Large-scale genomic analyses with machine learning uncover predictive patterns associated with fungal phytopathogenic lifestyles and traits.

Invasive plant pathogenic fungi have a global impact, with devastating economic and environmental effects on crops and forests. Biosurveillance, a critical component of threat mitigation, requires risk prediction based on fungal lifestyles and traits. Recent studies have revealed distinct genomic patterns associated with specific groups of plant pathogenic fungi. We sought to establish whether these phytopathogenic genomic patterns hold across diverse taxonomic and ecological groups from the Ascomycota and Basidiomycota, and furthermore, if those patterns can be used in a predictive capacity for biosurveillance. Using a supervised machine learning approach that integrates phylogenetic and genomic data, we analyzed 387 fungal genomes to test a proof-of-concept for the use of genomic signatures in predicting fungal phytopathogenic lifestyles and traits during biosurveillance activities. Our machine learning feature sets were derived from genome annotation data of carbohydrate-active enzymes (CAZymes), peptidases, secondary metabolite clusters (SMCs), transporters, and transcription factors. We found that machine learning could successfully predict fungal lifestyles and traits across taxonomic groups, with the best predictive performance coming from feature sets comprising CAZyme, peptidase, and SMC data. While phylogeny was an important component in most predictions, the inclusion of genomic data improved prediction performance for every lifestyle and trait tested. Plant pathogenicity was one of the best-predicted traits, showing the promise of predictive genomics for biosurveillance applications. Furthermore, our machine learning approach revealed expansions in the number of genes from specific CAZyme and peptidase families in the genomes of plant pathogens compared to non-phytopathogenic genomes (saprotrophs, endo- and ectomycorrhizal fungi). Such genomic feature profiles give insight into the evolution of fungal phytopathogenicity and could be useful to predict the risks of unknown fungi in future biosurveillance activities.

Motif discovery in heterogeneous sequence data.

This paper introduces the first integrated algorithm designed to discover novel motifs in heterogeneous sequence data, which is comprised of coregulated genes from a single genome together with the orthologs of these genes from other genomes. Results are presented for regulons in yeasts, worms, and mammals.

Comparative analyses of multi-species sequences from targeted genomic regions.

The systematic comparison of genomic sequences from different organisms represents a central focus of contemporary genome analysis. Comparative analyses of vertebrate sequences can identify coding and conserved non-coding regions, including regulatory elements, and provide insight into the forces that have rendered modern-day genomes. As a complement to whole-genome sequencing efforts, we are sequencing and comparing targeted genomic regions in multiple, evolutionarily diverse vertebrates. Here we report the generation and analysis of over 12 megabases (Mb) of sequence from 12 species, all derived from the genomic region orthologous to a segment of about 1.8 Mb on human chromosome 7 containing ten genes, including the gene mutated in cystic fibrosis. These sequences show conservation reflecting both functional constraints and the neutral mutational events that shaped this genomic region. In particular, we identify substantial numbers of conserved non-coding segments beyond those previously identified experimentally, most of which are not detectable by pair-wise sequence comparisons alone. Analysis of transposable element insertions highlights the variation in genome dynamics among these species and confirms the placement of rodents as a sister group to the primates.

Separating real motifs from their artifacts.

The typical output of many computational methods to identify binding sites is a long list of motifs containing some real motifs (those most likely to correspond to the actual binding sites) along with a large number of random variations of these. We present a statistical method to separate real motifs from their artifacts. This produces a short list of high quality motifs that is sufficient to explain the over-representation of all motifs in the given sequences. Using synthetic data sets, we show that the output of our method is very accurate. On various sets of upstream sequences in S. cerevisiae, our program identifies several known binding sites, as well as a number of significant novel motifs.

Is vaginal birth after cesarean safe? Experience at a community hospital.

OBJECTIVE: We sought to evaluate the effectiveness and safety of promoting a trial of labor after prior cesarean birth in a community hospital. STUDY DESIGN: A 4-year prospective cohort study was conducted of all patients who had prior cesarean births (N = 1481). A comparison of outcomes was performed between those who elected repeat cesarean delivery (n = 727) and those who attempted a trial of labor after previous cesarean(s) (n = 754). RESULTS: We found that the vaginal birth after cesarean attempt rate was 50.9% and declined significantly during the last 2 years of the study. The elective repeat cesarean rate was 49.1% and increased significantly during the last 2 years of the study. In addition, we found that neonatal outcomes were similar, with the exception of 2 neonatal deaths caused by uterine rupture. Twelve uterine ruptures occurred (1.6%), and 11 of the 12 ruptures involved either induction or augmentation of labor, or both. CONCLUSIONS: A trial of vaginal birth after cesarean is safe provided that induction of labor is not used. The uterine rupture rate of 1.6% is higher than reported in the literature; this may reflect underreporting by community hospitals.

Dimethyl sulfoxide affects the selection of splice sites.

Depending on the cell lines and cell types, dimethyl sulfoxide (Me2SO) can induce or block cell differentiation and apoptosis. Although Me2SO treatment alters many levels of gene expression, the molecular processes that are directly affected by Me2SO have not been clearly identified. Here, we report that Me2SO affects splice site selection on model pre-mRNAs incubated in a nuclear extract prepared from HeLa cells. A shift toward the proximal pair of splice sites was observed on pre-mRNAs carrying competing 5'-splice sites or competing 3'-splice sites. Because the activity of recombinant hnRNP A1 protein was similar when added to extracts containing or lacking Me2SO, the activity of endogenous A1 proteins is probably not affected by Me2SO. Notably, in a manner reminiscent of SR proteins, Me2SO activated splicing in a HeLa S100 extract. Moreover, the activity of recombinant SR proteins in splice site selection in vitro was improved by Me2SO. Polar solvents like DMF and formamide similarly modulated splice site selection in vitro but formamide did not activate a HeLa S100 extract. We propose that Me2SO improves ionic interactions between splicing factors that contain RS-domains. The direct impact of Me2SO on alternative splicing may explain, at least in part, the different and sometimes opposite effects of Me2SO on cell differentiation and apoptosis.

An exact algorithm to identify motifs in orthologous sequences from multiple species.

The identification of sequence motifs is a fundamental method for suggesting good candidates for biologically functional regions such as promoters, splice sites, binding sites, etc. We investigate the following approach to identifying motifs: given a collection of orthologous sequences from multiple species related by a known phylogenetic tree, search for motifs that are well conserved (according to a parsimony measure) in the species. We present an exact algorithm for solving this problem. We then discuss experimental results on finding promoters of the rbcS gene for a family of 10 plants, on finding promoters of the adh gene for 12 Drosophila species, and on finding promoters of several chloroplast encoded genes.

Phylogenetic invariants for genome rearrangements.

We review the combinatorial optimization problems in calculating edit distances between genomes and phylogenetic inference based on minimizing gene order changes. With a view to avoiding the computational cost and the "long branches attract" artifact of some tree-building methods, we explore the probabilization of genome rearrangement models prior to developing a methodology based on branch-length invariants. We characterize probabilistically the evolution of the structure of the gene adjacency set for reversals on unsigned circular genomes and, using a nontrivial recurrence relation, reversals on signed genomes. Concepts from the theory of invariants developed for the phylogenetics of homologous gene sequences can be used to derive a complete set of linear invariants for unsigned reversals, as well as for a mixed rearrangement model for signed genomes, though not for pure transposition or pure signed reversal models. The invariants are based on an extended Jukes-Cantor semigroup. We illustrate the use of these invariants to relate mitochondrial genomes from a number of invertebrate animals.

Gene order breakpoint evidence in animal mitochondrial phylogeny.

Multiple genome rearrangement methodology facilitates the inference of animal phylogeny from gene orders on the mitochondrial genome. The breakpoint distance is preferable to other, highly correlated but computationally more difficult, genomic distances when applied to these data. A number of theories of metazoan evolution are compared to phylogenies reconstructed by ancestral genome optimization, using a minimal total breakpoints criterion. The notion of unambiguously reconstructed segments is introduced as a way of extracting the invariant aspects of multiple solutions for a given ancestral genome; this enables a detailed reconstruction of the evolution of non-tRNA mitochondrial gene order.

Modulation of exon skipping by high-affinity hnRNP A1-binding sites and by intron elements that repress splice site utilization.

The RNA-binding protein hnRNP A1 is a splicing regulator produced by exclusion of alternative exon 7B from the A1 pre-mRNA. Each intron flanking exon 7B contains a high-affinity A1-binding site. The A1-binding elements promote exon skipping in vivo, activate distal 5' splice site selection in vitro and improve the responsiveness of pre-mRNAs to increases in the concentration of A1. Whereas the glycine-rich C-terminal domain of A1 is not required for binding, it is essential to activate the distal 5' splice site. Because A1 complexes can interact simultaneously with two A1-binding sites, we propose that an interaction between bound A1 proteins facilitates the pairing of distant splice sites. Based on the distribution of putative A1-binding sites in various pre-mRNAs, an A1-mediated change in pre-mRNA conformation may help define the borders of mammalian introns. We also identify an intron element which represses the 3' splice site of exon 7B. The activity of this element is mediated by a factor distinct from A1. Our results suggest that exon 7B skipping results from the concerted action of several intron elements that modulate splice site recognition and pairing.

Multiple genome rearrangement and breakpoint phylogeny.

Multiple alignment of macromolecular sequences generalizes from N = 2 to N > or = 3 the comparison of N sequences which have diverged through the local processes of insertion, deletion and substitution. Gene-order sequences diverge through non-local genome rearrangement processes such as inversion (or reversal) and transposition. In this paper we show which formulations of multiple alignment have counterparts in multiple rearrangement. Based on difficulties inherent in rearrangement edit-distance calculation and interpretation, we argue for the simpler "breakpoint analysis." Consensus-based multiple rearrangement of N > or = 3 orders can be solved exactly through reduction to instances of the Travelling Salesman Problem (TSP). We propose a branch-and-bound solution to TSP particularly suited to these instances. Simulations show how non-uniqueness of the solution is attenuated with increasing numbers of data genomes. Tree-based multiple alignment can be achieved to a great degree of accuracy by decomposing the tree into a number of overlapping 3-stars centered on the non-terminal nodes, and solving the consensus-based problem iteratively for these nodes until convergence. Accuracy improves with very careful initializations at the non-terminal nodes. The degree of non-uniqueness of solutions depends on the position of the node in the tree in terms of path length to the terminal vertices.

Idiopathic thrombocytopenic purpura (ITP): a historical odyssey.

Purpura has been recognized since ancient times and its clinical syndromes were refined by important observations in the sixteenth, seventeenth and eighteenth centuries. It required the development of adequate microscopes in the nineteenth century, however, to recognize the platelet, leading to the recognition of the thrombocytopenic component of ITP. The twentieth century brought recognition of the pathophysiology of the disorder and further defined the clinical states and treatments for ITP. The latter half of the twentieth century has focussed on the autoimmune components of ITP, initially on the humoral immune aspects and more recently on dysregulation of cellular immunity.

The history of idiopathic thrombocytopenic purpura (ITP).

Purpura, initially recognized in ancient times, was defined into clinical syndromes in the 16th, 17th and 18th centuries. With advances in microscope science in the nineteenth century, the platelet was identified, leading to the recognition of the thrombocytopenic component of idiopathic thrombocytopenic purpura (ITP). The 20th century brought recognition of the pathophysiology of the disorder and the clinical states were refined and treatments for ITP developed. The latter half of the 20th century has focused on the autoimmune components of ITP, attempting to develop diagnostic tests, apply new therapies, and elucidate the immune dysregulation associated with, and underlying, the disorder.

Phylogenetic Invariants for Metazoan Mitochondrial Genome Evolution.

The method of phylogenetic invariants was developed to apply to aligned sequence data generated, according to a stochastic substitution model, for N species related through an unknown phylogenetic tree. The invariants are functions of the probabilities of the observable N-tuples, which are identically zero, over all choices of branch length, for some trees. Evaluating the invariants associated with all possible trees, using observed N-tuple frequencies over all sequence positions, enables us to rapidly infer the generating tree. An aspect of evolution at the genomic level much studied recently is the rearrangements of gene order along the chromosome from one species to another. Instead of the substitutions responsible for sequence evolution, we examine the non-local processes responsible for genome rearrangements such as inversion of arbitrarily long segments of chromosomes. By treating the potential adjacency of each possible pair of genes as a position", an appropriate substitution" model can be recognized as governing the rearrangement process, and a probabilistically principled phylogenetic inference can be set up. We calculate the invariants for this process for N=5, and apply them to mitochondrial genome data from coelomate metazoans, showing how they resolve key aspects of branching order.

An intron element modulating 5' splice site selection in the hnRNP A1 pre-mRNA interacts with hnRNP A1.

The hnRNP A1 pre-mRNA is alternatively spliced to yield the A1 and A1b mRNAs, which encode proteins differing in their ability to modulate 5' splice site selection. Sequencing a genomic portion of the murine A1 gene revealed that the intron separating exon 7 and the alternative exon 7B is highly conserved between mouse and human. In vitro splicing assays indicate that a conserved element (CE1) from the central portion of the intron shifts selection toward the distal donor site when positioned in between the 5' splice sites of exon 7 and 7B. In vivo, the CE1 element promotes exon 7B skipping. A 17-nucleotide sequence within CE1 (CE1a) is sufficient to activate the distal 5' splice site. RNase T1 protection/immunoprecipitation assays indicate that hnRNP A1 binds to CE1a, which contains the sequence UAGAGU, a close match to the reported optimal A1 binding site, UAGGGU. Replacing CE1a by different oligonucleotides carrying the sequence UAGAGU or UAGGGU maintains the preference for the distal 5' splice site. In contrast, mutations in the AUGAGU sequence activate the proximal 5' splice site. In support of a direct role of the A1-CE1 interaction in 5'-splice-site selection, we observed that the amplitude of the shift correlates with the efficiency of A1 binding. Whereas addition of SR proteins abrogates the effect of CE1, the presence of CE1 does not modify U1 snRNP binding to competing 5' splice sites, as judged by oligonucleotide-targeted RNase H protection assays. Our results suggest that hnRNP A1 modulates splice site selection on its own pre-mRNA without changing the binding of U1 snRNP to competing 5' splice sites.

A highly stable duplex structure sequesters the 5' splice site region of hnRNP A1 alternative exon 7B.

Exon 7B in the hnRNP A1 pre-mRNA is alternatively spliced to yield A1 and A1(B), two proteins that differ in their ability to modulate 5' splice site selection. Sequencing the murine intron downstream of exon 7B revealed the existence of several regions of similarity to the corresponding human intron. In vitro splicing assays indicate that an 84-nt region (CE6IO) decreases splicing to the proximal 5' splice site in a pre-mRNA carrying the 5' splice sites of exon 7 and 7B. In vivo, the CE6IO element promotes exon 7B skipping in pre-mRNAs expressed from a mini-gene containing the hnRNP A1 alternative splicing unit. Using oligonucleotide-targeted RNase H cleavage assays, we provide support for the existence of highly stable base pairing interactions between CE6IO and the 5' splice site region of exon 7B. Duplex formation occurs in naked pre-mRNA, resists incubation in splicing extracts, and is associated with a reduction in the assembly of U1 snRNP-dependent complexes to the 5' splice site of exon 7B. Our results demonstrate that pre-mRNA secondary structure plays an important role in promoting exon 7B skipping in the A1 pre-mRNA.

Breakpoint Phylogenies.

We describe a number of heuristicsfor inferring the gene orders of the hypothetical ancestral genomes in a fixed phylogeny. The optimization criterion is the minimum number of breakpoints (pairs of genes adjacent in one genome but not the other) in the gene orders of two genomes connected by an edge of the tree, summed over all edges. The key to the method is an exact solution for trees with three leaves (the median problem) based on a reduction to the Traveling Salesman Problem.

Parametric genome rearrangement.

Algorithms inspired by comparative genomics calculate an edit distance between two linear orders based on elementary edit operations such as inversion, transposition and reciprocal translocation. All operations are generally assigned the same weight, simply by default, because no systematic empirical studies exist verifying whether algorithmic outputs involve realistic proportion of each. Nor do we have data on how weights should vary with the length of the inverted or transposed segment of the chromosome. In this paper, we present a rapid algorithm that allows each operation to take on a range of weights, producing an relatively tight upper bound on the distance between single-chromosome genomes, by means of a greedy search with look-ahead. The efficiency of this algorithm allows us to test random genomes for each parameter setting, to detect gene order similarity and to infer the parameter values most appropriate to the phylogenetic domain under study. We apply this method to genome segments in which the same gene order is conserved in Escherichia coli and Bacillus subtilis, as well as to the gene order in human versus Drosophila mitochondrial genomes. In both cases, we conclude that it is most appropriate to assign somewhat more than twice the weight to transpositions and inverted transpositions than to inversions. We also explore segment-length weighting for fungal mitochondrial gene orders.

A longitudinal cephalometric study of the soft tissue profile of short- and long-face syndromes from 7 to 17 years.

The longitudinal growth and development of the soft tissue drape for boys and girls with long and short vertical patterns was examined from age 7 to 17 years. The sample was taken from the Denver Growth Study and consisted of 32 subjects who were selected on the basis of their percentage of lower anterior vertical face height. All subjects were of northern European ancestry, and none had undergone orthodontic treatment. The sexual dimorphism was evident as anticipated for several soft tissue measurements. The boys showed continued growth through age 16 years in contrast to the girls who attained the adult size of the soft tissue integument around 14 years. A significant difference between vertical facial patterns was reported for all soft tissue variables with the exception of the soft tissue thickness at A point and the upper lip height. The boys and girls with long vertical patterns exhibited a thicker and longer soft tissue drape for the most variables when compared with those with short facial patterns. These soft tissue differences are believed to be compensatory mechanisms in long-face subjects, which may attempt to mask the vertical dysplasia, thereby producing a more normal facial profile. Individual growth assessments revealed that the perioral soft tissues follow a pattern similar to that of the mean group patterns. The subjects with long vertical facial patterns experienced their pubertal growth spurt earlier than the short-face subjects. This may have clinical implications in the timing of orthodontic intervention and treatment.