Conformational Effects on the Passive Membrane Permeability of Synthetic Macrocycles.

Macrocyclic compounds (MCs) can have complex conformational properties that affect pharmacologically important behaviors such as membrane permeability. We measured the passive permeability of 3600 diverse nonpeptidic MCs and used machine learning to analyze the results. Incorporating selected properties based on the three-dimensional (3D) conformation gave models that predicted permeability with Q2 = 0.81. A biased spatial distribution of polar versus nonpolar regions was particularly important for good permeability, consistent with a mechanism in which the initial insertion of nonpolar portions of a MC helps facilitate the subsequent membrane entry of more polar parts. We also examined effects on permeability of 800 substructural elements by comparing matched molecular pairs. Some substitutions were invariably beneficial or invariably deleterious to permeability, while the influence of others was highly contextual. Overall, the work provides insights into how the permeability of MCs is influenced by their 3D conformational properties and suggests design hypotheses for achieving macrocycles with high membrane permeability.

Canine ACL rupture: a spontaneous large animal model of human ACL rupture.

BACKGROUND: Anterior cruciate ligament (ACL) rupture in humans is a common condition associated with knee pain, joint instability, and secondary osteoarthritis (OA). Surgical treatment with an intraarticular graft provides reasonable outcomes at mid and long-term follow-up. Non-modifiable and modifiable factors influence risk of ACL rupture. The etiology, mechanobiology, causal biomechanics, and causal molecular pathways are not fully understood. The dog model has shared features of ACL rupture that make it a valuable spontaneous preclinical animal model. In this article, we review shared and contrasting features of ACL rupture in the two species and present information supporting spontaneous canine ACL rupture as a potentially useful preclinical model of human ACL rupture with a very large subject population. RESULTS: ACL rupture is more common in dogs than in humans and is diagnosed and treated using similar approaches to that of human patients. Development of OA occurs in both species, but progression is more rapid in the dog, and is often present at diagnosis. Use of client-owned dogs for ACL research could reveal impactful molecular pathways, underlying causal genetic variants, biomechanical effects of specific treatments, and opportunities to discover new treatment and prevention targets. Knowledge of the genetic contribution to ACL rupture is more advanced in dogs than in humans. In dogs, ACL rupture has a polygenetic architecture with moderate heritability. Heritability of human ACL rupture has not been estimated. CONCLUSION: This article highlights areas of One Health research that are particularly relevant to future studies using the spontaneous canine ACL rupture model that could fill gaps in current knowledge.

Defining and navigating macrocycle chemical space.

Macrocyclic compounds (MCs) are of growing interest for inhibition of challenging drug targets. We consider afresh what structural and physicochemical features could be relevant to the bioactivity of this compound class. Using these features, we performed Principal Component Analysis to map oral and non-oral macrocycle drugs and clinical candidates, and also commercially available synthetic MCs, in structure-property space. We find that oral MC drugs occupy defined regions that are distinct from those of the non-oral MC drugs. None of the oral MC regions are effectively sampled by the synthetic MCs. We identify 13 properties that can be used to design synthetic MCs that sample regions overlapping with oral MC drugs. The results advance our understanding of what molecular features are associated with bioactive and orally bioavailable MCs, and illustrate an approach by which synthetic chemists can better evaluate MC designs. We also identify underexplored regions of macrocycle chemical space.

Biologically Enhanced Genome-Wide Association Study Provides Further Evidence for Candidate Loci and Discovers Novel Loci That Influence Risk of Anterior Cruciate Ligament Rupture in a Dog Model.

Anterior cruciate ligament (ACL) rupture is a common condition that disproportionately affects young people, 50% of whom will develop knee osteoarthritis (OA) within 10 years of rupture. ACL rupture exhibits both hereditary and environmental risk factors, but the genetic basis of the disease remains unexplained. Spontaneous ACL rupture in the dog has a similar disease presentation and progression, making it a valuable genomic model for ACL rupture. We leveraged the dog model with Bayesian mixture model (BMM) analysis (BayesRC) to identify novel and relevant genetic variants associated with ACL rupture. We performed RNA sequencing of ACL and synovial tissue and assigned single nucleotide polymorphisms (SNPs) within differentially expressed genes to biological prior classes. SNPs with the largest effects were on chromosomes 3, 5, 7, 9, and 24. Selection signature analysis identified several regions under selection in ACL rupture cases compared to controls. These selection signatures overlapped with genome-wide associations with ACL rupture as well as morphological traits. Notable findings include differentially expressed ACSF3 with MC1R (coat color) and an association on chromosome 7 that overlaps the boundaries of SMAD2 (weight and body size). Smaller effect associations were within or near genes associated with regulation of the actin cytoskeleton and the extracellular matrix, including several collagen genes. The results of the current analysis are consistent with previous work published by our laboratory and others, and also highlight new genes in biological pathways that have not previously been associated with ACL rupture. The genetic associations identified in this study mirror those found in human beings, which lays the groundwork for development of disease-modifying therapies for both species.

Recapitulating the Binding Affinity of Nrf2 for KEAP1 in a Cyclic Heptapeptide, Guided by NMR, X-ray Crystallography, and Machine Learning.

Macrocycles, including macrocyclic peptides, have shown promise for targeting challenging protein-protein interactions (PPIs). One PPI of high interest is between Kelch-like ECH-Associated Protein-1 (KEAP1) and Nuclear Factor (Erythroid-derived 2)-like 2 (Nrf2). Guided by X-ray crystallography, NMR, modeling, and machine learning, we show that the full 20 nM binding affinity of Nrf2 for KEAP1 can be recapitulated in a cyclic 7-mer peptide, c[(D)-ß-homoAla-DPETGE]. This compound was identified from the Nrf2-derived linear peptide GDEETGE (KD = 4.3 µM) solely by optimizing the conformation of the cyclic compound, without changing any KEAP1 interacting residue. X-ray crystal structures were determined for each linear and cyclic peptide variant bound to KEAP1. Despite large variations in affinity, no obvious differences in the conformation of the peptide binding residues or in the interactions they made with KEAP1 were observed. However, analysis of the X-ray structures by machine learning showed that locations of strain in the bound ligand could be identified through patterns of subangstrom distortions from the geometry observed for unstrained linear peptides. We show that optimizing the cyclic peptide affinity was driven partly through conformational preorganization associated with a proline substitution at position 78 and with the geometry of the noninteracting residue Asp77 and partly by decreasing strain in the ETGE motif itself. This approach may have utility in dissecting the trade-off between conformational preorganization and strain in other ligand-receptor systems. We also identify a pair of conserved hydrophobic residues flanking the core DxETGE motif which play a conformational role in facilitating the high-affinity binding of Nrf2 to KEAP1.

Analysis of copy number variation in dogs implicates genomic structural variation in the development of anterior cruciate ligament rupture.

Anterior cruciate ligament (ACL) rupture is an important condition of the human knee. Second ruptures are common and societal costs are substantial. Canine cranial cruciate ligament (CCL) rupture closely models the human disease. CCL rupture is common in the Labrador Retriever (5.79% prevalence), ~100-fold more prevalent than in humans. Labrador Retriever CCL rupture is a polygenic complex disease, based on genome-wide association study (GWAS) of single nucleotide polymorphism (SNP) markers. Dissection of genetic variation in complex traits can be enhanced by studying structural variation, including copy number variants (CNVs). Dogs are an ideal model for CNV research because of reduced genetic variability within breeds and extensive phenotypic diversity across breeds. We studied the genetic etiology of CCL rupture by association analysis of CNV regions (CNVRs) using 110 case and 164 control Labrador Retrievers. CNVs were called from SNPs using three different programs (PennCNV, CNVPartition, and QuantiSNP). After quality control, CNV calls were combined to create CNVRs using ParseCNV and an association analysis was performed. We found no strong effect CNVRs but found 46 small effect (max(T) permutation P<0.05) CCL rupture associated CNVRs in 22 autosomes; 25 were deletions and 21 were duplications. Of the 46 CCL rupture associated CNVRs, we identified 39 unique regions. Thirty four were identified by a single calling algorithm, 3 were identified by two calling algorithms, and 2 were identified by all three algorithms. For 42 of the associated CNVRs, frequency in the population was <10% while 4 occurred at a frequency in the population ranging from 10-25%. Average CNVR length was 198,872bp and CNVRs covered 0.11 to 0.15% of the genome. All CNVRs were associated with case status. CNVRs did not overlap previous canine CCL rupture risk loci identified by GWAS. Associated CNVRs contained 152 annotated genes; 12 CNVRs did not have genes mapped to CanFam3.1. Using pathway analysis, a cluster of 19 homeobox domain transcript regulator genes was associated with CCL rupture (P = 6.6E-13). This gene cluster influences cranial-caudal body pattern formation during embryonic limb development. Clustered genes were found in 3 CNVRs on chromosome 14 (HoxA), 28 (NKX6-2), and 36 (HoxD). When analysis was limited to deletion CNVRs, the association was strengthened (P = 8.7E-16). This study suggests a component of the polygenic risk of CCL rupture in Labrador Retrievers is associated with small effect CNVs and may include aspects of stifle morphology regulated by homeobox domain transcript regulator genes.

Bayesian and Machine Learning Models for Genomic Prediction of Anterior Cruciate Ligament Rupture in the Canine Model.

Anterior cruciate ligament (ACL) rupture is a common, debilitating condition that leads to early-onset osteoarthritis and reduced quality of human life. ACL rupture is a complex disease with both genetic and environmental risk factors. Characterizing the genetic basis of ACL rupture would provide the ability to identify individuals that have high genetic risk and allow the opportunity for preventative management. Spontaneous ACL rupture is also common in dogs and shows a similar clinical presentation and progression. Thus, the dog has emerged as an excellent genomic model for human ACL rupture. Genome-wide association studies (GWAS) in the dog have identified a number of candidate genetic variants, but research in genomic prediction has been limited. In this analysis, we explore several Bayesian and machine learning models for genomic prediction of ACL rupture in the Labrador Retriever dog. Our work demonstrates the feasibility of predicting ACL rupture from SNPs in the Labrador Retriever model with and without consideration of non-genetic risk factors. Genomic prediction including non-genetic risk factors approached clinical relevance using multiple linear Bayesian and non-linear models. This analysis represents the first steps toward development of a predictive algorithm for ACL rupture in the Labrador Retriever model. Future work may extend this algorithm to other high-risk breeds of dog. The ability to accurately predict individual dogs at high risk for ACL rupture would identify candidates for clinical trials that would benefit both veterinary and human medicine.

Late-onset laryngeal paralysis: Owner perception of quality of life and cause of death.

BACKGROUND: Late-onset laryngeal paralysis (LoLP) is an idiopathic disease of older dogs, and is common in the Labrador Retriever. Owner perspective of how LoLP affects their pet's quality of life (QOL), the degree to which LoLP is perceived to be a life-limiting disease, and how a glottic opening procedure affects these perceptions is not known. OBJECTIVES: (a) To determine owner's perception of late-onset laryngeal paralysis (LoLP) with respect to their dog's QOL; (b) To determine whether LoLP is considered by owners to be a life-limiting disease; (c) To evaluate whether a glottic opening procedure altered QOL and perceived cause of death in affected dogs. METHODS: Owners of Labrador Retrievers with LoLP completed a questionnaire. Questions were asked pertaining to a dog's LoLP, including clinical progression and perception of cause of death, and whether a glottic opening procedure was undertaken. Owners also completed a pet-owner administered QOL survey. RESULTS: Seventy-six owners participated. Overall, 94% of owners felt their dog's LoLP affected QOL, and 47% of owners felt LoLP was a large contributing factor in their dog's death. Dogs that underwent a glottic opening procedure were reported to have a better QOL, and the contribution of LoLP towards their death was less than dogs that did not have surgery. CONCLUSION: Owners of Labrador Retrievers with LoLP perceive LoLP to be a life-limiting disease that negatively impacts their dog's QOL. Arytenoid lateralization surgery had a positive impact on QOL in affected dogs.

Contribution of Habitual Activity to Cruciate Ligament Rupture in Labrador Retrievers.

OBJECTIVE: The aim of this study was to describe the contribution of signalment and habitual activity in the development of cruciate ligament rupture (CR) in Labrador Retrievers. STUDY DESIGN: Four hundred and twelve client-owned purebred Labrador Retrievers were recruited. Dogs were assigned either as affected with CR or as controls based on signalment, physical examination and radiographic evidence of disease. Clients were asked to complete a questionnaire related to signalment, concurrent disease and a questionnaire pertaining to their dog's activity before development of CR or general activity during the dog's most active years. RESULTS: Habitual activity was not significantly different between dogs affected with CR and controls. There was no significant difference in neuter status or body weight between CR affected dogs and controls. Labrador Retrievers with a yellow coat, and Labradors that did not maintain an optimal body weight in the opinion of their veterinarian were at increased risk of developing CR. CONCLUSIONS: Habitual activity level is not a risk factor for development of CR in Labrador Retrievers. Our study did not show neuter status, sex or body weight to be risk factors for CR. However, coat colour and not sustaining optimal body condition are significant risk factors for CR.

Multivariate genome-wide association analysis identifies novel and relevant variants associated with anterior cruciate ligament rupture risk in the dog model.

BACKGROUND: Anterior cruciate ligament rupture (ACLR) is a debilitating and potentially life-changing condition in humans, as there is a high prevalence of early-onset osteoarthritis after injury. Identification of high-risk individuals before they become patients is important, as post-treatment lifetime burden of ACLR in the USA ranges from $7.6 to $17.7 billion annually. ACLR is a complex disease with multiple risk factors including genetic predisposition. Naturally occurring ACLR in the dog is an excellent model for human ACLR, as risk factors and disease characteristics in humans and dogs are similar. In a univariate genome-wide association study (GWAS) of 237 Labrador Retrievers, we identified 99 ACLR candidate loci. It is likely that additional variants remain to be identified. Joint analysis of multiple correlated phenotypes is an underutilized technique that increases statistical power, even when only one phenotype is associated with the trait. Proximal tibial morphology has been shown to affect ACLR risk in both humans and dogs. In the present study, tibial plateau angle (TPA) and relative tibial tuberosity width (rTTW) were measured on bilateral radiographs from purebred Labrador Retrievers that were recruited to our initial GWAS. We performed a multivariate genome wide association analysis of ACLR status, TPA, and rTTW. RESULTS: Our analysis identified 3 loci with moderate evidence of association that were not previously associated with ACLR. A locus on Chr1 associated with both ACLR and rTTW is located within ROR2, a gene important for cartilage and bone development. A locus on Chr4 associated with both ACLR and TPA resides within DOCK2, a gene that has been shown to promote immune cell migration and invasion in synovitis, an important predictor of ACLR. A third locus on Chr23 associated with only ACLR is located near a long non-coding RNA (lncRNA). LncRNA's are important for regulation of gene transcription and translation. CONCLUSIONS: These results did not overlap with our previous GWAS, which is reflective of the different methods used, and supports the need for further work. The results of the present study are highly relevant to ACLR pathogenesis, and identify potential drug targets for medical treatment.

Genome-wide association analysis in dogs implicates 99 loci as risk variants for anterior cruciate ligament rupture.

Anterior cruciate ligament (ACL) rupture is a common condition that can be devastating and life changing, particularly in young adults. A non-contact mechanism is typical. Second ACL ruptures through rupture of the contralateral ACL or rupture of a graft repair is also common. Risk of rupture is increased in females. ACL rupture is also common in dogs. Disease prevalence exceeds 5% in several dog breeds, ~100 fold higher than human beings. We provide insight into the genetic etiology of ACL rupture by genome-wide association study (GWAS) in a high-risk breed using 98 case and 139 control Labrador Retrievers. We identified 129 single nucleotide polymorphisms (SNPs) within 99 risk loci. Associated loci (P<5E-04) explained approximately half of phenotypic variance in the ACL rupture trait. Two of these loci were located in uncharacterized or non-coding regions of the genome. A chromosome 24 locus containing nine genes with diverse functions met genome-wide significance (P = 3.63E-0.6). GWAS pathways were enriched for c-type lectins, a gene set that includes aggrecan, a gene set encoding antimicrobial proteins, and a gene set encoding membrane transport proteins with a variety of physiological functions. Genotypic risk estimated for each dog based on the risk contributed by each GWAS locus showed clear separation of ACL rupture cases and controls. Power analysis of the GWAS data set estimated that ~172 loci explain the genetic contribution to ACL rupture in the Labrador Retriever. Heritability was estimated at 0.48. We conclude ACL rupture is a moderately heritable highly polygenic complex trait. Our results implicate c-type lectin pathways in ACL homeostasis.

Variance associated with walking velocity during force platform gait analysis of a heterogeneous sample of clinically normal dogs.

OBJECTIVE To determine whether walking at specific ranges of absolute and relative (V*) velocity would aid efficient capture of gait trial data with low ground reaction force (GRF) variance in a heterogeneous sample of dogs. ANIMALS 17 clinically normal dogs of various breeds, ages, and sexes. PROCEDURES Each dog was walked across a force platform at its preferred velocity, with controlled acceleration within 0.5 m/s2. Ranges in V* were created for height at the highest point of the shoulders (withers; WHV*). Variance effects from 8 walking absolute velocity ranges and associated WHV* ranges were examined by means of repeated-measures ANCOVA. RESULTS The individual dog effect provided the greatest contribution to variance. Narrow velocity ranges typically resulted in capture of a smaller percentage of valid trials and were not consistently associated with lower variance. The WHV* range of 0.33 to 0.46 allowed capture of valid trials efficiently, with no significant effects on peak vertical force and vertical impulse. CONCLUSIONS AND CLINICAL RELEVANCE Dogs with severe lameness may be unable to trot or may have a decline in mobility with gait trial repetition. Gait analysis involving evaluation of individual dogs at their preferred absolute velocity, such that dogs are evaluated at a similar V*, may facilitate efficient capture of valid trials without significant effects on GRF. Use of individual velocity ranges derived from a WHV* range of 0.33 to 0.46 can account for heterogeneity and appears suitable for use in clinical trials involving dogs at a walking gait.

Nevus comedonicus in oral-facial-digital syndrome type 1: a new finding or overlapping syndromes?

We report a patient with oral-facial-digital syndrome type 1 (OFDS1) who exhibited features overlapping those of nevus comedonicus syndrome, an unusual presentation that may potentially represent a new variant of OFDS1. OFDS1 and nevus comedonicus syndrome represent two rare syndromes with numerous overlapping features that have yet to be described in relation to one another. The features present in our patient led us to propose the possibility of a new variant of OFDS1 in which nevus comedonicus represents a cutaneous manifestation of the syndrome. Knowledge of this potential relationship is important for identification and management of the syndromes' accompanying manifestations in affected patients and may offer further insight into crossroads of pathogenesis.

Optimising experimental design for high-throughput phenotyping in mice: a case study.

To further the functional annotation of the mammalian genome, the Sanger Mouse Genetics Programme aims to generate and characterise knockout mice in a high-throughput manner. Annually, approximately 200 lines of knockout mice will be characterised using a standardised battery of phenotyping tests covering key disease indications ranging from obesity to sensory acuity. From these findings secondary centres will select putative mutants of interest for more in-depth, confirmatory experiments. Optimising experimental design and data analysis is essential to maximise output using the resources with greatest efficiency, thereby attaining our biological objective of understanding the role of genes in normal development and disease. This study uses the example of the noninvasive blood pressure test to demonstrate how statistical investigation is important for generating meaningful, reliable results and assessing the design for the defined research objectives. The analysis adjusts for the multiple-testing problem by applying the false discovery rate, which controls the number of false calls within those highlighted as significant. A variance analysis finds that the variation between mice dominates this assay. These variance measures were used to examine the interplay between days, readings, and number of mice on power, the ability to detect change. If an experiment is underpowered, we cannot conclude whether failure to detect a biological difference arises from low power or lack of a distinct phenotype, hence the mice are subjected to testing without gain. Consequently, in confirmatory studies, a power analysis along with the 3Rs can provide justification to increase the number of mice used.

Testosterone, alcohol, and civil and rough conflict resolution strategies in lesbian couples.

The present study investigated the relations among testosterone level, acute alcohol consumption, and the use of violent (Rough) or non-violent (Civil) conflict resolution strategies in lesbian couples. The participants were 54 lesbian campers at a women's campground or spectators at a gay pride celebration who each provided a saliva sample for testosterone assay and completed a questionnaire. On the questionnaire, participants indicated whether they used Civil or Rough tactics to deal with domestic discord, and whether or not their use of these tactics varied with their use of alcohol. High testosterone women used Rough tactics equally when drinking as when not drinking, while low testosterone women used Rough tactics far more often when drinking than when not drinking. Alcohol appears to release violent tenden- cies in low testosterone women, who are characteristically restrained under sober conditions, but has little effect on high testosterone women.