of this effort one of our aims it to characterise the bacterial population of portuguese women both healthy and diagnosed with BV. As such we collected swab samples of vaginal fluids from protuguese women with the help of health professionals and using self collection. The swabs were collected and treated within 24 hours at the University of Minho for the characterization of the bacterial population present, by using conventional microbiological growth techniques, PNA-FISH microscopy and 16S PCR. It was found that about 20% of the samples tested possessed G. vaginalis and all possessed Lactobacillus spp. using all 3 identification techniques described. This result is consistent with previous reports of G. vaginalis prevalence althought slightly lower, and shows that traditional microbiological techniques, microscopy and molecular methods were consistent in terms of results

Unraveling the insights into phage endolysin association

In view of the abundacy of phages (1), even rare phage-induced events are frequent at the global level. They have a staggering ecological impact on the bacterial population and in the evolution of bacterial genomic structure upon virus-host interactions, acting as agents in the recycling of organic matter and presenting a valuable tool in molecular biology and epidemiology. Th focus on genomic research have revealed information on open reading frames of proteins of interest (2). Increasing interest has been given to phage (endo )lysins in molecular biology, biotechnology and medicine. Lysins are phage lytic enzymes that break down the peptidoglycan of the bacterial cell wall at the terminal stage of the phage reproduction cycle, to release the phage progeny with the consequent death of the bacterial cells (3). Despite the increasing number of genomes in Genbank:, no effort has been made so far to understand the relation between lysins and their phage family and host species, presenting challenges in their annotation, comparative analysis, and representation. The almost 700 complete phage genomes deposited in the NCBI database were searched for the presence of lysins by making use of the pfam ( 4) identified domains and BLAST comparison of putative/unidentified complete genome against known lysins. In approximately 5% of the phage genomes it was not possible to identify any lysin. The identified enzymes were used to construct a phylogenetic tree with Phylip (5), using Neighbor-Joining, Maximum Likelihood and Parsimony algorithms (6). From the resulting tree, we were able to present a phage-lysin characterization network analysis taking into account the lysin aa sequence and the different phage classes (Family/Genus) and host species to study their evolutionary stories. Regarding the phage families, muramidases, amidases and peptidases are the largest type of lysins in Myoviridae, Podoviridae and Siphoviridae phages respectively. Grouped data will also be used to identify conserved domains among lysins of different phages which will play an important role in the annotation of the unidentified lytic cassette of sequenced phages

Insights into phage endolysins

(Bacterio)phages are viruses that specifically infect bacteria, and thus are harmless to humans, animals, and plants. They are the most abundant microorganisms on the planet (estimated to be 1031 on Earth) in a ratio of 10 times more than bacteria (1). Consequently, even rare phage-induced events are frequent at the global level. Therefore, they have a staggering ecological impact on the bacterial population and in the evolution of bacterial genomic structure upon virus-host interactions, acting as agents in the recycling of organic matter and presenting a valuable tool in molecular biology and epidemiology. Regarding the diversity of phages, they can have different types of replication mechanisms, morphologies, nucleic acid composition and genome sizes. Over the last decade improvements on phages genome sequencing and progresses in genomic research have revealed information on open reading frames of proteins of interest (2). Increasing interest has been given to phage (endo)lysins in molecular biology, biotechnology and medicine. Lysins are phage lytic enzymes that break down the peptidoglycan of the bacterial cell wall at the terminal stage of the phage reproduction cycle, in order to release the phage progeny with the consequent death of the bacterial cells (3). The number of phage genomes deposited in GenBank has been increasing exponentially in the last years. However, no effort has been made so far to understand the relation between lysins and their phage family and host species, presenting challenges in their annotation, comparative analysis, and representation. The almost 700 complete phage genomes deposited in the NCBI database were searched for the presence of lysins by making use of the Pfam (4) identified domains and BLAST comparison of putative or unidentified complete genome against known lysins. In approximately 5% of the phage genomes it was not possible to identify any lysin. The identified enzymes were used to construct a phylogenetic tree with Phylip (5), using Neighbor-Joining, Maximum Likelihood and Parsimony algorithms (6). From the resulting tree, we were able to present a phage-lysin characterization network analysis taking into account the lysin aminoacid sequence and the different phage classes (Family/Genus) and host species to study their evolutionary stories. Regarding the phage families, muramidases, amidases and peptidases are the largest type of lysins in Myoviridae, Podoviridae and Siphoviridae phages respectively. Grouped data will also be used to identify conserved domains among lysins of different phages which will play an important role in the annotation of the still unidentified lytic cassette of phages with sequenced genomes

Molecular aspects and comparative genomics of bacteriophage endolysins

Phages are recognized as the most abundant and diverse entities on the planet. Their diversity is predominantly determined by their dynamic adaptation capacities, when confronted with different selective pressures in an endless cycle of co-evolution with a widespread group of bacterial hosts. At the end of the infection cycle, progeny virions are confronted with a rigid cell wall that hinders their release into the environment and the opportunity to start a new infection cycle. Consequently, phages encode hydrolytic enzymes, called endolysins, to digest the peptidoglycan. In this work, we bring to light all phage endolysins found in completely sequenced double stranded nucleic acid phage genomes and uncover clues that explain the phage-endolysin-host ecology that led phages to recruit unique and specialized endolysins.This work was supported by a grant from the Portuguese Foundation for Science and Technology in the scope of the projects PTDC/AGR-ALI/100492/2008 and PTDC/AGR-ALI/121057/2010. Hugo Oliveira, Luis D. R. Melo, and Silvio B. Santos acknowledge the FCT grants SFRH/BD/63734/2009, SFRH/BD/66166/2009, and SFRH/BPD/75311/2010, respectively

Comparison between classical and molecular (FISH and PCR) methods for Lactobacillus spp. detection in clinical samples

Lactobacillus species constitute the main beneficial bacteria in our body by inhibiting the growth from pathogenic microorganisms. Fluorescence In Situ Hybridization (FISH) is an ideal method for cultivation-independent detection of microorganisms in microbial communities or clinical samples. Therefore, the current aims of this research are to identify and discriminate Lactobacillus spp. contained in clinical samples by the use of PNA-FISH methodology. In spite this method is proved to be useful to visualize target cells in natural habitats, it wasn't possible to find a Lactobacillus spp. 16S conservative region that allowed an unique and efficient identification in clinical samples. To overcome this problem, we used morphological visualization to differentiate Lactobacillus genus from another relative genera of the same Lactobacillaceae order. In addition, we also needed to overcome some methodological limitations, such as minimizing probe penetration problems and increasing hybridization efficiencies. As a result, we investigated the effect of different pre-treatment procedures of the exopolymer cell walls prior to the hybridization step, such as, several types of fixation compounds (paraformaldehyde and ethanol percentages), buffer steps and enzymatic (lysozyme and protease) pre-treatment. Furthermore, we modified PNA FISH protocol in several steps, for instance, hybridization and washing steps. In resume, the use of PNA probe specific for Lactobacillus spp. in situ hybridization by fluorescence microscopy could be perfectly used to study the complex and spatial organization of vaginal microbial samples. To conclude, we validate Lactobacillus spp. PNA probe by FISH to quantify and characterize in mixed microbiologic populations present in clinical samples

Fluorescence in situ hybridization method using peptide nucleic acid probes for rapid detection of Lactobacillus and Gardnerella spp

Background: Bacterial vaginosis (BV) is a common vaginal infection occurring in women of reproductive age. It is widely accepted that the microbial switch from normal microflora to BV is characterized by a decrease in vaginal colonization by Lactobacillus species together with an increase of Gardnerella vaginalis and other anaerobes. Our goal was to develop and optimize a novel Peptide Nucleic Acid (PNA) Fluorescence in situ Hybridization assay (PNA FISH) for the detection of Lactobacillus spp. and G. vaginalis in mixed samples. Results: Therefore, we evaluated and validated two specific PNA probes by using 36 representative Lactobacillus strains, 22 representative G. vaginalis strains and 27 other taxonomically related or pathogenic bacterial strains commonly found in vaginal samples. The probes were also tested at different concentrations of G. vaginalis and Lactobacillus species in vitro, in the presence of a HeLa cell line. Specificity and sensitivity of the PNA probes were found to be 98.0% (95% confidence interval (CI), from 87.8 to 99.9%) and 100% (95% CI, from 88.0 to 100.0%), for Lactobacillus spp.; and 100% (95% CI, from 92.8 to 100%) and 100% (95% CI, from 81.5 to 100.0%) for G. vaginalis. Moreover, the probes were evaluated in mixed samples mimicking women with BV or normal vaginal microflora, demonstrating efficiency and applicability of our PNA FISH. Conclusions: This quick method accurately detects Lactobacillus spp. and G. vaginalis species in mixed samples, thus enabling efficient evaluation of the two bacterial groups, most frequently encountered in the vagina.This work was supported by European Union funds (FEDER/COMPETE) and by national funds (FCT) under the project with reference FCOMP-01-0124-FEDER-008991 (PTDC/BIA-MIC/098228/2008). AM acknowledges the FCT individual fellowship - SFRH/BD/62375/2009)