Potential Mechanisms for Cancer Resistance in Elephants and Comparative Cellular Response to DNA Damage in Humans.

IMPORTANCE: Evolutionary medicine may provide insights into human physiology and pathophysiology, including tumor biology. OBJECTIVE: To identify mechanisms for cancer resistance in elephants and compare cellular response to DNA damage among elephants, healthy human controls, and cancer-prone patients with Li-Fraumeni syndrome (LFS). DESIGN, SETTING, AND PARTICIPANTS: A comprehensive survey of necropsy data was performed across 36 mammalian species to validate cancer resistance in large and long-lived organisms, including elephants (n = 644). The African and Asian elephant genomes were analyzed for potential mechanisms of cancer resistance. Peripheral blood lymphocytes from elephants, healthy human controls, and patients with LFS were tested in vitro in the laboratory for DNA damage response. The study included African and Asian elephants (n = 8), patients with LFS (n = 10), and age-matched human controls (n = 11). Human samples were collected at the University of Utah between June 2014 and July 2015. EXPOSURES: Ionizing radiation and doxorubicin. MAIN OUTCOMES AND MEASURES: Cancer mortality across species was calculated and compared by body size and life span. The elephant genome was investigated for alterations in cancer-related genes. DNA repair and apoptosis were compared in elephant vs human peripheral blood lymphocytes. RESULTS: Across mammals, cancer mortality did not increase with body size and/or maximum life span (eg, for rock hyrax, 1% [95% CI, 0%-5%]; African wild dog, 8% [95% CI, 0%-16%]; lion, 2% [95% CI, 0%-7%]). Despite their large body size and long life span, elephants remain cancer resistant, with an estimated cancer mortality of 4.81% (95% CI, 3.14%-6.49%), compared with humans, who have 11% to 25% cancer mortality. While humans have 1 copy (2 alleles) of TP53, African elephants have at least 20 copies (40 alleles), including 19 retrogenes (38 alleles) with evidence of transcriptional activity measured by reverse transcription polymerase chain reaction. In response to DNA damage, elephant lymphocytes underwent p53-mediated apoptosis at higher rates than human lymphocytes proportional to TP53 status (ionizing radiation exposure: patients with LFS, 2.71% [95% CI, 1.93%-3.48%] vs human controls, 7.17% [95% CI, 5.91%-8.44%] vs elephants, 14.64% [95% CI, 10.91%-18.37%]; P < .001; doxorubicin exposure: human controls, 8.10% [95% CI, 6.55%-9.66%] vs elephants, 24.77% [95% CI, 23.0%-26.53%]; P < .001). CONCLUSIONS AND RELEVANCE: Compared with other mammalian species, elephants appeared to have a lower-than-expected rate of cancer, potentially related to multiple copies of TP53. Compared with human cells, elephant cells demonstrated increased apoptotic response following DNA damage. These findings, if replicated, could represent an evolutionary-based approach for understanding mechanisms related to cancer suppression.

Effects of DNA methylation on nucleosome stability.

Methylation of DNA at CpG dinucleotides represents one of the most important epigenetic mechanisms involved in the control of gene expression in vertebrate cells. In this report, we conducted nucleosome reconstitution experiments in conjunction with high-throughput sequencing on 572 KB of human DNA and 668 KB of mouse DNA that was unmethylated or methylated in order to investigate the effects of this epigenetic modification on the positioning and stability of nucleosomes. The results demonstrated that a subset of nucleosomes positioned by nucleotide sequence was sensitive to methylation where the modification increased the affinity of these sequences for the histone octamer. The features that distinguished these nucleosomes from the bulk of the methylation-insensitive nucleosomes were an increase in the frequency of CpG dinucleotides and a unique rotational orientation of CpGs such that their minor grooves tended to face toward the histones in the nucleosome rather than away. These methylation-sensitive nucleosomes were preferentially associated with exons as compared to introns while unmethylated CpG islands near transcription start sites became enriched in nucleosomes upon methylation. The results of this study suggest that the effects of DNA methylation on nucleosome stability in vitro can recapitulate what has been observed in the cell and provide a direct link between DNA methylation and the structure and function of chromatin.

Structure of Penaeus stylirostris densovirus, a shrimp pathogen.

Penaeus stylirostris densovirus (PstDNV), a pathogen of penaeid shrimp, causes significant damage to farmed and wild shrimp populations. In contrast to other parvoviruses, PstDNV probably has only one type of capsid protein that lacks the phospholipase A2 activity that has been implicated as a requirement during parvoviral host cell infection. The structure of recombinant virus-like particles, composed of 60 copies of the 37.5-kDa coat protein, the smallest parvoviral capsid protein reported thus far, was determined to 2.5-Å resolution by X-ray crystallography. The structure represents the first near-atomic resolution structure within the genus Brevidensovirus. The capsid protein has a ß-barrel "jelly roll" motif similar to that found in many icosahedral viruses, including other parvoviruses. The N-terminal portion of the PstDNV coat protein adopts a "domain-swapped" conformation relative to its twofold-related neighbor similar to the insect parvovirus Galleria mellonella densovirus (GmDNV) but in stark contrast to vertebrate parvoviruses. However, most of the surface loops have little structural resemblance to any of the known parvoviral capsid proteins.

Measuring fit of sequence data to phylogenetic model: gain of power using marginal tests.

Testing fit of data to model is fundamentally important to any science, but publications in the field of phylogenetics rarely do this. Such analyses discard fundamental aspects of science as prescribed by Karl Popper. Indeed, not without cause, Popper (Unended quest: an intellectual autobiography. Fontana, London, 1976) once argued that evolutionary biology was unscientific as its hypotheses were untestable. Here we trace developments in assessing fit from Penny et al. (Nature 297:197-200, 1982) to the present. We compare the general log-likelihood ratio (the G or G (2) statistic) statistic between the evolutionary tree model and the multinomial model with that of marginalized tests applied to an alignment (using placental mammal coding sequence data). It is seen that the most general test does not reject the fit of data to model (P approximately 0.5), but the marginalized tests do. Tests on pairwise frequency (F) matrices, strongly (P < 0.001) reject the most general phylogenetic (GTR) models commonly in use. It is also clear (P < 0.01) that the sequences are not stationary in their nucleotide composition. Deviations from stationarity and homogeneity seem to be unevenly distributed amongst taxa; not necessarily those expected from examining other regions of the genome. By marginalizing the 4( t ) patterns of the i.i.d. model to observed and expected parsimony counts, that is, from constant sites, to singletons, to parsimony informative characters of a minimum possible length, then the likelihood ratio test regains power, and it too rejects the evolutionary model with P << 0.001. Given such behavior over relatively recent evolutionary time, readers in general should maintain a healthy skepticism of results, as the scale of the systematic errors in published trees may really be far larger than the analytical methods (e.g., bootstrap) report.

LINE-1 elements: analysis by fluorescence in-situ hybridization and nucleotide sequences.

Long-interspersed nuclear element-1 (LINE-1) is a non-terminal repeat transposon that constitutes a major component of the mammalian genome. LINE-1 has a dynamic evolutionary history characterized by the rise, fall, and replacement of subfamilies. The distribution of LINE-1 elements can be viewed from a chromosomal perspective using fluorescence in-situ hybridization (FISH), as well as at the sequence level. We have designed LINE-1 primers from regions conserved among mouse, rat, rabbit, and human L1, which were able to amplify part of ORF2 from all eutherian (placental) mammals tested thus far. The product generated can be used as a FISH painting probe to examine the genomic distribution of L1 in different species. It can also be cloned and sequenced for phylogenetic analysis. Although FISH patterns resulting from LINE-1 chromosome painting and bioinformatic analyses have shown that this element accumulates in AT-rich regions of the genomes of mouse and human, our PCR amplified LINE-1 probe suggests that this is not a universal phenomenon, and that the patterns displayed in laurasiatherian, afrotherian and xenarthran species are less prominent. The "banding" like distribution of LINE-1 observed in human and mouse, therefore, appears to reflect aspects of genome architecture unique to Euarchontoglires (Supraprimates), the superordinal clade to which they belong. By sequencing the cloned amplicons used for FISH experiments and supplementing these with L1 sequences obtained from public databases, analysis by parsimony, distance-based, maximum likelihood, and "hierarchical Bayesian" or "marginal likelihood" methods provides a powerful adjunct to the FISH data. Using this approach, relatively intact LINE-1 from most placental orders tend to reflect accepted eutherian evolutionary relationships. This suggests that there were often only closely related copies active near branch points in the tree, that inactive copies tended to become extinct quite readily, and that for many orders recently active copies belong to a single lineage of this LINE.

TALI: local alignment of protein structures using backbone torsion angles.

UNLABELLED: Torsion angle alignment (TALI) is a novel approach to local structural motif alignment, based on backbone torsion angles (phi, psi) rather than the more traditional atomic distance matrices. Representation of a protein structure in the form of a sequence of torsion angles enables easy integration of sequence and structural information, and adopts mature techniques in sequence alignment to improve performance and alignment quality. We show that TALI is able to match local structural motifs as well as identify global structural similarity. TALI is also compared to other structure alignment methods such as DALI, CE, and SSM, as well as sequence alignment based on PSI-BLAST; TALI is shown to be equally successful as, or more successful than, these other methods when applied to challenging structural alignments. The inference of the evolutionary tree of class II aminoacyl-tRNA synthetase shows the potential for TALI in estimating protein structural evolution and in identifying structural divergence among homologous structures. AVAILABILITY: http://redcat.cse.sc.edu/index.php/ PROJECT: TALI/.

Robust time estimation reconciles views of the antiquity of placental mammals.

BACKGROUND: Molecular studies have reported divergence times of modern placental orders long before the Cretaceous-Tertiary boundary and far older than paleontological data. However, this discrepancy may not be real, but rather appear because of the violation of implicit assumptions in the estimation procedures, such as non-gradual change of evolutionary rate and failure to correct for convergent evolution. METHODOLOGY/PRINCIPAL FINDINGS: New procedures for divergence-time estimation robust to abrupt changes in the rate of molecular evolution are described. We used a variant of the multidimensional vector space (MVS) procedure to take account of possible convergent evolution. Numerical simulations of abrupt rate change and convergent evolution showed good performance of the new procedures in contrast to current methods. Application to complete mitochondrial genomes identified marked rate accelerations and decelerations, which are not obtained with current methods. The root of placental mammals is estimated to be approximately 18 million years more recent than when assuming a log Brownian motion model. Correcting the pairwise distances for convergent evolution using MVS lowers the age of the root about another 20 million years compared to using standard maximum likelihood tree branch lengths. These two procedures combined revise the root time of placental mammals from around 122 million years ago to close to 84 million years ago. As a result, the estimated distribution of molecular divergence times is broadly consistent with quantitative analysis of the North American fossil record and traditional morphological views. CONCLUSIONS/SIGNIFICANCE: By including the dual effects of abrupt rate change and directly accounting for convergent evolution at the molecular level, these estimates provide congruence between the molecular results, paleontological analyses and morphological expectations. The programs developed here are provided along with sample data that reproduce the results of this study and are especially applicable studies using genome-scale sequence lengths.

Evolutionary history of LINE-1 in the major clades of placental mammals.

BACKGROUND: LINE-1 constitutes an important component of mammalian genomes. It has a dynamic evolutionary history characterized by the rise, fall and replacement of subfamilies. Most data concerning LINE-1 biology and evolution are derived from the human and mouse genomes and are often assumed to hold for all placentals. METHODOLOGY: To examine LINE-1 relationships, sequences from the 3' region of the reverse transcriptase from 21 species (representing 13 orders across Afrotheria, Xenarthra, Supraprimates and Laurasiatheria) were obtained from whole genome sequence assemblies, or by PCR with degenerate primers. These sequences were aligned and analysed. PRINCIPAL FINDINGS: Our analysis reflects accepted placental relationships suggesting mostly lineage-specific LINE-1 families. The data provide clear support for several clades including Glires, Supraprimates, Laurasiatheria, Boreoeutheria, Xenarthra and Afrotheria. Within the afrotherian LINE-1 (AfroLINE) clade, our tree supports Paenungulata, Afroinsectivora and Afroinsectiphillia. Xenarthran LINE-1 (XenaLINE) falls sister to AfroLINE, providing some support for the Atlantogenata (Xenarthra+Afrotheria) hypothesis. SIGNIFICANCE: LINEs and SINEs make up approximately half of all placental genomes, so understanding their dynamics is an essential aspect of comparative genomics. Importantly, a tree of LINE-1 offers a different view of the root, as long edges (branches) such as that to marsupials are shortened and/or broken up. Additionally, a robust phylogeny of diverse LINE-1 is essential in testing that site-specific LINE-1 insertions, often regarded as homoplasy-free phylogenetic markers, are indeed unique and not convergent.

Phylogenetic methodology for detecting protein interactions.

Detecting protein-protein interactions and assigning proteins to functional complexes are key challenges of modern biology. The rise of genomics has lead to evidence that correlated patterns of presence/absence and/or fusing of proteins in any organism suggest these proteins interact. Unfortunately, methods based on such data work best with divergent genomes, whereas major sequencing efforts in vertebrates, for example, are yielding alignments of the same set of proteins sampled from the same set of taxa (species). Using vertebrate mitochondrial genomes to illustrate a novel method, we associate proteins based on vectors of their evolutionary tree edge (branch or internode) lengths. This approach is based on the expectation that molecular coevolution is greatest between proteins that interact in some way. Mitochondrial DNA-encoded proteins are associated into groups largely consistent with the complexes they come from. This association is apparently not due to the tree structure or mutation processes, leaving coevolution as the best explanation. We show that it is important that the tree used to derive the edge-length vector is estimated accurately in terms of both topology and edge lengths. Although more complex substitution models reduce systematic error, they also inflate stochastic error. This makes the use of less complex substitution models preferable in some circumstances. We describe a method to estimate correlations of pairwise evolutionary distances, which adjusts for non-independent correlations due to shared evolutionary history. Associations of proteins based on their edge-length vectors are visualized and assessed using a variety of hierarchical clustering and multidimensional scaling methods. New formula for estimating the fit of data to model, including the average percent standard deviation of distances on least squares trees, are presented. Use of edge-length vectors is compared and contrasted with correlated distance methods, correlated rates methods, and site-specific evidence of coevolution.

Measuring the fit of sequence data to phylogenetic model: allowing for missing data.

It is fundamentally important to assess the fit of data to model in phylogenetic and evolutionary studies. Phylogenetic methods using molecular sequences typically start with a multiple alignment. It is possible to measure the fit of data to model expectations of data, for example, via the likelihood-ratio (G) test or the X(2) test, if all sites in all sequences have an unambiguous residue. However, nearly all alignments of interest contain sites (columns of the alignment) with missing data, that is, ambiguous nucleotides, gaps, or unsequenced regions, which must presently be removed before using the above tests. Unfortunately, this is often either undesirable or impractical, as it will discard much of the data. Here, we show how iterative ML estimators may directly estimate the site-pattern probabilities for columns with missing data, given only standard i.i.d. assumptions. The optimization may use an EM or Newton algorithm, or any other hill-climbing approach. The resulting optimal likelihood under the unconstrained or multinomial model may be compared directly with the likelihood of the data coming from the model (a G statistic). Alternatively the modified observed and the expected frequencies of site patterns may be compared using a X(2) test. The distribution of such statistics is best assessed using appropriate simulations. The new method is applicable to models using codons or paired sites. The methods are also useful with Hadamard conjugations (spectral analysis) and are illustrated with these and with ML evolutionary models that allow site-rate variability.

Molecular systematics of primary reptilian lineages and the tuatara mitochondrial genome.

We provide phylogenetic analyses for primary Reptilia lineages including, for the first time, Sphenodon punctatus (tuatara) using data from whole mitochondrial genomes. Our analyses firmly support a sister relationship between Sphenodon and Squamata, which includes lizards and snakes. Using Sphenodon as an outgroup for select squamates, we found evidence indicating a sister relationship, among our study taxa, between Serpentes (represented by Dinodon) and Varanidae. Our analyses support monophyly of Archosauria, and a sister relationship between turtles and archosaurs. This latter relationship is congruent with a growing set of morphological and molecular analyses placing turtles within crown Diapsida and recognizing them as secondarily anapsid (lacking a skull fenestration). Inclusion of Sphenodon, as the only surviving member of Sphenodontia (with fossils from the mid-Triassic), helps to fill a sampling gap within previous analyses of reptilian phylogeny. We also report a unique configuration for the mitochondrial genome of Sphenodon, including two tRNA(Lys) copies and an absence of ND5, tRNA(His), and tRNA(Thr) genes.

Evaluating placental inter-ordinal phylogenies with novel sequences including RAG1, gamma-fibrinogen, ND6, and mt-tRNA, plus MCMC-driven nucleotide, amino acid, and codon models.

It is essential to test a priori scientific hypotheses with independent data, not least to partly negate factors such as gene-specific base composition biases misleading our models. Seven new gene segments and sequences plus Bayesian likelihood phylogenetic methods were used to compare and test five recent placental phylogenies. These five phylogenies are similar to each other, yet quite different from Fthose of previously proposed trees, and span Waddell et al. [Syst. Biol. 48 (1999) 1] to Murphy et al. [Science 294 (2001b) 2348]. Trees for RAG1, gamma-fibrinogen, ND6, mt-tRNA, mt-RNA, c-MYC, epsilon -globin, and GHR are significantly congruent with the four main groups of mammals common to the five phylogenies, i.e., Afrotheria, Laurasiatheria, Euarchontoglires, Xenarthra plus Boreoeutheria (Laurasiatheria plus Euarchontoglires). Where these five a priori phylogenies differ, remain areas generally hard to resolve with the new sequences. The root remains ambiguous and does not reject a basal Afrotheria (the Exafroplacentalia hypothesis), Afrotheria plus Xenarthra together with basal (Atlantogenata), or Epitheria (Xenarthra basal) convincingly. Good evidence is found that Eulipotyphla is monophyletic and is located at the base of Laurasiatheria. The shrew mole, Uropsilus, is found to cluster consistently with other moles, while Solenodon may be the sister taxa to all other eulipotyphlans. Support is found for a probable sister pairing of just hedgehogs/gymnures and shrews. Relationships within Afrotheria, except the Paenungulata clade, remain hard to resolve, although there is congruent support for Afroinsectiphillia (aardvark, elephant shrews, golden moles, and tenrecs). A first-time use is made of MCMC enacted general time-reversible (GTR) amino acid and codon-based models for general tree selection. Even with ND6, a GTR amino acid model provided resolution of fine features, such as the sister group relationship of walrus to Otatriidae, and with BRCA a more reasonable rooting. An extensive analysis of GHR sequences reveals strong congruence with prior phylogenies, including strong support for Eulipotyphla, and good resolution within Rodentia. A codon model gives a worse likelihood than a nucleotide model and sometimes switches support, e.g., with RAG1+gamma-fibrinogen from a hyrax-sirenian association to support for Tethytheria. An analysis of the concatenated data is in accordance with well-resolved features of the gene trees. Taken all together, this work suggests that we are on the right path finding strong confirmation of prior phylogenies. However, with the use of robust criteria for assessing trees (i.e., not Bayesian posteriors), it is apparent parts of the tree remain hard to resolve. Since our current models are far from fitting the sequence data, we should continue with our exploratory analyses to arrive at a refined set of hypotheses for future testing using more model independent characters (e.g., rare indels, gene rearrangement, and SINE data).

Pika and vole mitochondrial genomes increase support for both rodent monophyly and glires.

Complete mitochondrial genomes are reported for a pika (Ochotona collaris) and a vole (Volemys kikuchii) then analysed together with 35 other mitochondrial genomes from mammals. With standard phylogenetic methods the pika joins with the other lagomorph (rabbit) and the vole with the other murid rodents (rat and mouse). In addition, with hedgehog excluded, the seven rodent genomes consistently form a homogeneous group in the unrooted placental tree. Except for uncertainty of the position of tree shrew, the clade Glires (monophyletic rodents plus lagomorphs) is consistently found. The unrooted tree obtained by ProtML (Protein Maximum Likelihood, a program in MOLPHY) is compatible with a reclassification of mammals [Syst. Biol. 48, 1-5 (1999)] which is also supported by other recent studies. However, when this tree is rooted with marsupials plus platypus, the outgroup often joins the lineage leading to the three murid rodents, so the rodents are no longer monophyletic. Apart from misplacing the root, the presence of the outgroups also distorts other parts of the unrooted tree. Either constraining the tree to maintain rodents monophyletic, or omitting murids, maintains the ingroup tree and sees the outgroup join on the edge to Xenarthra, to Afrotheria, or to these two groups together. This emphasises the importance of carrying out both an unrooted and a rooted analysis. It is known from cancer research that murid rodents have reduced activity in some DNA repair mechanisms and this alters their substitution pattern - this may be the case for mitochnodrial DNA as well. Comparing nucleotide compositions may identify taxa that differ in aspects of their DNA repair mechanisms.

Very fast algorithms for evaluating the stability of ML and Bayesian phylogenetic trees from sequence data.

Evolutionary trees sit at the core of all realistic models describing a set of related sequences, including alignment, homology search, ancestral protein reconstruction and 2D/3D structural change. It is important to assess the stochastic error when estimating a tree, including models using the most realistic likelihood-based optimizations, yet computation times may be many days or weeks. If so, the bootstrap is computationally prohibitive. Here we show that the extremely fast "resampling of estimated log likelihoods" or RELL method behaves well under more general circumstances than previously examined. RELL approximates the bootstrap (BP) proportions of trees better that some bootstrap methods that rely on fast heuristics to search the tree space. The BIC approximation of the Bayesian posterior probability (BPP) of trees is made more accurate by including an additional term related to the determinant of the information matrix (which may also be obtained as a product of gradient or score vectors). Such estimates are shown to be very close to MCMC chain values. Our analysis of mammalian mitochondrial amino acid sequences suggest that when model breakdown occurs, as it typically does for sequences separated by more than a few million years, the BPP values are far too peaked and the real fluctuations in the likelihood of the data are many times larger than expected. Accordingly, several ways to incorporate the bootstrap and other types of direct resampling with MCMC procedures are outlined. Genes evolve by a process which involves some sites following a tree close to, but not identical with, the species tree. It is seen that under such a likelihood model BP (bootstrap proportions) and BPP estimates may still be reasonable estimates of the species tree. Since many of the methods studied are very fast computationally, there is no reason to ignore stochastic error even with the slowest ML or likelihood based methods.