Tree shape‐based approaches for the comparative study of cophylogeny

Cophylogeny is the congruence of phylogenetic relationships between two different groups of organisms due to their long‐term interaction. We investigated the use of tree shape distance measures to quantify the degree of cophylogeny. We implemented a reverse‐time simulation model of pathogen phylogenies within a fixed host tree, given cospeciation probability, host switching, and pathogen speciation rates. We used this model to evaluate 18 distance measures between host and pathogen trees including two kernel distances that we developed for labeled and unlabeled trees, which use branch lengths and accommodate different size trees. Finally, we used these measures to revisit published cophylogenetic studies, where authors described the observed associations as representing a high or low degree of cophylogeny. Our simulations demonstrated that some measures are more informative than others with respect to specific coevolution parameters especially when these did not assume extreme values. For real datasets, trees’ associations projection revealed clustering of high concordance studies suggesting that investigators are describing it in a consistent way. Our results support the hypothesis that measures can be useful for quantifying cophylogeny. This motivates their usage in the field of coevolution and supports the development of simulation‐based methods, i.e., approximate Bayesian computation, to estimate the underlying coevolutionary parameters.     

Direct demonstration of the flexibility of the glycosylated proline-threonine linker in the Cellulomonas fimi Xylanase Cex through NMR spectroscopic analysis

The modular xylanase Cex (or CfXyn10A) from Cellulomonas fimi consists of an N-terminal catalytic domain and a C-terminal cellulose-binding domain, joined by a glycosylated proline-threonine (PT) linker. To characterize the conformation and dynamics of the Cex linker and the consequences of its modification, we have used NMR spectroscopy to study full-length Cex in its nonglycosylated ( approximately 47 kDa) and glycosylated ( approximately 51 kDa) forms. The PT linker lacks any predominant structure in either form as indicated by random coil amide chemical shifts. Furthermore, heteronuclear (1)H-(15)N nuclear Overhauser effect relaxation measurements demonstrate that the linker is flexible on the ns-to-ps time scale and that glycosylation partially dampens this flexibility. The catalytic and cellulose-binding domains also exhibit identical amide chemical shifts whether in isolation or in the context of either unmodified or glycosylated full-length Cex. Therefore, there are no noncovalent interactions between the two domains of Cex or between either domain and the linker. This conclusion is supported by the distinct (15)N relaxation properties of the two domains, as well as their differential alignment within a magnetic field by Pf1 phage particles. These data demonstrate that the PT linker is a flexible tether, joining the structurally independent catalytic and cellulose-binding domains of Cex in an ensemble of conformations; however, more extended forms may predominate because of restrictions imparted by the alternating proline residues. This supports the postulate that the binding-domain anchors Cex to the surface of cellulose, whereas the linker provides flexibility for the catalytic domain to hydrolyze nearby hemicellulose (xylan) chains.     

An in vivo study of common carp (Cyprinus carpio L.) liver during prolonged hypoxia

Hypoxia induced apoptosis has been studied extensively in many mammalian cell lines but there are only a few studies using whole animal models. We investigated the response of the intact liver to hypoxia in a hypoxia tolerant fish, the carp (Cyprinus carpio, L). We exposed carp to hypoxia for up to 42 days, using oxygen level (0.5 mgO₂/L) that were slightly higher than the critical oxygen level of carp. There was extensive DNA damage in liver cells, especially during the first week of exposure, indicated by a massive TUNEL signal. However there was no change in cell proliferation, cell number or size, no increase in caspase-3 activity, no increase in single stranded DNA and this, combined with a number of other observations, led us to conclude there was no increase in apoptosis in the liver during hypoxia. There was up-regulation of some anti-apoptotic genes and proteins (Bcl-2, HSP70, p27) and down-regulation of some pro-apoptotic genes (Tetraspanin 5 and Cell death activator). The cells appeared to enter cell cycle arrest, presumably to allow repair of damaged DNA. As there was no change in cell proliferation and cell number, the damaged cells were not entering apoptosis and must have recovered during prolonged hypoxia.     

An evolutionary-network model reveals stratified interactions in the V3 loop of the HIV-1 envelope

The third variable loop (V3) of the human immunodeficiency virus type 1 (HIV-1) envelope is a principal determinant of antibody neutralization and progression to AIDS. Although it is undoubtedly an important target for vaccine research, extensive genetic variation in V3 remains an obstacle to the development of an effective vaccine. Comparative methods that exploit the abundance of sequence data can detect interactions between residues of rapidly evolving proteins such as the HIV-1 envelope, revealing biological constraints on their variability. However, previous studies have relied implicitly on two biologically unrealistic assumptions: (1) that founder effects in the evolutionary history of the sequences can be ignored, and; (2) that statistical associations between residues occur exclusively in pairs. We show that comparative methods that neglect the evolutionary history of extant sequences are susceptible to a high rate of false positives (20%–40%). Therefore, we propose a new method to detect interactions that relaxes both of these assumptions. First, we reconstruct the evolutionary history of extant sequences by maximum likelihood, shifting focus from extant sequence variation to the underlying substitution events. Second, we analyze the joint distribution of substitution events among positions in the sequence as a Bayesian graphical model, in which each branch in the phylogeny is a unit of observation. We perform extensive validation of our models using both simulations and a control case of known interactions in HIV-1 protease, and apply this method to detect interactions within V3 from a sample of 1,154 HIV-1 envelope sequences. Our method greatly reduces the number of false positives due to founder effects, while capturing several higher-order interactions among V3 residues. By mapping these interactions to a structural model of the V3 loop, we find that the loop is stratified into distinct evolutionary clusters. We extend our model to detect interactions between the V3 and C4 domains of the HIV-1 envelope, and account for the uncertainty in mapping substitutions to the tree with a parametric bootstrap.     

Shape of Ocr, the gene 0.3 protein of bacteriophage T7: modeling based on light scattering experiments

Ocr, the first protein expressed by bacteriophage T7, inhibits type Iota DNA restriction enzymes by preventing them from binding to DNA. This inhibition allows the phage to successfully infect the host. The shape of ocr is modeled on the basis of static and dynamic light scattering measurements. The static light scattering data confirm previous observations that ocr exists in solution as a dimer. The diffusion constant determined by dynamic light scattering indicates a nonspherical shape of the ocr dimer. Hydrodynamic models of ellipsoids are presented, and it is argued that ocr is best described by a prolate ellipsoid with dimensions of 10.4 nm by 2.6 nm. The size and shape predicted by this model are consistent with ocr acting as a mimic of the DNA structure bound by type Iota restriction enzymes.     

Expedited SAR study of an mGluR5 antagonists: generation of a focused library using a solution-phase Suzuki coupling methodology

The SAR of the lead compounds 2a and 2b was rapidly explored. Utilizing a parallel solution-phase Suzuki coupling approach, in tandem with strong cation exchange resin (SCX) purification afforded the desired focused library. The library was evaluated in vitro, a ninefold potency increase was achieved and the preference for ortho substitution of moderate steric bulk of the fourth, phenyl ring was identified. In addition, dimethylisoxazole, as a heterocyclic replacement for the phenylic ring of the lead compound, was also identified by this approach.     

2-(2-[3-(pyridin-3-yloxy)phenyl]-2H-tetrazol-5-yl) pyridine: a highly potent, orally active, metabotropic glutamate subtype 5 (mGlu5) receptor antagonist

Structure-activity relationship studies on 3-(5-pyridin-2-yl-2H-tetrazol-2-yl)benzonitrile 2 led to the discovery of 2-(2-[3-(pyridin-3-yloxy)phenyl]-2H-tetrazol-5-yl)pyridine (10)-a highly potent and selective mGlu5 receptor antagonist with good brain penetration and in vivo receptor occupancy in rat and cross-species oral bioavailability.     

Unique and conserved features of genome and proteome of SARS-coronavirus,an early split-off from the coronavirus group 2 lineage

The genome organization and expression strategy of the newly identified severe acute respiratory syndrome coronavirus (SARS-CoV) were predicted using recently published genome sequences. Fourteen putative open reading frames were identified, 12 of which were predicted to be expressed from a nested set of eight subgenomic mRNAs. The synthesis of these mRNAs in SARS-CoV-infected cells was confirmed experimentally. The 4382- and 7073 amino acid residue SARS-CoV replicase polyproteins are predicted to be cleaved into 16 subunits by two viral proteinases (bringing the total number of SARS-CoV proteins to 28). A phylogenetic analysis of the replicase gene, using a distantly related torovirus as an outgroup, demonstrated that, despite a number of unique features, SARS-CoV is most closely related to group 2 coronaviruses. Distant homologs of cellular RNA processing enzymes were identified in group 2 coronaviruses, with four of them being conserved in SARS-CoV. These newly recognized viral enzymes place the mechanism of coronavirus RNA synthesis in a completely new perspective. Furthermore, together with previously described viral enzymes, they will be important targets for the design of antiviral strategies aimed at controlling the further spread of SARS-CoV.