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

Functional Profiling of Transcription Factor Genes in Neurospora crassa.

Regulation of gene expression by DNA-binding transcription factors is essential for proper control of growth and development in all organisms. In this study, we annotate and characterize growth and developmental phenotypes for transcription factor genes in the model filamentous fungus Neurospora crassa We identified 312 transcription factor genes, corresponding to 3.2% of the protein coding genes in the genome. The largest class was the fungal-specific Zn2Cys6 (C6) binuclear cluster, with 135 members, followed by the highly conserved C2H2 zinc finger group, with 61 genes. Viable knockout mutants were produced for 273 genes, and complete growth and developmental phenotypic data are available for 242 strains, with 64% possessing at least one defect. The most prominent defect observed was in growth of basal hyphae (43% of mutants analyzed), followed by asexual sporulation (38%), and the various stages of sexual development (19%). Two growth or developmental defects were observed for 21% of the mutants, while 8% were defective in all three major phenotypes tested. Analysis of available mRNA expression data for a time course of sexual development revealed mutants with sexual phenotypes that correlate with transcription factor transcript abundance in wild type. Inspection of this data also implicated cryptic roles in sexual development for several cotranscribed transcription factor genes that do not produce a phenotype when mutated

Algorithmic Debugging of Real-World Haskell Programs: Deriving Dependencies from the Cost Centre Stack

Existing algorithmic debuggers for Haskell require a transformation of all modules in a program, even libraries that the user does not want to debug and which may use language features not supported by the debugger. This is a pity, because a promising ap- proach to debugging is therefore not applicable to many real-world programs. We use the cost centre stack from the Glasgow Haskell Compiler profiling environment together with runtime value observations as provided by the Haskell Object Observation Debugger (HOOD) to collect enough information for algorithmic debugging. Program annotations are in suspected modules only. With this technique algorithmic debugging is applicable to a much larger set of Haskell programs. This demonstrates that for functional languages in general a simple stack trace extension is useful to support tasks such as profiling and debugging

Predicting protein function with hierarchical phylogenetic profiles: The Gene3D phylo-tuner method applied to eukaryotic Genomes

"Phylogenetic profiling'' is based on the hypothesis that during evolution functionally or physically interacting genes are likely to be inherited or eliminated in a codependent manner. Creating presence-absence profiles of orthologous genes is now a common and powerful way of identifying functionally associated genes. In this approach, correctly determining orthology, as a means of identifying functional equivalence between two genes, is a critical and nontrivial step and largely explains why previous work in this area has mainly focused on using presence-absence profiles in prokaryotic species. Here, we demonstrate that eukaryotic genomes have a high proportion of multigene families whose phylogenetic profile distributions are poor in presence-absence information content. This feature makes them prone to orthology mis-assignment and unsuited to standard profile-based prediction methods. Using CATH structural domain assignments from the Gene3D database for 13 complete eukaryotic genomes, we have developed a novel modification of the phylogenetic profiling method that uses genome copy number of each domain superfamily to predict functional relationships. In our approach, superfamilies are subclustered at ten levels of sequence identity from 30% to 100% - and phylogenetic profiles built at each level. All the profiles are compared using normalised Euclidean distances to identify those with correlated changes in their domain copy number. We demonstrate that two protein families will "auto-tune'' with strong co-evolutionary signals when their profiles are compared at the similarity levels that capture their functional relationship. Our method finds functional relationships that are not detectable by the conventional presence - absence profile comparisons, and it does not require a priori any fixed criteria to define orthologous genes

Functional Genomics Profiling of Bladder Urothelial Carcinoma MicroRNAome as a Potential Biomarker.

Though bladder urothelial carcinoma is the most common form of bladder cancer, advances in its diagnosis and treatment have been modest in the past few decades. To evaluate miRNAs as putative disease markers for bladder urothelial carcinoma, this study develops a process to identify dysregulated miRNAs in cancer patients and potentially stratify patients based on the association of their microRNAome phenotype to genomic alterations. Using RNA sequencing data for 409 patients from the Cancer Genome Atlas, we examined miRNA differential expression between cancer and normal tissues and associated differentially expressed miRNAs with patient survival and clinical variables. We then correlated miRNA expressions with genomic alterations using the Wilcoxon test and REVEALER. We found a panel of six miRNAs dysregulated in bladder cancer and exhibited correlations to patient survival. We also performed differential expression analysis and clinical variable correlations to identify miRNAs associated with tobacco smoking, the most important risk factor for bladder cancer. Two miRNAs, miR-323a and miR-431, were differentially expressed in smoking patients compared to nonsmoking patients and were associated with primary tumor size. Functional studies of these miRNAs and the genomic features we identified for potential stratification may reveal underlying mechanisms of bladder cancer carcinogenesis and further diagnosis and treatment methods for urothelial bladder carcinoma

Development of personalised functional foods needs metabolic profiling

Purpose of review There is growing interest in applying metabolic profiling technologies to food science as this approach is now embedded into the foodomics toolbox. This review aims at exploring how metabolic profiling can be applied to the development of functional foods. Recent findings One of the biggest challenges of modern nutrition is to propose a healthy diet to populations worldwide that must suit high inter-individual variability driven by complex gene-nutrient-environment interactions. Although a number of functional foods are now proposed in support of a healthy diet, a one-size-fits-all approach to nutrition is inappropriate and new personalised functional foods are necessary. Metabolic profiling technologies can assist at various levels of the development of functional foods, from screening for food composition to identification of new biomarkers of food intake to support diet intervention and epidemiological studies. Summary Modern ‘omics’ technologies, including metabolic profiling, will support the development of new personalised functional foods of high relevance to twenty-first-century medical challenges such as controlling the worldwide spread of metabolic disorders and ensuring healthy ageing

Identifying opportunities for dynamic circuit specialization

This work describes the identification of designs that benefit from a Dynamic Circuit Specialization (DCS) implementation on FPGAs. In DCS, the circuit is specialized for slowly changing inputs, called parameters. For certain applications or cores, a DCS implementation is faster and smaller than the original implementation. However, the best DCS implementation can be hard to identify, as it requires the designer to be familiar with both the design and DCS. In this paper, we present a profiling tool to aid the designer in analyzing the feasibility of a DCS implementation. It automatically provides a functional density estimate for the most interesting DCS implementations

Discovery of noncanonical translation initiation sites through mass spectrometric analysis of protein N termini

Translation initiation generally occurs at AUG codons in eukaryotes, although it has been shown that non-AUG or non-canonical translation initiation can also occur. However, the evidence for noncanonical translation initiation sites (TISs) is largely indirect and based on ribosome profiling (Ribo-seq) studies. Here, using a strategy specifically designed to enrich N termini of proteins, we demonstrate that many human proteins are translated at noncanonical TISs. The large majority of TISs that mapped to 5' untranslated regions were noncanonical and led to N-terminal extension of annotated proteins or translation of upstream small open reading frames (uORF). It has been controversial whether the amino acid corresponding to the start codon is incorporated at the TIS or methionine is still incorporated. We found that methionine was incorporated at almost all noncanonical TISs identified in this study. Comparison of the TISs determined through mass spectrometry with ribosome profiling data revealed that about two-thirds of the novel annotations were indeed supported by the available ribosome profiling data. Sequence conservation across species and a higher abundance of noncanonical TISs than canonical ones in some cases suggests that the noncanonical TISs can have biological functions. Overall, this study provides evidence of protein translation initiation at noncanonical TISs and argues that further studies are required for elucidation of functional implications of such noncanonical translation initiation

Structural and functional glycosphingolipidomics by glycoblotting with aminooxy-functionalized gold nanoparticle

Glycosphingolipids (GSLs) synthesized in Golgi apparatus by sequential transfer of sugar residues to a ceramide lipid anchor are ubiquitously distributing on vertebrate plasma membranes. Standardized method allowing for high throughput structural profiling and functional characterization of living cell surface GSLs is of growing importance because they function as crucial signal transduction molecules in various processes of dynamic cellular recognitions. However, methods are not available for amplification of GSLs, while the genomic scale PCR amplification permits large-scale mammalian proteomic analysis.　Here we communicate such an approach to a novel "omics", namely glycosphingolipidomics based on the glycoblotting method. The method, which involves selective ozonolysis of the C-C double bond in ceramide moiety and subsequent enrichment of generated GSL-aldehydes by chemical ligation using aminooxy-functionalized gold nanoparticle (aoGNP) should be of widespread utility for identifying and characterizing whole GSLs present in the living cell surfaces. The present protocol using glycoblotting permitted MALDI-TOFMS-based high throughput structural profiling of mouse brain gangliosides such as GM1, GD1a/GD1b, and GT1b for adult or GD3 in case for embryonic mouse. When mouse melanoma B16 cells were subjected to this protocol, it was demonstrated that gangliosides enriched from the plasma membranes are only GM3 bearing microheteogeneity in the structure of N-acyl chain. Surface plasmon resonance analysis revealed that aoGNP displaying whole GSLs blotted from mouse B16 melanoma cell surfaces can be used directly for monitoring specific interaction with self-assembled monolayer (SAM) of Gg3Cer (gangliotriaosylceramide). Our results indicate that GSL-selective enrichment onto aoGNP from living cell surfaces allows for rapid reconstruction of plasma membrane models mimicking intact GSL-microdomain feasible for further structural and functional characterization