Molecular Substitution Research Articles

The Ibr‐7 derivative of ibrutinib exhibits enhanced cytotoxicity against non‐small cell lung cancer cells via targeting of mTORC1/S6 signaling

Ibrutinib is a small molecule drug that targets Bruton's tyrosine kinase in B‐cell malignancies and is highly efficient at killing mantle cell lymphoma and chronic lymphocytic leukemia. However, the anti‐cancer activity of ibrutinib against solid tumors, such as non‐small cell lung cancer (NSCLC), remains low. To improve the cytotoxicity of ibrutinib towards lung cancer, we synthesized a series of ibrutinib derivatives, of which Ibr‐7 exhibited superior anti‐cancer activity to ibrutinib, especially against epithelial growth factor receptor (EGFR) wild‐type NSCLC cell lines. Ibr‐7 was observed to dramatically suppress the mammalian target of Rapamycin complex 1 (mTORC1)/S6 signaling pathway, which is only slightly affected by ibrutinib, thus accounting for the superior anti‐cancer activity of Ibr‐7 towards NSCLC. Ibr‐7 was shown to overcome the elevation of Mcl‐1 caused by ABT‐199 mono‐treatment, and thus exhibited a significant synergistic effect when combined with ABT‐199. In conclusion, we used a molecular substitution method to generate a novel ibrutinib derivative, termed Ibr‐7, which exhibits enhanced anti‐cancer activity against NSCLC cells as compared with the parental compound.

Plastid phylogenomic insights into the evolution of Caryophyllales

The Caryophyllales includes 40 families and 12,500 species, representing a large and diverse clade of angiosperms. Collectively, members of the clade grow on all continents and in all terrestrial biomes and often occupy extreme habitats (e.g., xeric, salty). The order is characterized by many taxa with unusual adaptations including carnivory, halophytism, and multiple origins of C4 photosynthesis. However, deep phylogenetic relationships within the order have long been problematic due to putative rapid divergence. To resolve the deep-level relationships of Caryophyllales, we performed phylogenomic analyses of all 40 families of Caryophyllales. We time-calibrated the molecular phylogeny of this clade, and evaluated putative correlations among plastid structural changes and rates of molecular substitution. We recovered a well-resolved and well-supported phylogeny of the Caryophyllales that was largely congruent with previous estimates of this order. Our results provide improved support for the phylogenetic position of several key families within this clade. The crown age of Caryophyllales was estimated at ca. 114.4 million years ago (Ma), with periods of rapid divergence in the mid-Cretaceous. A strong, positive correlation between nucleotide substitution rate and plastid structural changes was detected. Our study highlights the importance of broad taxon sampling in phylogenomic inference and provides a firm basis for future investigations of molecular, morphological, and ecophysiological evolution in Caryophyllales.

Anthracene-Based Highly Selective and Sensitive Fluorescent "Turn-on" Chemodosimeter for Hg2.

Three π-extended anthracene-bearing thioacetals (1–3) have been synthesized, and their fluorescence “turn-on” responses to Hg2+ ions are studied. The chemodosimetric fluorescence-sensing behavior and their resulting hydrolysis via a desulfurization reaction mechanism leads to the formation of highly fluorescent respective aldehyde substitutions. Furthermore, this mechanism was supported by increase in the quantum yields of their resulting aldehydes and is correlated to their molecular substitution. The chemosensors 1–3 have exhibited to be promising receptors toward Hg2+ ions in the presence of other competitive metal ions. Moreover, the detection limits of 1–3 have been found to be in the nanomolar range (94, 59, and 235, respectively). Fluorescence microscopic imaging studies show that 1–2 have been found to be effective for fluorescence imaging in live cells. Moreover, compounds 1–3 act as potential candidates for the detection of Hg2+ in environmental and biological systems as well as real samples.

Malate Dehydrogenase 2 (MDH2) as a New Diabetogene Causing Hyperglycemia in Families with Multigenerational Diabetes

We are pursuing the identification of new diabetogenes by whole exome sequencing in families with multigenerational diabetes that do not carry MODY-gene mutations. Here we report two gain-of- function mutations (c.154 C>T p.Arg52Cys and c.478 G>A p.Val160Met) in the gene coding for Malate Dehydrogenase 2 (MDH2) that we have identified as segregating with hyperglycemia in two such unrelated families. The MDH2 enzyme is localized to the mitochondria where it catalyzes the reversible oxidation of malate to oxaloacetate, utilizing the NAD/NADH cofactor system in the tricarboxylic acid cycle. MDH2 also plays a pivotal role in the malate-aspartate shuttle that operates in the metabolic coordination between cytosol and mitochondria. By molecular dynamic simulation, both the Arg52Cys and Val160Met substitutions were found to severely alter the atomic dynamics of the MDH2 structure. MDH2 enzymatic activity was significantly increased (p<0.01) in HepG2 cells transfected with either MDH2_T154 or MDH2_A478 as compared to WT_MDH2, despite no differences in MDH2 protein expression levels. Increased MDH2 activity is known to decrease NAD/NADH ratio—a biochemical alteration reported to affect both insulin signaling and secretion. In genetically modified C. elegans strains obtained by means of CRISPR/Cas9, the Arg52Cys mutant exhibited a significantly (p<0.05) prolonged lifespan and increased Paraquat resistance and lipid storage when compared to WT strains. These changes resemble those reported for C. elegans with defective insulin signaling—a condition which in humans predisposes to both insulin resistance and inadequate insulin secretion. In conclusion, our data point to MDH2 as a new diabetogene, heterozygous mutations of which cause hyperglycemia in families presenting with multigenerational diabetes. Disclosure P. Jungtrakoon: None. S. Pezzilli: None. A. Marucci: None. L. Pannone: None. E. Flex: None. T. Biagini: None. P. Buranasupkajorn: None. O. Ludovico: None. L. Mercuri: None. T. Hastings: None. R. Di Paola: None. F. Alberico: None. C. Mendonca: None. J. Ceron: None. M. Porta de la Riva: None. L. Marselli: None. T. Mazza: None. S. Martinelli: None. V. Trischitta: None. A. Doria: Research Support; Self; Sanofi-Aventis. S. Prudente: None.

Recipes for improper ferroelectricity in molecular perovskites

The central goal of crystal engineering is to control material function via rational design of structure. A particularly successful realisation of this paradigm is hybrid improper ferroelectricity in layered perovskite materials, where layering and cooperative octahedral tilts combine to break inversion symmetry. However, in the parent family of inorganic ABX3 perovskites, symmetry prevents hybrid coupling to polar distortions. Here, we use group-theoretical analysis to uncover a profound enhancement of the number of improper ferroelectric coupling schemes available to molecular perovskites. This enhancement arises because molecular substitution diversifies the range of distortions possible. Not only do our insights rationalise the emergence of polarisation in previously studied materials, but we identify the fundamental importance of molecular degrees of freedom that are straightforwardly controlled from a synthetic viewpoint. We envisage that the crystal design principles we develop here will enable targeted synthesis of a large family of new acentric functional materials.

Neutral Theory and the Somatic Evolution of Cancer.

Kimura's neutral theory argued that positive selection was not responsible for an appreciable fraction of molecular substitutions. Correspondingly, quantitative analysis reveals that the vast majority of substitutions in cancer genomes are not detectably under selection. Insights from the somatic evolution of cancer reveal that beneficial substitutions in cancer constitute a small but important fraction of the molecular variants. The molecular evolution of cancer community will benefit by incorporating the neutral theory of molecular evolution into their understanding and analysis of cancer evolution-and accepting the use of tractable, predictive models, even when there is some evidence that they are not perfect.

A revised phylogeny of macropathine cave crickets (Orthoptera: Rhaphidophoridae) uncovers a paraphyletic Australian fauna

Australian cave crickets are members of the subfamily Macropathinae (Orthoptera: Rhaphidophoridae). The subfamily is thought to have originated prior to the tectonic separation of the supercontinent Gondwana based on distributions of extant lineages and molecular phylogenetic evidence, although the Australian fauna have been underrepresented in previous studies. The current study augments existing multigene data (using 12S, 16S, and 28S rRNA genes) to investigate the placement of the Australian representatives within the Macropathinae and to assess divergence dates of select clades. Results suggest that the endemic Tasmanian genus Parvotettix is the sister lineage to the remaining members of the subfamily, an outcome that presents a paraphyletic Australian fauna in contrast to previous studies. All other Australian taxa represented in this study (Micropathus and Novotettix) emerged as a sister group to the New Zealand and South American macropathine lineages. Estimation of phylogenetic divergence ages among the aforementioned clades were calibrated using two methods, in absence of suitable fossil records: (i) tectonic events depicting the fragmentation of Gondwanan landmasses that invoke vicariant scenarios of present day geographic distributions; and (ii) molecular evolutionary rates. Geological calibrations place the median age of the most recent common ancestor of extant macropathines at ∼125 to ∼165 Ma, whereas analyses derived from molecular substitution rates suggest a considerably younger origin of ∼32 Ma. This phylogenetic study represents the most rigorous taxonomic sampling of the Australian cave cricket fauna to date and stresses the influence of lineage representation on biogeographic inference.

Cocrystals Help Break the “Rules” of Isostructurality: Solid Solutions and Polymorphism in the Malic/Tartaric Acid System

Crystalline solid solutions have the potential to afford tunable materials for pharmaceutical and technological applications. Unfortunately, these poorly understood phases are difficult to obtain and, hence, to study. In fact, commonly accepted empirical rules prescribe that only molecules of similar size and electron distribution are mutually soluble in the solid state. Here, despite the evident structural and electronic differences, the enantiomers of malic acid and tartaric acid are crystallized together in a variable stoichiometric ratio to produce both cocrystals and solid solutions. In some cases, physical mixtures are observed. The composition and polymorphism of the crystalline products are explained by DFT-d molecular substitution calculations for the cocrystallized molecules in different (known) structures. At the same time, from a crystal engineering perspective, the behavior of this complex system is rationalized thanks to the existence of intermediate cocrystal forms that merge the structural...

Nuclear Excluded Autism-Associated Phosphatase and Tensin Homolog Mutations Dysregulate Neuronal Growth

BackgroundPhosphatase and tensin homolog (PTEN) negatively regulates downstream protein kinase B signaling, resulting in decreased cellular growth and proliferation. PTEN is mutated in a subset of children with autism spectrum disorder (ASD); however, the mechanism by which specific point mutations alter PTEN function is largely unknown. Here, we assessed how ASD-associated single-nucleotide variations in PTEN (ASD-PTEN) affect function. MethodsWe used viral-mediated molecular substitution of human PTEN into Pten knockout mouse neurons and assessed neuronal morphology to determine the functional impact of ASD-PTEN. We employed molecular cloning to examine how PTEN’s stability, subcellular localization, and catalytic activity affect neuronal growth. ResultsWe identified a set of ASD-PTEN mutations displaying altered lipid phosphatase function and subcellular localization. We demonstrated that wild-type PTEN can rescue the neuronal hypertrophy, while PTEN H93R, F241S, D252G, W274L, N276S, and D326N failed to rescue this hypertrophy. A subset of these mutations lacked nuclear localization, prompting us to examine the role of nuclear PTEN in regulating neuronal growth. We found that nuclear PTEN alone is sufficient to regulate soma size. Furthermore, forced localization of the D252G and W274L mutations into the nucleus partially restores regulation of soma size. ConclusionsASD-PTEN mutations display decreased stability, catalytic activity, and/or altered subcellular localization. Mutations lacking nuclear localization uncover a novel mechanism whereby lipid phosphatase activity in the nucleus can regulate mammalian target of rapamycin signaling and neuronal growth.

Proton-transfer inhibition by molecular anion substitutions in Photosystem II

Proton-transfer inhibition by molecular anion substitutions in Photosystem II

Structural organization of cycloartane-based saponins in the genus Astragalus (Fabaceae)

Cycloartane represents the most common aglycone in saponins of Astragalus genus. These saponins revealed high structural diversity through continuous elucidation of new molecules. Traditionally, saponins’ diversity was updated through reviews listing molecules with respect to their pharmacological activities and/or producing plant species. Beyond the long lists of reviewed molecules, this work provides statistical classification of 178 Astragalus saponins published between 1983 and 2014, leading to different molecular clusters grouping structurally close saponins and separating dissimilar ones. Clustering highlighted hierarchical factors governing structural diversification of cycloartane-based saponins in Astragalus, including rare aglycone forms, rare substitution positions and relative substitution levels of the carbon C3. Rare cycloartane forms were due to different epoxydations occurring at different degrees and positions in the lateral part of cycloartane. In common cycloartane forms, rare saponins were due to rare substituted positions consisting of atypical hydroxylations. Within the most common saponins, several classes were highlighted by the variations of C3-substitution levels in both epoxydated and not epoxydated cycloartane. Structural variations within and between saponin classes provided a strong basis for highlighting significant effects of cycloartane forms and C3-substitution levels on molecular ramification, elongation levels and substitution types (glycosylation and acetylation). Finally, these two key structural factors seemed to influence compartmental distribution of saponins in Astragalus genus.

Aggregation and rheological behavior of soluble dietary fibers from wheat bran

The present study assesses the aggregation behavior of wheat bran arabinoxylan-rich soluble dietary fiber (SDF) fractions with diverse molecular weight and substitution in order to provide useful information to prevent the formation of a block network. In the present work, dynamic and static light scattering, diffusing wave spectroscopy, small amplitude dynamic rheology, atomic force microscopy, and the water-holding and swelling capacities were evaluated to assess the SDF aggregation behavior induced by intrinsic and extrinsic factors. Furthermore, the rheological behavior was explained by the physically cross-linked or interpenetrating hydrocolloid network established during SDF self-aggregation, dependent on its molecular structure. The results indicated that the SDF fractions exhibiting a high molecular weight and a lower substitution degree and di-substituted ratio led to more significant aggregation due to the formation of disordered tangles coupled with a more solid-like behavior. The obtained information will prove useful for the development of more stable and compatible SDF fractions.

The Past Sure is Tense: On Interpreting Phylogenetic Divergence Time Estimates.

Divergence time estimation-the calibration of a phylogeny to geological time-is an integral first step in modeling the tempo of biological evolution (traits and lineages). However, despite increasingly sophisticated methods to infer divergence times from molecular genetic sequences, the estimated age of many nodes across the tree of life contrast significantly and consistently with timeframes conveyed by the fossil record. This is perhaps best exemplified by crown angiosperms, where molecular clock (Triassic) estimates predate the oldest (Early Cretaceous) undisputed angiosperm fossils by tens of millions of years or more. While the incompleteness of the fossil record is a common concern, issues of data limitation and model inadequacy are viable (if underexplored) alternative explanations. In this vein, Beaulieu et al. (2015) convincingly demonstrated how methods of divergence time inference can be misled by both (i) extreme state-dependent molecular substitution rate heterogeneity and (ii) biased sampling of representative major lineages. These results demonstrate the impact of (potentially common) model violations. Here, we suggest another potential challenge: that the configuration of the statistical inference problem (i.e., the parameters, their relationships, and associated priors) alone may preclude the reconstruction of the paleontological timeframe for the crown age of angiosperms. We demonstrate, through sampling from the joint prior (formed by combining the tree (diversification) prior with the calibration densities specified for fossil-calibrated nodes) that with no data present at all, that an Early Cretaceous crown angiosperms is rejected (i.e., has essentially zero probability). More worrisome, however, is that for the 24 nodes calibrated by fossils, almost all have indistinguishable marginal prior and posterior age distributions when employing routine lognormal fossil calibration priors. These results indicate that there is inadequate information in the data to over-rule the joint prior. Given that these calibrated nodes are strategically placed in disparate regions of the tree, they act to anchor the tree scaffold, and so the posterior inference for the tree as a whole is largely determined by the pseudodata present in the (often arbitrary) calibration densities. We recommend, as for any Bayesian analysis, that marginal prior and posterior distributions be carefully compared to determine whether signal is coming from the data or prior belief, especially for parameters of direct interest. This recommendation is not novel. However, given how rarely such checks are carried out in evolutionary biology, it bears repeating. Our results demonstrate the fundamental importance of prior/posterior comparisons in any Bayesian analysis, and we hope that they further encourage both researchers and journals to consistently adopt this crucial step as standard practice. Finally, we note that the results presented here do not refute the biological modeling concerns identified by Beaulieu et al. (2015). Both sets of issues remain apposite to the goals of accurate divergence time estimation, and only by considering them in tandem can we move forward more confidently.

Structural studies and nociceptive activity of a native lectin from Platypodium elegans seeds (nPELa)

A native lectin (nPELa), purified from seeds of the species Platypodium elegans, Dalbergieae tribe, was crystallized and structurally characterized by X-ray diffraction crystallography and bioinformatics tools. The obtained crystals diffracted to 1.6Å resolution, and nPELa structure were solved through molecular substitution. In addition, nPELa has a metal binding site and a conserved carbohydrate recognition domain (CRD) similar to other Dalbergieae tribe lectins, such as PAL (Pterocarpus angolensis) and CTL (Centrolobium tomentosum). Molecular docking analysis indicated high affinity of this lectin for different mannosides, mainly trimannosides, formed by α-1,3 or α-1,6 glycosidic bond, as evidenced by the obtained scores. In addition, molecular dynamics simulations were performed to demonstrate the structural behavior of nPELa in aqueous solution. In solution, nPELa was highly stable, and structural modifications in its carbohydrate recognition site allowed interaction between the lectin and the different ligands. Different modifications were observed during simulations for each one of the glycans, which included different hydrogen bonds and hydrophobic interactions through changes in the relevant residues. In addition, nPELa was evaluated for its nociceptive activity in mice and was reported to be the first lectin of the Dalbergieae tribe to show CRD-dependent hypernociceptive activity.

Phylogenetic estimates of diversification rate are affected by molecular rate variation.

Molecular phylogenies are increasingly being used to investigate the patterns and mechanisms of macroevolution. In particular, node heights in a phylogeny can be used to detect changes in rates of diversification over time. Such analyses rest on the assumption that node heights in a phylogeny represent the timing of diversification events, which in turn rests on the assumption that evolutionary time can be accurately predicted from DNA sequence divergence. But there are many influences on the rate of molecular evolution, which might also influence node heights in molecular phylogenies, and thus affect estimates of diversification rate. In particular, a growing number of studies have revealed an association between the net diversification rate estimated from phylogenies and the rate of molecular evolution. Such an association might, by influencing the relative position of node heights, systematically bias estimates of diversification time. We simulated the evolution of DNA sequences under several scenarios where rates of diversification and molecular evolution vary through time, including models where diversification and molecular evolutionary rates are linked. We show that commonly used methods, including metric-based, likelihood and Bayesian approaches, can have a low power to identify changes in diversification rate when molecular substitution rates vary. Furthermore, the association between the rates of speciation and molecular evolution rate can cause the signature of a slowdown or speedup in speciation rates to be lost or misidentified. These results suggest that the multiple sources of variation in molecular evolutionary rates need to be considered when inferring macroevolutionary processes from phylogenies.

Extreme anoxia tolerance in crucian carp and goldfish through neofunctionalization of duplicated genes creating a new ethanol-producing pyruvate decarboxylase pathway

Without oxygen, most vertebrates die within minutes as they cannot meet cellular energy demands with anaerobic metabolism. However, fish of the genus Carassius (crucian carp and goldfish) have evolved a specialized metabolic system that allows them to survive prolonged periods without oxygen by producing ethanol as their metabolic end-product. Here we show that this has been made possible by the evolution of a pyruvate decarboxylase, analogous to that in brewer’s yeast and the first described in vertebrates, in addition to a specialized alcohol dehydrogenase. Whole-genome duplication events have provided additional gene copies of the pyruvate dehydrogenase multienzyme complex that have evolved into a pyruvate decarboxylase, while other copies retained the essential function of the parent enzymes. We reveal the key molecular substitution in duplicated pyruvate dehydrogenase genes that underpins one of the most extreme hypoxic survival strategies among vertebrates and that is highly deleterious in humans.

Genomic Signature of an Avian Lilliput Effect across the K-Pg Extinction

Survivorship following major mass extinctions may be associated with a decrease in body size-a phenomenon called the Lilliput Effect. Body size is a strong predictor of many life history traits (LHTs), and is known to influence demography and intrinsic biological processes. Pronounced changes in organismal size throughout Earth history are therefore likely to be associated with concomitant genome-wide changes in evolutionary rates. Here, we report pronounced heterogeneity in rates of molecular evolution (varying up to $\sim$20-fold) across a large-scale avian phylogenomic data set and show that nucleotide substitution rates are strongly correlated with body size and metabolic rate. We also identify potential body size reductions associated with the Cretaceous-Paleogene (K-Pg) transition, consistent with a Lilliput Effect in the wake of that mass extinction event. We posit that selection for reduced body size across the K-Pg extinction horizon may have resulted in transient increases in substitution rate along the deepest branches of the extant avian tree of life. This "hidden" rate acceleration may result in both strict and relaxed molecular clocks over-estimating the age of the avian crown group through the relationship between life history and demographic parameters that scale with molecular substitution rate. If reductions in body size (and/or selection for related demographic parameters like short generation times) are a common property of lineages surviving mass extinctions, this phenomenon may help resolve persistent divergence time debates across the tree of life. Furthermore, our results suggest that selection for certain LHTs may be associated with deterministic molecular evolutionary outcomes.

Body mass is less important than bird order in determining the molecular rate for bird mitochondrial DNA.

A negative relationship between body mass and molecular evolution rates has been suggested, and recently a correlation equation has been published based on mitochondrial genomic data of 475 bird species and their body masses. Here, we re-analysed these data and show that the bird order as a proxy of monophyletic groups was a stronger predictor of the molecular rate than the body mass. We provide order-specific molecular substitution rates. Only three orders (Galliformes, Gruiformes, Pelecaniformes) showed a very clear negative correlation, and specific correlation equations are given for these. The molecular rates of bird orders differed strongly at similar mean body masses, and we suggest that the previously described trend across all birds may arise as smaller species also tend to have characteristic life histories, namely faster turnover of generations, higher fecundity and shorter lifespans.

Testing for the Occurrence of Selective Episodes During the Divergence of Otophysan Fishes: Insights from Mitogenomics

How natural selection shapes biodiversity constitutes a topic of renewed interest during the last few decades. The division Otophysi comprises approximately two-thirds of freshwater fish diversity and probably underwent an extensive adaptive radiation derived from a single invasion of the supercontinent Pangaea, giving place to the evolution of the main five Otophysan lineages during a short period of time. Little is known about the factors involved in the processes that lead to lineage diversification among this group of fishes and identifying directional selection acting over protein-coding genes could offer clues about the processes acting on species diversification. The main objective of this study was to explore the otophysan mitochondrial genome evolution, in order to account for the possible signatures of selective events in this lineage, and to explore for the functional connotations of these molecular substitutions. Mainly, three different approaches were used: the "ω-based" BS-REL and MEME methods, implemented in the DATAMONKEY web server, and analysis of selection on amino acid properties, implemented in the software TreeSAAP. We found evidence of selective episodes along several branches of the evolutionary history of othophysan fishes. Analyses carried out using the BS-REL algorithm suggest episodic diversifying selection at basal branches of the otophysan lineage, which was also supported by analyses implemented in MEME and TreeSAAP. These results suggest that throughout the Siluriformes radiation, an important number of adaptive changes occurred in their mitochondrial genome. The metabolic consequences and ecological correlates of these molecular substitutions should be addressed in future studies.

Molecular substitution rate increases with latitude in butterflies: evidence for a trans‐glacial latitudinal layering of populations?

A well‐documented consequence of repeated global ice ages is the negative relationship between latitude and intraspecific genetic diversity. However, little is known about additional effects of such major climatic events on population genetic structure. Here we studied the phylogeographic structure of five lycaenid butterfly species with varied ecological adaptations, sampled across a latitudinal gradient in the Holarctic region. We found a positive correlation between latitude and substitution rate of mitochondrial DNA sequences in all species investigated. We propose that this result is the signal of increased genetic drift and founder effects during post‐glacial recolonization of northern populations. Given that phylogenetic branch length is the result of a cumulative process over evolutionary time, we hypothesize that a latitudinal layering of populations has generally been maintained during repeated cycles of glaciation, possibly due to a neutral spatial effect and/or local adaptive advantage. This trans‐glacial latitudinal layering could be viewed as a particular case of the more general phenomenon of intraspecific structuring that is created and maintained in a fluctuating environmental gradient.