Statistics and truth in phylogenomics

Phylogenomics refers to the inference of historical relationships among species using genome-scale sequence data and to the use of phylogenetic analysis to infer protein function in multigene families. With rapidly decreasing sequencing costs, phylogenomics is becoming synonymous with evolutionary analysis of genome-scale and taxonomically densely sampled data sets. In phylogenetic inference applications, this translates into very large data sets that yield evolutionary and functional inferences with extremely small variances and high statistical confidence (P value). However, reports of highly significant P values are increasing even for contrasting phylogenetic hypotheses depending on the evolutionary model and inference method used, making it difficult to establish true relationships. We argue that the assessment of the robustness of results to biological factors, that may systematically mislead (bias) the outcomes of statistical estimation, will be a key to avoiding incorrect phylogenomic inferences. In fact, there is a need for increased emphasis on the magnitude of differences (effect sizes) in addition to the P values of the statistical test of the null hypothesis. On the other hand, the amount of sequence data available will likely always remain inadequate for some phylogenomic applications, for example, those involving episodic positive selection at individual codon positions and in specific lineages. Again, a focus on effect size and biological relevance, rather than the P value, may be warranted. Here, we present a theoretical overview and discuss practical aspects of the interplay between effect sizes, bias, and P values as it relates to the statistical inference of evolutionary truth in phylogenomics.     

Pharmacologic treatment of hyperthyroidism during pregnancy

Clinical hyperthyroidism has been associated with an increased risk of maternal, fetal, and neonatal complications. The available antithyroid drugs are methimazole/carbimazole and propylthiouracil. Several case reports and some epidemiologic studies suggest that methimazole/carbimazole exposure during the first trimester of pregnancy is associated with an increased risk of congenital malformations, including ectodermal anomalies, choanal atresia, esophageal atresia, and omphalocele. However, the absolute risk appears to be very small, and it remains unclear whether the association is driven by the maternal disease, the medication, or the combination of both factors. Propylthiouracil exposure has not been associated with an increased risk of congenital malformations and is the recommended drug during the first trimester of pregnancy. Since propylthiouracil-induced hepatotoxicity has been reported in approximately 0.1% of exposed adults and the number of case-reports of severe liver injury is increasing, treatment with low dose methimazole during the second and third trimesters should be considered. Until now, there has been no evidence that children prenatally exposed to methimazole/carbimazole or propylthiouracil have an increased risk of neurodevelopmental delay.     

Molecular evidence for an Asian origin of monitor lizards followed by Tertiary dispersals to Africa and Australasia

Monitor lizards are emblematic reptiles that are widely distributed in the Old World. Although relatively well studied in vertebrate research, their biogeographic history is still controversial. We constructed a molecular dataset for 54 anguimorph species, including representatives of all families with detailed sampling of the Varanidae (38 species). Our results are consistent with an Asian origin of the Varanidae followed by a dispersal to Africa 41 (49-33) Ma, possibly via an Iranian route. Another major event was the dispersal of monitors to Australia in the Late Eocene-Oligocene 32 (39-26) Ma. This divergence estimate adds to the suggestion that Australia was colonized by several squamate lineages prior to the collision of the Australian plate with the Asian plate starting 25 Ma.     

Redox properties of heme peroxidases

Peroxidases are heme enzymes found in bacteria, fungi, plants and animals, which exploit the reduction of hydrogen peroxide to catalyze a number of oxidative reactions, involving a wide variety of organic and inorganic substrates. The catalytic cycle of heme peroxidases is based on three consecutive redox steps, involving two high-valent intermediates (Compound I and Compound II), which perform the oxidation of the substrates. Therefore, the thermodynamics and the kinetics of the catalytic cycle are influenced by the reduction potentials of three redox couples, namely Compound I/Fe³⁺, Compound I/Compound II and Compound II/Fe³⁺. In particular, the oxidative power of heme peroxidases is controlled by the (high) reduction potential of the latter two couples. Moreover, the rapid H₂O₂-mediated two-electron oxidation of peroxidases to Compound I requires a stable ferric state in physiological conditions, which depends on the reduction potential of the Fe³⁺/Fe²⁺ couple. The understanding of the molecular determinants of the reduction potentials of the above redox couples is crucial for the comprehension of the molecular determinants of the catalytic properties of heme peroxidases.This review provides an overview of the data available on the redox properties of Fe³⁺/Fe²⁺, Compound I/Fe³⁺, Compound I/Compound II and Compound II/Fe³⁺ couples in native and mutated heme peroxidases. The influence of the electron donor properties of the axial histidine and of the polarity of the heme environment is analyzed and the correlation between the redox properties of the heme group with the catalytic activity of this important class of metallo-enzymes is discussed.     

CDKN2A mutations and MC1R variants in Italian patients with single or multiple primary melanoma

We evaluated the contribution of germline CDKN2A mutations and MC1R variants to the development of melanoma in a hospital-based study of single (SPM, n = 398) and multiple primary melanoma (MPM, n = 95). The overall frequency of CDKN2A mutations was 15.2%, and four-fold higher in MPM than in SPM cases (OR = 4.27; 95% CI 2.43-7.53). The likelihood of identifying a CDKN2A mutation increased with family history of melanoma and younger age at diagnosis in MPM cases. Compared to SPM patients, the risk of harboring a CDKN2A mutation rose as the number of primary melanomas increased and was not influenced by family history. The G101W and E27X founder mutations were the most common. Several other mutations (W15X, Q50X, R58X, A68L, A127P and H142R) were detected for the first time in Italian patients. One novel mutation, T77A, was identified. Several non-coding variants with unknown functional significance were also found (5'UTR -25C > T, -21C > T, -67G > C, IVS1 +37G > C); the novel 5'UTR -21C > T variant was not detected in controls. The CDKN2A A148T polymorphism was more frequent in MPM patients than in the control population (15.7% versus 6.6%). Compared to the SPM patients, MPM cases had a 2-fold increased probability of being MC1R variant carriers and a higher probability of carrying two or more variants. No specific association was observed between the type of variant and the number of melanomas, suggesting that the number rather than the type of MC1R variant increases the risk of MPM. We observed no interaction between CDKN2A status and the presence of MC1R variants. The high frequency of CDKN2A mutations in our MPM cases, independent of their family history, is of relevance to genetic counseling and testing in our population.     

Conservation of the free energy change of the alkaline isomerization in mitochondrial and bacterial cytochromes c

The thermodynamic parameters of the alkaline transition for oxidized native yeast iso-1 cytochrome c and Rhodopseudomonas palustris cytochrome c(2) (cytc(2)) have been determined through direct electrochemistry experiments carried out at variable pH and temperature and compared to those for horse and beef heart cytochromes c. We have found that both transition enthalpy and entropy are remarkably species dependent, following the order R. palustris cytc(2) > beef (horse) heart cytc>yeast iso-1 cytc. Considering the high homology at the heme-protein interface in the native species, this variability is likely to be mainly determined by differences in the structural and solvation properties and the relative abundance of the various alkaline conformers. Notably, changes in transition enthalpy and entropy among these cytochromes c are compensative and result in small variations in the free energy change of the process (which amounts approximately to +50 kJ mol(-1)) and consequently in the apparent pK(a) value. This compensation indicates that solvent reorganization effects play an important role in the thermodynamics of the transition. This mechanism is functional to ensure a relatively high pK(a) value for the alkaline transition, which is needed to preserve His,Met ligation to the heme iron in cytochrome c at physiological pH and temperature, hence the E(o) value required for the biological function.     

Thermodynamics of the acid transition in blue copper proteins

The thermodynamic parameters of the conformational transition occurring at low pH (acid transition, AT) in blue copper proteins, involving protonation and detachment from the Cu(I) ion of one histidine ligand, have been determined electrochemically for spinach and cucumber plastocyanins, Rhus vernicifera stellacyanin, cucumber basic protein (CBP), and Paracoccus versutus amicyanin. These data were obtained from direct protein electrochemistry experiments carried out at varying pH and temperature. For all species but CBP, the overall conformational change turns out to be exothermic. The entropy change is remarkably species-dependent. In particular, we found that (i) the balance of bond breaking/formation favors the acid transition in plastocyanins, which show remarkably negative DeltaH degrees '(AT) values, and (ii) the transition enthalpy turns out to be much less negative (or even positive) for the two phytocyanins (stellacyanin and CBP): for these species, the transition turns out to be observable thanks to the favorable (positive) entropy change. Thus, it is apparent that the thermodynamic "driving force" for this transition is enthalpic for the plastocyanins and entropic for the phytocyanins. Amicyanin is an intermediate case in which both enthalpic and entropic terms favor the transition. Under the assumption that the transition entropy originates from solvent reorganization effects, which are known to involve compensative enthalpy and entropy changes, the free energy change of the transition would also correspond to the enthalpy change due to bond breaking/formation in the first coordination sphere of the metal and in its immediate environment. Indeed, this term turns out to be very similar for the proteins investigated, in line with the conservation of the Cu(I)-His bond strengths in these species, except for amicyanin, for which the greater exothermicity of the transition can be ascribed to peculiar features of the active site.     

Enthalpic and entropic contributions to the mutational changes in the reduction potential of azurin

The changes in the reduction potential of Pseudomonas aeruginosa and Alcaligenes denitrificans azurins following point mutations and residue ionizations were factorized into the enthalpic and entropic contributions through variable temperature direct electrochemistry experiments. The effects on the reduction enthalpy due to changes in the first coordination sphere of the copper ion, as in the Met121Gln and Met121His variants of A. denitrificans azurin, insertion of a net charge and alteration in the solvation properties and electrostatic potential in proximity of the metal site, as in the Met44Lys and His35Leu variants of P. aeruginosa azurin, respectively, and proton uptake/release in wild-type and mutated species could invariably be accounted for on the basis of simple coordination chemistry and/or electrostatic considerations. The concomitant changes in reduction entropy were found in general to contribute to the E degrees ' variation to a lesser extent as compared to the enthalpy changes. However, their effects were by no means negligible and in some instances were found to heavily contribute to (or even become the main determinant of) the observed change in reduction potential. Several lines of evidence indicate that the entropic effects are notably influenced by reduction-induced solvent reorganization effects. In particular, protein reduction tends to be favored on entropic grounds with increasing exposure of the copper site to the solvent. Moreover, enthalpy-entropy compensation phenomena are invariably observed when residue mutation or pH-induced conformational changes modify the solvent accessibility of the metal site or alter the H-bonding network in the hydration shell of the molecule. Therefore, in these cases, caution must be used in making predictions of E degrees ' changes simply based on Coulombic or coordination chemistry arguments.     

Free energy of transition for the individual alkaline conformers of yeast iso-1-cytochrome c

Direct protein electrochemistry was used to obtain the thermodynamic parameters of transition from the native (state III) to the alkaline (state IV) conformer for untrimethylated Saccharomyces cerevisiae iso-1-cytochrome c expressed in E. coli and its single and multiple lysine-depleted variants. In these variants, one or more of the lysine residues involved in axial Met substitution (Lys72, Lys73, and Lys79) was mutated to alanine. The aim of this work is to determine the thermodynamic affinity of each of the substituting lysines for the heme iron and evaluate the interplay of enthalpic and entropic factors. The equilibrium constants for the deprotonation reaction of Lys72, 73, and 79 were computed for the minimized MD average structures of the wild-type and mutated proteins, applying a modified Tanford-Kirkwood calculation. Solvent accessibility calculations for the substituting lysines in all variants were also performed. The transition enthalpy and entropy values within the protein series show a compensatory behavior, typical of a process involving extensive solvent reorganization effects. The experimental and theoretical data indicate that Lys72 most readily deprotonates and replaces M80 as the axial heme iron ligand, whereas Lys73 and Lys79 show comparably higher pKa values and larger transition free energies. A good correlation is found within the series between the lowest calculated Lys pKa value and the corresponding experimental pKa value, which can be interpreted as indicative of the deprotonating lysine itself acting as the triggering group for the conformational transition. The triple Lys to Ala mutant, in which no lysine residues are available for heme iron binding, features transition thermodynamics consistent with a hydroxide ion replacing the axial methionine ligand.