Respiratory virus transmission dynamics determine timing of asthma exacerbation peaks: Evidence from a population-level model

Asthma exacerbations exhibit a consistent annual pattern, closely mirroring the school calendar. Although respiratory viruses—the “common cold” viruses—are implicated as a principal cause, there is little evidence to link viral prevalence to seasonal differences in risk. We jointly fit a common cold transmission model and a model of biological and environmental exacerbation triggers to estimate effects on hospitalization risk. Asthma hospitalization rate, influenza prevalence, and air quality measures are available, but common cold circulation is not; therefore, we generate estimates of viral prevalence using a transmission model. Our deterministic multivirus transmission model includes transmission rates that vary when school is closed. We jointly fit the two models to 7 y of daily asthma hospitalizations in adults and children (66,000 events) in eight metropolitan areas. For children, we find that daily viral prevalence is the strongest predictor of asthma hospitalizations, with transmission reduced by 45% (95% credible interval =41–49%) during school closures. We detect a transient period of nonspecific immunity between infections lasting 19 (17–21) d. For adults, hospitalizations are more variable, with influenza driving wintertime peaks. Neither particulate matter nor ozone was an important predictor, perhaps because of the large geographic area of the populations. The school calendar clearly and predictably drives seasonal variation in common cold prevalence, which results in the “back-to-school” asthma exacerbation pattern seen in children and indirectly contributes to exacerbation risk in adults. This study provides a framework for anticipating the seasonal dynamics of common colds and the associated risks for asthmatics.Asthma is a chronic airway condition with increasing prevalence in many countries (1, 2). Exacerbations, the worsening of asthma symptoms, are a growing public health concern, resulting in millions of missed work and school days and $50 billion in direct healthcare costs in the United States each year (3–5). Prior studies have examined environmental correlates of asthma exacerbations, including air quality measures (6–8), whereas others have considered the role of respiratory virus infections in triggering asthma exacerbation (9–13). However, none have simultaneously considered both infectious and noninfectious factors that potentially influence the large-scale spatiotemporal dynamics of asthma exacerbations.Asthma-related hospitalizations exhibit an extraordinarily consistent seasonal pattern from year to year (14). In children, this pattern strongly reflects the school calendar (15–17). A wave of asthma exacerbations in children ensues shortly after the return to school after summer break (shown in Fig. 1 for Texas in mid-August). The return-to-school peak has been termed the “September epidemic of asthma” (14) and noted in the United Kingdom (18), Canada (17, 19), and elsewhere (20). Asthma hospitalizations also seem to rise after the 2-wk winter holiday (late December through early January) and 1-wk spring break (late March) (Fig. 1).Open in a separate windowFig. 1.Daily number of asthma hospitalizations. Total hospitalizations in the eight largest metropolitan areas in Texas from August of 2004 to August of 2005, where markers indicate the first day of the month. Daily count values (light gray) and a spline-smoothed value (dark gray) in (A) children ages 5–18 y old and (B) adults ages 19–55 y old. In 2004, most Texas schools started in mid-August, took a 2-wk winter break in late December to early January, and took a 1-wk spring break in late March.Respiratory virus infections, including those responsible for the common cold, are known to cause exacerbations in asthmatic children and to a lesser extent, adults suffering from respiratory diseases (11, 21, 22). In particular, rhinovirus has been widely implicated in asthma exacerbations and wheezing-related hospitalizations (13, 16, 23–29). Although asthma is not infectious, these aggravating viruses are infectious. Consequently, the dynamics of asthma hospitalizations can seem as if children are serving as transmission vectors for exacerbations (19). Data on the prevalence of these common viruses are infrequently available and are never available for large sample sizes.Common cold viruses spread rampantly—typically causing two to four relatively mild infections in adults and three to eight infections in children annually (30, 31). Although asthmatics tend to experience more severe and prolonged illness on infection, studies suggest that the frequency of infection is similar for asthmatics and nonasthmatics (21). However, little is known about the transmission dynamics of these viruses or the extent to which they account for the complex annual cycles of asthma exacerbations. Mathematical models of viral transmission are widely used for estimating epidemiological parameters, such as transmission rates, from disease surveillance data (32–34). Such data are rare for common colds, because most infections are subclinical, never entering the healthcare system. Here, we exploit asthmatics as a “sentinel” population for the common cold to infer the transmission dynamics of these viruses.Although viral infections are an important trigger for asthma exacerbations, they are not the only cause of asthma hospitalizations. In particular, poor air quality is thought to be a critical risk factor, and the link between pollution and asthma has been studied extensively (8, 35–39). Elevated particulate matter and ozone levels have both been associated with increased asthma exacerbations and hospitalization events.By fitting a mathematical model of viral transmission jointly with a model of noninfectious drivers to asthma hospitalization data from eight metropolitan populations in Texas, we are able to both estimate epidemiological characteristics of common cold viruses and rigorously assess the relative contributions of proposed infectious and noninfectious drivers of asthma exacerbations. Our analysis provides insight into the dynamics of common cold viruses and a robust framework for predicting times of heightened risk and thus, key periods for clinical intervention in the growing population of asthmatic people.     

Self-assembly of amphiphilic Janus dendrimers into uniform onion-like dendrimersomes with predictable size and number of bilayers

A constitutional isomeric library synthesized by a modular approach has been used to discover six amphiphilic Janus dendrimer primary structures, which self-assemble into uniform onion-like vesicles with predictable dimensions and number of internal bilayers. These vesicles, denoted onion-like dendrimersomes, are assembled by simple injection of a solution of Janus dendrimer in a water-miscible solvent into water or buffer. These dendrimersomes provide mimics of double-bilayer and multibilayer biological membranes with dimensions and number of bilayers predicted by the Janus compound concentration in water. The simple injection method of preparation is accessible without any special equipment, generating uniform vesicles, and thus provides a promising tool for fundamental studies as well as technological applications in nanomedicine and other fields.Most living organisms contain single-bilayer membranes composed of lipids, glycolipids, cholesterol, transmembrane proteins, and glycoproteins (1). Gram-negative bacteria (2, 3) and the cell nucleus (4), however, exhibit a strikingly special envelope that consists of a concentric double-bilayer membrane. More complex membranes are also encountered in cells and their various organelles, such as multivesicular structures of eukaryotic cells (5) and endosomes (6), and multibilayer structures of endoplasmic reticulum (7, 8), myelin (9, 10), and multilamellar bodies (11, 12). This diversity of biological membranes inspired corresponding biological mimics. Liposomes (Fig. 1) self-assembled from phospholipids are the first mimics of single-bilayer biological membranes (13–16), but they are polydisperse, unstable, and permeable (14). Stealth liposomes coassembled from phospholipids, cholesterol, and phospholipids conjugated with poly(ethylene glycol) exhibit improved stability, permeability, and mechanical properties (17–20). Polymersomes (21–24) assembled from amphiphilic block copolymers exhibit better mechanical properties and permeability, but are not always biocompatible and are polydisperse. Dendrimersomes (25–28) self-assembled from amphiphilic Janus dendrimers and minidendrimers (26–28) have also been elaborated to mimic single-bilayer biological membranes. Amphiphilic Janus dendrimers take advantage of multivalency both in their hydrophobic and hydrophilic parts (23, 29–32). Dendrimersomes are assembled by simple injection (33) of a solution of an amphiphilic Janus dendrimer (26) in a water-soluble solvent into water or buffer and produce uniform (34), impermeable, and stable vesicles with excellent mechanical properties. In addition, their size and properties can be predicted by their primary structure (27). Amphiphilic Janus glycodendrimers self-assemble into glycodendrimersomes that mimic the glycan ligands of biological membranes (35). They have been demonstrated to be bioactive toward biomedically relevant bacterial, plant, and human lectins, and could have numerous applications in nanomedicine (20).Open in a separate windowFig. 1.Strategies for the preparation of single-bilayer vesicles and multibilayer onion-like vesicles.More complex and functional cell mimics such as multivesicular vesicles (36, 37) and multibilayer onion-like vesicles (38–40) have also been discovered. Multivesicular vesicles compartmentalize a larger vesicle (37) whereas multibilayer onion-like vesicles consist of concentric alternating bilayers (40). Currently multibilayer vesicles are obtained by very complex and time-consuming methods that do not control their size (39) and size distribution (40) in a precise way. Here we report the discovery of “single–single” (28) amphiphilic Janus dendrimer primary structures that self-assemble into uniform multibilayer onion-like dendrimersomes (Fig. 1) with predictable size and number of bilayers by simple injection of their solution into water or buffer.     

The evolution of self-control

Cognition presents evolutionary research with one of its greatest challenges. Cognitive evolution has been explained at the proximate level by shifts in absolute and relative brain volume and at the ultimate level by differences in social and dietary complexity. However, no study has integrated the experimental and phylogenetic approach at the scale required to rigorously test these explanations. Instead, previous research has largely relied on various measures of brain size as proxies for cognitive abilities. We experimentally evaluated these major evolutionary explanations by quantitatively comparing the cognitive performance of 567 individuals representing 36 species on two problem-solving tasks measuring self-control. Phylogenetic analysis revealed that absolute brain volume best predicted performance across species and accounted for considerably more variance than brain volume controlling for body mass. This result corroborates recent advances in evolutionary neurobiology and illustrates the cognitive consequences of cortical reorganization through increases in brain volume. Within primates, dietary breadth but not social group size was a strong predictor of species differences in self-control. Our results implicate robust evolutionary relationships between dietary breadth, absolute brain volume, and self-control. These findings provide a significant first step toward quantifying the primate cognitive phenome and explaining the process of cognitive evolution.Since Darwin, understanding the evolution of cognition has been widely regarded as one of the greatest challenges for evolutionary research (1). Although researchers have identified surprising cognitive flexibility in a range of species (2–40) and potentially derived features of human psychology (41–61), we know much less about the major forces shaping cognitive evolution (62–71). With the notable exception of Bitterman’s landmark studies conducted several decades ago (63, 72–74), most research comparing cognition across species has been limited to small taxonomic samples (70, 75). With limited comparable experimental data on how cognition varies across species, previous research has largely relied on proxies for cognition (e.g., brain size) or metaanalyses when testing hypotheses about cognitive evolution (76–92). The lack of cognitive data collected with similar methods across large samples of species precludes meaningful species comparisons that can reveal the major forces shaping cognitive evolution across species, including humans (48, 70, 89, 93–98).To address these challenges we measured cognitive skills for self-control in 36 species of mammals and birds (Fig. 1 and Tables S1–S4) tested using the same experimental procedures, and evaluated the leading hypotheses for the neuroanatomical underpinnings and ecological drivers of variance in animal cognition. At the proximate level, both absolute (77, 99–107) and relative brain size (108–112) have been proposed as mechanisms supporting cognitive evolution. Evolutionary increases in brain size (both absolute and relative) and cortical reorganization are hallmarks of the human lineage and are believed to index commensurate changes in cognitive abilities (52, 105, 113–115). Further, given the high metabolic costs of brain tissue (116–121) and remarkable variance in brain size across species (108, 122), it is expected that the energetic costs of large brains are offset by the advantages of improved cognition. The cortical reorganization hypothesis suggests that selection for absolutely larger brains—and concomitant cortical reorganization—was the predominant mechanism supporting cognitive evolution (77, 91, 100–106, 120). In contrast, the encephalization hypothesis argues that an increase in brain volume relative to body size was of primary importance (108, 110, 111, 123). Both of these hypotheses have received support through analyses aggregating data from published studies of primate cognition and reports of “intelligent” behavior in nature—both of which correlate with measures of brain size (76, 77, 84, 92, 110, 124).Open in a separate windowFig. 1.A phylogeny of the species included in this study. Branch lengths are proportional to time except where long branches have been truncated by parallel diagonal lines (split between mammals and birds ∼292 Mya).With respect to selective pressures, both social and dietary complexities have been proposed as ultimate causes of cognitive evolution. The social intelligence hypothesis proposes that increased social complexity (frequently indexed by social group size) was the major selective pressure in primate cognitive evolution (6, 44, 48, 50, 87, 115, 120, 125–141). This hypothesis is supported by studies showing a positive correlation between a species’ typical group size and the neocortex ratio (80, 81, 85–87, 129, 142–145), cognitive differences between closely related species with different group sizes (130, 137, 146, 147), and evidence for cognitive convergence between highly social species (26, 31, 148–150). The foraging hypothesis posits that dietary complexity, indexed by field reports of dietary breadth and reliance on fruit (a spatiotemporally distributed resource), was the primary driver of primate cognitive evolution (151–154). This hypothesis is supported by studies linking diet quality and brain size in primates (79, 81, 86, 142, 155), and experimental studies documenting species differences in cognition that relate to feeding ecology (94, 156–166).Although each of these hypotheses has received empirical support, a comparison of the relative contributions of the different proximate and ultimate explanations requires (i) a cognitive dataset covering a large number of species tested using comparable experimental procedures; (ii) cognitive tasks that allow valid measurement across a range of species with differing morphology, perception, and temperament; (iii) a representative sample within each species to obtain accurate estimates of species-typical cognition; (iv) phylogenetic comparative methods appropriate for testing evolutionary hypotheses; and (v) unprecedented collaboration to collect these data from populations of animals around the world (70).Here, we present, to our knowledge, the first large-scale collaborative dataset and comparative analysis of this kind, focusing on the evolution of self-control. We chose to measure self-control—the ability to inhibit a prepotent but ultimately counterproductive behavior—because it is a crucial and well-studied component of executive function and is involved in diverse decision-making processes (167–169). For example, animals require self-control when avoiding feeding or mating in view of a higher-ranking individual, sharing food with kin, or searching for food in a new area rather than a previously rewarding foraging site. In humans, self-control has been linked to health, economic, social, and academic achievement, and is known to be heritable (170–172). In song sparrows, a study using one of the tasks reported here found a correlation between self-control and song repertoire size, a predictor of fitness in this species (173). In primates, performance on a series of nonsocial self-control control tasks was related to variability in social systems (174), illustrating the potential link between these skills and socioecology. Thus, tasks that quantify self-control are ideal for comparison across taxa given its robust behavioral correlates, heritable basis, and potential impact on reproductive success.In this study we tested subjects on two previously implemented self-control tasks. In the A-not-B task (27 species, n = 344), subjects were first familiarized with finding food in one location (container A) for three consecutive trials. In the test trial, subjects initially saw the food hidden in the same location (container A), but then moved to a new location (container B) before they were allowed to search (Movie S1). In the cylinder task (32 species, n = 439), subjects were first familiarized with finding a piece of food hidden inside an opaque cylinder. In the following 10 test trials, a transparent cylinder was substituted for the opaque cylinder. To successfully retrieve the food, subjects needed to inhibit the impulse to reach for the food directly (bumping into the cylinder) in favor of the detour response they had used during the familiarization phase (Movie S2).Thus, the test trials in both tasks required subjects to inhibit a prepotent motor response (searching in the previously rewarded location or reaching directly for the visible food), but the nature of the correct response varied between tasks. Specifically, in the A-not-B task subjects were required to inhibit the response that was previously successful (searching in location A) whereas in the cylinder task subjects were required to perform the same response as in familiarization trials (detour response), but in the context of novel task demands (visible food directly in front of the subject).     

Solid-phase peptide synthesis and solid-phase fragment coupling mediated by isonitriles

The synthesis of polypeptides on solid phase via mediation by isonitriles is described. The acyl donor is a thioacid, which presumably reacts with the isonitrile to generate a thio-formimidate carboxylate mixed anhydride intermediate. Applications of this chemistry to reiterative solid-phase peptide synthesis as well as solid-phase fragment coupling are described.Amide bond formations are arguably among the most important constructions in organic chemistry (1, 2). The centrality of the amide linkage, as found in polypeptides and proteins, in the maintenance of life hardly needs restatement. Numerous strategies, resulting in a vast array of protocols to synthesize biologically active polypeptides and proteins, have been demonstrated (3, 4). Central to reiterative polypeptide bond formations was the discovery and remarkable development of solid-phase peptide synthesis (SPPS) (5, 6). The extraordinary impact of SPPS in fostering enhanced access to homogeneous polypeptides is clear to everyone in the field.As we have described elsewhere, by classical, mechanistic reasoning, we were led to conjecture about some hitherto-unexplored possibilities relevant to the chemistry of isonitriles (7–14). It was anticipated that isonitriles might be able to mediate the acylation of amines, thus giving rise to amides (15). Early experiments focused on free carboxylic acids as the acylating agents. As our studies progressed, it was found that the combination of thioacids, amines, and isonitriles leads to the efficient formation of amide bonds under stoichiometric or near-stoichiometric conditions (7–13, 16, 17). Although there remain unresolved issues of detail and nuance, the governing mechanism for amide formation under these conditions involves reaction of the thioacid, 1, with an isonitrile, 2, to generate a thio-formimidate carboxylate mixed anhydride (thio-FCMA), 3, which is intercepted by the “acyl-accepting” amine to generate amide, 5, and thioformamide, 6 (Fig. 1). The efficiency of the amidation was further improved through the use of hydroxybenzotriazole (HOBt) (18), which could well give rise to HOBt ester 7, although this pathway has not been mechanistically proven.Open in a separate windowFig. 1.Isonitrile-mediated amidation; structure of OT.The potentialities of the isonitrile-mediated amidation method were foreshadowed via its application to the synthesis of cyclosporine (19). The power of the method was particularly well demonstrated in the context of our recent total synthesis of oxytocin (OT) (20), wherein isonitrile mediation was used in each of the peptide bond constructions, leading to the synthesis of the hormone in high yield and excellent purity. This nonapeptide is involved in a range of biological functions including parturition and lactation (21, 22). Signaling of OT to its receptor (OTR) is apparently an important factor in quality maintenance of various CNS functions (23). The ability to synthesize such modestly sized, but bio-impactful peptides in both native (wild-type) form, and as strategically modified variants, is one of the current missions of our laboratory, with the objective of possible applications to the very serious problem of autism (24–26).     

De novo production of the plant-derived alkaloid strictosidine in yeast

The monoterpene indole alkaloids are a large group of plant-derived specialized metabolites, many of which have valuable pharmaceutical or biological activity. There are ∼3,000 monoterpene indole alkaloids produced by thousands of plant species in numerous families. The diverse chemical structures found in this metabolite class originate from strictosidine, which is the last common biosynthetic intermediate for all monoterpene indole alkaloid enzymatic pathways. Reconstitution of biosynthetic pathways in a heterologous host is a promising strategy for rapid and inexpensive production of complex molecules that are found in plants. Here, we demonstrate how strictosidine can be produced de novo in a Saccharomyces cerevisiae host from 14 known monoterpene indole alkaloid pathway genes, along with an additional seven genes and three gene deletions that enhance secondary metabolism. This system provides an important resource for developing the production of more complex plant-derived alkaloids, engineering of nonnatural derivatives, identification of bottlenecks in monoterpene indole alkaloid biosynthesis, and discovery of new pathway genes in a convenient yeast host.Monoterpene indole alkaloids (MIAs) are a diverse family of complex nitrogen-containing plant-derived metabolites (1, 2). This metabolite class is found in thousands of plant species from the Apocynaceae, Loganiaceae, Rubiaceae, Icacinaceae, Nyssaceae, and Alangiaceae plant families (2, 3). Many MIAs and MIA derivatives have medicinal properties; for example, vinblastine, vincristine, and vinflunine are approved anticancer therapeutics (4, 5). These structurally complex compounds can be difficult to chemically synthesize (6, 7). Consequently, industrial production relies on extraction from the plant, but these compounds are often produced in small quantities as complex mixtures, making isolation challenging, laborious, and expensive (8–10). Reconstitution of plant pathways in microbial hosts is proving to be a promising approach to access plant-derived compounds as evidenced by the successful production of terpenes, flavonoids, and benzylisoquinoline alkaloids in microorganisms (11–19). Microbial hosts can also be used to construct hybrid biosynthetic pathways to generate modified natural products with potentially enhanced bioactivities (8, 20, 21). Across numerous plant species, strictosidine is believed to be the core scaffold from which all 3,000 known MIAs are derived (1, 2). Strictosidine undergoes a variety of redox reactions and rearrangements to form the thousands of compounds that comprise the MIA natural product family (Fig. 1) (1, 2). Due to the importance of strictosidine, the last common biosynthetic intermediate for all known MIAs, we chose to focus on heterologous production of this complex molecule (1). Therefore, strictosidine reconstitution represents the necessary first step for heterologous production of high-value MIAs.Open in a separate windowFig. 1.Strictosidine, the central intermediate in monoterpene indole alkaloid (MIA) biosynthesis, undergoes a series of reactions to produce over 3,000 known MIAs such as vincristine, quinine, and strychnine.     

Detection of genomic variations and DNA polymorphisms and impact on analysis of meiotic recombination and genetic mapping

DNA polymorphisms are important markers in genetic analyses and are increasingly detected by using genome resequencing. However, the presence of repetitive sequences and structural variants can lead to false positives in the identification of polymorphic alleles. Here, we describe an analysis strategy that minimizes false positives in allelic detection and present analyses of recently published resequencing data from Arabidopsis meiotic products and individual humans. Our analysis enables the accurate detection of sequencing errors, small insertions and deletions (indels), and structural variants, including large reciprocal indels and copy number variants, from comparisons between the resequenced and reference genomes. We offer an alternative interpretation of the sequencing data of meiotic products, including the number and type of recombination events, to illustrate the potential for mistakes in single-nucleotide polymorphism calling. Using these examples, we propose that the detection of DNA polymorphisms using resequencing data needs to account for nonallelic homologous sequences.DNA polymorphisms are ubiquitous genetic variations among individuals and include single nucleotide polymorphisms (SNPs), insertions and deletions (indels), and other larger rearrangements (1–3) (Fig. 1 A and B). They can have phenotypic consequences and also serve as molecular markers for genetic analyses, facilitating linkage and association studies of genetic diseases, and other traits in humans (4–6), animals, plants, (7–10) and other organisms. Using DNA polymorphisms for modern genetic applications requires low-error, high-throughput analytical strategies. Here, we illustrate the use of short-read next-generation sequencing (NGS) data to detect DNA polymorphisms in the context of whole-genome analysis of meiotic products.Open in a separate windowFig. 1.(A) SNPs and small indels between two ecotype genomes. (B) Possible types of SVs. Col genotypes are marked in blue and Ler in red. Arrows indicate DNA segments involved in SVs between the two ecotypes. (C) Meiotic recombination events including a CO and a GC (NCO). Centromeres are denoted by yellow dots.There are many methods for detecting SNPs (11–14) and structural variants (SVs) (15–25), including NGS, which can capture nearly all DNA polymorphisms (26–28). This approach has been widely used to analyze markers in crop species such as rice (29), genes associated with diseases (6, 26), and meiotic recombination in yeast and plants (30, 31). However, accurate identification of DNA polymorphisms can be challenging, in part because short-read sequencing data have limited information for inferring chromosomal context.Genomes usually contain repetitive sequences that can differ in copy number between individuals (26–28, 31); therefore, resequencing analyses must account for chromosomal context to avoid mistaking highly similar paralogous sequences for polymorphisms. Here, we use recently published datasets to describe several DNA sequence features that can be mistaken as allelic (32, 33) and describe a strategy for differentiating between repetitive sequences and polymorphic alleles. We illustrate the effectiveness of these analyses by examining the reported polymorphisms from the published datasets.Meiotic recombination is initiated by DNA double-strand breaks (DSBs) catalyzed by the topoisomerase-like SPORULATION 11 (SPO11). DSBs are repaired as either crossovers (COs) between chromosomes (Fig. 1C), or noncrossovers (NCOs). Both COs and NCOs can be accompanied by gene conversion (GC) events, which are the nonreciprocal transfer of sequence information due to the repair of heteroduplex DNA during meiotic recombination. Understanding the control of frequency and distribution of CO and NCO (including GC) events has important implications for human health (including cancer and aneuploidy), crop breeding, and the potential for use in genome engineering. COs can be detected relatively easily by using polymorphic markers in the flanking sequences, but NCO products can only be detected if they are accompanied by a GC event. Because GCs associated with NCO result in allelic changes at polymorphic sites without exchange of flanking sequences, they are more difficult to detect. Recent advances in DNA sequencing have made the analysis of meiotic NCOs more feasible (30–32, 34); however, SVs present a challenge in these analyses. We recommend a set of guidelines for detection of DNA polymorphisms by using genomic resequencing short-read datasets. These measures improve the accuracy of a wide range of analyses by using genomic resequencing, including estimation of COs, NCOs, and GCs.     

Understanding the kinetic mechanism of RNA single base pair formation

RNA functions are intrinsically tied to folding kinetics. The most elementary step in RNA folding is the closing and opening of a base pair. Understanding this elementary rate process is the basis for RNA folding kinetics studies. Previous studies mostly focused on the unfolding of base pairs. Here, based on a hybrid approach, we investigate the folding process at level of single base pairing/stacking. The study, which integrates molecular dynamics simulation, kinetic Monte Carlo simulation, and master equation methods, uncovers two alternative dominant pathways: Starting from the unfolded state, the nucleotide backbone first folds to the native conformation, followed by subsequent adjustment of the base conformation. During the base conformational rearrangement, the backbone either retains the native conformation or switches to nonnative conformations in order to lower the kinetic barrier for base rearrangement. The method enables quantification of kinetic partitioning among the different pathways. Moreover, the simulation reveals several intriguing ion binding/dissociation signatures for the conformational changes. Our approach may be useful for developing a base pair opening/closing rate model.RNAs perform critical cellular functions at the level of gene expression and regulation (1–4). RNA functions are determined not only by RNA structure or structure motifs [e.g., tetraloop hairpins (5, 6)] but also by conformational distributions and dynamics and kinetics of conformational changes. For example, riboswitches can adopt different conformations in response to specific conditions of the cellular environment (7, 8). Understanding the kinetics, such as the rate and pathways for the conformational changes, is critical for deciphering the mechanism of RNA function (9–19). Extensive experimental and theoretical studies on RNA folding kinetics have provided significant insights into the kinetic mechanism of RNA functions (19–36). However, due to the complexity of the RNA folding energy landscape (37–46) and the limitations of experimental tools (47–55), many fundamental problems, including single base flipping and base pair formation and fraying, remain unresolved. These unsolved fundamental problems have hampered our ability to resolve other important issues, such as RNA hairpin and larger structure folding kinetics. Several key questions remain unanswered, such as whether the hairpin folding is rate-limited by the conformational search of the native base pairs, whose formation leads to fast downhill folding of the whole structure, or by the breaking of misfolded base pairs before refolding to the native structure (18, 19, 54–73).Motivated by the need to understand the basic steps of nucleic acids folding, Hagan et al. (74) performed forty-three 200-ps unfolding trajectories at 400 K and identified both on- and off-pathway intermediates and two dominant unfolding pathways for a terminal C-G base pair in a DNA duplex. In one of the pathways, base pairing and stacking interactions are broken concomitantly, whereas in the other pathway, base stacking is broken after base pairing is disrupted. Furthermore, the unfolding requires that the Cyt diffuse away from the pairing Gua to a distance such that the C-G hydrogen bond cannot reform easily. More recently, Colizzi and Bussi (75) performed molecular dynamics (MD) pulling simulations for an RNA duplex and construct free energy landscape from the pulling simulation. The simulation showed that the base pair opening reaction starts with the unbinding of the 5′-base, followed by the unbinding of the 3′-base (i.e., the 5′-base is less stable than the 3′-base). These previous unfolding simulations offered significant insights into the pathways and transition states. However, as shown below, several important issues remain.One intriguing problem is the rate model for base pairing. There are currently three main types of models. In the first type of model, the barrier ΔG+‡ for closing a base pair is dominated by the entropic cost ΔS for positioning the nucleotides to the base-paired configuration and the barrier ΔG−‡ for opening a base pair is the enthalpic cost ΔH for disrupting the hydrogen bonds and base stacking interactions (18, 59, 60). In the second type of model, ΔG+‡ is the net free energy change for base pairing ΔG = ΔH ? TΔS and ΔG−‡ is zero (76, 77). In the third type of model, ΔG±‡=±ΔG/2 is used (78). In addition to the above three main types, other models, such as more sophisticated hybrid rate models, have been proposed (29).In this paper, we report a hybrid method (see Fig. 1) to investigate the single base pairing process. In contrast to the previous simulations for temperature- or force-induced unfolding reactions, we directly model the folding process here (i.e., the base pair closing process). Specifically, we use MD simulations to identify the conformational clusters. Based on the network of the conformational clusters as a reduced conformational ensemble, we apply kinetic Monte Carlo (KMC) and master equation (ME) methods to elucidate the detailed roles of base pairing and stacking interactions, as well as the roles of water and ions (79–82). The study reveals previously unidentified kinetics pathways, misfolded states, and rate-limiting steps. A clear understanding of the microscopic details of the elementary kinetic move is a prerequisite for further rigorous study of large-scale RNA kinetic studies. The method described here may provide a feasible way to develop a rate model for the base pair/stack-based kinetic move set. Furthermore, the mechanism of RNA single base folding may provide useful insights into many biologically significant processes, such as nucleotide flipping (83) in helicases and base pair fraying (84) (as the possible first step for nucleic duplex melting in nucleic acid enzymatic processes).Open in a separate windowFig. 1.(A) Folding of a single nucleotide (G1, red) from the unfolded (Left) to the native folded (Right) state. (B) Exhaustive sampling for the (discrete) conformations of the G1 nucleotide (Right) through enumeration of the torsion angles (formed by the blue bonds). (C) Schematic plot shows the trajectories on the energy landscape (depicted with two reaction coordinates for clarity) explored by the MD simulations. The lines, open circles, and hexagons denote the trajectories; the initial states; and the (centroid structures of the) clusters, respectively. (D) Conformational network based on six clusters. (E) The rmsds to the different clusters provide information about the structural changes in a MD trajectory.     

Contingency and entrenchment in protein evolution under purifying selection

The phenotypic effect of an allele at one genetic site may depend on alleles at other sites, a phenomenon known as epistasis. Epistasis can profoundly influence the process of evolution in populations and shape the patterns of protein divergence across species. Whereas epistasis between adaptive substitutions has been studied extensively, relatively little is known about epistasis under purifying selection. Here we use computational models of thermodynamic stability in a ligand-binding protein to explore the structure of epistasis in simulations of protein sequence evolution. Even though the predicted effects on stability of random mutations are almost completely additive, the mutations that fix under purifying selection are enriched for epistasis. In particular, the mutations that fix are contingent on previous substitutions: Although nearly neutral at their time of fixation, these mutations would be deleterious in the absence of preceding substitutions. Conversely, substitutions under purifying selection are subsequently entrenched by epistasis with later substitutions: They become increasingly deleterious to revert over time. Our results imply that, even under purifying selection, protein sequence evolution is often contingent on history and so it cannot be predicted by the phenotypic effects of mutations assayed in the ancestral background.Whether a heritable mutation is advantageous or deleterious to an organism often depends on the evolutionary history of the population. A mutation that is beneficial at the time of its introduction may confer its beneficial effect only in the presence of other potentiating or permissive mutations (1–9). Thus, the fate of a mutation arising in a population may be contingent on previous mutations (10–13). Conversely, once a mutation has fixed in a population, the mutation becomes part of the genetic background onto which subsequent modifications are introduced. Because the beneficial effects of the subsequent modifications may depend on the focal mutation, as time passes reversion of the focal mutation may become increasingly deleterious, leading to a type of evolutionary conservatism, or entrenchment (14–18).In the context of protein evolution, the effects of contingency and entrenchment are most easily studied by considering a sequence of single amino acid changes (19) that extends both forward and backward in time from some focal substitution. To assess the roles of contingency and entrenchment we can study the degree to which each focal substitution was facilitated by previous substitutions, and the degree to which the focal substitution influences the subsequent course of evolution (Fig. 1A).Open in a separate windowFig. 1.(A) A schematic model indicating how a focal substitution may be contingent on prior substitutions and may constrain future substitutions along an evolutionary trajectory, owing to epistasis. (B) A model of protein evolution under weak mutation and purifying selection for thermodynamic stability. Starting from the wild-type sequence of argT we propose 10 random 1-aa point mutations. For each of the proposed mutants we compute its predicted stability (ΔG) using FoldX, and its associated fitness. The fitness function is assumed to be either Gaussian or semi-Gaussian, with a maximum at the wild-type stability. One of the proposed mutants fixes in the population, based on its relative fixation probability under the Moran model with effective population size N_e. This process is iterated for 30 consecutive substitutions to produce an evolutionary trajectory. We simulate 100 replicate trajectories, each initiated at the wild-type argT sequence.Dependencies within a sequence of substitutions are closely connected to the concept of epistasis—that is, the idea that the phenotypic effect of a mutation at a particular genetic site may depend on the genetic background in which it arises (20–24). In the absence of epistasis, a mutation has the same effect regardless of its context and therefore regardless of any prior history or subsequent evolution. By contrast, in the presence of epistasis, each substitution may be contingent on the entire prior history of the protein, and it may constrain all subsequent evolution.The potential for epistasis to play an important role in evolution, including protein evolution, has not been overlooked by researchers (1, 8, 25–34), nor have the concepts of contingency (3, 4, 9, 12, 35–38) and, more recently, entrenchment (18, 39, 40). However, most studies have addressed the role of epistasis in the context of adaptive evolution (1–9, 27, 30, 31, 36, 38), whereas the consequences of epistasis under purifying selection have received less attention (18, 41–44). Indeed, although some more sophisticated models have been proposed (e.g., refs. 45–50), all commonly used phylogenetic models of long-term protein evolution assume that epistasis is absent so that sites evolve independently (51–56).Here we explore the relationships between epistasis, contingency, and entrenchment under long-term purifying selection on protein stability. Our analysis combines computational models for protein structures with population-genetic models for evolutionary dynamics. We use a force-field-based model, FoldX (57), to characterize the effects of point mutations on a protein’s stability and fitness. This approach allows us to simulate evolutionary trajectories of protein sequences under purifying selection, by the sequential fixation of nearly neutral mutations. We can then dissect the epistatic relationships between these substitutions by systematically inserting or reverting particular substitutions at various time points along the evolutionary trajectory.Our analysis considers epistasis both at the level of protein stability and at the level of fitness. Whereas empirical studies in diverse proteins have demonstrated that the stability effects of point mutations are typically additive across sites (58, 59), in this study we are specifically interested in epistasis for stability among the mutations that fix during evolution. Even if most random mutations are virtually additive in their effects on stability, the mutations that fix under purifying selection are highly nonrandom, and so there is reason to suspect that epistasis for stability may be enriched among such mutations. Moreover, because the mapping from stability to fitness is itself nonlinear (18, 26, 60, 61) and because selection is sensitive to selection coefficients as small as the inverse of the population size (62), even slight variation in the stability effects of mutations across different genetic backgrounds may be sufficient to influence the course of evolution.Using the computational approach summarized above, we will demonstrate that the nearly neutral mutations that fix under purifying selection are, indeed, often epistatic with each other for both stability and fitness. In particular, we find that each mutation that fixes is typically permitted to fix by the presence of preceding substitutions—that is, most substitutions would be too deleterious to fix were it not for epistasis with preceding substitutions. Conversely, we also find that mutations that fix typically become entrenched over time by epistasis—so that a substitution that was nearly neutral when it fixed becomes increasingly deleterious to revert as subsequent substitutions accumulate (18, 39). These results imply an important role for epistasis in shaping the course of sequence evolution in a protein under selection to maintain thermodynamic stability.     

Single-molecule spectroscopy reveals how calmodulin activates NO synthase by controlling its conformational fluctuation dynamics

Mechanisms that regulate the nitric oxide synthase enzymes (NOS) are of interest in biology and medicine. Although NOS catalysis relies on domain motions, and is activated by calmodulin binding, the relationships are unclear. We used single-molecule fluorescence resonance energy transfer (FRET) spectroscopy to elucidate the conformational states distribution and associated conformational fluctuation dynamics of the two electron transfer domains in a FRET dye-labeled neuronal NOS reductase domain, and to understand how calmodulin affects the dynamics to regulate catalysis. We found that calmodulin alters NOS conformational behaviors in several ways: It changes the distance distribution between the NOS domains, shortens the lifetimes of the individual conformational states, and instills conformational discipline by greatly narrowing the distributions of the conformational states and fluctuation rates. This information was specifically obtainable only by single-molecule spectroscopic measurements, and reveals how calmodulin promotes catalysis by shaping the physical and temporal conformational behaviors of NOS.Although proteins adopt structures determined by their amino acid sequences, they are not static objects and fluctuate among ensembles of conformations (1). Transitions between these states can occur on a variety of length scales (Å to nm) and time scales (ps to s) and have been linked to functionally relevant phenomena such as allosteric signaling, enzyme catalysis, and protein–protein interactions (2–4). Indeed, protein conformational fluctuations and dynamics, often associated with static and dynamic inhomogeneity, are thought to play a crucial role in biomolecular functions (5–11). It is difficult to characterize such spatially and temporally inhomogeneous dynamics in bulk solution by an ensemble-averaged measurement, especially in proteins that undergo multiple-conformation transformations. In such cases, single-molecule spectroscopy is a powerful approach to analyze protein conformational states and dynamics under physiological conditions, and can provide a molecular-level perspective on how a protein’s structural dynamics link to its functional mechanisms (12–21).A case in point is the nitric oxide synthase (NOS) enzymes (22–24), whose nitric oxide (NO) biosynthesis involves electron transfer reactions that are associated with relatively large-scale movement (tens of Å) of the enzyme domains (Fig. 1A). During catalysis, NADPH-derived electrons first transfer into an FAD domain and an FMN domain in NOS that together comprise the NOS reductase domain (NOSr), and then transfer from the FMN domain to a heme group that is bound in a separate attached “oxygenase” domain, which then enables NO synthesis to begin (22, 25–27). The electron transfers into and out of the FMN domain are the key steps for catalysis, and they appear to rely on the FMN domain cycling between electron-accepting and electron-donating conformational states (28, 29) (Fig. 1B). In this model, the FMN domain is suggested to be highly dynamic and flexible due to a connecting hinge that allows it to alternate between its electron-accepting (FAD→FMN) or closed conformation and electron-donating (FMN→heme) or open conformation (Fig. 1 A and B) (28, 30–36). In the electron-accepting closed conformation, the FMN domain interacts with the NADPH/FAD domain (FNR domain) to receive electrons, whereas in the electron donating open conformation the FMN domain has moved away to expose the bound FMN cofactor so that it may transfer electrons to a protein acceptor like the NOS oxygenase domain, or to a generic protein acceptor like cytochrome c. In this way, the reductase domain structure cycles between closed and open conformations to deliver electrons, according to a conformational equilibrium that determines the movements and thus the electron flux capacity of the FMN domain (25, 28, 32, 34, 35, 37). A similar conformational switching mechanism is thought to enable electron transfer through the FMN domain in the related flavoproteins NADPH-cytochrome P450 reductase and methionine synthase reductase (38–42).Open in a separate windowFig. 1.(A) The nNOSr ribbon structure (from PDB: 1TLL) showing bound FAD (yellow) in FNR domain (green), FMN (orange) in FMN domain (yellow), connecting hinge (blue), and the Cy3–Cy5 label positions (pink) and distance (42 Å, dashed line). (B) Cartoon of an equilibrium between the FMN-closed and FMN-open states, with Cy dye label positions indicated. (C) Cytochrome c reductase activity of nNOSr proteins in their CaM-bound and CaM-free states. Color scheme of bar graphs: Black, WT nNOSr unlabeled; Red, Cys-lite (CL) nNOSr unlabeled; Blue, E827C/Q1268C CL nNOSr unlabeled; and Dark cyan, E827C/Q1268C CL nNOSr labeled.NOS enzymes also contain a calmodulin (CaM) binding domain that is located just before the N terminus of the FMN domain (Fig. 1B), and this provides an important layer of regulation (25, 27). CaM binding to NOS enzymes increases electron transfer from NADPH through the reductase domain and also triggers electron transfer from the FMN domain to the NOS heme as is required for NO synthesis (31, 32). The ability of CaM, or similar signaling proteins, to regulate electron transfer reactions in enzymes is unusual, and the mechanism is a topic of interest and intensive study. It has long been known that CaM binding alters NOSr structure such that, on average, it populates a more open conformation (43, 44). Recent equilibrium studies have detected a buildup of between two to four discreet conformational populations in NOS enzymes and in related flavoproteins, and in some cases, have also estimated the distances between the bound FAD and FMN cofactors in the different species (26, 36, 37, 39, 40), and furthermore, have confirmed that CaM shifts the NOS population distribution toward more open conformations (34, 36, 45). Although valuable, such ensemble-averaged results about conformational states cannot explain how electrons transfer through these enzymes, or how CaM increases the electron flux in NOS, because answering these questions requires a coordinate understanding of the dynamics of the conformational fluctuations. Indeed, computer modeling has indicated that a shift toward more open conformations as is induced by CaM binding to nNOS should, on its own, actually diminish electron flux through nNOS and through certain related flavoproteins (38). Despite its importance, measuring enzyme conformational fluctuation dynamics is highly challenging, and as far as we know, there have been no direct measures on the NOS enzymes or on related flavoproteins, nor studies on how CaM binding might influence the conformational fluctuation dynamics in NOS.To address this gap, we used single-molecule fluorescence energy resonance transfer (FRET) spectroscopy to characterize individual molecules of nNOSr that had been labeled at two specific positions with Cyanine 3 (Cy3) donor and Cyanine 5 (Cy5) acceptor dye molecules, regarding their conformational states distribution and the associated conformational fluctuation dynamics, which in turn enabled us to determine how CaM binding impacts both parameters. This work provides a unique perspective and a novel study of the NOS enzymes and within the broader flavoprotein family, which includes the mammalian enzymes methionine synthase reductase (MSR) and cytochrome P450 reductase (CPR), and reveals how CaM’s control of the conformational behaviors may regulate the electron transfer reactions of NOS catalysis.     

Robust efficiency and actuator saturation explain healthy heart rate control and variability

The correlation of healthy states with heart rate variability (HRV) using time series analyses is well documented. Whereas these studies note the accepted proximal role of autonomic nervous system balance in HRV patterns, the responsible deeper physiological, clinically relevant mechanisms have not been fully explained. Using mathematical tools from control theory, we combine mechanistic models of basic physiology with experimental exercise data from healthy human subjects to explain causal relationships among states of stress vs. health, HR control, and HRV, and more importantly, the physiologic requirements and constraints underlying these relationships. Nonlinear dynamics play an important explanatory role––most fundamentally in the actuator saturations arising from unavoidable tradeoffs in robust homeostasis and metabolic efficiency. These results are grounded in domain-specific mechanisms, tradeoffs, and constraints, but they also illustrate important, universal properties of complex systems. We show that the study of complex biological phenomena like HRV requires a framework which facilitates inclusion of diverse domain specifics (e.g., due to physiology, evolution, and measurement technology) in addition to general theories of efficiency, robustness, feedback, dynamics, and supporting mathematical tools.Biological systems display a variety of well-known rhythms in physiological signals (1–6), with particular patterns of variability associated with a healthy state (2–6). Decades of research demonstrate that heart rate (HR) in healthy humans has high variability, and loss of this high HR variability (HRV) is correlated with adverse states such as stress, fatigue, physiologic senescence, or disease (6–13). The dominant approach to analysis of HRV has been to focus on statistics and patterns in HR time series that have been interpreted as fractal, chaotic, scale-free, critical, etc. (6–17). The appeal of time series analysis is understandable as it puts HRV in the context of a broad and popular approach to complex systems (5, 18), all while requiring minimal attention to domain-specific (e.g., physiological) details. However, despite intense research activity in this area, there is limited consensus regarding causation or mechanism and minimal clinical application of the observed phenomena (10). This paper takes a completely different approach, aiming for more fundamental rigor (19–24) and methods that have the potential for clinical relevance. Here we use and model data from experimental studies of exercising healthy athletes, to add simple physiological explanations for the largest source of HRV and its changes during exercise. We also present methods that can be used to systematically pursue further explanations about HRV that can generalize to less healthy subjects.Fig. 1 shows the type of HR data analyzed, collected from healthy young athletes (n = 5). The data display responses to changes in muscle work rate on a stationary bicycle during mostly aerobic exercise. Fig. 1A shows three separate exercise sessions with identical workload fluctuations about three different means. With proper sleep, hydration, nutrition, and prevention from overheating, trained athletes can maintain the highest workload in Fig. 1 for hours and the lower and middle levels almost indefinitely. This ability requires robust efficiency: High workloads are sustained while robustly maintaining metabolic homeostasis, a particularly challenging goal in the case of the relatively large, metabolically demanding, and fragile human brain.Open in a separate windowFig. 1.HR responses to simple changes in muscle work rate on a stationary bicycle: Each experimental subject performed separate stationary cycle exercises of ∼10 min for each workload profile, with different means but nearly identical square wave fluctuations around the mean. A typical result is shown from subject 1 for three workload profiles with time on the horizontal axis (zoomed in to focus on a 6-min window). (A) HR (red) and workload (blue); linear local piecewise static fits (black) with different parameters for each exercise. The workload units (most strenuous exercise on top of graph) are shifted and scaled so that the blue curves are also the best global linear fit. (B) Corresponding dynamics fits, either local piecewise linear (black) or global linear (blue). Note that, on all time scales, mean HR increases and variability (HRV) goes down with the increasing workload. Breathing was spontaneous (not controlled).Whereas mean HR in Fig. 1A increases monotonically with workloads, both slow and fast fluctuations (i.e., HRV) in HR are saturating nonlinear functions of workloads, meaning that both high- and low-frequency HRV component goes down. Results from all subjects showed qualitatively similar nonlinearities (SI Appendix). We will argue that this saturating nonlinearity is the simplest and most fundamental example of change in HRV in response to stressors (11, 12, 25) [exercise in the experimental case, but in general also fatigue, dehydration, trauma, infection, even fear and anxiety (6–9, 11, 12, 25)].Physiologists have correlated HRV and autonomic tone (7, 11, 12, 14), and the (im)balance between sympathetic stimulation and parasympathetic withdrawal (12, 26–28). The alternation in autonomic control of HR (more sympathetic and less parasympathetic tone during exercise) serves as an obvious proximate cause for how the HRV changes as shown in Fig. 1, but the ultimate question remains as to why the system is implemented this way. It could be an evolutionary accident, or could follow from hard physiologic tradeoff requirements on cardiovascular control, as work in other systems suggests (1). Here, the explanation of HRV similarly involves hard physiological tradeoffs in robust efficiency and employs the mathematical tools necessary to make this explanation rigorous in the context of large measurement and modeling uncertainties.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Lagging adaptation to warming climate in Arabidopsis thaliana

If climate change outpaces the rate of adaptive evolution within a site, populations previously well adapted to local conditions may decline or disappear, and banked seeds from those populations will be unsuitable for restoring them. However, if such adaptational lag has occurred, immigrants from historically warmer climates will outperform natives and may provide genetic potential for evolutionary rescue. We tested for lagging adaptation to warming climate using banked seeds of the annual weed Arabidopsis thaliana in common garden experiments in four sites across the species’ native European range: Valencia, Spain; Norwich, United Kingdom; Halle, Germany; and Oulu, Finland. Genotypes originating from geographic regions near the planting site had high relative fitness in each site, direct evidence for broad-scale geographic adaptation in this model species. However, genotypes originating in sites historically warmer than the planting site had higher average relative fitness than local genotypes in every site, especially at the northern range limit in Finland. This result suggests that local adaptive optima have shifted rapidly with recent warming across the species’ native range. Climatic optima also differed among seasonal germination cohorts within the Norwich site, suggesting that populations occurring where summer germination is common may have greater evolutionary potential to persist under future warming. If adaptational lag has occurred over just a few decades in banked seeds of an annual species, it may be an important consideration for managing longer-lived species, as well as for attempts to conserve threatened populations through ex situ preservation.Rapid climate change has already caused species range shifts and local extinctions (1) and is predicted to have greater future impacts (2). As the suitable climate space for a species shifts poleward (3), populations previously well adapted to the historical climate in a particular region may experience strong selection to adapt to rapidly warming local temperatures (4–10). Rapid evolutionary response to climate change has already been observed (11, 12), but it remains unclear whether evolutionary response can keep pace with rapidly changing local adaptive optima (6, 8, 13–15). If local adaptation is slower than the rate of climate change, the average fitness of local populations may decline over time (7, 14, 16, 17), possibly resulting in local extinctions and range collapse at the warmer margin. Where such lag exists, we expect that local seeds banked for conservation may no longer be well adapted to their sites of origin (18). However, such adaptational lag may be mitigated by migration or gene flow from populations in historically warmer sites if those populations are better adapted to current conditions in a site than local populations (8, 13, 19, 20). Although adaptational lag has been predicted (4–6, 8, 14, 15, 19, 21, 22), the distinctive signature of mismatch between local population performance and current climate optima has not yet been explicitly demonstrated in nature.Despite evidence for local adaptation in many organisms (23), there have been few explicit tests for the role of specific climate factors in shaping local fitness optima (4, 9, 13). Such tests require growing many genotypes from populations spanning a range of climates in common gardens across a species’ range to decouple climate of origin from geographic variation in other selective factors (4, 6, 14). If adaptation to local climate has occurred, then genotypes from climates similar to each planting site are expected to have high fitness in that site relative to genotypes from dissimilar climates (6). However, if local adaptive optima have shifted with rapid warming trends over the last 50 y, we expect that banked genotypes from historically warmer climates will have higher fitness within a site than banked genotypes of local origin (6, 21, 22).We tested for lagging adaptation to climate using Arabidopsis thaliana, a naturally inbreeding annual species that inhabits a broad climate space across its native Eurasian range (24). A. thaliana exhibits strong circumstantial evidence of climate adaptation, including geographic clines in ecologically important life-history traits (25–28) and in candidate genes associated with these traits (29, 30), as well as genome-wide associations of single nucleotide polymorphisms with climatic factors (31–34). To test explicitly for local adaptation to climate we measured the lifetime fitness of more than 230 accessions from banked seeds originating from a broad range of climates in replicated field experiments in four sites across the species’ native climate range (Fig. 1). We observed that genotypes originating in historically warmer climates outperformed local genotypes, particularly at the northern range limit.Open in a separate windowFig. 1.Map of common garden sites and sites of origin of the 241 native A. thaliana accessions represented in our experiments.     

Human genome–guided identification of memory-modulating drugs

In the last decade there has been an exponential increase in knowledge about the genetic basis of complex human traits, including neuropsychiatric disorders. It is not clear, however, to what extent this knowledge can be used as a starting point for drug identification, one of the central hopes of the human genome project. The aim of the present study was to identify memory-modulating compounds through the use of human genetic information. We performed a multinational collaborative study, which included assessment of aversive memory—a trait central to posttraumatic stress disorder—and a gene-set analysis in healthy individuals. We identified 20 potential drug target genes in two genomewide-corrected gene sets: the neuroactive ligand–receptor interaction and the long-term depression gene set. In a subsequent double-blind, placebo-controlled study in healthy volunteers, we aimed at providing a proof of concept for the genome-guided identification of memory modulating compounds. Pharmacological intervention at the neuroactive ligand–receptor interaction gene set led to significant reduction of aversive memory. The findings demonstrate that genome information, along with appropriate data mining methodology, can be used as a starting point for the identification of memory-modulating compounds.Recent advances in human genetics have led to an unprecedented rate of discovery of genes related to complex human disease, including neuropsychiatric disorders (1–3). The human genome–based gain of knowledge is certainly expected to have a large impact on drug discovery in complex human disease (4–6). It is, however, still not clear to what extent this knowledge can be used as a starting point for the identification of druggable molecular pathways of complex traits (7), including mental disorders (8).Genomewide association studies (GWASs) using single-marker statistics have been very successful in identifying trait-associated single-gene loci (9). It is, however, widely accepted that single marker–based analyses have limited power to identify the genetic basis of a given trait, as for example, many loci will fail to reach stringent genomewide significance threshold, despite the fact that they may be genuinely associated with the trait. Triggered by statistical approaches for the analysis of gene expression, gene set–based analytical methods have recently become available. These methods aim at identifying biologically meaningful sets of genes associated with a certain trait, rather than focusing on a single GWAS gene locus (10). By taking into account prior biological knowledge, gene set–based approaches examine whether test statistics for a group of related genes have consistent deviation from chance (10). As shown recently in studies on autism (11), bipolar disorder (12, 13), attention deficit hyperactivity disorder (ADHD) (14), and schizophrenia (15), such approaches can convincingly identify convergent molecular pathways relevant to neuropsychiatry. Importantly, the identification of groups of functionally related genes is likely to facilitate drug discovery, because the most significant single loci from a GWAS might not be the best candidates for therapeutic intervention (7, 10).In the present study, we focused on emotionally aversive memory—a trait central to anxiety disorders such as posttraumatic stress disorder (PTSD) (16–23). Strong memory for emotionally arousing events can be seen as a primarily adaptive phenomenon, which helps us to remember vital information (e.g., dangerous situations). In case of an extremely aversive event, however, this mechanism can also lead to intrusive and persistent traumatic memories, thereby contributing to the development and symptoms of PTSD (18–22). Symptoms related to aversive memory include intrusive daytime recollections, traumatic nightmares, and flashbacks in which components of the event are relived. Aversive memory is a genetically complex trait as shown both in healthy subjects and in traumatized individuals (17, 23). Furthermore, we recently reported evidence suggesting a genetic link between the predisposition to build strong aversive memories and the risk for PTSD (16).Based on these observations, we developed a process (Fig. 1) aimed at identifying gene sets related to human aversive memory, followed by a pharmacological intervention study as proof-of-concept for the genome-guided identification of memory-modulating drugs.Open in a separate windowFig. 1.Drug discovery process.     

Multiaddressable molecular rectangles with reversible host–guest interactions: Modulation of pH-controlled guest release and capture

A series of multiaddressable platinum(II) molecular rectangles with different rigidities and cavity sizes has been synthesized by endcapping the U-shaped diplatinum(II) terpyridine moiety with various bis-alkynyl ligands. The studies of the host–guest association with various square planar platinum(II), palladium(II), and gold(III) complexes and the related low-dimensional gold(I) complexes, most of which are potential anticancer therapeutics, have been performed. Excellent guest confinement and selectivity of the rectangular architecture have been shown. Introduction of pH-responsive functionalities to the ligand backbone generates multifunctional molecular rectangles that exhibit reversible guest release and capture on the addition of acids and bases, indicating their potential in controlled therapeutics delivery on pH modulation. The reversible host–guest interactions are found to be strongly perturbed by metal–metal and π–π interactions and to a certain extent, electrostatic interactions, giving rise to various spectroscopic changes depending on the nature of the guest molecules. Their binding mode and thermodynamic parameters have been determined by 2D NMR and van’t Hoff analysis and supported by computational study.The study of metal–metal interactions has drawn enormous attention since the past two decades because of the intriguing spectroscopic and photophysical properties arising from the close proximity of the metal centers (1, 2). Square planar d⁸ platinum(II) complexes with coordination unsaturation are one of the important classes of metal complexes that have been extensively explored because of their capability to exhibit metal–metal interactions and display rich photophysical properties (3–26). Platinum(II) terpyridine complexes have been found to exhibit rich polymorphism in the solid state (16–20) owing to their square planar coordination geometry, which permits facile access to Pt(II)···Pt(II) interactions as well as π–π interactions between the chromophores. It was not until 2001 that the first successful synthesis of platinum(II) terpyridine alkynyl complexes, which possess enhanced solubility and luminescence compared with the chloro counterpart, was reported (16). Additional efforts have been devoted to the use of the system to respond to external stimuli, such as variation in solvent composition (17, 18), pH (19, 20), temperature (21, 22), addition of ionic (24–26), and polymeric species (27, 28), in which spectral changes induced by strong Pt(II)···Pt(II) and π−π interactions have been displayed.In the past few decades, enormous efforts have been devoted to the construction of molecular architectures by fusing the organic framework to the transition metal centers through self-assembly processes (29–57). There has been continuous interest in the construction of stimuli-responsive metallosupramolecular architectures with diverse sizes, shapes, and symmetries to rationalize the criteria for molecular recognition and impart them on unique areas of applications, such as stereoselective guest encapsulation and molecular transporting devices (45–65). Although such a variety of metal–organic macrocyclic architectures has been reported, those involving the use of noncovalent interactions other than those of hydrogen bonding, donor–acceptor, electrostatic, and hydrophobic–hydrophobic interactions as well as luminescence changes that depend on the nature of the guests, which would be attractive for chemo- and biosensing, have been rare and are rather underexplored. Examples of such systems that can exhibit reversible host–guest association are also limited.Since the discovery of anticancer properties of cisplatin in 1969 (58), the coordination chemistry and the development of related species with enhanced properties and reduced cytotoxicity have received enormous attention. Although the potency and cytotoxicity studies are important, the availability of the drugs and their transport and release to the site of action are equally important. Thus, the design of smart drug delivery systems has been an area of growing interest. The first phosphorescent molecular tweezers making use of the alkynylplatinum(II) terpyridine moiety have been reported by our group to show their host–guest interactions with transition metal complexes (57). However, the opened structures of the tweezers have limited their selectivity and functionality. To accomplish the controlled drug delivery functionalities, the first main strategy is to rigidify the molecular architecture of the host from tweezers to a rectangle, so that the guest molecules would be better accommodated within the cavity, which may lead to a more selective encapsulation of guests within a definite size and steric environment. The possibility of introducing responsive functionalities into the molecular rectangles, which may serve as models for the study of on-demand controlled guest capture and release systems, has also been explored. pH-sensitive pyridine moieties have, therefore, been incorporated into the backbone of the rectangle to modulate the reversible host–guest interaction within the constrained rectangle environment on protonation/deprotonation of the pyridine nitrogen atom to achieve multiaddressable functions that would not have been readily achievable with the molecular tweezers structure. Additionally, the use of various platinum and gold complexes as guest molecules, which have been shown to display anticancer therapeutic behavior (58–65), may lead to the design of a smart multiaddressable molecular rectangle system that could capture and release specific guest molecules under different pH conditions to achieve proof-of-principle on-demand controlled drug delivery. Herein, the design and synthesis of a series of alkynylplatinum(II) terpyridine molecular rectangles (Fig. 1) with different geometries, topologies and electronic properties are reported. Moreover, the encapsulation of various guest molecules is also investigated in detail to provide a proof-of-principle model for the design of artificial drug delivery systems with the modulation of drug release by pH.Open in a separate windowFig. 1.Molecular structures of rectangles 1−4.     

Tautomerism provides a molecular explanation for the mutagenic properties of the anti-HIV nucleoside 5-aza-5,6-dihydro-2′-deoxycytidine

Viral lethal mutagenesis is a strategy whereby the innate immune system or mutagenic pool nucleotides increase the error rate of viral replication above the error catastrophe limit. Lethal mutagenesis has been proposed as a mechanism for several antiviral compounds, including the drug candidate 5-aza-5,6-dihydro-2′-deoxycytidine (KP1212), which causes A-to-G and G-to-A mutations in the HIV genome, both in tissue culture and in HIV positive patients undergoing KP1212 monotherapy. This work explored the molecular mechanism(s) underlying the mutagenicity of KP1212, and specifically whether tautomerism, a previously proposed hypothesis, could explain the biological consequences of this nucleoside analog. Establishing tautomerism of nucleic acid bases under physiological conditions has been challenging because of the lack of sensitive methods. This study investigated tautomerism using an array of spectroscopic, theoretical, and chemical biology approaches. Variable temperature NMR and 2D infrared spectroscopic methods demonstrated that KP1212 existed as a broad ensemble of interconverting tautomers, among which enolic forms dominated. The mutagenic properties of KP1212 were determined empirically by in vitro and in vivo replication of a single-stranded vector containing a single KP1212. It was found that KP1212 paired with both A (10%) and G (90%), which is in accord with clinical observations. Moreover, this mutation frequency is sufficient for pushing a viral population over its error catastrophe limit, as observed before in cell culture studies. Finally, a model is proposed that correlates the mutagenicity of KP1212 with its tautomeric distribution in solution.Many viruses exhibit a high mutation rate when replicating their genomes, enabling quick adaptation to both changing cellular environments and therapeutics (1–5). Mammalian innate immune systems have developed a mechanism to exploit this high mutation rate against the virus; in a phenomenon termed “lethal mutagenesis,” (6–14) the immune system employs nucleic acid-modifying enzymes (e.g., APOBEC and ADAR) to increase the viral mutation rate sharply, stressing the functional gene product repertoire of the virus to the point that the viral population collapses (15–17). Several antiviral agents are proposed to work at least in part by a chemical version of lethal mutagenesis [e.g., ribavirin against hepatitis C virus (18–22), 5-hydroxy-2′-deoxycytidine against HIV (7), and T-705 against influenza viruses (23)]. When a sufficient number of these mutagenic nucleoside analogs is incorporated into viral genomes, the analogs increase the viral mutation rate above the error catastrophe limit, the rate above which no viable progeny are produced (6, 24–27). This work aimed to understand the molecular basis underlying the biological phenomenon of lethal mutagenesis induced by mutagenic nucleotides.The nucleoside analog 5-aza-5,6-dihydro-2′-deoxycytidine (KP1212) (Fig. 1A) is specifically designed to induce lethal mutagenesis in HIV (28–30). KP1212, the only anti-HIV drug candidate in clinical trials to use this mechanism, has been shown to increase the mutation rate of HIV both in cell culture and in isolates from humans undergoing monotherapy (28, 29). The mutagenic properties of KP1212 in cell culture reveal that it is likely to base pair promiscuously with A and G, and that the progressive acquisition of mutations (primarily A-to-G and G-to-A transitions) precedes population collapse (Fig. 1B) (29). These data are supported by biochemical experiments performed using purified polymerases that establish the ability of KP1212 to pair with either A or G, both when the modified base enters DNA from the nucleotide pool and when it acts as a template base (30). Understanding the chemical and structural basis of mutagenesis of this drug candidate is critical for both its future clinical progress and the development of new therapeutic agents that work by the principle of lethal mutagenesis.Open in a separate windowFig. 1.Schematic presentation of KP1212''s mutagenic effect on viruses. (A) KP1212 exists as an array of different tautomeric forms, whereas cytosine almost exclusively exists as one form, the canonical keto-amino tautomer. (B) The deoxynucleotide analog of KP1212 is incorporated by viral polymerases, causing G-to-A and A-to-G mutations during viral replication. KP1212 is a poor substrate for human polymerases, which provides selectivity in its action against the virus. The progressive acquisition of mutations in the viral genome leads to viral population collapse.Tautomerism of KP1212 leading to viral mutagenesis has been proposed by us and others (29, 30) to be the basis for the clinical activity of this drug candidate. There are, however, no direct data to support that view. Tautomerism as the basis of mutagenesis of natural bases has long been proposed (31–35), and substantiated in part by experimental evidence of minor tautomeric forms of both canonical bases (36–38) and certain base analogs (e.g., 5-hydroxy-2′-deoxycytidine) (39). In a search for a chemical rationale to explain the ambiguous pairing of KP1212 during replication, the present study revealed that the compound readily adopts multiple tautomeric forms, some of which were unexpected. Previously, spectroscopic methods (e.g., UV, Raman, NMR) have been used to study tautomerism of nucleobases (37, 39, 40). In the current work, we also used a battery of spectroscopic tools (1D, 2D, and variable temperature NMR; FTIR; and 2D IR) (41) to quantify and structurally characterize the array of tautomers exhibited by KP1212. Tautomer interconversion equilibria deconvoluted from NMR spectra provided data on the relative levels of tautomers in solution. In parallel with the spectroscopic studies, the qualitative and quantitative features of KP1212 mutagenesis were directly determined by inserting the KP1212 base into a single-stranded viral vector and measuring the intrinsic mutagenic properties of the base, both in vitro and in vivo. Finally, a model is proposed that correlates the mutagenic and clinical properties of KP1212 with its ability to exist as multiple tautomers.     

Asymmetrical effects of unilateral right or left amygdala damage on auditory cortical processing of vocal emotions

We tested whether human amygdala lesions impair vocal processing in intact cortical networks. In two functional MRI experiments, patients with unilateral amygdala resection either listened to voices and nonvocal sounds or heard binaural vocalizations with attention directed toward or away from emotional information on one side. In experiment 1, all patients showed reduced activation to voices in the ipsilesional auditory cortex. In experiment 2, emotional voices evoked increased activity in both the auditory cortex and the intact amygdala for right-damaged patients, whereas no such effects were found for left-damaged amygdala patients. Furthermore, the left inferior frontal cortex was functionally connected with the intact amygdala in right-damaged patients, but only with homologous right frontal areas and not with the amygdala in left-damaged patients. Thus, unilateral amygdala damage leads to globally reduced ipsilesional cortical voice processing, but only left amygdala lesions are sufficient to suppress the enhanced auditory cortical processing of vocal emotions.Socially relevant and emotionally charged stimuli evoke increased activation in sensory cortices, both during the visual processing of emotional pictures or facial expressions (1, 2) and during the auditory processing of vocally expressed emotions (3–5). Such increases are assumed to be remotely driven by the amygdala, which is critically involved in decoding the emotional value of stimuli (6–8). Moreover, these effects seem to be predominantly (although not exclusively) mediated by ipsilateral anatomical (9) and functional connections between amygdala and sensory areas (10, 11).In line with this view, recent studies conducted in patients with amygdala lesions reported impairments in the recognition of facial expressions (12), emotional words (13), or vocal emotions (14, 15). Furthermore, studies in both human patients (16, 17) and monkeys (18) showed significant changes in visual cortical activations to facial expressions following lesions of the amygdala. These changes in cortical processing are assumed to be remotely driven by the impaired emotional processing in the amygdala (10, 16). Distant effects of amygdala damage have also been observed for visual stimuli in cats (19) and for auditory stimuli in rats (8). However, other results have challenged this view, with some studies reporting no impairment in recognition (20–25) or changes in cortical processing for emotional stimuli in patients with amygdala lesions (26). Notably, Edmiston et al. (26) observed normal visual increases in response to emotional scenes for patients with unilateral amygdala resection, arguing against a direct role for the amygdala in modulating activity in sensory cortical areas. However, in that study (26), such increases could be related to attentional effects driven by greater interest or complexity of emotional scenes (27, 28).Thus, evidence for impaired cortical responses to emotional stimuli after unilateral amygdala damage in humans remains inconsistent. In addition, unlike in rodents (8), to date, no study has investigated how the cortical processing of emotionally salient auditory stimuli might be affected by amygdala lesions in humans. Here, we tested for the first time, to our knowledge, whether unilateral amygdala damage in patients with left or right medial temporal lobe (MTL) lesions would modify auditory responses in intact cortical areas to voices and vocally expressed emotions. Previous studies consistently found differential activity in several subregions of auditory cortex in response to vocal emotions (29, 30), as well as in the amygdala (4, 5, 29, 31, 32), especially for angry voices (3–5). These auditory effects predominate in the superior temporal gyrus (STG) and superior temporal sulcus (STS), attributed to the processing of emotional valence in the amygdala (10) and presumably mediated by direct anatomical connections between the latter and auditory cortex (9, 33). Previous studies also consistently reported a response to emotional voices in the inferior frontal cortex, which may support higher level categorization processes (34) and thus constitutes an important component of the distributed network involved in detecting and decoding vocal emotions (29, 35).We therefore hypothesized that cortical processing of human vocalizations in general, and of vocal emotions in particular, might be impaired in patients with lesions to the amygdala. This impairment is thought to result from a reduced emotional decoding of affective vocal cues in the amygdala, which is generally sensitive to emotional cues in voices (4, 5, 29, 31, 36) and usually is assumed to enhance cortical processing remotely (10, 16, 19). We also hypothesized that left and right amygdala lesions might have different effects. Whereas a right MTL lesion may strongly impair the processing of facial expressions due to well-known hemispheric asymmetries in face processing (12, 17, 37), the left amygdala seems to be more strongly involved in the decoding of emotional cues expressed in speech (13) or speech-like material (3, 4, 29). In two experiments, we tested brain responses to human vocalizations in general (experiment 1) and to emotional vocalizations embedded in pseudolanguage (experiment 2) (Fig. 1) while 10 patients with unilateral left amygdala lesions and 10 patients with unilateral right amygdala lesions (SI Results, Fig. S1A, and Table S1) underwent functional MRI (fMRI) scanning. In experiment 2, emotional voices were presented in either the attended or unattended ear during a dichotic listening task (38). We expected, first, that vocalizations, as socially salient stimuli, would generally produce weaker cortical processing in interconnected regions due to unilateral amygdala damage (experiment 1) (39). Second, in keeping with predominant left amygdala activity in healthy individuals during the processing of vocal emotions, we expected more severe impairment in cortical processing of emotional cues in patients with left amygdala lesions compared with right amygdala lesions (experiment 2) (29, 40).Open in a separate windowFig. 1.Experiment 2 included three emotion conditions, with angry voices presented in the left or right ear or neither. On an trials, an angry voice was heard on the task-relevant side, whereas on na trials, an angry voice was heard on the task-irrelevant side. On nn trials, neutral voices were presented to each ear. (A) Examples show all three conditions when attention was focused on the right ear. The same trials were also performed when attention was focused on the left ear (not shown here). (B) RTs and error rates for gender decisions on the attended voice revealed a main effect for the factor condition, indicating increased RTs and error rates during the an condition, as indicated by the asterisks.