Experiments and a model examining learning in the area-restricted search behavior of ferrets (Mustela putorius furo)

Area-restricted searches have been described as important componentsof the foraging behavior of many organisms. It is unclear, however,whether individual foragers can use learning to fine-tune theirsearches, or even whether these searches are efficiently performed.I used a simulation model to make qualitative predictions aboutsearch behavior in a laboratory system. The simulation modelindicates that the sinuosity and path length of searches stronglyaffect search efficiency. The model predicts that, for a rate-maximizingforager, path length should increase and search sinuosity shoulddecrease as prey become less clumped. Foraging animals may thereforebe selected to learn the path length and sinuosity of searchesin response to changing degrees of dumping of prey. These predictionswere tested in a laboratory system involving ferrets (Mustelaputorius furo) foraging for oil-drop "prey items." Search pathschanged in a graded manner to experimental manipulations ofthe dumping of prey. As predicted by the model, ferrets learnedto perform longer and less sinuous search paths as prey becameless clumped. This study provides the first evidence that area-restrictedsearch behavior is learned and can be fine-tuned to efficientlyexploit different spatial distributions of food.     

A genome-wide search for genes predisposing to familial psoriasis by using a stratification approach

We have performed a genome scan, using markers spaced by 10 cM, in the search for psoriasis-susceptibility loci. The family material of 134 affected sibling pairs was ascertained on the basis of a population genetic study in which 65% of the probands had two healthy parents. Genotyping results were analyzed for non-random excessive allele-sharing between sib pairs by using GENEHUNTER ver 1.1. A stratification approach was applied to increase the homogeneity of the material by means of an operational definition of joint complaints among affected individuals. Significant linkage to the human leukocyte antigen region on chromosome 6p in a cohort including 42 families without joint complaints (nonparametric linkage score of 2.83, P=0.002) strongly supported the validity of this operational definition as it replicated results from an earlier linkage report with similar stratification criteria. New candidate regions on chromosomes 3 and 15 were identified. The highest non-parametric linkage values in this study, 2.96 (P=0.0017) and 2.89 (P=0.0020), were reached on chromosome 15 in a subgroup with joint complaints and on chromosome 3 in a subgroup without joint complaints. In addition, confirmation of previously reported loci was established on chromosomes 4q, 6p, and 17q. This study indicates that distinct disease loci might be involved in psoriasis etiology for various phenotypes.     

Proximity of an ARS consensus sequence to a replication origin of pea (Pisum sativum)

The replication origin (ori-r9) of the 9.0 kb rDNA repeats of pea (Pisum sativum, cv. Alaska) was cloned and found to reside in a 1.5 kb fragment of the non-transcribed spacer region located between the 25 S and 18 S genes. Labeled rDNA rich in replication forks, from cells positioned at the G₁/S phase boundary, was used to map ori-r9 by hybridization procedures. Ori-r9 is in a 210-base fragment that is 1.6 kb from the 5 end of the 18 S gene and about 1.5 kb from the 3 end of the 25 S gene. The same procedures, using labeled synthetic ARS consensus sequence as a probe, showed than an ARS consensus sequence is located 3 to ori-r9 in a 710-base fragment. An ARS consensus sequence is, therefore, adjacent to ori-r9 but not coincidental with it.     

Molecular approach to the phylogenetics of sea spiders (Arthropoda: Pycnogonida) using partial sequences of nuclear ribosomal DNA

The phylogenetic relationships among major evolutionary lineages of the sea spiders (subphylum Pycnogonida) were investigated using partial sequences of nuclear DNA, 18S, and 28S ribosomal genes. Topological differences were obtained with separate analyses of 18S and 28S, and estimates of phylogeny were found to be significantly different between a combined molecular data set (18S and 28S) and a subset of a morphological data matrix analyzed elsewhere. Colossendeidae played a major role in the conflicts; it was closely related to Callipallenidae or Nymphonidae with 18S or 28S, respectively, but related to Ammotheidae according to morphological characters. Austrodecidae was defined as a basal taxon for Pycnogonida by these molecular data. The 18S sequences were surprisingly conserved among pycnogonid taxa, suggesting either an unusual case of slow evolution of the gene, or an unexpected recent divergence of pycnogonid lineages. Notwithstanding difficulties such as non-optimal taxon sampling, this is the first attempt to reconstruct the pycnogonid phylogeny based on DNA. Continued studies of sequences and other characters should increase the reliability of the analyses and our understanding of the phylogenetics of sea spiders.     

High-rate partial nitritation using porous poly(vinyl alcohol) sponge

Poly(vinyl alcohol) (PVA) has been utilized as a support material for the immobilization of nitrifying bacteria without the comprehensive survey of partial nitritation. In the present study, the activities of nitrifiers and the maximum nitrogen conversion rate of partial nitritation with PVA sponge-cubes were specified according to different conditions. The selective enrichment of ammonia-oxidizing bacteria (AOB) on PVA sponge-cubes was achieved by the competition between AOB and nitrite-oxidizing bacteria for dissolved oxygen. The efficiency of ammonia oxidation was proportional to the concentration of HCO₃ ^? with the molar ratio of HCO₃ ^?-C/NH₄ ⁺-N = 1.91 and a half of the ratio was applied to the further experiments to ensure stable partial nitritation. The maximum nitrogen conversion rate of partial nitritation was dependent on the volume, not the size of sponge-cubes. The partial nitritation showed the superior rate performance of 3.09 kg N/m³ day with the packing ratio of 32 % of 5 × 5 × 5 mm³ PVA sponge-cubes.     

Single-strand-binding factor(s) which interact with ARS1 of Saccharomyces cerevisiae

Since plasmids containing autonomously replicating sequence(s) (ARS) can transform Saccharomyces cerevisiae cells at high frequency, ARS are considered to be the replication origins of chromosomes. To study the mechanism of initiation of eukaryotic chromosomal replication, we examined protein factors which interact with the ARS1 region located near the centromere of chromosome IV in S. cerevisiae. Using the gel-shift assay, we found protein factors which bound to a single-stranded, 97-bp fragment of the ARS1 region containing the core consensus. Competition experiments with various oligodeoxyribonucleotides (oligos) suggest that a site recognized by the factor(s) was within the element containing the core consensus and adjacent close matches to the core consensus of the minus strand. Indeed, when the oligo containing the minus strand of this element was used as a probe, two oligo-protein complexes were detected. Mutations in the core consensus reduced these binding activities. When the plus-strand oligo of the same region was used as a probe, a retarded band was also detected, but with less specific binding. Considering the fact that the core consensus and close matches to the core consensus are important for ARS function, these results imply that the protein factors detected in this experiment may participate in DNA replication.     

Prediction of clinical outcome with microarray data: a partial least squares discriminant analysis (PLS-DA) approach

Partial least squares discriminant analysis (PLS-DA) is a partial least squares regression of a set Y of binary variables describing the categories of a categorical variable on a set X of predictor variables. It is a compromise between the usual discriminant analysis and a discriminant analysis on the significant principal components of the predictor variables. This technique is specially suited to deal with a much larger number of predictors than observations and with multicollineality, two of the main problems encountered when analysing microarray expression data. We explore the performance of PLS-DA with published data from breast cancer (Perou et al. 2000). Several such analyses were carried out: (1) before vs after chemotherapy treatment, (2) estrogen receptor positive vs negative tumours, and (3) tumour classification. We found that the performance of PLS-DA was extremely satisfactory in all cases and that the discriminant cDNA clones often had a sound biological interpretation. We conclude that PLS-DA is a powerful yet simple tool for analysing microarray data.     

Isolation and partial characterization of apolipoprotein (a) from human lipoprotein (a)

A procedure was developed for the dissociation of apolipoprotein (a) (apo (a)) from pure human lipoprotein (a) (Lp(a)) prepared by density gradient ultracentrifugation and gel filtration. Lp(a) was ultracentrifuged through a layer of saline which was adjusted to a density of 1.182 g/mL and contained 30 mM dithiothreitol (50 mM) and phenylmethylsulfonyl fluoride (1.25 mM). Following centrifugation, the lipid and apolipoprotein B (apo B) were recovered as a lipoprotein (Lp(a) B) in the supernatant fraction, while the apo (a) was recovered as a lipid-poor protein pellet. An investigation of the supernatant lipoprotein by electron microscopy and compositional analysis revealed that it was similar in size and composition to low density lipoprotein (LDL) isolated from the same density range and contained apo B100 with an amino acid and carbohydrate composition which was similar to apo B from LDL. Estimates of the apparent molecular weight of the apo (a) varied amongst individuals but was always greater than apo B100 (congruent to 450,000). The amino acid composition of apo (a), which was very distinct from apo B, was characterized by a higher content of serine, threonine, proline, and tyrosine, but lower amounts of isoleucine, phenylalanine, and lysine when compared with apo B of Lp(a) or LDL. The apo (a) contained a much higher proportion of carbohydrate, in particular N-acetylgalactosamine, galactose, and N-acetylneuraminic acid (which were three- to six-fold higher) than the apo B of Lp(a). It is concluded that apo (a) is distinct from other apolipoproteins owing to its low avidity for lipid and the nature of the interaction with apo B. Lp(a) consists of an LDL-like particle with a carbohydrate-rich apo (a) attached to the surface of apo B.     

Gene network homology in prokaryotes using a similarity search approach: queries of quorum sensing signal transduction

Bacterial cell-cell communication is mediated by small signaling molecules known as autoinducers. Importantly, autoinducer-2 (AI-2) is synthesized via the enzyme LuxS in over 80 species, some of which mediate their pathogenicity by recognizing and transducing this signal in a cell density dependent manner. AI-2 mediated phenotypes are not well understood however, as the means for signal transduction appears varied among species, while AI-2 synthesis processes appear conserved. Approaches to reveal the recognition pathways of AI-2 will shed light on pathogenicity as we believe recognition of the signal is likely as important, if not more, than the signal synthesis. LMNAST (Local Modular Network Alignment Similarity Tool) uses a local similarity search heuristic to study gene order, generating homology hits for the genomic arrangement of a query gene sequence. We develop and apply this tool for the E. coli lac and LuxS regulated (Lsr) systems. Lsr is of great interest as it mediates AI-2 uptake and processing. Both test searches generated results that were subsequently analyzed through a number of different lenses, each with its own level of granularity, from a binary phylogenetic representation down to trackback plots that preserve genomic organizational information. Through a survey of these results, we demonstrate the identification of orthologs, paralogs, hitchhiking genes, gene loss, gene rearrangement within an operon context, and also horizontal gene transfer (HGT). We found a variety of operon structures that are consistent with our hypothesis that the signal can be perceived and transduced by homologous protein complexes, while their regulation may be key to defining subsequent phenotypic behavior.     

Sequence specific resonance assignment via Multicanonical Monte Carlo search using an ABACUS approach

ABACUS [Grishaev et al. (2005) Proteins 61:36-43] is a novel protocol for automated protein structure determination via NMR. ABACUS starts from molecular fragments defined by unassigned J-coupled spin-systems and involves a Monte Carlo stochastic search in assignment space, probabilistic sequence selection, and assembly of fragments into structures that are used to guide the stochastic search. Here, we report further development of the two main algorithms that increase the flexibility and robustness of the method. Performance of the BACUS [Grishaev and Llinás (2004) J Biomol NMR 28:1-101] algorithm was significantly improved through use of sequential connectivities available from through-bond correlated 3D-NMR experiments, and a new set of likelihood probabilities derived from a database of 56 ultra high resolution X-ray structures. A Multicanonical Monte Carlo procedure, Fragment Monte Carlo (FMC), was developed for sequence-specific assignment of spin-systems. It relies on an enhanced assignment sampling and provides the uncertainty of assignments in a quantitative manner. The efficiency of the protocol was validated on data from four proteins of between 68-116 residues, yielding 100% accuracy in sequence specific assignment of backbone and side chain resonances.     

Phylogeny of Eunicida (Annelida) and exploring data congruence using a partition addition bootstrap alteration (PABA) approach

Even though relationships within Annelida are poorly understood, Eunicida is one of only a few major annelid lineages well supported by morphology. The seven recognized eunicid families possess sclerotized jaws that include mandibles and a maxillary apparatus. The maxillary apparatuses vary in shape and number of elements, and three main types are recognized in extant taxa: ctenognath, labidognath, and prionognath. Ctenognath jaws are usually considered to represent the plesiomorphic state of Eunicida, whereas taxa with labidognath and prionognath are thought to form a derived monophyletic assemblage. However, this hypothesis has never been tested in a statistical framework even though it holds considerable importance for understanding annelid phylogeny and possibly lophotrochozoan evolution because Eunicida has the best annelid fossil record. Therefore, we used maximum likelihood and Bayesian inference approaches to reconstruct Eunicida phylogeny using sequence data from nuclear 18S and 28S rDNA genes and mitochondrial 16S rDNA and cytochrome c oxidase subunit I genes. Additionally, we conducted three different tests to investigate suitability of combining data sets. Incongruence length difference (ILD) and Shimodaira-Hasegawa (SH) test comparisons of resultant trees under different data partitions have been widely used previously but do not give a good indication as to which nodes may be causing the conflict. Thus, we developed a partition addition bootstrap alteration (PABA) approach that evaluates congruence or conflict for any given node by determining how bootstrap scores are altered when different data partitions are added. PABA shows the contribution of each partition to the phylogeny obtained in the combined analysis. Generally, the ILD test performed worse than the other approaches in detecting incongruence. Both PABA and the SH approach indicated the 28S and COI data sets add conflicting signal, but PABA is more informative for elucidating which data partition may be misleading at a given node. All our analyses indicate that the monophyly of the labidognath/prionognath taxa and even a labidognath clade (i.e., a "Eunicidae"/Onuphidae/Lumbrineridae clade) is significantly rejected. We show that the definition of both the labidognath and ctenognath jaw type does not address adequately the variation within Eunicida and thus misleads our current evolutionary understanding. Based on the presented results a symmetric maxillary apparatus with a carrier and four to six maxillae is most likely the plesiomorphic condition for Eunicida. [COI; conflicting data; fossil record; ILD; Jaw Evolution; molecular phylogeny; rDNA; SH test.].     

Molecular cloning and characterization of ARS elements from the mud loach (Misgurnus mizolepis)

Autonomously replicating sequences (ARSs) are thought to occur within, or adjacent to, the matrix attachment regions (MARs). To identify fish ARSs, MARs of the mud loach fish were obtained from nuclear matrices using a modified LIS method. These DNA fragments were screened for their ability to act as ARSs by being cloned into the ARS cloning vector, pURY19, and transformed into Saccharomyces cerevisiae. Sixteen ARSs were isolated, most of which were more efficient in transformation than the positive control vector, pURY19-2 microm, which contained the 2 microm circle origin of yeast. In particular, one clone, pURY19-ARS223, was 18 times more efficient in back-transforming E. coli than the positive control vector. Therefore, ARS223, which has strong ARS activity in yeast, could be a good candidate for inclusion in expression vehicles that are used to transfect fish cell lines or embryos. A DNA sequence analysis showed that the essential ARS elements contain potential ARS consensus sequences, and are predicted to have hairpin loop structures, or curved or kinked DNA. In addition, the MAR-Finder program suggested that ARSs also contain MAR motifs. These include AT tracts, ORI patterns, kinked DNA, ATC tracts, and Topoisomerase II consensus sequences. The in vitro matrix binding assay confirmed that all of the cloned ARSs could associate with the nuclear matrix. This indicates that ARSs elements may be located in or near the MARs. This is the first study that has identified and characterized ARSs in fish.     

Surrogate variable analysis using partial least squares (SVA-PLS) in gene expression studies

MOTIVATION: In a typical gene expression profiling study, our prime objective is to identify the genes that are differentially expressed between the samples from two different tissue types. Commonly, standard analysis of variance (ANOVA)/regression is implemented to identify the relative effects of these genes over the two types of samples from their respective arrays of expression levels. But, this technique becomes fundamentally flawed when there are unaccounted sources of variability in these arrays (latent variables attributable to different biological, environmental or other factors relevant in the context). These factors distort the true picture of differential gene expression between the two tissue types and introduce spurious signals of expression heterogeneity. As a result, many genes which are actually differentially expressed are not detected, whereas many others are falsely identified as positives. Moreover, these distortions can be different for different genes. Thus, it is also not possible to get rid of these variations by simple array normalizations. This both-way error can lead to a serious loss in sensitivity and specificity, thereby causing a severe inefficiency in the underlying multiple testing problem. In this work, we attempt to identify the hidden effects of the underlying latent factors in a gene expression profiling study by partial least squares (PLS) and apply ANCOVA technique with the PLS-identified signatures of these hidden effects as covariates, in order to identify the genes that are truly differentially expressed between the two concerned tissue types. RESULTS: We compare the performance of our method SVA-PLS with standard ANOVA and a relatively recent technique of surrogate variable analysis (SVA), on a wide variety of simulation settings (incorporating different effects of the hidden variable, under situations with varying signal intensities and gene groupings). In all settings, our method yields the highest sensitivity while maintaining relatively reasonable values for the specificity, false discovery rate and false non-discovery rate. Application of our method to gene expression profiling for acute megakaryoblastic leukemia shows that our method detects an additional six genes, that are missed by both the standard ANOVA method as well as SVA, but may be relevant to this disease, as can be seen from mining the existing literature.     

The Marker State Space (MSS) Method for Classifying Clinical Samples

The development of accurate clinical biomarkers has been challenging in part due to the diversity between patients and diseases. One approach to account for the diversity is to use multiple markers to classify patients, based on the concept that each individual marker contributes information from its respective subclass of patients. Here we present a new strategy for developing biomarker panels that accounts for completely distinct patient subclasses. Marker State Space (MSS) defines “marker states” based on all possible patterns of high and low values among a panel of markers. Each marker state is defined as either a case state or a control state, and a sample is classified as case or control based on the state it occupies. MSS was used to define multi-marker panels that were robust in cross validation and training-set/test-set analyses and that yielded similar classification accuracy to several other classification algorithms. A three-marker panel for discriminating pancreatic cancer patients from control subjects revealed subclasses of patients based on distinct marker states. MSS provides a straightforward approach for modeling highly divergent subclasses of patients, which may be adaptable for diverse applications.