Biology subjects

Davis, I.

Publications and source records attributed to Davis, I..

At least 19 recordsLinked to original sources

bioRxiv2026

The Effects of Prelimbic Inhibition on Time-based Intervention and Impulsive Choice

Impulsive choice is the suboptimal preference for a smaller-sooner (SS, "impulsive") option over a larger-later (LL, "self-controlled") option. Fixed-interval (FI) training delivers delayed-reinforcement trials to increase LL choices and improve FI timing precision. While there are plenty of studies exploring the neurobiological factors underlying impulsive choice, it is unknown what neurobiological changes account for the FI training effects. The prelimbic cortex (PL) region is implicated in both impulsive choice and timing. To investigate the role of the PL, we used designer receptors exclusively activated by designer drugs (GiDREADDs) to reversibly inhibit the PL during either the FI training phase or the follow-up impulsive choice task in male and female Sprague-Dawley rats. Compared to a control group, the GiDREADDs rats showed reduced LL choices when CNO was administered during the FI training or impulsive choice tasks. GiDREADDS did not alter response rates or latency to choose. Overall, these data demonstrate that inhibition of the PL increases impulsive choice and may block the effect of the FI training to improve self-control.

animal behavior and cognition↗

bioRxiv2024

XRN1 supplies free nucleotides to feed alphavirus replication

Several RNA viruses induce widespread degradation of cellular mRNAs upon infection; however, the biological significance and mechanistic details of this phenomenon remain unknown. Here, we make use of a model alphavirus, Sindbis virus (SINV), to fill this knowledge gap. We found that SINV triggers cellular RNA decay through the exonuclease XRN1 and the 5-to-3 degradation machinery (5-3DM). These proteins accumulate at viral replication organelles (VROs) and interact with the non-structural protein 1 (nsP1), bringing mRNA degradation into proximity with vRNA synthesis. Our data suggest that monophosphate nucleotides released by cellular RNA decay are recycled through the salvage pathway to feed viral replications. Our work thus reveals a fundamental connection between cellular mRNA degradation and viral replication via nucleotides repurposing. Research highlightsO_LI5-3 RNA decay is essential for the replication of a wide range of viruses. C_LIO_LIXRN1 directly interacts with transcripts which are degraded during infection. C_LIO_LIRNA decay factors and salvage pathway members localise to viral factories. C_LIO_LISupplying nucleosides to several 5-3DM deficient cells facilitates SINV infection. C_LI

biochemistry↗

bioRxiv2024

Imp and Syp in vivo temporal RNA interactomes uncover networks of temporal regulators of Drosophila brain development.

Temporal patterning of neural progenitors is an evolutionarily conserved mechanism generating neural diversity. In Drosophila, post-embryonic neurogenesis requires the RNA-binding proteins (RBPs) Imp/IGF2BP and Syp/SYNCRIP. However, how they co-achieve their function is not well understood. Here, we elucidate the in vivo temporal RNA interactome landscapes of Imp and Syp during larval brain development. Imp and Syp bind a highly overlapping set of conserved mRNAs encoding proteins involved in neurodevelopment. We identify transcripts differentially occupied by Imp/Syp over time, featuring a network of known and novel candidate temporal regulators that are post-transcriptionally regulated by Imp/Syp. Furthermore, the physical and co-evolutionary relationships between Imp and Syp binding sites reveal a combinatorial, rather than competitive, mode of molecular interplay. Our study establishes a new in vivo framework for dissecting the temporal co-regulation of RBP networks as well as providing a resource for understanding neural fate specification.

cell biology↗

bioRxiv2023

Deep super-resolution imaging of thick tissue using structured illumination with adaptive optics

Three-dimensional structured illumination microscopy (3D-SIM) doubles the resolution of fluorescence imaging in all directions and increases the image contrast. However, 3D-SIM has not been widely applied to imaging deep in thick tissues due to its sensitivity to sample-induced aberrations, making the method difficult to apply beyond 10 {micro}m in depth. Furthermore, 3D-SIM has not been available in an upright configuration, limiting its use for live imaging while manipulating the specimen, for example with electrophysiology. Here, we have overcome these barriers by developing a novel upright 3D-SIM system (termed Deep3DSIM) that incorporates adaptive optics for aberration correction and remote focusing, reducing artefacts, improving contrast, restoring resolution, and removing the need to move the specimen or the objective lens in volume imaging. These advantages are equally applicable to inverted 3D-SIM systems. We demonstrate high-quality 3D-SIM imaging in various samples, including an example of imaging 130 {micro}m into Drosophila brain.

cell biology↗

bioRxiv2023

Incorporation of genome-bound cellular proteins into HIV-1 particles regulates viral infection

The initial steps of the human immunodeficiency virus 1 (HIV-1) lifecycle are regulated by cellular RNA-binding proteins (RBPs). To understand the scope of these early host-virus interactions, we developed in virion RNA interactome capture (ivRIC), which allowed the comprehensive and systematic profiling of the proteins that interact with the HIV-1 genomic (g)RNA inside viral particles. ivRIC identified 104 cellular RBPs within the encapsidated HIV-1 ribonucleoprotein, many of which are typically found in the cellular nucleus. Notably, these nuclear RBPs interact with the HIV-1 RBP Rev, suggesting that they associate with HIV-1 gRNA during its nuclear life. Functional assays show that ivRBPs are important for HIV-1, including PURA and PURB, which control viral gene expression and infectivity through interaction with critical sequences in the gRNA. Our characterisation of the composition of the encapsidated ribonucleoprotein of HIV-1 uncovers new host-virus interactions that invokes new mechanisms for controlling HIV-1 infection.

molecular biology↗

bioRxiv2022

Sex-specific deubiquitylation drives immune-related neurodegeneration in Drosophila

Risk of neurodegenerative disease such as late onset Alzheimers is linked to aberrant ubiquitinylation and accumulation of non-degraded proteins in brain cells. A glial network of innate immune genes modulates inflammatory responses to such protein deposition. However, vulnerability differs between the sexes. Here, we show that the Drosophila homologue of the deubiquitylase Trabid can align the sex-specific aspects of neurodegenerative phenotypes with changes in ubiquitylation and inflammatory activity. An enzymatically null Trabid in flies, caused sex-specific changes in locomotion, sleep patterns, brain histology and ultimately, lifespan. These changes were underscored by altered ubiquitin and proteome enrichment profiles and the same enzymatic activity as its human counterpart. When the sex-determination gene transformer was silenced in astrocytes or immunocompetent tissues, sex differences were significantly reduced. Our results indicate that Trabid underscores sex-specificity in disease neurology, by controlling the balance between ubiquitylation and inflammation.

immunology↗

bioRxiv2022

Dynamically regulated transcription factors are encoded by highly unstable mRNAs in the Drosophila larval brain

The level of each RNA species depends on the balance between its rates of production and decay. Although previous studies have measured RNA decay across the genome in tissue culture and single-celled organisms, few experiments have been performed in intact complex tissues and organs. It is therefore unclear whether the determinants of RNA decay found in cultured cells are preserved in an intact tissue, and whether they differ between neighboring cell types and are regulated during development. To address these questions, we measured RNA synthesis and decay rates genome wide via metabolic labeling of whole cultured Drosophila larval brains using 4-thiouridine. Our analysis revealed that decay rates span a range of more than 100-fold, and that RNA stability is linked to gene function, with mRNAs encoding transcription factors being much less stable than mRNAs involved in core metabolic functions. Surprisingly, among transcription factor mRNAs there was a clear demarcation between more widely used transcription factors and those that are expressed only transiently during development. mRNAs encoding transient transcription factors are among the least stable in the brain. These mRNAs are characterized by epigenetic silencing in most cell types, as shown by their enrichment with the histone modification H3K27me3. Our data suggests the presence of an mRNA destabilizing mechanism targeted to these transiently expressed transcription factors to allow their levels to be regulated rapidly with high precision. Our study also demonstrates a general method for measuring mRNA transcription and decay rates in intact organs or tissues, offering insights into the role of mRNA stability in the regulation of complex developmental programs.

biochemistry↗

bioRxiv2022

Mammalian glial protrusion transcriptomes predict localization of Drosophila glial transcripts required for synaptic plasticity

The polarization of cells often involves the transport of specific mRNAs and their localized translation in distal projections. Neurons and glia both contain long cytoplasmic processes with important functions. mRNA localization has been studied extensively in neurons, but very little in glia, especially in intact nervous systems. Here, we predicted 1,700 localized Drosophila glial transcripts by extrapolating from our meta-analysis of 8 existing studies characterizing the localized transcriptomes and translatomes of synaptically-associated mammalian glia. We tested these predictions in glia of the neuromuscular junction of Drosophila larvae and found that localization to mammalian glia is a strong predictor of mRNA localization of the high confidence Drosophila homologues. We further showed that some of these localized transcripts are required in glia for plasticity of the neuromuscular junction synapses. We conclude that peripheral glial mRNA localization is a common and conserved phenomenon and propose that it is likely to be functionally important.

cell biology↗

bioRxiv2022

PRC2 disruption in cerebellar progenitors produces cerebellar hypoplasia and aberrant myoid differentiation without blocking medulloblastoma growth

We show that the Polycomb Repressive Complex 2 (PRC2) maintains neural identity in Sonic Hedgehog (SHH)-driven cerebellar granule neuron progenitors (CGNPs) and SHH-driven medulloblastoma, a cancer of CGNPs. Proliferating CGNPs and medulloblastoma cells pass the neural fate commitment to their progeny through epigenetic mechanisms. The PRC2 mediates epigenetic regulation through histone methylation, and PRC2 inhibitors have been proposed for medulloblastoma therapy. We investigated PRC2 function in CGNPs and medulloblastoma by conditionally deleting PRC2 components Eed or Ezh2 in CGNPs and analyzing cerebellar growth, cellular gene expression (scRNA-seq), and tumorigenesis in medulloblastoma-prone Smo-mutant mice. Eed-deleted CGNPs showed reduced growth, with decreased proliferation, increased apoptosis and inappropriate myoid differentiation. Ezh2-deleted CGNPs also showed myoid differentiation without reduced growth. Eed-deleted and Ezh2-deleted medulloblastomas similarly demonstrated myoid differentiation, but progressed more rapidly than PRC2-intact controls. The PRC2 thus maintained neural fate in CGNPs and medulloblastoma, but PRC2 disruption did not block SHH medulloblastoma progression.

neuroscience↗

bioRxiv2022

Systematic analysis of YFP gene traps reveals common discordance between mRNA and protein across the nervous system

While post-transcriptional control is thought to be required at the periphery of neurons and glia, its extent is unclear. Here, we investigate systematically the spatial distribution and expression of mRNA at single molecule sensitivity and their corresponding proteins of 200 YFP trap protein trap lines across the intact Drosophila nervous system. 98% of the genes studied showed discordance between the distribution of mRNA and the proteins they encode in at least one region of the nervous system. These data suggest that post-transcriptional regulation is very common, helping to explain the complexity of the nervous system. We also discovered that 68.5% of these genes have transcripts present at the periphery of neurons, with 9.5% at the glial periphery. Peripheral transcripts include many potential new regulators of neurons, glia and their interactions. Our approach is applicable to most genes and tissues and includes powerful novel data annotation and visualisation tools for post-transcriptional regulation. Brief outlineA novel high resolution and sensitive approach to systematically co-visualise the distribution of mRNAs and proteins in the intact nervous system reveals that post-transcriptional regulation of gene expression is very common. The rich data landscape is provided as a browsable resource (link), using Zegami, a cloud-based data exploration platform (link). Our solution provides a paradigm for the characterisation of post-transcriptional regulation of most genes and model systems. HighlightsO_LI196/200 (98%) Drosophila genes show discordant RNA and protein expression in at least one nervous system region C_LIO_LI137/200 (68.5%) mRNAs are present in at least one synaptic compartment C_LIO_LINovel localised mRNA and protein discovered in periphery of glial processes C_LIO_LINew paradigm for analysis of post-transcriptional regulation and data exploration C_LI

cell biology↗

bioRxiv2021

Compositional analysis of Sindbis virus ribonucleoproteins reveals an extensive co-opting of key nuclear RNA-binding proteins

RNA is a central molecule for RNA viruses, acting as mRNA and genome. However, the interactions that viral (v)RNA establishes with the host cell is only starting to be elucidated. Here, we determine with unprecedented depth the composition of the ribonucleoproteins (RNPs) of the prototypical arthropod-borne Sindbis virus (SINV) using viral RNA interactome capture. We show that SINV RNAs engage with hundreds of cellular proteins and pathways, including a group of nuclear RNA-binding proteins (RBPs) with unknown roles in infection. Combining subcellular fractionation and proteomics with several orthogonal approaches, we demonstrate that these nuclear RBPs are selectively redistributed to the cytoplasm after infection, where they associate with the viral replication organelles. These nuclear RBPs potently supress viral gene expression, with activities spanning viral species and families. Our study provides a comprehensive and systematic analysis of SINV RNP composition, revealing a network of nuclear RBPs with moonlighting antiviral function. Research highlightsO_LISINV RNAs interact with over four hundred cellular RBPs C_LIO_LISINV induces selective cytoplasmic translocation of a subset of nuclear RBPs C_LIO_LIThese nuclear RBPs display potent antiviral effects C_LIO_LIThe SF3B complex binds to SINV RNA and supresses infection in a splicing-independent manner C_LI

microbiology↗

bioRxiv2021

Absolute quantitation of individual SARS-CoV-2 RNA molecules: a new paradigm for infection dynamics and variant differences

Despite an unprecedented global research effort on SARS-CoV-2, early replication events remain poorly understood. Given the clinical importance of emergent viral variants with increased transmission, there is an urgent need to understand the early stages of viral replication and transcription. We used single molecule fluorescence in situ hybridisation (smFISH) to quantify positive sense RNA genomes with 95% detection efficiency, while simultaneously visualising negative sense genomes, sub-genomic RNAs and viral proteins. Our absolute quantification of viral RNAs and replication factories revealed that SARS-CoV-2 genomic RNA is long-lived after entry, suggesting that it avoids degradation by cellular nucleases. Moreover, we observed that SARS-CoV-2 replication is highly variable between cells, with only a small cell population displaying high burden of viral RNA. Unexpectedly, the B.1.1.7 variant, first identified in the UK, exhibits significantly slower replication kinetics than the Victoria strain, suggesting a novel mechanism contributing to its higher transmissibility with important clinical implications. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/450133v2_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@10f7bf1org.highwire.dtl.DTLVardef@192214dorg.highwire.dtl.DTLVardef@c84916org.highwire.dtl.DTLVardef@1366287_HPS_FORMAT_FIGEXP M_FIG C_FIG In briefBy detecting nearly all individual SARS-CoV-2 RNA molecules, we quantified viral replication and defined cell susceptibility to infection. We discovered that a minority of cells show significantly elevated viral RNA levels and observed slower replication kinetics for the Alpha variant relative to the Victoria strain. Highlights O_LISingle molecule quantification of SARS-CoV-2 replication uncovers early infection kinetics C_LIO_LIThere is substantial heterogeneity between cells in rates of SARS-CoV-2 replication C_LIO_LIGenomic RNA is stable and persistent during the initial stages of infection C_LIO_LIB.1.1.7 variant replicates more slowly than the Victoria strain C_LI

microbiology↗

bioRxiv2021

Python-Microscope: high performance control of arbitrarily complex and scalable bespoke microscopes

Custom built microscopes often require control of multiple hardware devices and precise hardware coordination. It is also desirable to have a solution that is scalable to complex systems and translatable between components from different manufacturers. Here we report Python-Microscope, a free and open source Python library for high performance control of arbitrarily complex and scalable custom microscope systems. Python-Microscope offers simple to use Python-based tools, abstracting differences between physical devices by providing a defined interface for different device types. Concrete implementations are provided for a range of specific hardware and a framework exists for further expansion. Python-Microscope supports the distribution of devices over multiple computers while maintaining synchronisation via highly precise hardware triggers. We discuss the architecture choices of Python-Microscope that overcome the performance problems often raised against Python and demonstrate the different use cases that drove its design: its integration in user facing projects, namely in the Microscope-Cockpit project; in controlling complex microscopes at high speed while using the Python programming language; and as a microscope simulation tool for software development.

biophysics↗

bioRxiv2021

Microscope-Cockpit: Python-based bespoke microscopy for bio-medical science

We have developed "Microscope-Cockpit" (Cockpit), a highly adaptable open source user-friendly Python-based GUI environment for precision control of both simple and elaborate bespoke microscope systems. The user environment allows next-generation near-instantaneous navigation of the entire slide landscape for efficient selection of specimens of interest and automated acquisition without the use of eyepieces. Cockpit uses "Python-Microscope" (Microscope) for high-performance coordinated control of a wide range of hardware devices using open source software. Microscope also controls complex hardware devices such as deformable mirrors for aberration correction and spatial light modulators for structured illumination via abstracted device models. We demonstrate the advantages of the Cockpit platform using several bespoke microscopes, including a simple widefield system and a complex system with adaptive optics and structured illumination. A key strength of Cockpit is its use of Python, which means that any microscope built with Cockpit is ready for future customisation by simply adding new libraries, for example machine learning algorithms to enable automated microscopy decision making while imaging. HighlightsO_LIUser-friendly setup and use for simple to complex bespoke microscopes. C_LIO_LIFacilitates collaborations between biomedical scientists and microscope technologists. C_LIO_LITouchscreen for near-instantaneous navigation of specimen landscape. C_LIO_LIUses Python-Microscope, for abstracted open source hardware device control. C_LIO_LIWell-suited for user training of AI-algorithms for automated microscopy. C_LI

biophysics↗

bioRxiv2020

Global analysis of protein-RNA interactions in SARS-CoV-2 infected cells reveals key regulators of infection

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19. SARS-CoV-2 relies on cellular RNA-binding proteins (RBPs) to replicate and spread, although which RBPs control SARS-CoV-2 infection remains largely unknown. Here, we employ a multi-omic approach to identify systematically and comprehensively which cellular and viral RBPs are involved in SARS-CoV-2 infection. We reveal that the cellular RNA-bound proteome is remodelled upon SARS-CoV-2 infection, having widespread effects on RNA metabolic pathways, non-canonical RBPs and antiviral factors. Moreover, we apply a new method to identify the proteins that directly interact with viral RNA, uncovering dozens of cellular RBPs and six viral proteins. Amongst them, several components of the tRNA ligase complex, which we show regulate SARS-CoV-2 infection. Furthermore, we discover that available drugs targeting host RBPs that interact with SARS-CoV-2 RNA inhibit infection. Collectively, our results uncover a new universe of host-virus interactions with potential for new antiviral therapies against COVID-19.

microbiology↗

bioRxiv2020

Dendritic localization of mRNA in Drosophila Mushroom Body Output Neurons

Memory-relevant neuronal plasticity is believed to require local translation of new proteins at synapses. Understanding this process requires the visualization of the relevant mRNAs within these neuronal compartments. Here we used single-molecule fluorescence in situ hybridization (smFISH) to localize mRNAs at subcellular resolution in the adult Drosophila brain. mRNAs for subunits of nicotinic acetylcholine receptors and kinases could be detected within the dendrites of co-labelled Mushroom Body Output Neurons (MBONs) and their relative abundance showed cell-specificity. Moreover, aversive olfactory learning produced a transient increase in the level of CaMKII mRNA within the dendritic compartments of the {gamma}5{beta}'2a MBONs. Localization of specific mRNAs in MBONs before and after learning represents a critical step towards deciphering the role of dendritic translation in the neuronal plasticity underlying behavioural change in Drosophila.

neuroscience↗

bioRxiv2020

Wavefront-sensorless adaptive optics with a laser-free spinning disk confocal microscope

Adaptive optics is being applied widely to a range of microscopies in order to improve imaging quality in the presence of specimen-induced aberrations. We present here the first implementation of wavefront-sensorless adaptive optics for a laser-free, aperture correlation, spinning disk microscope. This widefield method provides confocal-like optical sectioning through use of a patterned disk in the illumination and detection paths. Like other high-resolution microscopes, its operation is compromised by aberrations due to refractive index mismatch and variations within the specimen. Correction of such aberrations shows improved signal level, contrast and resolution.

cell biology↗

bioRxiv2020

Democratising "Microscopi": a 3D printed automated XYZT fluorescence imaging system for teaching, outreach and fieldwork

Commercial fluorescence microscope stands and fully automated XYZt fluorescence imaging systems are generally beyond the limited budgets available for teaching and outreach. We have addressed this problem by developing "Microscopi", an accessible, affordable, DIY automated imaging system that is built from 3D printed and commodity off-the-shelf hardware, including electro-mechanical, computer and optical components. Our design features automated sample navigation and image capture with a simple web-based graphical user interface, accessible with a tablet or other mobile device. The light path can easily be switched between different imaging modalities. The open source Python-based control software allows the hardware to be driven as an integrated imaging system. Furthermore, the microscope is fully customisable, which also enhances its value as a learning tool. Here, we describe the basic design and demonstrate imaging performance for a range of easily sourced specimens. HighlightsO_LIPortable, low cost, self-build from 3D printed and commodity components C_LIO_LIMultimodal imaging: bright field, dark field, pseudo-phase and fluorescence C_LIO_LIAutomated XYZt imaging from a tablet or smartphone via a simple GUI C_LIO_LIWide ranging applications in teaching, outreach and fieldwork C_LIO_LIOpen source hardware and software design, allowing user modification C_LI

scientific communication and education↗