Atomic Level Characterisation of Millisecond-Time Scale Protein Motions through a Combined Molecular Simulations and NMR Approach, bioRxiv, 2018-12-09
AbstractAdvances in biomolecular sciences are closely linked to our ability to chart the energy landscapes of biomolecules with atomic details. Here we validate a new paradigm to characterise thermodynamics and kinetics of millisecond timescale conformational transitions between ground state and transient excited states in the enzyme cyclophilin A (CypA). We describe a novel methodology that combines molecular dynamics simulations and Markov State modelling with NMR measurements to provide atomic-level insights into the nature of CypA transient conformational states. The computed conformational ensembles also enabled the predictive design and experimental validation of a single-site mutant that dramatically perturbs millisecond timescale loop motions, converting a CypA excited state into the ground state. The resulting models open up new horizons for targeting CypA with inhibitors and pave the way towards rational design of protein energy landscapes for protein engineering and drug discovery purposes.
biorxiv biophysics 0-100-users 2018Dynamic design manipulation of millisecond timescale motions on the energy landscape of Cyclophilin A, bioRxiv, 2018-12-09
AbstractProteins need to interconvert between many conformations in order to function, many of which are formed transiently, and sparsely populated. Particularly when the lifetimes of these states approach the millisecond timescale, identifying the relevant structures and the mechanism by which they inter-convert remains a tremendous challenge. Here we introduce a novel combination of accelerated MD (aMD) simulations and Markov State modelling (MSM) to explore these ‘excited’ conformational states. Applying this to the highly dynamic protein CypA, a protein involved in immune response and associated with HIV infection, we identify five principally populated conformational states and the atomistic mechanism by which they interconvert. A rational design strategy predicted that the mutant D66A should stabilise the minor conformations and substantially alter the dynamics whereas the similar mutant H70A should leave the landscape broadly unchanged. These predictions are confirmed using CPMG and R1ρ solution state NMR measurements. By accurately and reliably exploring functionally relevant, but sparsely populated conformations with milli-second lifetimes in silico, our aMDMSM method has tremendous promise for the design of dynamic protein free energy landscapes for both protein engineering and drug discovery.
biorxiv biophysics 0-100-users 2018Chemogenetic ligands for translational neurotheranostics, bioRxiv, 2018-12-08
AbstractDesigner Receptors Exclusively Activated by Designer Drugs (DREADDs) are a popular chemogenetic technology for manipulation of neuronal activity in uninstrumented awake animals with potential for precision medicine-based clinical theranostics. DREADD ligands developed to date are not appropriate for such translational applications. The prototypical DREADD agonist clozapine N-oxide (CNO) lacks brain entry and converts to clozapine. The second-generation DREADD agonist, Compound 21 (C21), was developed to overcome these limitations. We found that C21 has low brain penetrance, weak affinity, and low in vivo DREADD occupancy. To address these drawbacks, we developed two new DREADD agonists, JHU37152 and JHU37160, and the first dedicated positron emission tomography (PET) DREADD radiotracer, [18F]JHU37107. JHU37152 and JHU37160 exhibit high in vivo DREADD potency. [18F]JHU37107 combined with PET allows for DREADD detection in locally-targeted neurons and at their long-range projections, enabling for the first time, noninvasive and longitudinal neuronal projection mapping and potential for neurotheranostic applications.
biorxiv neuroscience 0-100-users 2018A comparison of gene expression and DNA methylation patterns across tissues and species, bioRxiv, 2018-12-05
AbstractPreviously published comparative functional genomic data sets from primates using frozen tissue samples, including many data sets from our own group, were collected and analyzed using non-optimal study designs and analysis approaches. In addition, when samples from multiple tissues were studied in a comparative framework, individual and tissue were confounded. We designed a multi-tissue comparative study of gene expression and DNA methylation in primates that minimizes confounding effects by using a balanced design with respect to species, tissues, and individuals. We also developed a comparative analysis pipeline that minimizes biases due to sequence divergence. We thus present the most comprehensive catalog of similarities and differences in gene expression and methylation levels between livers, kidneys, hearts, and lungs, in humans, chimpanzees, and rhesus macaques. We estimate that overall, only between 7 to 11% (depending on the tissue) of inter-species differences in gene expression levels can be accounted for by corresponding differences in promoter DNA methylation. However, gene expression divergence in conserved tissue-specific genes can be explained by corresponding inter-species methylation changes more often. We end the paper by providing recommendations for effective study design and best practices for meta-data recording for comparative functional genomic studies in primates.
biorxiv genomics 0-100-users 2018Natural selection contributed to immunological differences between human hunter-gatherers and agriculturalists, bioRxiv, 2018-12-05
AbstractThe shift from a hunter-gatherer (HG) to an agricultural (AG) mode of subsistence is believed to have been associated with profound changes in the burden and diversity of pathogens across human populations. Yet, the extent to which the advent of agriculture may have impacted the evolution of the human immune system remains unknown. Here we present a comparative study of variation in the transcriptional responses of peripheral blood mononuclear cells to bacterial and viral stimuli between Batwa rainforest hunter-gatherers and Bakiga agriculturalists from Uganda. We observed increased divergence between hunter-gatherers and agriculturalists in the transcriptional response to viruses compared to that for bacterial stimuli. We demonstrate that a significant fraction of these transcriptional differences are under genetic control, and we show that positive natural selection has helped to shape population differences in immune regulation. Across the set of genetic variants underlying inter-population immune response differences, however, the signatures of positive selection were disproportionately observed in the rainforest hunter-gatherers. This result is counter to expectations based on the popularized notion that shifts in pathogen exposure due to the advent of agriculture imposed radically heightened selective pressures in agriculturalist populations.
biorxiv evolutionary-biology 0-100-users 2018Non-academic employability of life science PhDs the importance of training beyond the bench, bioRxiv, 2018-12-05
ABSTRACTTo better understand how PhD graduates have prepared for the non-academic job market, we surveyed life science PhD and postdoctoral graduates from the University of Toronto who were employed in non-academic sectors. We also surveyed life science PhD and postdoctoral trainees to assess their engagement in career preparation activities. PhD professionals employed in non-academic sectors had engaged in various career preparation activities during their training. Some activities had a higher perceived impact on the path to employment than others. Trainees had also engaged in such activities, but those rated by professionals as having a highly positive impact on their path to employment were engaged in by only a minority of trainees. The proportion of trainees who wished to work in a non-academic sector was higher among those who were closer to program completion. Like professionals, many trainees reported facing barriers to pursuing career development activities. Our findings suggest that PhD trainees seeking to work in non-academic sectors should engage in career preparation activities, particularly those that involve experiential learning. By supporting co-curricular programming and reducing barriers to participation in career development activities, academic administrators and faculty have the opportunity to support trainees’ professional development beyond the laboratory.
biorxiv scientific-communication-and-education 0-100-users 2018