Development of a statistically robust quantification method for microorganisms in mixtures using oligonucleotide microarrays

High-density oligonucleotide arrays can be extremely useful for identifying and quantifying specific targets (i.e., ribosomal RNA of microorganisms) in mixtures. However, current array identification schemes are severely compromised by nonspecific hybridization, resulting in numerous false-positive and false-negative calls, they lack an adequate internal control for assessing the quality of identification, and are dependent on amplification of specific target sequences which introduce biases. We have developed a novel approach for the routine quantification and identification of metabolically active microorganisms in mixed samples. The advantage of our approach over conventional ones is that it avoids designing, optimizing, validating, and selecting oligonucleotide probes for arrays; also, nonspecific hybridization is no longer a problem. The basic principle of the approach is that a fluorescence pattern of a mixed sample is a superposition of the fluorescent patterns for each target. The superposition can be quantitatively deconvoluted in terms of concentrations of each microbe. We demonstrated the utility of our approach by extracting rRNA from three microorganisms, making test mixtures, labeling the rRNA, and hybridizing each test mixture to DNA oligonucleotide (20-mers, n=346,608) arrays. Comparison of known concentrations of individual targets in mixtures to those estimated by the solution revealed highly consistent results. The goodness-of-fit of the solution revealed that about 90% of the variability in the data could be explained. A new analytical approach for microbial identification and quantification has been presented in this report. Our findings demonstrate that including signal intensity values from all duplexes on the array, which are essentially nonspecific to the target organisms, significantly improved predictions of known microbial targets. To our knowledge, this is the first study to report this phenomenon. In addition, we demonstrate that the method is a self-sufficient analytical procedure since it provides statistical confidence of the quantification.     

Structures of the Cd44-hyaluronan complex provide insight into a fundamental carbohydrate-protein interaction

Regulation of transient interactions between cells and the ubiquitous matrix glycosaminoglycan hyaluronan is crucial to such fundamental processes as embryonic development and leukocyte homing. Cd44, the primary cell surface receptor for hyaluronan, binds ligand via a lectin-like fold termed the Link module, but only after appropriate functional activation. The molecular details of the Cd44-hyaluronan interaction and hence the structural basis for this activation are unknown. Here we present the first crystal structure of Cd44 complexed with hyaluronan. This reveals that the interaction with hyaluronan is dominated by shape and hydrogen-bonding complementarity and identifies two conformational forms of the receptor that differ in orientation of a crucial hyaluronan-binding residue (Arg45, equivalent to Arg41 in human CD44). Measurements by NMR indicate that the conformational transition can be induced by hyaluronan binding, providing further insight into possible mechanisms for regulation of Cd44.     

An essential role for IL-18 in CD8 T cell-mediated suppression of IgE responses

The ability of CD8 T cells to suppress IgE responses is well established. Previously, we demonstrated that CD8 T cells inhibit IgE responses via the induction of IL-12, which promotes Th1 and suppresses Th2 responses. In this study, we show that IL-18 also plays an essential role in IgE suppression. In vitro, IL-18 synergized with IL-12 to promote Th1/T cytotoxic 1 and inhibit Th2/T cytotoxic 2 differentiation. OVA-specific TCR transgenic (OT-I) CD8 cells induced both IL-12 and IL-18 when cultured with OVA(257-264) peptide-pulsed dendritic cells. In vivo, IL-18(-/-) mice exhibited higher IgE and IgG1 levels compared with wild-type mice after immunization with OVA/alum. Furthermore, adoptive transfer of CD8 T cells from OVA-primed mice suppressed IgE responses in OVA/alum-immunized mice, but not in IL-18(-/-) mice. IgE suppression in IL-18(-/-) mice was restored if CD8 T cells were coadoptively transferred with IL-18-competent wild-type bone marrow dendritic cell progenitors, demonstrating an essential role of IL-18 in CD8 T cell-mediated suppression of IgE responses. The data suggest that CD8 T cells induce IL-18 production during a cognate interaction with APCs that synergizes with IL-12 to promote immune deviation away from the allergic phenotype. Our data identify IL-18 induction as a potentially useful target in immunotherapy of allergic disease.     

Molecular cloning, characterisation and ligand-bound structure of an azoreductase from Pseudomonas aeruginosa

The gene PA0785 from Pseudomonas aeruginosa strain PAO1, which is annotated as a probable acyl carrier protein phosphodiesterase (acpD), has been cloned and heterologously overexpressed in Escherichia coli. The purified recombinant enzyme exhibits activity corresponding to that of azoreductase but not acpD. Each recombinant protein molecule has an estimated molecular mass of 23,050 Da and one non-covalently bound FMN as co-factor. This enzyme, now identified as azoreductase 1 from Pseudomonas aeruginosa (paAzoR1), is a flavodoxin-like protein with an apparent molecular mass of 110 kDa as determined by gel-filtration chromatography, indicating that the protein is likely to be tetrameric in solution. The three-dimensional structure of paAzoR1, in complex with the substrate methyl red, was solved at a resolution of 2.18 A by X-ray crystallography. The protein exists as a dimer of dimers in the crystal lattice, with two spatially separated active sites per dimer, and the active site of paAzoR1 was shown to be a well-conserved hydrophobic pocket formed between two monomers. The paAzoR1 enzyme is able to reduce different classes of azo dyes and activate several azo pro-drugs used in the treatment of inflammatory bowel disease (IBD). During azo reduction, FMN serves as a redox centre in the electron-transferring system by mediating the electron transfer from NAD(P)H to the azo substrate. The spectral properties of paAzoR1 demonstrate the hydrophobic interaction between FMN and the active site in the protein. The structure of the ligand-bound protein also highlights the pi-stacking interactions between FMN and the azo substrate.     

Do studies of mechanical restitution tell us anything about the mechanism of Ca2+ release in myocardial cells?

We present information from the literature which is discordant with the idea that the trigger Ca2+ for Ca2(+)-induced Ca2+ release from the myocardial sarcoplasmic reticulum (SR) is carried by the inward current. In addition, evidence is emerging to show that recovery of contractility with increasing interval after an excitation-contraction-relaxation cycle (mechanical restitution) begins with repolarization of the sarcolemma, not mechanical relaxation (SR Ca2+ uptake). These two phenomena, if confirmed, would be compatible with a sarcolemmal source of trigger Ca2+.     

Disruption of ER‐mitochondria tethering and signalling in C9orf72‐associated amyotrophic lateral sclerosis and frontotemporal dementia

Hexanucleotide repeat expansions in C9orf72 are the most common cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The mechanisms by which the expansions cause disease are not properly understood but a favoured route involves its translation into dipeptide repeat (DPR) polypeptides, some of which are neurotoxic. However, the precise targets for mutant C9orf72 and DPR toxicity are not fully clear, and damage to several neuronal functions has been described. Many of these functions are regulated by signalling between the endoplasmic reticulum (ER) and mitochondria. ER‐mitochondria signalling requires close physical contacts between the two organelles that are mediated by the VAPB‐PTPIP51 ‘tethering’ proteins. Here, we show that ER‐mitochondria signalling and the VAPB‐PTPIP51 tethers are disrupted in neurons derived from induced pluripotent stem (iPS) cells from patients carrying ALS/FTD pathogenic C9orf72 expansions and in affected neurons in mutant C9orf72 transgenic mice. In these mice, disruption of the VAPB‐PTPIP51 tethers occurs prior to disease onset suggesting that it contributes to the pathogenic process. We also show that neurotoxic DPRs disrupt the VAPB‐PTPIP51 interaction and ER‐mitochondria contacts and that this may involve activation of glycogen synthase kinases‐3β (GSK3β), a known negative regulator of VAPB‐PTPIP51 binding. Finally, we show that these DPRs disrupt delivery of Ca²⁺ from ER stores to mitochondria, which is a primary function of the VAPB‐PTPIP51 tethers. This delivery regulates a number of key neuronal functions that are damaged in ALS/FTD including bioenergetics, autophagy and synaptic function. Our findings reveal a new molecular target for mutant C9orf72‐mediated toxicity.     

Flow Cytometry for Determination of the Efficacy of Contact Lens Disinfecting Solutions against Acanthamoeba spp.

Flow cytometric analyses of cellular staining with fluorescent viability dyes and direct microscopic observations of methylene blue exclusion were compared for evaluation of the effects of a chlorhexidine gluconate-based contact lens disinfectant solution and a polyhexamethylene biguanide solution against cysts and trophozoites of Acanthamoeba castellanii and Acanthamoeba polyphaga. The flow cytometric procedure with propidium iodide (used to stain dead cells) indicated that more than 90% of trophozoites of both species (inocula of 10⁵ to 10⁶/ml) at 22°C lost their viability after 4 h of exposure to chlorhexidine. When propidium iodide was used in combination with fluorescein diacetate (for live cells), the apparent number of propidium iodide-stained cells was reduced, but the relative efficacies of the two biguanide solutions appeared unchanged from those evident with the single dyes; the chlorhexidine solution was more effective than the polyhexamethylene biguanide solution. Similar data were obtained with the more cumbersome methylene blue exclusion procedure. Flow cytometric analyses provided a statistically reproducible and rapid procedure for determining the relative antiamoebal efficacies of the disinfecting solutions.     

Lowland boreal forests characterization in Algonquin Provincial Park relative to beaver (Castor canadensis) foraging and edaphic factors

Beaver (Caster canadensis) foraging and edaphic conditions can modify the vegetational characteristics of woody plant community in lowland boreal forests. Effective management of these areas requires an understanding of the relative contribution of these factors in shaping the woody plant community structure. Our objective was to quantify the effects of herbivory by beavers and edaphic conditions on woody plant community organization of lowland boreal forests surrounding beaver ponds. Woody vegetation and soils were sampled at 15 ponds occupied by beavers and one other pond abandoned by them in southern Algonquin Park, Ontario. We measured spatial variation in plant diversity, foraging rates and sapling recruitment of trees and shrubs along gradients of beaver foraging intensity and soil moisture, P, K, Mg, and pH. Beavers fed preferentially on a small number of deciduous species and the number of cut stems declined sharply with increasing distance from ponds. Conifers increased in relative dominance to deciduous species in the presence of beavers. Plant species richness and stem and basal area diversity peaked at intermediate distances (about 25 m) from ponds. Sapling recruitment by non-preferred species was positively related to foraging intensity. Total stem abundance and basal area and sapling recruitment by four preferred species (Populus tremuloides, Acer rubrum, Acer saccharum and Corylus cornuta) were negatively related to foraging intensity. However, by including Alnus rugosa and Salix bebbiana (also preferred by beavers) these patterns changed, becoming positively related to foraging intensity. There was also a pronounced gradient in soil moisture, which also decreased with distance from ponds. The other measured edaphic variables did not vary consistently with distance from ponds. Sapling recruitment in mesic versus xeric species varied consistently with hydrid conditions along the moisture gradient, such that variation in moisture also could produce the observed pattern of plant diversity. Diversity patterns changed three years after beaver abandonment of a pond, though sapling recruitment patterns in preferred and non-preferred species around the abandoned pond were similar to the occupied ponds. These observations suggest spatial variation in woody plant richness and diversity could be determined by combined effects of both herbivory (disturbance by beavers) and variable responses of different species to edaphic conditions.     

Consistent probabilistic outputs for protein function prediction

Background:

The wide availability of genome-scale data for several organisms has stimulated interest in computational approaches to gene function prediction. Diverse machine learning methods have been applied to unicellular organisms with some success, but few have been extensively tested on higher level, multicellular organisms. A recent mouse function prediction project (MouseFunc) brought together nine bioinformatics teams applying a diverse array of methodologies to mount the first large-scale effort to predict gene function in the laboratory mouse.

Results:

In this paper, we describe our contribution to this project, an ensemble framework based on the support vector machine that integrates diverse datasets in the context of the Gene Ontology hierarchy. We carry out a detailed analysis of the performance of our ensemble and provide insights into which methods work best under a variety of prediction scenarios. In addition, we applied our method to Saccharomyces cerevisiae and have experimentally confirmed functions for a novel mitochondrial protein.

Conclusion:

Our method consistently performs among the top methods in the MouseFunc evaluation. Furthermore, it exhibits good classification performance across a variety of cellular processes and functions in both a multicellular organism and a unicellular organism, indicating its ability to discover novel biology in diverse settings.