High-Precision Automated Reconstruction of Neurons with Flood-filling Networks, bioRxiv, 2017-10-10
AbstractReconstruction of neural circuits from volume electron microscopy data requires the tracing of complete cells including all their neurites. Automated approaches have been developed to perform the tracing, but without costly human proofreading their error rates are too high to obtain reliable circuit diagrams. We present a method for automated segmentation that, like the majority of previous efforts, employs convolutional neural networks, but contains in addition a recurrent pathway that allows the iterative optimization and extension of the reconstructed shape of individual neural processes. We used this technique, which we call flood-filling networks, to trace neurons in a data set obtained by serial block-face electron microscopy from a male zebra finch brain. Our method achieved a mean error-free neurite path length of 1.1 mm, an order of magnitude better than previously published approaches applied to the same dataset. Only 4 mergers were observed in a neurite test set of 97 mm path length.
biorxiv neuroscience 0-100-users 2017Predictive Coding of Novel versus Familiar Stimuli in the Primary Visual Cortex, bioRxiv, 2017-10-04
AbstractTo explore theories of predictive coding, we presented mice with repeated sequences of images with novel images sparsely substituted. Under these conditions, mice could be rapidly trained to lick in response to a novel image, demonstrating a high level of performance on the first day of testing. Using 2-photon calcium imaging to record from layer 23 neurons in the primary visual cortex, we found that novel images evoked excess activity in the majority of neurons. When a new stimulus sequence was repeatedly presented, a majority of neurons had similarly elevated activity for the first few presentations, which then decayed to almost zero activity. The decay time of these transient responses was not fixed, but instead scaled with the length of the stimulus sequence. However, at the same time, we also found a small fraction of the neurons within the population (∼2%) that continued to respond strongly and periodically to the repeated stimulus. Decoding analysis demonstrated that both the transient and sustained responses encoded information about stimulus identity. We conclude that the layer 23 population uses a two-channel predictive code a dense transient code for novel stimuli and a sparse sustained code for familiar stimuli. These results extend and unify existing theories about the nature of predictive neural codes.
biorxiv neuroscience 0-100-users 2017Directed evolution of TurboID for efficient proximity labeling in living cells and organisms, bioRxiv, 2017-10-03
AbstractProtein interaction networks and protein compartmentation underlie every signaling process and regulatory mechanism in cells. Recently, proximity labeling (PL) has emerged as a new approach to study the spatial and interaction characteristics of proteins in living cells. However, the two enzymes commonly used for PL come with tradeoffs – BioID is slow, requiring tagging times of 18-24 hours, while APEX peroxidase uses substrates that have limited cell permeability and high toxicity. To address these problems, we used yeast display-based directed evolution to engineer two mutants of biotin ligase, TurboID and miniTurbo, with much greater catalytic efficiency than BioID, and the ability to carry out PL in cells in much shorter time windows (as little as 10 minutes) with non-toxic and easily deliverable biotin. In addition to shortening PL time by 100-fold and increasing PL yield in cell culture, TurboID enabled biotin-based PL in new settings, including yeast, Drosophila, and C. elegans.
biorxiv bioengineering 0-100-users 2017Reprogramming human T cell function and specificity with non-viral genome targeting, bioRxiv, 2017-09-01
Human T cells are central to physiological immune homeostasis, which protects us from pathogens without collateral autoimmune inflammation. They are also the main effectors in most current cancer immunotherapy strategies1. Several decades of work have aimed to genetically reprogram T cells for therapeutic purposes2–5, but as human T cells are resistant to most standard methods of large DNA insertion these approaches have relied on recombinant viral vectors, which do not target transgenes to specific genomic sites6, 7. In addition, the need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells through homology-directed repair (HDR), but to date in human T cells this still requires viral transduction8, 9. Here, we developed a non-viral, CRISPR-Cas9 genome targeting system that permits the rapid and efficient insertion of individual or multiplexed large (>1 kilobase) DNA sequences at specific sites in the genomes of primary human T cells while preserving cell viability and function. We successfully tested the potential therapeutic use of this approach in two settings. First, we corrected a pathogenic IL2RA mutation in primary T cells from multiple family members with monogenic autoimmune disease and demonstrated enhanced signalling function. Second, we replaced the endogenous T cell receptor (TCR) locus with a new TCR redirecting T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognized the tumour antigen, with concomitant cytokine release and tumour cell killing. Taken together, these studies provide preclinical evidence that non-viral genome targeting will enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells.
biorxiv genetics 0-100-users 2017Patterns of structural variation in human cancer, bioRxiv, 2017-08-28
ABSTRACTA key mutational process in cancer is structural variation, in which rearrangements delete, amplify or reorder genomic segments ranging in size from kilobases to whole chromosomes. We developed methods to group, classify and describe structural variants, applied to >2,500 cancer genomes. Nine signatures of structural variation emerged. Deletions have trimodal size distribution; assort unevenly across tumour types and patients; enrich in late-replicating regions; and correlate with inversions. Tandem duplications also have trimodal size distribution, but enrich in early-replicating regions, as do unbalanced translocations. Replication-based mechanisms of rearrangement generate varied chromosomal structures with low-level copy number gains and frequent inverted rearrangements. One prominent structure consists of 1-7 templates copied from distinct regions of the genome strung together within one locus. Such ‘cycles of templated insertions’ correlate with tandem duplications, frequently activating the telomerase gene, TERT, in liver cancer. Cancers access many rearrangement processes, flexibly sculpting the genome to maximise oncogenic potential.
biorxiv cancer-biology 0-100-users 2017High Aspect Ratio Nanomaterials Enable Delivery of Functional Genetic Material Without DNA Integration in Mature Plants, bioRxiv, 2017-08-23
Genetic engineering of plants is at the core of sustainability efforts, natural product synthesis, and agricultural crop engineering. The plant cell wall is a barrier that limits the ease and throughput with which exogenous biomolecules can be delivered to plants. Current delivery methods either suffer from host range limitations, low transformation efficiencies, tissue damage, or unavoidable DNA integration into the host genome. Here, we demonstrate efficient diffusion-based biomolecule delivery into tissues and organs of intact plants of several species with a suite of pristine and chemically-functionalized high aspect ratio nanomaterials. Efficient DNA delivery and strong protein expression without transgene integration is accomplished in Nicotiana benthamiana (Nb), Eruca sativa (arugula), Triticum aestivum (wheat) and Gossypium hirsutum (cotton) leaves and arugula protoplasts. We also demonstrate a second nanoparticle-based strategy in which small interfering RNA (siRNA) is delivered to Nb leaves and silence a gene with 95% efficiency. We find that nanomaterials not only facilitate biomolecule transport into plant cells but also protect polynucleotides from nuclease degradation. Our work provides a tool for species-independent and passive delivery of genetic material, without transgene integration, into plant cells for diverse biotechnology applications.
biorxiv plant-biology 0-100-users 2017