A promoter-level mammalian expression atlas.

Regulated transcription controls the diversity, developmental pathways and spatial organization of the hundreds of cell types that make up a mammal. Using single-molecule cDNA sequencing, we mapped transcription start sites (TSSs) and their usage in human and mouse primary cells, cell lines and tissues to produce a comprehensive overview of mammalian gene expression across the human body. We find that few genes are truly 'housekeeping', whereas many mammalian promoters are composite entities composed of several closely separated TSSs, with independent cell-type-specific expression profiles. TSSs specific to different cell types evolve at different rates, whereas promoters of broadly expressed genes are the most conserved. Promoter-based expression analysis reveals key transcription factors defining cell states and links them to binding-site motifs. The functions of identified novel transcripts can be predicted by coexpression and sample ontology enrichment analyses. The functional annotation of the mammalian genome 5 (FANTOM5) project provides comprehensive expression profiles and functional annotation of mammalian cell-type-specific transcriptomes with wide applications in biomedical research.

In silico screening for candidate chassis strains of free fatty acid-producing cyanobacteria.

BACKGROUND: Finding a source from which high-energy-density biofuels can be derived at an industrial scale has become an urgent challenge for renewable energy production. Some microorganisms can produce free fatty acids (FFA) as precursors towards such high-energy-density biofuels. In particular, photosynthetic cyanobacteria are capable of directly converting carbon dioxide into FFA. However, current engineered strains need several rounds of engineering to reach the level of production of FFA to be commercially viable; thus new chassis strains that require less engineering are needed. Although more than 120 cyanobacterial genomes are sequenced, the natural potential of these strains for FFA production and excretion has not been systematically estimated. RESULTS: Here we present the FFA SC (FFASC), an in silico screening method that evaluates the potential for FFA production and excretion of cyanobacterial strains based on their proteomes. A literature search allowed for the compilation of 64 proteins, most of which influence FFA production and a few of which affect FFA excretion. The proteins are classified into 49 orthologous groups (OGs) that helped create rules used in the scoring/ranking of algorithms developed to estimate the potential for FFA production and excretion of an organism. Among 125 cyanobacterial strains, FFASC identified 20 candidate chassis strains that rank in their FFA producing and excreting potential above the specifically engineered reference strain, Synechococcus sp. PCC 7002. We further show that the top ranked cyanobacterial strains are unicellular and primarily include Prochlorococcus (order Prochlorales) and marine Synechococcus (order Chroococcales) that cluster phylogenetically. Moreover, two principal categories of enzymes were shown to influence FFA production the most: those ensuring precursor availability for the biosynthesis of lipids, and those involved in handling the oxidative stress associated to FFA synthesis. CONCLUSION: To our knowledge FFASC is the first in silico method to screen cyanobacteria proteomes for their potential to produce and excrete FFA, as well as the first attempt to parameterize the criteria derived from genetic characteristics that are favorable/non-favorable for this purpose. Thus, FFASC helps focus experimental evaluation only on the most promising cyanobacteria.

Hi-Jack: a novel computational framework for pathway-based inference of host-pathogen interactions.

MOTIVATION: Pathogens infect their host and hijack the host machinery to produce more progeny pathogens. Obligate intracellular pathogens, in particular, require resources of the host to replicate. Therefore, infections by these pathogens lead to alterations in the metabolism of the host, shifting in favor of pathogen protein production. Some computational identification of mechanisms of host-pathogen interactions have been proposed, but it seems the problem has yet to be approached from the metabolite-hijacking angle. RESULTS: We propose a novel computational framework, Hi-Jack, for inferring pathway-based interactions between a host and a pathogen that relies on the idea of metabolite hijacking. Hi-Jack searches metabolic network data from hosts and pathogens, and identifies candidate reactions where hijacking occurs. A novel scoring function ranks candidate hijacked reactions and identifies pathways in the host that interact with pathways in the pathogen, as well as the associated frequent hijacked metabolites. We also describe host-pathogen interaction principles that can be used in the future for subsequent studies. Our case study on Mycobacterium tuberculosis (Mtb) revealed pathways in human-e.g. carbohydrate metabolism, lipids metabolism and pathways related to amino acids metabolism-that are likely to be hijacked by the pathogen. In addition, we report interesting potential pathway interconnections between human and Mtb such as linkage of human fatty acid biosynthesis with Mtb biosynthesis of unsaturated fatty acids, or linkage of human pentose phosphate pathway with lipopolysaccharide biosynthesis in Mtb. AVAILABILITY AND IMPLEMENTATION: Datasets and codes are available at http://cloud.kaust.edu.sa/Pages/Hi-Jack.aspx

Bioprospecting Red Sea Coastal Ecosystems for Culturable Microorganisms and Their Antimicrobial Potential.

Microorganisms that inhabit unchartered unique soil such as in the highly saline and hot Red Sea lagoons on the Saudi Arabian coastline, represent untapped sources of potentially new bioactive compounds. In this study, a culture-dependent approach was applied to three types of sediments: mangrove mud (MN), microbial mat (MM), and barren soil (BS), collected from Rabigh harbor lagoon (RHL) and Al-Kharrar lagoon (AKL). The isolated bacteria were evaluated for their potential to produce bioactive compounds. The phylogenetic characterization of 251 bacterial isolates based on the 16S rRNA gene sequencing, supported their assignment to five different phyla: Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, and Planctomycetes. Fifteen putative novel species were identified based on a 16S rRNA gene sequence similarity to other strain sequences in the NCBI database, being ≤98%. We demonstrate that 49 of the 251 isolates exhibit the potential to produce antimicrobial compounds. Additionally, at least one type of biosynthetic gene sequence, responsible for the synthesis of secondary metabolites, was recovered from 25 of the 49 isolates. Moreover, 10 of the isolates had a growth inhibition effect towards Staphylococcus aureus, Salmonella typhimurium and Pseudomonas syringae. We report the previously unknown antimicrobial activity of B. borstelensis, P. dendritiformis and M. salipaludis against all three indicator pathogens. Our study demonstrates the evidence of diverse cultured microbes associated with the Red Sea harbor/lagoon environments and their potential to produce antimicrobial compounds.

A novel mercuric reductase from the unique deep brine environment of Atlantis II in the Red Sea.

A unique combination of physicochemical conditions prevails in the lower convective layer (LCL) of the brine pool at Atlantis II (ATII) Deep in the Red Sea. With a maximum depth of over 2000 m, the pool is characterized by acidic pH (5.3), high temperature (68 °C), salinity (26%), low light levels, anoxia, and high concentrations of heavy metals. We have established a metagenomic dataset derived from the microbial community in the LCL, and here we describe a gene for a novel mercuric reductase, a key component of the bacterial detoxification system for mercuric and organomercurial species. The metagenome-derived gene and an ortholog from an uncultured soil bacterium were synthesized and expressed in Escherichia coli. The properties of their products show that, in contrast to the soil enzyme, the ATII-LCL mercuric reductase is functional in high salt, stable at high temperatures, resistant to high concentrations of Hg(2+), and efficiently detoxifies Hg(2+) in vivo. Interestingly, despite the marked functional differences between the orthologs, their amino acid sequences differ by less than 10%. Site-directed mutagenesis and kinetic analysis of the mutant enzymes, in conjunction with three-dimensional modeling, have identified distinct structural features that contribute to extreme halophilicity, thermostability, and high detoxification capacity, suggesting that these were acquired independently during the evolution of this enzyme. Thus, our work provides fundamental structural insights into a novel protein that has undergone multiple biochemical and biophysical adaptations to promote the survival of microorganisms that reside in the extremely demanding environment of the ATII-LCL.

Dragon TIS Spotter: an Arabidopsis-derived predictor of translation initiation sites in plants.

SUMMARY: In higher eukaryotes, the identification of translation initiation sites (TISs) has been focused on finding these signals in cDNA or mRNA sequences. Using Arabidopsis thaliana (A.t.) information, we developed a prediction tool for signals within genomic sequences of plants that correspond to TISs. Our tool requires only genome sequence, not expressed sequences. Its sensitivity/specificity is for A.t. (90.75%/92.2%), for Vitis vinifera (66.8%/94.4%) and for Populus trichocarpa (81.6%/94.4%), which suggests that our tool can be used in annotation of different plant genomes. We provide a list of features used in our model. Further study of these features may improve our understanding of mechanisms of the translation initiation. AVAILABILITY AND IMPLEMENTATION: Our tool is implemented as an artificial neural network. It is available as a web-based tool and, together with the source code, the list of features, and data used for model development, is accessible at http://cbrc.kaust.edu.sa/dts.

DAMPD: a manually curated antimicrobial peptide database.

The demand for antimicrobial peptides (AMPs) is rising because of the increased occurrence of pathogens that are tolerant or resistant to conventional antibiotics. Since naturally occurring AMPs could serve as templates for the development of new anti-infectious agents to which pathogens are not resistant, a resource that contains relevant information on AMP is of great interest. To that extent, we developed the Dragon Antimicrobial Peptide Database (DAMPD, http://apps.sanbi.ac.za/dampd) that contains 1232 manually curated AMPs. DAMPD is an update and a replacement of the ANTIMIC database. In DAMPD an integrated interface allows in a simple fashion querying based on taxonomy, species, AMP family, citation, keywords and a combination of search terms and fields (Advanced Search). A number of tools such as Blast, ClustalW, HMMER, Hydrocalculator, SignalP, AMP predictor, as well as a number of other resources that provide additional information about the results are also provided and integrated into DAMPD to augment biological analysis of AMPs.

Dragon PolyA Spotter: predictor of poly(A) motifs within human genomic DNA sequences.

MOTIVATION: Recognition of poly(A) signals in mRNA is relatively straightforward due to the presence of easily recognizable polyadenylic acid tail. However, the task of identifying poly(A) motifs in the primary genomic DNA sequence that correspond to poly(A) signals in mRNA is a far more challenging problem. Recognition of poly(A) signals is important for better gene annotation and understanding of the gene regulation mechanisms. In this work, we present one such poly(A) motif prediction method based on properties of human genomic DNA sequence surrounding a poly(A) motif. These properties include thermodynamic, physico-chemical and statistical characteristics. For predictions, we developed Artificial Neural Network and Random Forest models. These models are trained to recognize 12 most common poly(A) motifs in human DNA. Our predictors are available as a free web-based tool accessible at http://cbrc.kaust.edu.sa/dps. Compared with other reported predictors, our models achieve higher sensitivity and specificity and furthermore provide a consistent level of accuracy for 12 poly(A) motif variants. CONTACT: vladimir.bajic@kaust.edu.sa SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Importance of the genetic diversity within the Mycobacterium tuberculosis complex for the development of novel antibiotics and diagnostic tests of drug resistance.

Despite being genetically monomorphic, the limited genetic diversity within the Mycobacterium tuberculosis complex (MTBC) has practical consequences for molecular methods for drug susceptibility testing and for the use of current antibiotics and those in clinical trials. It renders some representatives of MTBC intrinsically resistant against one or multiple antibiotics and affects the spectrum and consequences of resistance mutations selected for during treatment. Moreover, neutral or silent changes within genes responsible for drug resistance can cause false-positive results with hybridization-based assays, which have been recently introduced to replace slower phenotypic methods. We discuss the consequences of these findings and propose concrete steps to rigorously assess the genetic diversity of MTBC to support ongoing clinical trials.

Conotoxins that confer therapeutic possibilities.

Cone snails produce a distinctive repertoire of venom peptides that are used both as a defense mechanism and also to facilitate the immobilization and digestion of prey. These peptides target a wide variety of voltage- and ligand-gated ion channels, which make them an invaluable resource for studying the properties of these ion channels in normal and diseased states, as well as being a collection of compounds of potential pharmacological use in their own right. Examples include the United States Food and Drug Administration (FDA) approved pharmaceutical drug, Ziconotide (Prialt(®); Elan Pharmaceuticals, Inc.) that is the synthetic equivalent of the naturally occurring ω-conotoxin MVIIA, whilst several other conotoxins are currently being used as standard research tools and screened as potential therapeutic drugs in pre-clinical or clinical trials. These developments highlight the importance of driving conotoxin-related research. A PubMed query from 1 January 2007 to 31 August 2011 combined with hand-curation of the retrieved articles allowed for the collation of 98 recently identified conotoxins with therapeutic potential which are selectively discussed in this review. Protein sequence similarity analysis tentatively assigned uncharacterized conotoxins to predicted functional classes. Furthermore, conotoxin therapeutic potential for neurodegenerative disorders (NDD) was also inferred.

Polymorphisms in isoniazid and prothionamide resistance genes of the Mycobacterium tuberculosis complex.

Sequence analyses of 74 strains that encompassed major phylogenetic lineages of the Mycobacterium tuberculosis complex revealed 10 polymorphisms in mshA (Rv0486) and four polymorphisms in inhA (Rv1484) that were not responsible for isoniazid or prothionamide resistance. Instead, some of these mutations were phylogenetically informative. This genetic diversity must be taken into consideration for drug development and for the design of molecular tests for drug resistance.

Impact of Fgd1 and ddn diversity in Mycobacterium tuberculosis complex on in vitro susceptibility to PA-824.

PA-824 is a promising drug candidate for the treatment of tuberculosis (TB). It is in phase II clinical trials as part of the first newly designed regimen containing multiple novel antituberculosis drugs (PA-824 in combination with moxifloxacin and pyrazinamide). However, given that the genes involved in resistance against PA-824 are not fully conserved in the Mycobacterium tuberculosis complex (MTBC), this regimen might not be equally effective against different MTBC genotypes. To investigate this question, we sequenced two PA-824 resistance genes (fgd1 [Rv0407] and ddn [Rv3547]) in 65 MTBC strains representing major phylogenetic lineages. The MICs of representative strains were determined using the modified proportion method in the Bactec MGIT 960 system. Our analysis revealed single-nucleotide polymorphisms in both genes that were specific either for several genotypes or for individual strains, yet none of these mutations significantly affected the PA-824 MICs (≤ 0.25 µg/ml). These results were supported by in silico modeling of the mutations identified in Fgd1. In contrast, "Mycobacterium canettii" strains displayed a higher MIC of 8 µg/ml. In conclusion, we found a large genetic diversity in PA-824 resistance genes that did not lead to elevated PA-824 MICs. In contrast, M. canettii strains had MICs that were above the plasma concentrations of PA-824 documented so far in clinical trials. As M. canettii is also intrinsically resistant against pyrazinamide, new regimens containing PA-824 and pyrazinamide might not be effective in treating M. canettii infections. This finding has implications for the design of multiple ongoing clinical trials.

Recently confirmed apoptosis-inducing lead compounds isolated from marine sponge of potential relevance in cancer treatment.

Despite intense efforts to develop non-cytotoxic anticancer treatments, effective agents are still not available. Therefore, novel apoptosis-inducing drug leads that may be developed into effective targeted cancer therapies are of interest to the cancer research community. Targeted cancer therapies affect specific aberrant apoptotic pathways that characterize different cancer types and, for this reason, it is a more desirable type of therapy than chemotherapy or radiotherapy, as it is less harmful to normal cells. In this regard, marine sponge derived metabolites that induce apoptosis continue to be a promising source of new drug leads for cancer treatments. A PubMed query from 01/01/2005 to 31/01/2011 combined with hand-curation of the retrieved articles allowed for the identification of 39 recently confirmed apoptosis-inducing anticancer lead compounds isolated from the marine sponge that are selectively discussed in this review.

Transcriptional response of Rhodococcus aetherivorans I24 to polychlorinated biphenyl-contaminated sediments.

We used a microarray targeting 3,524 genes to assess the transcriptional response of the actinomycete Rhodococcus aetherivorans I24 in minimal medium supplemented with various substrates (e.g., PCBs) and in both PCB-contaminated and non-contaminated sediment slurries. Relative to the reference condition (minimal medium supplemented with glucose), 408 genes were upregulated in the various treatments. In medium and in sediment, PCBs elicited the upregulation of a common set of 100 genes, including gene-encoding chaperones (groEL), a superoxide dismutase (sodA), alkyl hydroperoxide reductase protein C (ahpC), and a catalase/peroxidase (katG). Analysis of the R. aetherivorans I24 genome sequence identified orthologs of many of the genes in the canonical biphenyl pathway, but very few of these genes were upregulated in response to PCBs or biphenyl. This study is one of the first to use microarrays to assess the transcriptional response of a soil bacterium to a pollutant under conditions that more closely resemble the natural environment. Our results indicate that the transcriptional response of R. aetherivorans I24 to PCBs, in both medium and sediment, is primarily directed towards reducing oxidative stress, rather than catabolism.

Metagenomics as a preliminary screen for antimicrobial bioprospecting.

Since the composition of soil directs the diversity of the contained microbiome and its potential to produce bioactive compounds, many studies has been focused on sediment types with unique features characteristic of extreme environments. However, not much is known about the potential of microbiomes that inhabit the highly saline and hot Red Sea lagoons. This case study explores mangrove mud and the microbial mat of sediments collected from the Rabigh harbor lagoon and Al Kharrar lagoon for antimicrobial bioprospecting. Rabigh harbor lagoon appears the better location, and the best sediment type for this purpose is mangrove mud. On the other hand, Al Kharrar lagoon displayed increased anaerobic hydrocarbon degradation and an abundance of bacterial DNA associated with antibiotic resistance. Moreover, our findings show an identical shift in phyla associated with historic hydrocarbon contamination exposure reported in previous studies (that is, enrichment of Gamma- and Delta-proteobacteria), but we also report that bacterial DNA sequences associated with antibiotic synthesis enzymes are derived from Gamma-, Delta- and Alpha-proteobacteria. This suggests that selection pressure associated with hydrocarbon contamination tend to enrich the bacterial classes DNA associated with antibiotic synthesis enzymes. Although Actinobacteria tends to be the common target for research when it comes to antimicrobial bioprospecting, our study suggests that Firmicutes (Bacilli and Clostridia), Bacteroidetes, Cyanobacteria, and Proteobacteria should be antimicrobial bioprospecting targets as well. To the best of our knowledge, this is the first metagenomic study that analyzed the microbiomes in Red Sea lagoons for antimicrobial bioprospecting.

Rhizosphere microbiome metagenomics of gray mangroves (Avicennia marina) in the Red Sea.

Mangroves are unique, and endangered, coastal ecosystems that play a vital role in the tropical and subtropical environments. A comprehensive description of the microbial communities in these ecosystems is currently lacking, and additional studies are required to have a complete understanding of the functioning and resilience of mangroves worldwide. In this work, we carried out a metagenomic study by comparing the microbial community of mangrove sediment with the rhizosphere microbiome of Avicennia marina, in northern Red Sea mangroves, along the coast of Saudi Arabia. Our results revealed that rhizosphere samples presented similar profiles at the taxonomic and functional levels and differentiated from the microbiome of bulk soil controls. Overall, samples showed predominance by Proteobacteria, Bacteroidetes and Firmicutes, with high abundance of sulfate reducers and methanogens, although specific groups were selectively enriched in the rhizosphere. Functional analysis showed significant enrichment in 'metabolism of aromatic compounds', 'mobile genetic elements', 'potassium metabolism' and 'pathways that utilize osmolytes' in the rhizosphere microbiomes. To our knowledge, this is the first metagenomic study on the microbiome of mangroves in the Red Sea, and the first application of unbiased 454-pyrosequencing to study the rhizosphere microbiome associated with A. marina. Our results provide the first insights into the range of functions and microbial diversity in the rhizosphere and soil sediments of gray mangrove (A. marina) in the Red Sea.