LoTo: a graphlet based method for the comparison of local topology between gene regulatory networks.

One of the main challenges of the post-genomic era is the understanding of how gene expression is controlled. Changes in gene expression lay behind diverse biological phenomena such as development, disease and the adaptation to different environmental conditions. Despite the availability of well-established methods to identify these changes, tools to discern how gene regulation is orchestrated are still required. The regulation of gene expression is usually depicted as a Gene Regulatory Network (GRN) where changes in the network structure (i.e., network topology) represent adjustments of gene regulation. Like other networks, GRNs are composed of basic building blocks; small induced subgraphs called graphlets. Here we present LoTo, a novel method that using Graphlet Based Metrics (GBMs) identifies topological variations between different states of a GRN. Under our approach, different states of a GRN are analyzed to determine the types of graphlet formed by all triplets of nodes in the network. Subsequently, graphlets occurring in a state of the network are compared to those formed by the same three nodes in another version of the network. Once the comparisons are performed, LoTo applies metrics from binary classification problems calculated on the existence and absence of graphlets to assess the topological similarity between both network states. Experiments performed on randomized networks demonstrate that GBMs are more sensitive to topological variation than the same metrics calculated on single edges. Additional comparisons with other common metrics demonstrate that our GBMs are capable to identify nodes whose local topology changes between different states of the network. Notably, due to the explicit use of graphlets, LoTo captures topological variations that are disregarded by other approaches. LoTo is freely available as an online web server at http://dlab.cl/loto.

Graphlet Based Metrics for the Comparison of Gene Regulatory Networks.

Understanding the control of gene expression remains one of the main challenges in the post-genomic era. Accordingly, a plethora of methods exists to identify variations in gene expression levels. These variations underlay almost all relevant biological phenomena, including disease and adaptation to environmental conditions. However, computational tools to identify how regulation changes are scarce. Regulation of gene expression is usually depicted in the form of a gene regulatory network (GRN). Structural changes in a GRN over time and conditions represent variations in the regulation of gene expression. Like other biological networks, GRNs are composed of basic building blocks called graphlets. As a consequence, two new metrics based on graphlets are proposed in this work: REConstruction Rate (REC) and REC Graphlet Degree (RGD). REC determines the rate of graphlet similarity between different states of a network and RGD identifies the subset of nodes with the highest topological variation. In other words, RGD discerns how th GRN was rewired. REC and RGD were used to compare the local structure of nodes in condition-specific GRNs obtained from gene expression data of Escherichia coli, forming biofilms and cultured in suspension. According to our results, most of the network local structure remains unaltered in the two compared conditions. Nevertheless, changes reported by RGD necessarily imply that a different cohort of regulators (i.e. transcription factors (TFs)) appear on the scene, shedding light on how the regulation of gene expression occurs when E. coli transits from suspension to biofilm. Consequently, we propose that both metrics REC and RGD should be adopted as a quantitative approach to conduct differential analyses of GRNs. A tool that implements both metrics is available as an on-line web server (http://dlab.cl/loto).

Target genes of Dpp/BMP signaling pathway revealed by transcriptome profiling in the early D.melanogaster embryo.

In the early Drosophila melanogaster embryo, the gene regulatory network controlled by Dpp signaling is involved in the subdivision of dorsal ectoderm into the presumptive dorsal epidermis and amnioserosa. In this work, we aimed to identify new Dpp downstream targets involved in dorsal ectoderm patterning. We used oligonucleotide D. melanogaster microarrays to identify the set of genes that are differential expressed between wild type embryos and embryos that overexpress Dpp (nos-Gal4>UAS-dpp) during early stages of embryo development. By using this approach, we identified 358 genes whose relative abundance significantly increased in response to Dpp overexpression. Among them, we found the entire set of known Dpp target genes that function in dorsal ectoderm patterning (zen, doc, hnt, pnr, ush, tup, and others) in addition to several up-regulated genes of unknown functions. Spatial expression pattern of up-regulated genes in response to Dpp overexpression as well as their opposing transcriptional responses to Dpp loss- and gain-of-function indicated that they are new candidate target genes of Dpp signaling pathway. We further analyse one of the candidate genes, CG13653, which is expressed at the dorsal-most cells of the embryo during a restricted period of time. CG13653 orthologs were not detected in basal lineages of Dipterans, which unlike D. melanogaster develop two extra-embryonic membranes, amnion and serosa. We characterized the enhancer region of CG13653 and revealed that CG13653 is directly regulated by Dpp signaling pathway.

Changes in Olfactory Receptor Expression Are Correlated With Odor Exposure During Early Development in the zebrafish (Danio rerio).

We have previously shown that exposure to phenyl ethyl alcohol (PEA) causes an increase in the expression of the transcription factor otx2 in the olfactory epithelium (OE) of juvenile zebrafish, and this change is correlated with the formation of an odor memory of PEA. Here, we show that the changes in otx2 expression are specific to ßPEA: exposure to αPEA did not affect otx2 expression. We identified 34 olfactory receptors (ORs) representing 16 families on 4 different chromosomes as candidates for direct regulation of OR expression via Otx2. Subsequent in silico analysis uncovered Hnf3b binding sites closely associated with Otx2 binding sites in the regions flanking the ORs. Analysis by quantitative polymerase chain reaction and RNA-seq of OR expression in developing zebrafish exposed to different isoforms of PEA showed that a subset of ORs containing both Otx2/Hnf3b binding sites were downregulated only in ßPEA-exposed juveniles and this change persisted through adult life. Localization of OR expression by in situ hybridization indicates the downregulation occurs at the level of RNA and not the number of cells expressing a given receptor. Finally, analysis of immediate early gene expression in the OE did not reveal changes in c-fos expression in response to either αPEA or ßPEA.

Transcriptional response of Atlantic salmon families to Piscirickettsia salmonis infection highlights the relevance of the iron-deprivation defence system.

BACKGROUND: Piscirickettsiosis or Salmonid Rickettsial Septicaemia (SRS) is a bacterial disease that has a major economic impact on the Chilean salmon farming industry. Despite the fact that Piscirickettsia salmonis has been recognized as a major fish pathogen for over 20 years, the molecular strategies underlying the fish response to infection and the bacterial mechanisms of pathogenesis are poorly understood. We analysed and compared the head kidney transcriptional response of Atlantic salmon (Salmo salar) families with different levels of susceptibility to P. salmonis infection in order to reveal mechanisms that might confer infection resistance. RESULTS: We ranked forty full-sibling Atlantic salmon families according to accumulated mortality after a challenge with P. salmonis and selected the families with the lowest and highest cumulative mortalities for microarray gene expression analysis. A comparison of the response to P. salmonis infection between low and high susceptibility groups identified biological processes presumably involved in natural resistance to the pathogen. In particular, expression changes of genes linked to cellular iron depletion, as well as low iron content and bacterial load in the head kidney of fish from low susceptibility families, suggest that iron-deprivation is an innate immunity defence mechanism against P. salmonis. To complement these results, we predicted a set of iron acquisition genes from the P. salmonis genome. Identification of putative Fur boxes and expression of the genes under iron-depleted conditions revealed that most of these genes form part of the Fur regulon of P. salmonis. CONCLUSIONS: This study revealed, for the first time, differences in the transcriptional response to P. salmonis infection among Atlantic salmon families with varied levels of susceptibility to the infection. These differences correlated with changes in the abundance of transcripts encoding proteins directly and indirectly involved in the immune response; changes that highlighted the role of nutritional immunity through iron deprivation in host defence mechanisms against P. salmonis. Additionally, we found that P. salmonis has several mechanisms for iron acquisition, suggesting that this bacterium can obtain iron from different sources, including ferric iron through capturing endogenous and exogenous siderophores and ferrous iron. Our results contribute to determining the underlying resistance mechanisms of Atlantic salmon to P. salmonis infection and to identifying future treatment strategies.

Life-giving caspases: revealing new roles during mouse embryo preimplantation development.

Caspases, cystein proteases traditionally related to programmed cell death, have recently been found to be involved in vital processes such as cell proliferation, adhesion and differentiation. Although caspases are expressed in mouse embryos before the blastocyst stage, their role is unclear, since apoptosis does not occur significantly before implantation. In this work, we have used mouse preimplantation development as a model to evaluate the existence of non-lethal caspase activities. The use of specific caspase inhibitors during in vitro embryo culture showed that caspase 8 activity, but not caspase 2 or 9, was relevant for development. The inhibition of caspase 8 affected the compaction of morulae and the progression to the blastocyst stage. In agreement with these results, caspase 8 was expressed in mouse embryos, as shown by indirect immunofluorescence and RT-PCR. An in silico approach was used to find putative caspase targets expressed in mouse preimplantation embryos. Large-scale management of sequence data from mouse embryos was used to predict caspase substrates by tools matrix-based on known cleavage sites. A total of 510 potential caspase targets expressed in mouse embryos were identified by this procedure. The functional characterization of these proteins by Gene Onthology associations showed that many of these putative caspase targets were previously related to non-apoptotic functions and only 63 had been previously reported to be actually cleaved by caspases. Interestingly, eleven knockout mice models for caspase substrates identified in our work, i.e. catenin alpha and beta, geminin, pescadillo, calpain-2, have preimplantation lethal phenotypes. This work supports the involvement of caspases in vital functions during mouse preimplantation development and proposes a model in which the regulated cleavage of caspase substrates could account for this role.

Genome analysis and detection of a Chilean isolate of Grapevine leafroll associated virus-3.

The complete genome of the Chilean isolate Cl-766 of Grapevine leafroll-associated virus-3 (GLRaV-3) has been sequenced. This is the first genome sequence obtained from a GLRaV-3 isolate of the Southern hemisphere. The genomic RNA of 17,919 nucleotides contains 13 open reading frames (ORFs) with 5' and 3' untranslated regions (UTR) of 158 and 277 nucleotides, respectively. Comparison with NY1, the only isolate with complete genomic sequence available today, shows 97.6% nucleotide identity between the two isolates. Examination of the genome variability shows that most of the genetic diversity is concentrated in ORF1a. Three additional isolates from different geographic regions of Chile were partially sequenced as well, one which showed sequence divergence with respect to the other local and foreign isolates, indicative of different evolutionary constrains. Immunodetection systems were developed using monoclonal and polyclonal antibodies produced against the recombinant major coat protein of GLRaV-3, providing sensitive and specific detection using a triple antibody sandwich-enzyme linked immunosorbent assay (TAS-ELISA) and an immunocapture-reverse transcription-polymerase chain reaction (IC-RT-PCR) assay.

The N-homologue LRR domain adopts a folding which explains the TMV-Cg-induced HR-like response in sensitive tobacco plants.

Following leaf infection with the tobacco mosaic virus (TMV), Nicotiana species that carry the disease resistance N gene develop a hypersensitive response (HR) that blocks the systemic movement of the virus. TMV-sensitive tobacco plants that lack the N gene develop classical disease symptoms following infection with most of the tobamoviruses. However, upon infection with TMV-Cg, these plants display a HR-like response that is unable to limit viral spread. We previously identified the NH gene in sensitive plants; this gene is homologous to the resistance N gene and both belong to the TIR/NBS/LRR family. Isolation and analysis of the NH transcript enabled the prediction of the amino acid sequence in which we detected a leucine-rich repeat domain, proposed to be involved in pathogen recognition. This domain is found in four of five classes of pathogen resistant proteins, in which sequence and structural changes may generate different specificities. In order to study the possible functional role of the LRR domain in the HR-like response, we developed a comparative three-dimensional model for the NH and N gene products, by means of functional and structural domains recognition, secondary structure prediction, domain assignment through profile Hidden Markov Models (HMM) and molecular dynamics (MD) simulations. Based on our results we postulate that the NH protein could adopt a LRR fold with a functional role in the HR-like response. Our two reliable LRR three-dimensional models (N-LRR, NH-LRR) can be used as structural frameworks for future experiments in which the structure-function relationships regarding the protein-protein interaction process may be revealed. Evolutionary aspects of the N and NH genes in Nicotiana species are also discussed.

Identification of NPR1-dependent and independent genes early induced by salicylic acid treatment in Arabidopsis.

Salicylic acid (SA) plays a crucial role in stress resistance in plants by modifying the expression of a battery of genes. In this paper, we report the identification of a group of early SA-regulated genes of Arabidopsis (activated between 0.5-2.5 h), using the cDNA-amplified fragment length polymorphism technique (cDNA-AFLP). Using 128 different primer combinations, we identified several genes based on their differential expression during SA treatment. Among these, we identified 12 genes up-regulated by SA whose patterns of induction were confirmed by Northern analysis. The identified genes can be grouped into two functional groups: Group 1: genes involved in cell protection (i.e. glycosyltransferases, glutathion S-transferases), and Group 2: genes involved in signal transduction (protein kinases and transcription factors). We also evaluated NPR1 requirement for the induction of the 12 up-regulated genes, and found that only those belonging to Group 2 require this co-activator for their expression. In silico analysis of the promoter sequences of the up-regulated genes, allowed us to identify putative cis-elements over-represented in these genes. Interestingly, as-1-like elements, previously characterized as SA-responsive elements, were specifically over-represented in Group 1 genes. The identification of early SA-regulated genes is an important step towards understanding the complex role of this hormone in plant stress resistance.

Evaluation of the BIOCEN GC agar medium base in antimicrobial susceptibility testing of Neisseria gonorrhoeae.

BACKGROUND: Since 1995, the Cuban Reference Laboratory for Neisseria has been monitoring the antibiotic susceptibility of gonococci, following the methodology of the National Committee for Clinical Laboratory Standards, which uses GC agar medium base supplemented with 1% Vitox. We evaluated three lots of GC agar medium base produced by BIOCEN, Cuba, in antibiotic susceptibility testing of reference and wild strains of gonococci. METHODS: The susceptibilities to five antibiotics were evaluated five times on three lots of GC agar medium base from BIOCEN. Four and one gonococcal reference strains were tested by MIC dilution and disc diffusion methods, respectively. Later, the antimicrobial susceptibilities of ten wild Neisseria gonorrhoeae strains were tested in triplicate. As internal control, a GC agar medium from Difco was used. RESULTS: All antibiotic MICs obtained on four lots of GC agar medium from different manufacturers fell within the proposed quality control limits for reference strains analyzed. The disc diffusion data for the reference strain of N. gonorrhoeae ATCC 49226 to five antibiotics provided essentially identical results in all lots of GC agar medium base. For wild strains of gonococci, identical modal MIC values and zone size diameters within a 3-mm range were observed in all the antibiotics tested. CONCLUSIONS: Excellent agreement in susceptibility testing methods among different lots of GC agar medium base from BIOCEN and Difco was obtained for all reference and wild gonococcal strains and antibiotics tested. We proposed that GC medium from BIOCEN can be used in antimicrobial susceptibility testing of N. gonorrhoeae by MIC dilution and disc diffusion tests.