Prokaryotic nanocompartments form synthetic organelles in a eukaryote, bioRxiv, 2018-01-07
AbstractCompartmentalization of proteins into organelles is a promising strategy for enhancing the productivity of engineered eukaryotic organisms. However, approaches that co-opt endogenous organelles may be limited by the potential for unwanted crosstalk and disruption of native metabolic functions. Here, we present the construction of synthetic non-endogenous organelles in the eukaryotic yeast Saccharomyces cerevisiae, based on the prokaryotic family of self-assembling proteins known as encapsulins. We establish that encapsulins self-assemble to form nanoscale compartments in yeast, and that heterologous proteins can be selectively targeted for compartmentalization. Housing destabilized proteins within encapsulin compartments affords protection against proteolytic degradation in vivo, while the interaction between split protein components is enhanced upon co-localization within the compartment interior. Furthermore, encapsulin compartments can support enzymatic catalysis, with substrate turnover observed for an encapsulated yeast enzyme. Encapsulin compartments therefore represent a modular platform, orthogonal to existing organelles, for programming synthetic compartmentalization in eukaryotes.
biorxiv synthetic-biology 0-100-users 2018Graph theory approaches to functional network organization in brain disorders A critique for a brave new small-world, bioRxiv, 2018-01-06
AbstractOver the past two decades, resting-state functional connectivity (RSFC) methods have provided new insights into the network organization of the human brain. Studies of brain disorders such as Alzheimer’s disease or depression have adapted tools from graph theory to characterize differences between healthy and patient populations. Here, we conducted a review of clinical network neuroscience, summarizing methodological details from 106 RSFC studies. Although this approach is prevalent and promising, our review identified four challenges. First, the composition of networks varied remarkably in terms of region parcellation and edge definition, which are fundamental to graph analyses. Second, many studies equated the number of connections across graphs, but this is conceptually problematic in clinical populations and may induce spurious group differences. Third, few graph metrics were reported in common, precluding meta-analyses. Fourth, some studies tested hypotheses at one level of the graph without a clear neurobiological rationale or considering how findings at one level (e.g., global topology) are contextualized by another (e.g., modular structure). Based on these themes, we conducted network simulations to demonstrate the impact of specific methodological decisions on case-control comparisons. Finally, we offer suggestions for promoting convergence across clinical studies in order to facilitate progress in this important field.
biorxiv neuroscience 0-100-users 2018Identification of Pre-Existing Adaptive Immunity to Cas9 Proteins in Humans, bioRxiv, 2018-01-06
AbstractThe CRISPR-Cas9 system has proven to be a powerful tool for genome editing, allowing for the precise modification of specific DNA sequences within a cell. Many efforts are currently underway to use the CRISPR-Cas9 system for the therapeutic correction of human genetic diseases. The most widely used homologs of the Cas9 protein are derived from the bacteria Staphylococcus aureus (S. aureus) and Streptococcus pyogenes (S. pyogenes). Based on the fact that these two bacterial species cause infections in the human population at high frequencies, we looked for the presence of pre-existing adaptive immune responses to their respective Cas9 homologs, SaCas9 (S. aureus homolog of Cas9) and SpCas9 (S. pyogenes homolog of Cas9). To determine the presence of anti-Cas9 antibodies, we probed for the two homologs using human serum and were able to detect antibodies against both, with 79% of donors staining against SaCas9 and 65% of donors staining against SpCas9. Upon investigating the presence of antigen-specific T-cells against the two homologs in human peripheral blood, we found anti-SaCas9 T-cells in 46% of donors. Upon isolating, expanding, and conducting antigen re-stimulation experiments on several of these donors’ anti-SaCas9 T-cells, we observed an SaCas9-specific response confirming that these T-cells were antigen-specific. We were unable to detect antigen-specific T-cells against SpCas9, although the sensitivity of the assay precludes us from concluding that such T-cells do not exist. Together, this data demonstrates that there are pre-existing humoral and cell-mediated adaptive immune responses to Cas9 in humans, a factor which must be taken into account as the CRISPR-Cas9 system moves forward into clinical trials.
biorxiv immunology 500+-users 2018Protein-coding variation and introgression of regulatory alleles drive plumage pattern diversity in the rock pigeon, bioRxiv, 2018-01-06
ABSTRACTBirds and other vertebrates display stunning variation in pigmentation patterning, yet the genes controlling this diversity remain largely unknown. Rock pigeons (Columba livia) are fundamentally one of four color pattern phenotypes, in decreasing order of melanism T-check, checker, bar (ancestral), or barless. Using whole-genome scans, we identified NDP as a candidate gene for this variation. Allele-specific expression differences in NDP indicate cis-regulatory differences between ancestral and melanistic alleles. Sequence comparisons suggest that derived alleles originated in the speckled pigeon (Columba guinea), providing a striking example of introgression of alleles that are favored by breeders and are potentially advantageous in the wild. In contrast, barless rock pigeons have an increased incidence of vision defects and, like two human families with hereditary blindness, carry start-codon mutations in NDP. In summary, we find unexpected links between color pattern, introgression, and vision defects associated with regulatory and coding variation at a single locus.
biorxiv evolutionary-biology 0-100-users 2018Genome-wide association study of 1 million people identifies 111 loci for atrial fibrillation, bioRxiv, 2018-01-05
SummaryTo understand the genetic variation underlying atrial fibrillation (AF), the most common cardiac arrhythmia, we performed a genome-wide association study (GWAS) of > 1 million people, including 60,620 AF cases and 970,216 controls. We identified 163 independent risk variants at 111 loci and prioritized 165 candidate genes likely to be involved in AF. Many of the identified risk variants fall near genes where more deleterious mutations have been reported to cause serious heart defects in humans or mice (MYH6, NKX2-5, PITX2, TBC1D32, TBX5),1,2 or near genes important for striated muscle function and integrity (e.g. MYH7, PKP2, SSPN, SGCA). Experiments in rabbits with heart failure and left atrial dilation identified a heterogeneous distributed molecular switch from MYH6 to MYH7 in the left atrium, which resulted in contractile and functional heterogeneity and may predispose to initiation and maintenance of atrial arrhythmia.
biorxiv genetics 100-200-users 2018Acoustically Targeted Chemogenetics for Noninvasive Control of Neural Circuits, bioRxiv, 2018-01-02
ABSTRACTNeurological and psychiatric diseases often involve the dysfunction of specific neural circuits in particular regions of the brain. Existing treatments, including drugs and implantable brain stimulators, aim to modulate the activity of these circuits, but are typically not cell type-specific, lack spatial targeting or require invasive procedures. Here, we introduce an approach to modulating neural circuits noninvasively with spatial, cell-type and temporal specificity. This approach, called acoustically targeted chemogenetics, or ATAC, uses transient ultrasonic opening of the blood brain barrier to transduce neurons at specific locations in the brain with virally-encoded engineered G-protein-coupled receptors, which subsequently respond to systemically administered bio-inert compounds to activate or inhibit the activity of these neurons. We demonstrate this concept in mice by using ATAC to noninvasively modify and subsequently activate or inhibit excitatory neurons within the hippocampus, showing that this enables pharmacological control of memory formation. This technology allows a brief, noninvasive procedure to make one or more specific brain regions capable of being selectively modulated using orally bioavailable compounds, thereby overcoming some of the key limitations of conventional brain therapies.
biorxiv neuroscience 100-200-users 2018