Mapping the Constrained Coding Regions in the Human Genome to Their Corresponding Proteins.

Constrained Coding Regions (CCRs) in the human genome have been derived from DNA sequencing data of large cohorts of healthy control populations, available in the Genome Aggregation Database (gnomAD) [1]. They identify regions depleted of protein-changing variants and thus identify segments of the genome that have been constrained during human evolution. By mapping these DNA-defined regions from genomic coordinates onto the corresponding protein positions and combining this information with protein annotations, we have explored the distribution of CCRs and compared their co-occurrence with different protein functional features, previously annotated at the amino acid level in public databases. As expected, our results reveal that functional amino acids involved in interactions with DNA/RNA, protein-protein contacts and catalytic sites are the protein features most likely to be highly constrained for variation in the control population. More surprisingly, we also found that linear motifs, linear interacting peptides (LIPs), disorder-order transitions upon binding with other protein partners and liquid-liquid phase separating (LLPS) regions are also strongly associated with high constraint for variability. We also compared intra-species constraints in the human CCRs with inter-species conservation and functional residues to explore how such CCRs may contribute to the analysis of protein variants. As has been previously observed, CCRs are only weakly correlated with conservation, suggesting that intraspecies constraints complement interspecies conservation and can provide more information to interpret variant effects.

Structural analysis of pathogenic missense mutations in GABRA2 and identification of a novel de novo variant in the desensitization gate.

BACKGROUND: Cys-loop receptors control neuronal excitability in the brain and their dysfunction results in numerous neurological disorders. Recently, six missense variants in GABRA2, a member of this family, have been associated with early infantile epileptic encephalopathy (EIEE). We identified a novel de novo missense variant in GABRA2 in a patient with EIEE and performed protein structural analysis of the seven variants. METHODS: The novel variant was identified by trio whole-genome sequencing. We performed protein structural analysis of the seven variants, and compared them to previously reported pathogenic mutations at equivalent positions in other Cys-loop receptors. Additionally, we studied the distribution of disease-associated variants in the transmembrane helices of these proteins. RESULTS: The seven variants are in the transmembrane domain, either close to the desensitization gate, the activation gate, or in inter-subunit interfaces. Six of them have pathogenic mutations at equivalent positions in other Cys-loop receptors, emphasizing the importance of these residues. Also, pathogenic mutations are more common in the pore-lining helix, consistent with this region being highly constrained for variation in control populations. CONCLUSION: Our study reports a novel pathogenic variant in GABRA2, characterizes the regions where pathogenic mutations are in the transmembrane helices, and underscores the value of considering sequence, evolutionary, and structural information as a strategy for variant interpretation of novel missense mutations.

Real-time particle pollution sensing using machine learning.

Particle pollution is a global health challenge that is linked to around three million premature deaths per year. There is therefore great interest in the development of sensors capable of precisely quantifying both the number and type of particles. Here, we demonstrate an approach that leverages machine learning in order to identify particulates directly from their scattering patterns. We show the capability for producing a 2D sample map of spherical particles present on a coverslip, and also demonstrate real-time identification of a range of particles including those from diesel combustion.

Interpretation of in Vitro Metabolic Stability Studies for Racemic Mixtures.

In early drug discovery, where chiral syntheses may not yet have been elucidated or enantiomeric separation is not feasible, screening of racemates in metabolic stability assays may offer a pragmatic approach. To assess the risk of incorrectly deprioritizing enantiomers due to misclassification of apparent in vitro intrinsic clearance (CLintapp), we evaluated (1) theoretical simulations; (2) literature on enantiomeric CLintapp differences; (3) historic MSD data; and (4) new data on enantiomers with high eudysmic ratios and low predicted three-dimensional similarity. Overall, the results suggested minimal risk of not progressing an enantiomer due to an appreciably different (>3-fold) racemate CLintapp.

A Pharmacokinetic Modeling Approach to Predict the Contribution of Active Metabolites to Human Efficacious Dose.

A preclinical drug candidate, MRK-1 (Merck candidate drug parent compound), was found to elicit tumor regression in a mouse xenograft model. Analysis of samples from these studies revealed significant levels of two circulating metabolites, whose identities were confirmed by comparison with authentic standards using liquid chromatography-tandem mass spectrometry. These metabolites were found to have an in vitro potency similar to that of MRK-1 against the pharmacological target and were therefore thought to contribute to the observed efficacy. To predict this contribution in humans, a pharmacokinetic (PK) modeling approach was developed. At the mouse efficacious dose, the areas under the plasma concentration time curves (AUCs) of the active metabolites were normalized by their in vitro potency compared with MRK-1. These normalized metabolite AUCs were added to that of MRK-1 to yield a composite efficacious unbound AUC, expressed as "parent drug equivalents," which was used as the target AUC for predictions of the human efficacious dose. In vitro and preclinical PK studies afforded predictions of the PK of MRK-1 and the two active metabolites in human as well as the relative pathway flux to each metabolite. These were used to construct a PK model (Berkeley Madonna, version 8.3.18; Berkeley Madonna Inc., University of California, Berkeley, CA) and to predict the human dose required to achieve the target parent equivalent exposure. These predictions were used to inform on the feasibility of the human dose in terms of size, frequency, formulation, and likely safety margins, as well as to aid in the design of preclinical safety studies.

Potential DNA binding and nuclease functions of ComEC domains characterized in silico.

Bacterial competence, which can be natural or induced, allows the uptake of exogenous double stranded DNA (dsDNA) into a competent bacterium. This process is known as transformation. A multiprotein assembly binds and processes the dsDNA to import one strand and degrade another yet the underlying molecular mechanisms are relatively poorly understood. Here distant relationships of domains in Competence protein EC (ComEC) of Bacillus subtilis (Uniprot: P39695) were characterized. DNA-protein interactions were investigated in silico by analyzing models for structural conservation, surface electrostatics and structure-based DNA binding propensity; and by data-driven macromolecular docking of DNA to models. Our findings suggest that the DUF4131 domain contains a cryptic DNA-binding OB fold domain and that the ß-lactamase-like domain is the hitherto cryptic competence nuclease. Proteins 2016; 84:1431-1442. © 2016 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

Wolves adapt territory size, not pack size to local habitat quality.

1. Although local variation in territorial predator density is often correlated with habitat quality, the causal mechanism underlying this frequently observed association is poorly understood and could stem from facultative adjustment in either group size or territory size. 2. To test between these alternative hypotheses, we used a novel statistical framework to construct a winter population-level utilization distribution for wolves (Canis lupus) in northern Ontario, which we then linked to a suite of environmental variables to determine factors influencing wolf space use. Next, we compared habitat quality metrics emerging from this analysis as well as an independent measure of prey abundance, with pack size and territory size to investigate which hypothesis was most supported by the data. 3. We show that wolf space use patterns were concentrated near deciduous, mixed deciduous/coniferous and disturbed forest stands favoured by moose (Alces alces), the predominant prey species in the diet of wolves in northern Ontario, and in proximity to linear corridors, including shorelines and road networks remaining from commercial forestry activities. 4. We then demonstrate that landscape metrics of wolf habitat quality - projected wolf use, probability of moose occupancy and proportion of preferred land cover classes - were inversely related to territory size but unrelated to pack size. 5. These results suggest that wolves in boreal ecosystems alter territory size, but not pack size, in response to local variation in habitat quality. This could be an adaptive strategy to balance trade-offs between territorial defence costs and energetic gains due to resource acquisition. That pack size was not responsive to habitat quality suggests that variation in group size is influenced by other factors such as intraspecific competition between wolf packs.

Space-use behaviour of woodland caribou based on a cognitive movement model.

Movement patterns offer a rich source of information on animal behaviour and the ecological significance of landscape attributes. This is especially useful for species occupying remote landscapes where direct behavioural observations are limited. In this study, we fit a mechanistic model of animal cognition and movement to GPS positional data of woodland caribou (Rangifer tarandus caribou; Gmelin 1788) collected over a wide range of ecological conditions. The model explicitly tracks individual animal informational state over space and time, with resulting parameter estimates that have direct cognitive and ecological meaning. Three biotic landscape attributes were hypothesized to motivate caribou movement: forage abundance (dietary digestible biomass), wolf (Canis lupus; Linnaeus, 1758) density and moose (Alces alces; Linnaeus, 1758) habitat. Wolves are the main predator of caribou in this system and moose are their primary prey. Resulting parameter estimates clearly indicated that forage abundance is an important driver of caribou movement patterns, with predator and moose avoidance often having a strong effect, but not for all individuals. From the cognitive perspective, our results support the notion that caribou rely on limited sensory inputs from their surroundings, as well as on long-term spatial memory, to make informed movement decisions. Our study demonstrates how sensory, memory and motion capacities may interact with ecological fitness covariates to influence movement decisions by free-ranging animals.

Design, synthesis, and structure-activity relationships of indole-3-heterocycles as agonists of the CB1 receptor.

Novel indole-3-heterocycles were designed and synthesized and found to be potent CB1 receptor agonists. Starting from a microsomally unstable lead 1, a bioisostere approach replacing a piperazine amide was undertaken. This was found to be a good strategy for improving stability both in vitro and in vivo. This led to the discovery of 24, which had an increased duration of action in the mouse tail flick test in comparison to the lead 1.

Mortality risk increases with natal dispersal distance in American martens.

The assumption that mortality risk increases with dispersal distance has rarely been tested. We compared patterns of natal dispersal in the American marten (Martes americana) between a large regenerating forest landscape and an uncut landscape that was dominated by more mature forest to test whether mortality risk increased with dispersal distance, and whether variation in mortality risk influenced dispersal distance. Mortality risk increased with dispersal distance in both landscape treatments, but the distance-dependent increase in mortality in the regenerating landscape was twice that in the uncut landscape. Differences in body condition, supported by other data on foraging efficiency, suggested that juveniles from the regenerating landscape were less able to cope with the energetic demands of dispersal compared with juveniles from older forests. Juveniles travelled shorter distances in the regenerating versus uncut landscape. These results implied that dispersal was costly in terms of juvenile survival and that mean dispersal distance was shaped, in part, by mortality risk.

Habitat-mediated variation in predation risk by the American marten.

The probability of prey encounter, attack, capture, and kill are often hypothesized to depend on habitat structure, but field evidence in terrestrial systems is rare. We tested whether predation efficiency by the American marten (Martes americana) and fear of predation by their primary prey, the red-backed vole (Clethrionomys gapperi), differed between 20- to 50-year-old regenerating forest stands and older uncut stands. Our results showed that the frequency of prey encounter, prey attack, and prey kill were higher in old uncut forests, despite the fact that small-mammal density was similar to that in younger logged forests. These differences in predation efficiency were linked to higher abundance of coarse woody debris, which seems to offer sensory cues to martens, thereby increasing the odds of hunting success. Red-backed voles in regenerating forest stands exhibited increased wariness compared to voles living in old uncut forest, suggestive of a behavioral response to habitat-mediated variation in predation risk.

The potential influence of CO2, as an agent for euthanasia, on the pharmacokinetics of basic compounds in rodents.

Rodent tissue distribution and pharmacokinetic studies were performed on basic compounds Org A and Org B in support of central nervous system drug discovery programs. A consistent observation from these studies was that drug concentrations in plasma obtained by cardiac puncture after CO(2) euthanasia were markedly higher compared with those from other sampling methods (serial sampling, isoflurane anesthesia, or cervical dislocation). Further investigations demonstrated that CO(2) euthanasia led to a reduction in blood pH in both rats and mice, which was not observed with the other sampling methods. The use of CO(2) euthanasia resulted in a decrease in the brain/plasma ratio of Org B, largely as a result of increased plasma concentrations. The pharmacokinetics of a basic drug, raloxifene, in rat were also influenced by sampling technique. CO(2) euthanasia before sampling, resulted in a 2- to 3-fold increase in the area under the drug concentration-time curve, a decrease in plasma clearance, and a decrease in the steady-state volume of distribution compared with isoflurane anesthesia. It is proposed that a decrease in the pH of blood relative to that of other tissues, as a consequence of CO(2) exposure, results in a redistribution of basic compounds out of the tissues, leading to higher concentrations in plasma.