Biology subjects

Wong, W.

Publications and source records attributed to Wong, W..

68 records · Page 4Linked to original sources

bioRxiv2020

Scalable recombinase-based gene expression cascades

Temporal modulation of multiple genes underlies sophisticated biological phenomena. However, there are few scalable and generalizable gene circuit architectures for the programming of sequential genetic perturbations. We describe a modular recombinase-based gene circuit architecture, comprising tandem gene perturbation cassettes (GPCs), that enables the sequential expression of multiple genes in a defined temporal order by alternating treatment with just two orthogonal ligands. We used tandem GPCs to sequentially express single-guide RNAs to encode transcriptional cascades and trigger the sequential accumulation of mutations. We built an all-in-one gene circuit that sequentially edits genomic loci, synchronizes cells at a specific stage within a gene expression cascade, and deletes itself for safety. Tandem GPCs offer a multi-tiered cellular programming tool for modeling multi-stage genetic changes, such as tumorigenesis and cellular differentiation.Competing Interest StatementThe authors have declared no competing interest.View Full Text

synthetic biology↗

bioRxiv2020

Spiny mice (Acomys) exhibit attenuated hallmarks of aging and rapid cell turnover after UV exposure in the skin epidermis

The study of long-lived and regenerative animal models has revealed diverse protective responses to stressors such as aging and tissue injury. Spiny mice (Acomys) are a unique mammalian model of skin regeneration, but their response to other types of physiological skin damage have not been investigated. In this study, we examine how spiny mice skin responds to acute UVB damage or chronological aging compared to non-regenerative C57Bl/6 mice (M. musculus). We find that, compared to M. musculus, the skin epidermis in A. cahirinus experiences a similar UVB-induced increase in basal cell proliferation but exhibits increased epidermal turnover. Notably, A. cahirinus uniquely form a suprabasal layer co-expressing Keratin 14 and Keratin 10 after UVB exposure concomitant with reduced epidermal inflammatory signaling and reduced markers of DNA damage. In the context of aging, old M. musculus animals exhibit typical hallmarks including epidermal thinning, increased inflammatory signaling and senescence. However, these age-related changes are absent in old A. cahirinus skin. Overall, we find that A. cahirinus have evolved novel responses to skin damage that reveals new aspects of its regenerative phenotype.

cell biology↗

bioRxiv2020

A mechanistic model of the BLADE platform predicts performance characteristics of 256 different synthetic DNA recombination circuits

Boolean logic and arithmetic through DNA excision (BLADE) is a recently developed platform for implementing inducible and logical control over gene expression in mammalian cells, which has the potential to revolutionise cell engineering for therapeutic applications. This 2-input 2-output platform can implement 256 different logical circuits that exploit the specificity and stability of DNA recombination. Here, we develop the first mechanistic mathematical model of the 2-input BLADE platform based on Cre- and Flp-mediated DNA excision. After calibrating the model on experimental data from two circuits, we demonstrate close agreement between model outputs and data on the other 111 circuits that have so far been experimentally constructed using the 2-input BLADE platform. Model simulations of the remaining 143 circuits that have yet to be tested experimentally predict excellent performance of the 2-input BLADE platform across the range of possible circuits. Circuits from both the tested and untested subsets that perform less well consist of a disproportionally high number of STOP sequences. Model predictions suggested that circuit performance declines with a decrease in recombinase expression and new experimental data was generated that confirms this relationship.

synthetic biology↗

bioRxiv2020

Chandelier cell anatomy and function reveal a variably distributed but common signal

The activity and connectivity of inhibitory cells has a profound impact on the operation of neuronal networks. While the average connectivity of many inhibitory cell types has been characterized, we still lack an understanding of how individual interneurons distribute their synapses onto their targets and how heterogeneous the inhibition is onto different individual excitatory neurons. Here, we use large-scale volumetric electron microscopy (EM) and functional imaging to address this question for chandelier cells in layer 2/3 of mouse visual cortex. Using dense morphological reconstructions from EM, we mapped the complete chandelier input onto 153 pyramidal neurons. We find that the number of input synapses is highly variable across the population, but the variability is correlated with structural features of the target neuron: soma depth, soma size, and the number of perisomatic synapses received. Functionally, we found that chandelier cell activity in vivo was highly correlated and tracks pupil diameter, a proxy for arousal state. We propose that chandelier cells provide a global signal whose strength is individually adjusted for each target neuron. This approach, combining comprehensive structural analysis with functional recordings of identified cell types, will be a powerful tool to uncover the wiring rules across the diversity of cortical cell types.

neuroscience↗

bioRxiv2020

CDeep3M-Preview: Online segmentation using the deep neural network model zoo

Sharing deep neural networks and testing the performance of trained networks typically involves a major initial commitment towards one algorithm, before knowing how the network will perform on a different dataset. Here we release a free online tool, CDeep3M-Preview, that allows end-users to rapidly test the performance of any of the pre-trained neural network models hosted on the CIL-CDeep3M modelzoo. This feature makes part of a set of complementary strategies we employ to facilitate sharing, increase reproducibility and enable quicker insights into biology. Namely we: (1) provide CDeep3M deep learning image segmentation software through cloud applications (Colab and AWS) and containerized installations (Docker and Singularity) (2) co-hosting trained deep neural networks with the relevant microscopy images and (3) providing a CDeep3M-Preview feature, enabling quick tests of trained networks on user provided test data or any of the publicly hosted large datasets. The CDeep3M-modelzoo and the cellimagelibrary.org are open for contributions of both, trained models as well as image datasets by the community and all services are free of charge.

neuroscience↗

bioRxiv2020

EASE: EM-Assisted Source Extraction from calcium imaging data

Combining two-photon calcium imaging (2PCI) and electron microscopy (EM) provides arguably the most powerful current approach for connecting function to structure in neural circuits. Recent years have seen dramatic advances in obtaining and processing CI and EM data separately. In addition, several joint CI-EM datasets (with CI performed in vivo, followed by EM reconstruction of the same volume) have been collected. However, no automated analysis tools yet exist that can match each signal extracted from the CI data to a cell segment extracted from EM; previous efforts have been largely manual and focused on analyzing calcium activity in cell bodies, neglecting potentially rich functional information from axons and dendrites. There are two major roadblocks to solving this matching problem: first, dense EM reconstruction extracts orders of magnitude more segments than are visible in the corresponding CI field of view, and second, due to optical constraints and non-uniform brightness of the calcium indicator in each cell, direct matching of EM and CI spatial components is nontrivial. In this work we develop a pipeline for fusing CI and densely-reconstructed EM data. We model the observed CI data using a constrained nonnegative matrix factorization (CNMF) framework, in which segments extracted from the EM reconstruction serve to initialize and constrain the spatial components of the matrix factorization. We develop an efficient iterative procedure for solving the resulting combined matching and matrix factorization problem and apply this procedure to joint CI-EM data from mouse visual cortex. The method recovers hundreds of dendritic components from the CI data, visible across multiple functional scans at different depths, matched with densely-reconstructed three-dimensional neural segments recovered from the EM volume. We publicly release the output of this analysis as a new gold standard dataset that can be used to score algorithms for demixing signals from 2PCI data. Finally, we show that this database can be exploited to (1) learn a mapping from 3d EM segmentations to predict the corresponding 2d spatial components estimated from CI data, and (2) train a neural network to denoise these estimated spatial components. This neural network denoiser is a stand-alone module that can be dropped in to enhance any existing 2PCI analysis pipeline.

neuroscience↗

bioRxiv2020

A Universal Law of Sensory Adaptation

Measurements of the peripheral sensory adaptation response were compared to a simple mathematical relationship involving the spontaneous, peak and steady-state activities. This relationship is based on the geometric mean and is found to be obeyed to good approximation in peripheral sensory units showing a sustained response to prolonged stimulation. From an extensive review of past studies, the geometric mean relationship is shown to be independent of modality and is satisfied in a wide range of animal species. The consilience of evidence, from nearly one hundred years of experiments beginning with the work of Edgar Adrian, suggests that this is a fundamental result of neurophysiology.

neuroscience↗

bioRxiv2019

Binary and analog variation of synapses between cortical pyramidal neurons

Learning from experience depends at least in part on changes in neuronal connections. We present the largest map of connectivity to date between cortical neurons of a defined type (L2/3 pyramidal cells), which was enabled by automated analysis of serial section electron microscopy images with improved handling of image defects. We used the map to identify constraints on the learning algorithms employed by the cortex. Previous cortical studies modeled a continuum of synapse sizes (Arellano et al. 2007) by a log-normal distribution (Loewenstein, Kuras, and Rumpel 2011; de Vivo et al. 2017; Santuy et al. 2018). A continuum is consistent with most neural network models of learning, in which synaptic strength is a continuously graded analog variable. Here we show that synapse size, when restricted to synapses between L2/3 pyramidal cells, is well-modeled by the sum of a binary variable and an analog variable drawn from a log-normal distribution. Two synapses sharing the same presynaptic and postsynaptic cells are known to be correlated in size (Sorra and Harris 1993; Koester and Johnston 2005; Bartol et al. 2015; Kasthuri et al. 2015; Dvorkin and Ziv 2016; Bloss et al. 2018; Motta et al. 2019). We show that the binary variables of the two synapses are highly correlated, while the analog variables are not. Binary variation could be the outcome of a Hebbian or other synaptic plasticity rule depending on activity signals that are relatively uniform across neuronal arbors, while analog variation may be dominated by other influences. We discuss the implications for the stability-plasticity dilemma.

neuroscience↗

bioRxiv2019

Light-Inducible Recombinases for Bacterial Optogenetics

Optogenetic tools can provide direct and programmable control of gene expression. Light-inducible recombinases, in particular, offer a powerful method for achieving precise spatiotemporal control of DNA modification. However, to-date this technology has been largely limited to eukaryotic systems. Here, we develop optogenetic recombinases for Escherichia coli which activate in response to blue light. Our approach uses a split recombinase coupled with photodimers, where blue light brings the split protein together to form a functional recombinase. We tested both Cre and Flp recombinases, Vivid and Magnet photodimers, and alternative protein split sites in our analysis. The optimal configuration, Opto-Cre-Vvd, exhibits strong blue light-responsive excision and low ambient light sensitivity. For this system we characterize the effect of light intensity and the temporal dynamics of light-induced recombination. These tools expand the microbial optogenetic toolbox, offering the potential for precise control of DNA excision with light-inducible recombinases in bacteria.

bioengineering↗

bioRxiv2019

The Dream Catcher experiment: Blinded analyses disconfirm markers of dreaming consciousness in EEG spectral power

The Dream Catcher test defines the criteria for a genuine discovery of the neural constituents of phenomenal consciousness. Passing the test implies that some patterns of purely brain-based data directly correspond to the subjective features of phenomenal experience, which would help to bridge the explanatory gap between consciousness and brain. Here, we conducted the Dream Catcher test for the first time in a graded and simplified form, capturing its core idea. The experiment involved a Data Team, who measured participants brain activity during sleep and collected dream reports, and a blinded Analysis Team, who was challenged to predict better than chance, based solely on brain measurements, whether or not a participant had a dream experience. Using a serial-awakening paradigm, the Data Team prepared 54 one-minute polysomnograms of NREM sleep--27 of dreamful sleep (3 from each of the 9 participants) and 27 of dreamless sleep--redacting from them all associated participant and dream information. The Analysis Team attempted to classify each recording as either dreamless or dreamful using an unsupervised machine learning classifier, based on hypothesis-driven, extracted features of EEG spectral power and electrode location. The procedure was repeated over five iterations with a gradual removal of blindness. At no level of blindness did the Analysis Team perform significantly better than chance, suggesting that EEG spectral power does not carry any signatures of phenomenal consciousness. Furthermore, we demonstrate an outright failure to replicate key findings of recently reported correlates of dreaming consciousness.\n\nHighlightsThe first reported attempt of the Dream Catcher test.\nThe correlates of conscious experience may not lie in EEG spectral power.\nReported markers of NREM dreaming consciousness misperformed in a blinded setting.\nThose markers also could not be confirmed in an unblinded setting.

neuroscience↗

bioRxiv2018

Strength of T cell signaling regulates HIV-1 replication and establishment of latency

A major barrier to curing HIV is the long-lived latent reservoir that supports re-emergence of HIV upon treatment interruption. Targeting this reservoir will require mechanistic insights into the establishment and maintenance of HIV latency. Whether T cell signaling at the time of HIV-1 infection influences productive replication or latency is not fully understood. We used a panel of chimeric antigen receptors (CARs) with different ligand binding affinities to induce a range of signaling strengths to model differential T cell receptor signaling at the time of HIV-1 infection. Stimulation of T cell lines or primary CD4+ T cells expressing chimeric antigen receptors supported HIV-1 infection regardless of affinity for ligand; however, only signaling by the highest affinity receptor facilitated HIV-1 expression. Activation of chimeric antigen receptors that had intermediate and low binding affinities did not support provirus transcription, suggesting that a minimal signal is required for optimal HIV-1 expression. In addition, strong signaling at the time of infection produced a latent population that was readily inducible, whereas latent cells generated in response to weaker signals were not easily reversed. Chromatin immunoprecipitation showed HIV-1 transcription was limited by transcriptional elongation and that robust signaling decreased the presence of negative elongation factor, a pausing factor, by more than 80%. These studies demonstrate that T cell signaling influences HIV-1 infection and the establishment of different subsets of latently infected cells, which may have implications for targeting the HIV reservoir.\n\nAuthor SummaryActivation of CD4+ T cells facilitates HIV-1 infection; however, whether there are minimal signals required for the establishment of infection, replication, and latency has not been explored. To determine how T cell signaling influences HIV-1 infection and the generation of latently infected cells, we used chimeric antigen receptors to create a tunable model. Stronger signals result in robust HIV-1 expression and an inducible latent population. Minimal signals predispose cells towards latent infections that are refractory to reversal. We discovered that repression of HIV-1 transcription immediately after infection is due to RNA polymerase II pausing and inefficient transcription elongation. These studies demonstrate that signaling events influence the course of HIV-1 infection and have implications for cure strategies. They also provide a mechanistic explanation for why a significant portion of the HIV latent reservoir is not responsive to latency reversing agents which function by modifiying chromatin.

microbiology↗

bioRxiv2017

The complement system supports normal postnatal development and gonadal function in both sexes

Male and female infertility are clinically managed and classified as distinct diseases, and relatively little is known about mechanisms of gonadal function common to both sexes. We used genome-wide genetic analysis on 74,896 women and men to find rare genetic variants that modulate gonadal function in both sexes. This uncovered an association with variants disrupting CSMD1, a complement regulatory protein located on 8p23, in a genomic region with an exceptional evolution. We found that Csmd1 knockout mice display a diverse array of gonadal defects in both sexes, and in females, impaired mammary gland development that leads to increased offspring mortality. The complement pathway is significantly disrupted in Csmd1 mice, and further disruption of the complement pathway from joint inactivation of C3 leads to more extreme reproductive defects. Our results can explain a novel human genetic association with infertility and implicate the complement system in the normal development of postnatal tissues.

developmental biology↗

bioRxiv2017

hmmIBD: software to infer pairwise identity by descent between haploid genotypes

SummaryWe introduce hmmIBD, software to estimate pairwise identity by decent between haploid genomes, such as those of the malaria parasite, sampled from one or more populations. We verified hmmIBD using simulated data, benchmarked it against a previously published method for detecting IBD within populations, and demonstrated its utility using Plasmodium falciparum data from Cambodia and Ghana.\n\nSupplementary informationSupplementary data include Appendices S1, S2 and S3, and are available online.\n\nAvailability and ImplemetationSource code written in C99/C11-compliant C and requiring no external libraries, is freely available for download at https://github.com/glipsnort/hmmIBD/releases, alongside test datasets.\n\nContactsfs@broadinstitute.org

bioinformatics↗

bioRxiv2017

The RacGAP βChimaerin is essential for cerebellar granule cell migration

During mammalian cerebellar development, postnatal granule cell progenitors proliferate in the outer part of the External Granule Layer (EGL). Postmitotic granule progenitors migrate tangentially in the inner EGL before switching to migrate radially inward, past the Purkinje cell layer, to achieve their final position in the mature Granule Cell Layer (GCL). Here, we show that the RacGAP {beta}-chimaerin is expressed by a small population of late-born, premigratory granule cells. {beta}-chimaerin deficiency causes a subset of granule cells to become arrested in the EGL, where they differentiate and form ectopic neuronal clusters. These clusters of granule cells are able to recruit aberrantly projecting mossy fibers. Collectively, these data suggest a role for {beta}-chimaerin as an intracellular mediator of Cerebellar Granule Cell radial migration.

neuroscience↗