Species-level functional profiling of metagenomes and metatranscriptomes.

Functional profiles of microbial communities are typically generated using comprehensive metagenomic or metatranscriptomic sequence read searches, which are time-consuming, prone to spurious mapping, and often limited to community-level quantification. We developed HUMAnN2, a tiered search strategy that enables fast, accurate, and species-resolved functional profiling of host-associated and environmental communities. HUMAnN2 identifies a community's known species, aligns reads to their pangenomes, performs translated search on unclassified reads, and finally quantifies gene families and pathways. Relative to pure translated search, HUMAnN2 is faster and produces more accurate gene family profiles. We applied HUMAnN2 to study clinal variation in marine metabolism, ecological contribution patterns among human microbiome pathways, variation in species' genomic versus transcriptional contributions, and strain profiling. Further, we introduce 'contributional diversity' to explain patterns of ecological assembly across different microbial community types

Aggregating sequences that occur in many proteins constitute weak spots of bacterial proteostasis

Aggregation is a sequence-specific process, nucleated by short aggregation-prone regions (APRs) that can be exploited to induce aggregation of proteins containing the same APR. Here, we find that most APRs are unique within a proteome, but that a small minority of APRs occur in many proteins. When aggregation is nucleated in bacteria by such frequently occurring APRs, it leads to massive and lethal inclusion body formation containing a large number of proteins. Buildup of bacterial resistance against these peptides is slow. In addition, the approach is effective against drug-resistant clinical isolates of Escherichiacoli and Acinetobacterbaumannii, reducing bacterial load in a murine bladder infection model. Our results indicate that redundant APRs are weak points of bacterial protein homeostasis and that targeting these may be an attractive antibacterial strategy

IUPHAR-DB: An Open-Access, Expert-Curated Resource for Receptor and Ion Channel Research

[Image: see text] This contribution highlights efforts by the International Union of Basic and Clinical Pharmacology (IUPHAR) Nomenclature Committee (NC-IUPHAR) to classify human receptors and ion channels, to document their properties, and to recommend ligands that are useful for characterization. This effort has inspired the creation of an online database (IUPHAR-DB), which is intended to provide free information to all scientists, summarized from primary literature by experts

SynSysNet:integration of experimental data on synaptic protein-protein interactions with drug-target relations

We created SynSysNet, available online at http://bioinformatics.charite.de/ synsysnet, to provide a platform that creates a comprehensive 4D network of synaptic interactions. Neuronal synapses are fundamental structures linking nerve cells in the brain and they are responsible for neuronal communication and information processing. These processes are dynamically regulated by a network of proteins. New developments in interaction prote-omics and yeast two-hybrid methods allow unbiased detection of interactors. The consolidation of data from different resources and methods is important to understand the relation to human behaviour and disease and to identify new therapeutic approaches. To this end, we established SynSysNet from a set of ∼1000 synapse specific proteins, their structures and small-molecule interactions. For two-thirds of these, 3D structures are provided (from Protein Data Bank and homology modelling). Drug-target interactions for 750 approved drugs and 50000 compounds, as well as 5000 experimentally validated protein-protein interactions, are included. The resulting interaction network and user-selected parts can be viewed interactively and exported in XGMML. Approximately 200 involved pathways can be explored regarding drug-target interactions. Homology-modelled structures are downloadable in Protein Data Bank format, and drugs are available as MOL-files. Protein-protein interactions and drug-target interactions can be viewed as networks; corresponding PubMed IDs or sources are given. © The Author(s) 2012

AKT overactivation can suppress DNA repair via p70S6 kinase-dependent downregulation of MRE11

Deregulated AKT kinase activity due to PTEN deficiency in cancer cells contributes to oncogenesis by incompletely understood mechanisms. Here, we show that PTEN deletion in HCT116 and DLD1 colon carcinoma cells leads to suppression of CHK1 and CHK2 activation in response to irradiation, impaired G2 checkpoint proficiency and radiosensitization. These defects are associated with reduced expression of MRE11, RAD50 and NBS1, components of the apical MRE11/RAD50/NBS1 (MRN) DNA damage response complex. Consistent with reduced MRN complex function, PTEN-deficient cells fail to resect DNA double-strand breaks efficiently after irradiation and show greatly diminished proficiency for DNA repair via the error-free homologous recombination (HR) repair pathway. MRE11 is highly unstable in PTEN-deficient cells but stability can be significantly restored by inhibiting mTORC1 or p70S6 kinase (p70S6K), downstream kinases whose activities are stimulated by AKT, or by mutating a residue in MRE11 that we show is phosphorylated by p70S6K in vitro. In primary human fibroblasts, activated AKT suppresses MRN complex expression to escalate RAS-induced DNA damage and thereby reinforce oncogene-induced senescence. Taken together, our data demonstrate that deregulation of the PI3K-AKT/ mTORC1/ p70S6K pathways, an event frequently observed in cancer, exert profound effects on genome stability via MRE11 with potential implications for tumour initiation and therapy

A novel transport mechanism for MOMP in Chlamydophila pneumoniae and its putative role in immune-therapy

Major outer membrane proteins (MOMPs) of Gram negative bacteria are one of the most intensively studied membrane proteins. MOMPs are essential for maintaining the structural integrity of bacterial outer membranes and in adaptation of parasites to their hosts. There is evidence to suggest a role for purified MOMP from Chlamydophila pneumoniae and corresponding MOMP-derived peptides in immune-modulation, leading to a reduced atherosclerotic phenotype in apoE−/− mice via a characteristic dampening of MHC class II activity. The work reported herein tests this hypothesis by employing a combination of homology modelling and docking to examine the detailed molecular interactions that may be responsible. A three-dimensional homology model of the C. pneumoniae MOMP was constructed based on the 14 transmembrane β-barrel crystal structure of the fatty acid transporter from Escherichia coli, which provides a plausible transport mechanism for MOMP. Ligand docking experiments were used to provide details of the possible molecular interactions driving the binding of MOMP-derived peptides to MHC class II alleles known to be strongly associated with inflammation. The docking experiments were corroborated by predictions from conventional immuno-informatic algorithms. This work supports further the use of MOMP in C. pneumoniae as a possible vaccine target and the role of MOMP-derived peptides as vaccine candidates for immune-therapy in chronic inflammation that can result in cardiovascular events

Whole genome sequence and manual annotation of Clostridium autoethanogenum, an industrially relevant bacterium

Clostridium autoethanogenum is an acetogenic bacterium capable of producing high value commodity chemicals and biofuels from the C1 gases present in synthesis gas. This common industrial waste gas can act as the sole energy and carbon source for the bacterium that converts the low value gaseous components into cellular building blocks and industrially relevant products via the action of the reductive acetyl-CoA (Wood-Ljungdahl) pathway. Current research efforts are focused on the enhancement and extension of product formation in this organism via synthetic biology approaches. However, crucial to metabolic modelling and directed pathway engineering is a reliable and comprehensively annotated genome sequence

A recombinant Fasciola gigantica 14-3-3 epsilon protein (rFg14-3-3e) modulates various functions of goat peripheral blood mononuclear cells

Background The molecular structure of Fasciola gigantica 14-3-3 protein has been characterized. However, the involvement of this protein in parasite pathogenesis remains elusive and its effect on the functions of innate immune cells is unknown. We report on the cloning and expression of a recombinant F. gigantica 14-3-3 epsilon protein (rFg14-3-3e), and testing its effects on specific functions of goat peripheral blood mononuclear cells (PBMCs). Methods rFg14-3-3e protein was expressed in Pichia pastoris. Western blot and immunofluorescence assay (IFA) were used to examine the reactivity of rFg14-3-3e protein to anti-F. gigantica and anti-rFg14-3-3e antibodies, respectively. Various assays were used to investigate the stimulatory effects of the purified rFg14-3-3e protein on specific functions of goat PBMCs, including cytokine secretion, proliferation, migration, nitric oxide (NO) production, phagocytosis, and apoptotic capabilities. Potential protein interactors of rFg14-3-3e were identified by querying the databases Intact, String, BioPlex and BioGrid. A Total Energy analysis of each of the identified interaction was performed. Gene Ontology (GO) enrichment analysis was conducted using Funcassociate 3.0. Results Sequence analysis revealed that rFg14-3-3e protein had 100% identity to 14-3-3 protein from Fasciola hepatica. Western blot analysis showed that rFg14-3-3e protein is recognized by sera from goats experimentally infected with F. gigantica and immunofluorescence staining using rat anti-rFg14-3-3e antibodies demonstrated the specific binding of rFg14-3-3e protein to the surface of goat PBMCs. rFg14-3-3e protein stimulated goat PBMCs to produce interleukin-10 (IL-10) and transforming growth factor beta (TGF-β), corresponding with low levels of IL-4 and interferon gamma (IFN-γ). Also, this recombinant protein promoted the release of NO and cell apoptosis, and inhibited the proliferation and migration of goat PBMCs and suppressed monocyte phagocytosis. Homology modelling revealed 65% identity between rFg14-3-3e and human 14-3-3 protein YWHAE. GO enrichment analysis of the interacting proteins identified terms related to apoptosis, protein binding, locomotion, hippo signalling and leukocyte and lymphocyte differentiation, supporting the experimental findings. Conclusions Our data suggest that rFg14-3-3e protein can influence various cellular and immunological functions of goat PBMCs in vitro and may be involved in mediating F. gigantica pathogenesis. Because of its involvement in F. gigantica recognition by innate immune cells, rFg14-3-3e protein may have applications for development of diagnostics and therapeutic interventions

SalmoNet, an integrated network of ten Salmonella enterica strains reveals common and distinct pathways to host adaptation

Salmonella enterica is a prominent bacterial pathogen with implications on human and animal health. Salmonella serovars could be classified as gastro-intestinal or extra-intestinal. Genome-wide comparisons revealed that extra-intestinal strains are closer relatives of gastro-intestinal strains than to each other indicating a parallel evolution of this trait. Given the complexity of the differences, a systems-level comparison could reveal key mechanisms enabling extra-intestinal serovars to cause systemic infections. Accordingly, in this work, we introduce a unique resource, SalmoNet, which combines manual curation, high-throughput data and computational predictions to provide an integrated network for Salmonella at the metabolic, transcriptional regulatory and protein-protein interaction levels. SalmoNet provides the networks separately for five gastro-intestinal and five extra-intestinal strains. As a multi-layered, multi-strain database containing experimental data, SalmoNet is the first dedicated network resource for Salmonella. It comprehensively contains interactions between proteins encoded in Salmonella pathogenicity islands, as well as regulatory mechanisms of metabolic processes with the option to zoom-in and analyze the interactions at specific loci in more detail. Application of SalmoNet is not limited to strain comparisons as it also provides a Salmonella resource for biochemical network modeling, host-pathogen interaction studies, drug discovery, experimental validation of novel interactions, uncovering new pathological mechanisms from emergent properties and epidemiological studies. SalmoNet is available at http://salmonet.org

High-throughput site-directed mutagenesis

Protein engineering has an array of uses: whether you are studying a disease mutation, removing undesirable sequences, adding stabilizing mutations for structural purposes, or simply dissecting protein function. Protein engineering is almost exclusively performed using site-directed mutagenesis (SDM) as this provides targeted modification of specific amino acids, as well as the option of rewriting the native sequence to include or exclude certain regions. Despite its widespread use, SDM has often proved to be a bottleneck, requiring precision manipulation on a sample-by-sample basis to make it work. When dealing with large volumes of samples it is not possible to use such a low-throughput approach. Here we describe a high-throughput (HTP) method for SDM, optimized and used by the Structural Genomics Consortium (SGC) to complement structural studies