FOLFCIS Treatment and Genomic Correlates of Response in Advanced Anal Squamous Cell Cancer

Background

Treatment of advanced anal squamous cell cancer (SCC) is usually with the combination of cisplatin and 5-fluorouracil, which is associated with heterogeneous responses across patients and significant toxicity. We examined the safety and efficacy of a modified schedule, FOLFCIS (leucovorin, fluorouracil, and cisplatin), and performed an integrated clinical and genomic analysis of anal SCC.

Phylogenomics with paralogs

Phylogenomics heavily relies on well-curated sequence data sets that comprise, for each gene, exclusively 1:1 orthologos. Paralogs are treated as a dangerous nuisance that has to be detected and removed. We show here that this severe restriction of the data sets is not necessary. Building upon recent advances in mathematical phylogenetics, we demonstrate that gene duplications convey meaningful phylogenetic information and allow the inference of plausible phylogenetic trees, provided orthologs and paralogs can be distinguished with a degree of certainty. Starting from tree-free estimates of orthology, cograph editing can sufficiently reduce the noise to find correct event-annotated gene trees. The information of gene trees can then directly be translated into constraints on the species trees. Although the resolution is very poor for individual gene families, we show that genome-wide data sets are sufficient to generate fully resolved phylogenetic trees, even in the presence of horizontal gene transfer.Molecular phylogenetics is primarily concerned with the reconstruction of evolutionary relationships between species based on sequence information. To this end, alignments of protein or DNA sequences are used, whose evolutionary history is believed to be congruent to that of the respective species. This property can be ensured most easily in the absence of gene duplications and horizontal gene transfer (HGT). Phylogenetic studies judiciously select families of genes that rarely exhibit duplications (such as rRNAs, most ribosomal proteins, and many of the housekeeping enzymes). In phylogenomics, elaborate automatic pipelines such as HaMStR (1), are used to filter genome-wide data sets to at least deplete sequences with detectable paralogs (homologs in the same species).In the presence of gene duplications, however, it becomes necessary to distinguish between the evolutionary history of genes (gene trees) and the evolutionary history of the species (species trees) in which these genes reside. Leaves of a gene tree represent genes. Their inner nodes represent two kinds of evolutionary events, namely the duplication of genes within a genome—giving rise to paralogs—and speciations, in which the ancestral gene complement is transmitted to two daughter lineages. Two genes are (co)orthologous if their last common ancestor in the gene tree represents a speciation event, whereas they are paralogous if their last common ancestor is a duplication event; see refs. 2 and 3 for a more recent discussion on orthology and paralogy relationships. Speciation events, in turn, define the inner vertices of a species tree. However, they depend on both the gene and the species phylogeny, as well as the reconciliation between the two. The latter identifies speciation vertices in the gene tree with a particular speciation event in the species tree and places the gene duplication events on the edges of the species tree. Intriguingly, it is nevertheless possible in practice to distinguish orthologs with acceptable accuracy without constructing either gene or species trees (4). Many tools of this type have become available over the last decade; see refs. 5 and 6 for a recent review. The output of such methods is an estimate Θ of the true orthology relation Θ^?, which can be interpreted as a graph G_Θ whose vertices are genes and whose edges connect estimated (co)orthologs.Recent advances in mathematical phylogenetics suggest that the estimated orthology relation Θ contains information on the structure of the species tree. To make this connection, we combine here three abstract mathematical results that are made precise in Materials and Methods below.

• i)Building upon the theory of symbolic ultrametrics (7), we showed that in the absence of horizontal gene transfer, the orthology relation of each gene family is a cograph (8). Cographs can be generated from the single-vertex graph K₁ by complementation and disjoint union (9). This special structure of cographs imposes very strong constraints that can be used to reduce the noise and inaccuracies of empirical estimates of orthology from pairwise sequence comparison. To this end, the initial estimate of G_Θ is modified to the closest correct orthology relation G_Θ^? in such a way that a minimal number of edges (i.e., orthology assignments) are introduced or removed. This amounts to solving the cograph-editing problem (10, 11).
• ii)It is well known that each cograph is equivalently represented by its cotree (9). The cotree is easily computed for a given cograph. In our context, the cotree of G_Θ^? is an incompletely resolved event-labeled gene tree. That is, in addition to the tree topology, we know for each internal branch point whether it corresponds to a speciation or a duplication event. Even though adjacent speciations or adjacent duplications cannot be resolved, the tree faithfully encodes the relative order of any pair of duplication and speciation (8). In the presence of horizontal gene transfer, G_Θ may deviate from the structural requirements of a cograph. Still, the situation can be described in terms of edge-colored graphs whose subgraphs are cographs (7, 8), so that the cograph structure remains an acceptable approximation.
• iii)Every triple (rooted binary tree on three leaves) in the cotree that has leaves from three species and is rooted in a speciation event also appears in the underlying species tree (12). Thus, the estimated orthology relation, after editing to a cograph and conversion to the equivalent event-labeled gene tree, provides much information on the species tree. This result allows us to collect, from the cotrees for each gene family, partial information on the underlying species tree. Interestingly, only gene families that harbor duplications, and thus have a nontrivial cotree, are informative. If no paralogs exist, then the orthology relation G_Θ is a clique (i.e., every family member is orthologous to every other family member) and the corresponding cotree is completely unresolved, and hence contains no triple. On the other hand, full resolution of the species tree is guaranteed if at least one duplication event between any two adjacent speciations is observable. The achievable resolution therefore depends on the frequency of gene duplications and the number of gene families.

Despite the variance reduction due to cograph editing, noise in the data, as well as the occasional introduction of contradictory triples as a consequence of horizontal gene transfer, is unavoidable. The species triples collected from the individual gene families thus will not always be congruent. A conceptually elegant way to deal with such potentially conflicting information is provided by the theory of supertrees in the form of the largest set of consistent triples (13, 14). The data will not always contain a sufficient set of duplication events to achieve full resolution. To this end, we consider trees with the property that the contraction of any edge leads to the loss of an input triple. There may be exponentially many alternative trees of this type. They can be listed efficiently using Semple’s algorithms (15). To reduce the solution space further, we search for a least resolved tree in the sense of ref. 16, i.e., a tree that has the minimum number of inner vertices. It constitutes one of the best estimates of the phylogeny without pretending a higher resolution than actually supported by the data. In SI Appendix, we discuss alternative choices.The mathematical reasoning summarized above, outlined in Materials and Methods, and presented in full detail in SI Appendix, directly translates into a computational workflow, Fig. 1. It entails three NP-hard combinatorial optimization problems: cograph editing (11), maximal consistent triple set (17–19), and least resolved supertree (16). We show here that they are nevertheless tractable in practice by formulating them as Integer Linear Programs (ILP) that can be solved for both artificial benchmark data sets and real-life data sets, comprising genome-scale protein sets for dozens of species, even in the presence of horizontal gene transfer.Open in a separate windowFig. 1.Outline of the computational framework. Starting from an estimated orthology relation Θ, its graph representation G_Θ is edited to obtain the closest cograph G_Θ^*, which, in turn, is equivalent to a (not necessarily fully resolved) gene tree T and an event labeling t. From (T, t), we extract the set ?? of all relevant species triples. As the triple set ?? need not be consistent, we compute the maximal consistent subset ??^? of ??. Finally, we construct a least resolved species tree from ??^?.     

Ultrastructural and microbiological analysis of the dentin layers affected by caries lesions in primary molars treated by minimal intervention

PURPOSE: The purpose of this in vivo study of primary teeth was to analyze the ultrastructure and microbiology of dentin layers affected by caries lesions before and after restorations with resin-modified glass ionomer. METHODS: Samples of carious dentin from primary teeth removed prior to restoration placement (baseline-0 day) were compared with samples taken after 30 and 60 days. Dentin from 8 primary molars was analyzed by scanning electron microscopy (SEM) and dentin from 22 primary molars was examined microbiologically to compare bacteria (total of viable counts, Streptococcus spp, Streptococcus mutans, Lactobacillus spp, and Actinomyces spp) before and after treatment (30 and 60 days). RESULTS: Baseline caries samples had enlarged dentinal tubules with bacteriol invasion. SEM samples after treatment suggest better tissue organization, with more compact collagen fibers arrangement and narrower dentinal tubules. The number of bacteria decreased in all samples at both 30 (98%) and 60 (96%) days, with all bacteria species showing similar trends. CONCLUSIONS: The minimal intervention approach is very effective to promote beneficial changes in the lesion environment and favorable conditions for the healing process in primary teeth.     

The fossilized birth–death process for coherent calibration of divergence-time estimates

Time-calibrated species phylogenies are critical for addressing a wide range of questions in evolutionary biology, such as those that elucidate historical biogeography or uncover patterns of coevolution and diversification. Because molecular sequence data are not informative on absolute time, external data—most commonly, fossil age estimates—are required to calibrate estimates of species divergence dates. For Bayesian divergence time methods, the common practice for calibration using fossil information involves placing arbitrarily chosen parametric distributions on internal nodes, often disregarding most of the information in the fossil record. We introduce the “fossilized birth–death” (FBD) process—a model for calibrating divergence time estimates in a Bayesian framework, explicitly acknowledging that extant species and fossils are part of the same macroevolutionary process. Under this model, absolute node age estimates are calibrated by a single diversification model and arbitrary calibration densities are not necessary. Moreover, the FBD model allows for inclusion of all available fossils. We performed analyses of simulated data and show that node age estimation under the FBD model results in robust and accurate estimates of species divergence times with realistic measures of statistical uncertainty, overcoming major limitations of standard divergence time estimation methods. We used this model to estimate the speciation times for a dataset composed of all living bears, indicating that the genus Ursus diversified in the Late Miocene to Middle Pliocene.A phylogenetic analysis of species has two goals: to infer the evolutionary relationships and the amount of divergence among species. Preferably, divergence is estimated in units proportional to time, thus revealing the times at which speciation events occurred. Once orthologous DNA sequences from the species have been aligned, both goals can be accomplished by assuming that nucleotide substitutions occur at the same rate in all lineages [the “molecular clock” assumption (1)] and that the time of at least one speciation event on the tree is known, i.e., one speciation event acts to “calibrate” the substitution rate.The goal of reconstructing rooted, time-calibrated phylogenies is complicated by substitution rates changing over the tree and by the difficulty of determining the date of any speciation event. Substitution rate variation among lineages is pervasive and has been accommodated in several ways. The most widely used method to account for rate heterogeneity is to assign an independent parameter to each branch of the tree. Branch lengths, then, are the product of substitution rate and time, and usually measured in units of expected number of substitutions per site. This solution allows estimation of the tree topology—which is informative about interspecies relationships—but does not attempt to estimate the rate and time separately. Thus, under this “unconstrained” parameterization, molecular sequence data allow inference of phylogenetic relationships and genetic distances among species, but the timing of speciation events is confounded in the branch-length parameter (2–4). Under a “relaxed-clock” model, substitution rates change over the tree in a constrained manner, thus separating the rate and time parameters associated with each branch and allowing inference of lineage divergence times. A considerable amount of effort has been directed at modeling lineage-specific substitution rate variation, with many different relaxed-clock models described in the literature (5–19). When such models are coupled with a model on the distribution of speciation events over time [e.g., the Yule model (20) or birth–death process (21)], molecular sequence data can inform the relative rates and node ages in a phylogenetic analysis.Estimates of branch lengths in units of absolute time (e.g., millions of years) are required for studies investigating comparative or biogeographical questions (e.g., refs. 22, 23). However, because commonly used diversification priors are imprecise on node ages, external information is required to infer the absolute timing of speciation events. Typically, a rooted time tree is calibrated by constraining the ages of a set of internal nodes. Age constraints may be derived from several sources, but the most common and reliable source of calibration information is the fossil record (24, 25). Despite the prevalence of these data in divergence time analyses, the problem of properly calibrating a phylogenetic tree has received less consideration than the problem of accommodating rate variation. Moreover, various factors may lead to substantial errors in parameter estimates (26–31). When estimating node ages, a calibration node must be identified for each fossil. For a given fossil, the calibration node is the node in the extant species tree that represents the most recent common ancestor (MRCA) of the fossil and a set of extant species. Based on the fossil, the calibration node’s age is estimated on an absolute timescale. Thus, fossil data typically can provide valid minimum-age constraints only on these nodes (24, 27), and erroneous conclusions may result if the calibration node is not specified properly (26).Bayesian inference methods are well adapted to accommodating uncertainty in calibration times by assuming that the age of the calibrated node is a random variable drawn from some parametric probability distribution (10, 14, 29, 31–35). Although this Bayesian approach properly propagates uncertainty in the calibration times through the analysis (reflected in the credible intervals on uncalibrated node ages), two problems remain unresolved.First, these approaches, as they commonly are applied, induce a probability distribution on the age of each calibrated node that comes from both the node-specific calibration prior and the tree-wide prior on node ages, leading to an incoherence in the model of branching times on the tree (35, 36). Typically, a birth–death process of cladogenesis is considered as the generating model for the tree and speciation times (20, 21, 37–40), serving as the tree-wide prior distribution on branch times in a Bayesian analysis. The speciation events acting as calibrations then are considered to be drawn from an additional, unrelated probability distribution intended to model uncertainty in the calibration time. This procedure results in overlaying two prior distributions for a calibration node: one from the tree prior and one from the calibration density (35, 41). Importantly, this incoherence is avoided by partitioning the nodes and applying a birth–death process to uncalibrated nodes conditioned on the calibrated nodes (32), although many divergence time methods do not use this approach. Nevertheless, a single model that acts as a prior on the speciation times for both calibrated and uncalibrated nodes is a better representation of the lineage diversification process and preferable as a prior on branching times when using fossil data.Second, the probability distributions used to model uncertainty in calibration times are poorly motivated. The standard practice in Bayesian divergence time methods is to model uncertainty in calibrated node ages by using simple probability distributions, such as the uniform, log-normal, gamma, or exponential distributions (29). When offset by a minimum age, these “calibration densities” (35) simply seek to characterize the age of the node with respect to its descendant fossil. However, the selection and parameterization of calibration priors rarely are informed by any biological process or knowledge of the fossil record (except see refs. 42–44). A probability model that acts as a fossil calibration prior should have parameters relevant to the preservation history of the group, such as the rate at which fossils occur in the rock record, a task that likely is difficult for most groups without an abundant fossil record (43, 45). Consequently, most biologists are faced with the challenge of choosing and parameterizing calibration densities without an explicit way to describe their prior knowledge about the calibration time. Thus, calibration priors often are specified based on arbitrary criteria or ad hoc validation methods (46), and ultimately, this may lead to arbitrary or ad hoc estimates of divergence times.We provide an alternative method for calibrating phylogenies with fossils. Because fossils and molecular sequences from extant species are different observations of the same diversification process, we use an explicit speciation–extinction–fossilization model to describe the distribution of speciation times and recovered fossils. This model—the fossilized birth–death (FBD) process—acts as a prior for divergence time dating. The parameters of the model—the speciation rate, extinction rate, fossil recovery rate, and proportion of sampled extant species—interact to inform the amount of uncertainty for every speciation event on the tree. These four parameters are the only quantities requiring prior assumptions, compared with assuming separate calibration densities for each fossil. Analyses of simulated data under the FBD model result in reliable estimates of absolute divergence times with realistic measures of statistical uncertainty. Moreover, node age estimates are robust to several biased sampling strategies of fossils and extant species—strategies that may be common practice or artifacts of fossil preservation but heavily violate assumptions of the model.     

Comparison of Porcine Epidemic Diarrhea Viruses from Germany and the United States, 2014

Since 2013, highly virulent porcine epidemic diarrhea virus has caused considerable economic losses in the United States. To determine the relation of US strains to those recently causing disease in Germany, we compared genomes and found that the strain from Germany is closely related to variants in the United States.