The high-affinity binding site for tricyclic antidepressants resides in the outer vestibule of the serotonin transporter

The structure of the bacterial leucine transporter from Aquifex aeolicus (LeuT(Aa)) has been used as a model for mammalian Na+/Cl--dependent transporters, in particular the serotonin transporter (SERT). The crystal structure of LeuT(Aa) liganded to tricyclic antidepressants predicts simultaneous binding of inhibitor and substrate. This is incompatible with the mutually competitive inhibition of substrates and inhibitors of SERT. We explored the binding modes of tricyclic antidepressants by homology modeling and docking studies. Two approaches were used subsequently to differentiate between three clusters of potential docking poses: 1) a diagnostic SERTY95F mutation, which greatly reduced the affinity for [H-3] imipramine but did not affect substrate binding; 2) competition binding experiments in the presence and absence of carbamazepine (i.e., a tricyclic imipramine analog with a short side chain that competes with [3H] imipramine binding to SERT). Binding of releasers (para-chloroamphetamine, methylene-dioxy-methamphetamine/ecstasy) and of carbamazepine were mutually exclusive, but Dixon plots generated in the presence of carbamazepine yielded intersecting lines for serotonin, MPP+, paroxetine, and ibogaine. These observations are consistent with a model, in which 1) the tricyclic ring is docked into the outer vestibule and the dimethyl-aminopropyl side chain points to the substrate binding site; 2) binding of amphetamines creates a structural change in the inner and outer vestibule that precludes docking of the tricyclic ring; 3) simultaneous binding of ibogaine (which binds to the inward-facing conformation) and of carbamazepine is indicative of a second binding site in the inner vestibule, consistent with the pseudosymmetric fold of monoamine transporters. This may be the second low-affinity binding site for antidepressants

Evolution of heterogeneous genome differentiation across multiple contact zones in a crow species complex

Uncovering the genetic basis of species diversification is a central goal in evolutionary biology. Yet, the link between the accumulation of genomic changes during population divergence and the evolutionary forces promoting reproductive isolation is poorly understood. Here, we analysed 124 genomes of crow populations with various degrees of genome-wide differentiation, with parallelism of a sexually selected plumage phenotype, and ongoing hybridization. Overall, heterogeneity in genetic differentiation along the genome was best explained by linked selection exposed on a shared genome architecture. Superimposed on this common background, we identified genomic regions with signatures of selection specific to independent phenotypic contact zones. Candidate pigmentation genes with evidence for divergent selection were only partly shared, suggesting context-dependent selection on a multigenic trait architecture and parallelism by pathway rather than by repeated single-gene effects. This study provides insight into how various forms of selection shape genome-wide patterns of genomic differentiation as populations diverge

Combination of short-read, long-read, and optical mapping assemblies reveals large-scale tandem repeat arrays with population genetic implications

Accurate and contiguous genome assembly is key to a comprehensive understanding of the processes shaping genomic diversity and evolution. Yet, it is frequently constrained by constitutive heterochromatin, usually characterized by highly repetitive DNA. As a key feature of genome architecture associated with centromeric and subtelomeric regions, it locally influences meiotic recombination. In this study, we assess the impact of large tandem repeat arrays on the recombination rate landscape in an avian speciation model, the Eurasian crow. We assembled two high-quality genome references using single-molecule real-time sequencing (long-read assembly [LR]) and single-molecule optical maps (optical map assembly [OM]). A three-way comparison including the published short-read assembly (SR) constructed for the same individual allowed assessing assembly properties and pinpointing misassemblies. By combining information from all three assemblies, we characterized 36 previously unidentified large repetitive regions in the proximity of sequence assembly breakpoints, the majority of which contained complex arrays of a 14-kb satellite repeat or its 1.2-kb subunit. Using whole-genome population resequencing data, we estimated the population-scaled recombination rate (ρ) and found it to be significantly reduced in these regions. These findings are consistent with an effect of low recombination in regions adjacent to centromeric or subtelomeric heterochromatin and add to our understanding of the processes generating widespread heterogeneity in genetic diversity and differentiation along the genome. By combining three different technologies, our results highlight the importance of adding a layer of information on genome structure that is inaccessible to each approach independently

From forensics to clinical research: expanding the variant calling pipeline for the precision ID mtDNA whole genome panel

Despite a multitude of methods for the sample preparation, sequencing, and data analysis of mitochondrial DNA (mtDNA), the demand for innovation remains, particularly in comparison with nuclear DNA (nDNA) research. The Applied Biosystems™ Precision ID mtDNA Whole Genome Panel (Thermo Fisher Scientific, USA) is an innovative library preparation kit suitable for degraded samples and low DNA input. However, its bioinformatic processing occurs in the enterprise Ion Torrent Suite™ Software (TSS), yielding BAM files aligned to an unorthodox version of the revised Cambridge Reference Sequence (rCRS), with a heteroplasmy threshold level of 10%. Here, we present an alternative customizable pipeline, the PrecisionCallerPipeline (PCP), for processing samples with the correct rCRS output after Ion Torrent sequencing with the Precision ID library kit. Using 18 samples (3 original samples and 15 mixtures) derived from the 1000 Genomes Project, we achieved overall improved performance metrics in comparison with the proprietary TSS, with optimal performance at a 2.5% heteroplasmy threshold. We further validated our findings with 50 samples from an ongoing independent cohort of stroke patients, with PCP finding 98.31% of TSS’s variants (TSS found 57.92% of PCP’s variants), with a significant correlation between the variant levels of variants found with both pipelines

Revising mtDNA haplotypes of the ancient Hungarian conquerors with next generation sequencing

As part of the effort to create a high resolution representative sequence database of the medieval Hungarian conquerors we have resequenced the entire mtDNA genome of 24 published ancient samples with Next Generation Sequencing, whose haplotypes had been previously determined with traditional PCR based methods. We show that PCR based methods are prone to erroneous haplotype or haplogroup determination due to ambiguous sequence reads, and many of the resequenced samples had been classified inaccurately. The SNaPshot method applied with published ancient DNA authenticity criteria is the most straightforward and cheapest PCR based approach for testing a large number of coding region SNP-s, which greatly facilitates correct haplogroup determination

Association of mitochondrial DNA copy number with metabolic syndrome and type 2 diabetes in 14 176 individuals

Background Mitochondria play an important role in cellular metabolism, and their dysfunction is postulated to be involved in metabolic disturbances. Mitochondrial DNA is present in multiple copies per cell. The quantification of mitochondrial DNA copy number (mtDNA-CN) might be used to assess mitochondrial dysfunction. Objectives We aimed to investigate the cross-sectional association of mtDNA-CN with type 2 diabetes and the potential mediating role of metabolic syndrome. Methods We examined 4812 patients from the German Chronic Kidney Disease (GCKD) study and 9364 individuals from the Cooperative Health Research in South Tyrol (CHRIS) study. MtDNA-CN was measured in whole blood using a plasmid-normalized qPCR-based assay. Results In both studies, mtDNA-CN showed a significant correlation with most metabolic syndrome parameters: mtDNA-CN decreased with increasing number of metabolic syndrome components. Furthermore, individuals with low mtDNA-CN had significantly higher odds of metabolic syndrome (OR = 1.025; 95% CI = 1.011–1.039, P = 3.19 × 10−4, for each decrease of 10 mtDNA copies) and type 2 diabetes (OR = 1.027; 95% CI = 1.012–1.041; P = 2.84 × 10−4) in a model adjusted for age, sex, smoking and kidney function in the meta-analysis of both studies. Mediation analysis revealed that the association of mtDNA-CN with type 2 diabetes was mainly mediated by waist circumference in the GCKD study (66%) and by several metabolic syndrome parameters, especially body mass index and triglycerides, in the CHRIS study (41%). Conclusions Our data show an inverse association of mtDNA-CN with higher risk of metabolic syndrome and type 2 diabetes. A major part of the total effect of mtDNA-CN on type 2 diabetes is mediated by obesity parameters

Discovery and population genomics of structural variation in a songbird genus

Structural variation (SV) constitutes an important type of genetic mutations providing the raw material for evolution. Here, we uncover the genome-wide spectrum of intra- and interspecific SV segregating in natural populations of seven songbird species in the genus Corvus. Combining short-read (N = 127) and long-read re-sequencing (N = 31), as well as optical mapping (N = 16), we apply both assembly- and read mapping approaches to detect SV and characterize a total of 220,452 insertions, deletions and inversions. We exploit sampling across wide phylogenetic timescales to validate SV genotypes and assess the contribution of SV to evolutionary processes in an avian model of incipient speciation. We reveal an evolutionary young (~530,000 years) cis-acting 2.25-kb LTR retrotransposon insertion reducing expression of the NDP gene with consequences for premating isolation. Our results attest to the wealth and evolutionary significance of SV segregating in natural populations and highlight the need for reliable SV genotyping