Biology subjects

Murphy, E.

Publications and source records attributed to Murphy, E..

32 records · Page 2Linked to original sources

bioRxiv2021

Direct RNA sequencing of Respiratory Syncytial Virus infected human cells generates a detailed overview of RSV polycistronic mRNA and transcript abundance

To characterize species of viral mRNA transcripts generated during respiratory syncytial virus (RSV) infection, human fibroblast-like MRC5 lung cells were infected with subgroup A RSV for 6, 16 and 24 hours. Total RNA was harvested and polyadenylated mRNA was enriched and sequenced by direct RNA sequencing on an Oxford nanopore device. This yielded over 150,000 direct mRNA transcript reads which were mapped to the viral genome and analysed to determine relative mRNA levels of viral genes using our in-house ORF-centric pipeline. We were also able to examine frequencies with which polycistronic readthrough mRNAs were generated and to assess the length of the polyadenylated tails for each group of transcripts. We show that there is a general but non-linear decline in gene transcript abundance across the viral genome, as predicted by the model of RSV gene transcription. However, the decline in transcript abundance is not consistent. We show that the polyadenylate tails generated by the viral polymerase are similar in length to those generated by the host cells polyadenylation machinery and broadly declined in length for most transcripts as infection progressed. Finally, we observed that the steady state abundance of transcripts with very short polyadenylate tails is much less for N, SH and G transcripts compared to NS1, NS2, P, M, F and M2 which may reflect differences in mRNA stability and/or translation rates.

microbiology↗

bioRxiv2021

Multiview super-resolution microscopy

We enhance the performance of confocal microscopy over imaging scales spanning tens of nanometers to millimeters in space and milliseconds to hours in time, improving volumetric resolution more than 10-fold while simultaneously reducing phototoxicity. We achieve these gains via an integrated, four-pronged approach: 1) developing compact line-scanners that enable sensitive, rapid, diffraction-limited imaging over large areas; 2) combining line-scanning with multiview imaging, developing reconstruction algorithms that improve resolution isotropy and recover signal otherwise lost to scattering; 3) adapting techniques from structured illumination microscopy, achieving super-resolution imaging in densely labeled, thick samples; 4) synergizing deep learning with these advances, further improving imaging speed, resolution and duration. We demonstrate these capabilities on more than twenty distinct fixed and live samples, including protein distributions in single cells; nuclei and developing neurons in Caenorhabditis elegans embryos, larvae, and adults; myoblasts in Drosophila wing imaginal disks; and mouse renal, esophageal, cardiac, and brain tissues.

bioengineering↗

bioRxiv2021

Minimal phrase composition revealed by intracranial recordings

The ability to comprehend phrases is an essential integrative property of the brain. Here we evaluate the neural processes that enable the transition from single word processing to a minimal compositional scheme. Previous research has reported conflicting timing effects of composition, and disagreement persists with respect to inferior frontal and posterior temporal contributions. To address these issues, 19 patients (10 male, 19 female) implanted with penetrating depth or surface subdural intracranial electrodes heard auditory recordings of adjective-noun, pseudoword-noun and adjective-pseudoword phrases and judged whether the phrase matched a picture. Stimulus-dependent alterations in broadband gamma activity, low frequency power and phase-locking values across the language-dominant left hemisphere were derived. This revealed a mosaic located in the posterior superior temporal sulcus (pSTS), in which closely neighboring cortical sites displayed exclusive sensitivity to either lexicality or phrase structure, but not both. Distinct timings were found for effects of phrase composition (210-300 ms) and pseudoword processing (approximately 300-700 ms), and these were localized to neighboring electrodes in pSTS. The pars triangularis and temporal pole encode anticipation of composition in broadband low frequencies, and both regions exhibit greater functional connectivity with pSTS during phrase composition. Our results suggest that the pSTS is a highly specialized region comprised of sparsely interwoven heterogeneous constituents that encodes both lower and higher level linguistic features. This hub in pSTS for minimal phrase processing may form the neural basis for the human-specific computational capacity for forming hierarchically organized linguistic structures. SignificanceLinguists have claimed that the integration of multiple words into a phrase demands a computational procedure distinct from single word processing. Here, we provide intracranial recordings from a large patient cohort, with high spatiotemporal resolution, to track the cortical dynamics of phrase composition. Epileptic patients volunteered to participate in a task in which they listened to phrases ("red boat"), word-pseudoword or pseudoword-word pairs (e.g., "red fulg"). At the onset of the second word in phrases, greater broadband high gamma activity was found in posterior superior temporal sulcus in electrodes that exclusively indexed phrasal meaning, and not lexical meaning. These results provide direct, high-resolution signatures of minimal phrase composition in humans, a potentially species-specific computational capacity.

neuroscience↗

bioRxiv2021

Auxin-triggered changes in the Arabidopsis root tip (phospho)proteome reveal novel root growth regulators

Auxin plays a dual role in growth regulation and, depending on the tissue and concentration of the hormone, it can either promote or inhibit division and expansion processes in plants. Recent studies revealed that, beyond transcriptional reprogramming, alternative auxin-controlled mechanisms regulate root growth. Here, we explored the impact of different auxin concentrations on the root tip proteome and phosphoproteome, generating a unique resource. From the phosphoproteome data we pinpointed (novel) growth regulators, such as the RALF34-THE1 module. Our results together with previously published studies suggest that auxin, H+-ATPases, cell wall modifications and cell wall sensing receptor-like kinases are tightly embedded in a pathway regulating cell elongation. Furthermore, our study assigned a novel role to MKK2 as a regulator of primary root growth and a (potential) regulator of auxin biosynthesis and signalling, and suggests the importance of the MKK2 Thr31 phosphorylation site for growth regulation in the Arabidopsis root tip. ONE SENTENCE SUMMARYAn auxin-triggered Arabidopsis root tip (phospho)proteome reveals novel root growth regulators

plant biology↗

bioRxiv2020

Deciphering the regulatory logic of a Drosophila enhancer through systematic sequence mutagenesis and quantitative image analysis

Transcriptional enhancers are short DNA sequences controlling the spatial activity, timing and levels of eukaryotic gene transcription. Their quantitative transcriptional output is thought to result from the number and organization of transcription factor binding sites (TFBSs). Yet, how the various aspects of regulatory information are encoded in enhancer sequences remains elusive. We addressed this question by quantifying the spatial activity of the yellow spot enhancer active in developing Drosophila wings. To identify which enhancer DNA sequence contributes to enhancer activity, we introduced systematic mutations along the enhancer. We developed an analytic framework that uses comprehensive descriptors to quantify reporter assay in transgenic flies and measure spatial variations in activity levels across the wing. Our analysis highlights an unexpected density of regulatory information in the spot enhancer sequence. Furthermore, it reveals an unanticipated regulatory logic underlying the activity of this enhancer, and how it reads the wing trans-regulatory landscape to encode a spatial pattern.

genetics↗

bioRxiv2020

ALKBH7 mediates necrosis via rewiring of glyoxal metabolism

Alkb homolog 7 (ALKBH7) is a mitochondrial -ketoglutarate dioxygenase required for necrotic cell death in response to DNA alkylating agents, but its physiologic role within tissues remains unclear. Herein, we show that ALKBH7 plays a key role in the regulation of dialdehyde metabolism, which impacts cardiac survival in response to ischemia-reperfusion (IR) injury. Using a multi-omics approach, we do not find evidence that ALKBH7 functions as a prolyl-hydroxylase. However, we do find that mice lacking ALKBH7 exhibit a significant increase in glyoxalase I (GLO-1), a dialdehyde detoxifying enzyme. Consistent with increased dialdehyde production, metabolomics analysis reveals rewiring of metabolic pathways related to the toxic glycolytic by-product methylglyoxal (MGO), as well as accelerated glycolysis and elevated levels of MGO protein adducts, in mice lacking ALKBH7. Consistent with roles for both necrosis and glycative stress in cardiac IR injury, hearts from male but not female Alkbh7-/- mice are protected against IR, although somewhat unexpectedly this protection does not appear to involve modulation of the mitochondrial permeability transition pore. Highlighting the importance of MGO metabolism for the observed protection, removal of glucose as a metabolic substrate or pharmacologic inhibition of GLO-1 both abrogate cardioprotection in ALKBH7 deficient mice. Integrating these observations, we propose that ALKBH7 plays a role in the regulation of glyoxal metabolism, and that protection against necrosis and IR injury bought on by ALKBH7 deficiency originates from hormetic signaling in response to elevated MGO stress.

cell biology↗

bioRxiv2020

A novel USP30 inhibitor recapitulates genetic loss of USP30 and sets the trigger for PINK1-PARKIN amplification of mitochondrial ubiquitylation.

The mitochondrial deubiquitylase USP30 negatively regulates the selective autophagy of damaged mitochondria. It has been proposed as an actionable target to alleviate the loss of function of the mitophagy pathway governed by the Parkinsons Disease associated genes PINK1 and PRKN. We present the characterisation of a N-cyano pyrrolidine derived compound, FT3967385, with high selectivity for USP30. The compound is well tolerated with no loss of total mitochondrial mass. We demonstrate that ubiquitylation of TOM20, a component of the outer mitochondrial membrane import machinery that directly interacts with USP30, represents a robust biomarker for both USP30 loss and inhibition. We have conducted proteomics analyses on a SHSY5Y neuroblastoma cell line model to directly compare the effects of genetic loss of USP30 with selective inhibition in an unbiased fashion. We have thereby identified a subset of ubiquitylation events consequent to mitochondrial depolarisation that are USP30 sensitive. Within responsive elements of the ubiquitylome, several components of the outer mitochondrial membrane transport (TOM) complex are most prominent. Thus, our data support a model whereby USP30 can regulate the availability of ubiquitin at the specific site of mitochondrial PINK1 accumulation following membrane depolarisation. In this model, USP30 deubiquitylation of TOM complex components dampens the trigger for the Parkin-dependent amplification of mitochondrial ubiquitylation leading to mitophagy. Accordingly, PINK1 generation of phospho-Ser65 Ubiquitin proceeds more rapidly and to a greater extent in cells either lacking USP30 or subject to USP30 inhibition.

cell biology↗

bioRxiv2020

AGO HITS-CLIP in Adipose Tissue Reveals miR-29 as a Post-Transcriptional Regulator of Leptin

MicroRNAs (miRNAs) are short, non-coding RNAs that associate with Argonaute (AGO) to regulate mRNA stability and translation. While individual miRNAs have been shown to play important roles in white and brown adipose tissue in normal physiology and disease1,2,3, a comprehensive analysis of miRNA activity in these tissues has not been performed. We used high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) to comprehensively characterize the network of high-confidence, in vivo mRNA:miRNA interactions across white and brown fat, revealing over 100,000 unique miRNA binding sites. Targets for each miRNA were ranked to generate a catalog of miRNA binding activity, and the miR-29 family emerged as a top regulator of adipose tissue gene expression. Among the top targets of miR-29 was leptin, an adipocyte-derived hormone that acts on the brain to regulate food intake and energy expenditure4. Two independent miR-29 binding sites in the leptin 3-UTR were validated using luciferase assays, and miR-29 gain and loss-of-function modulated leptin mRNA and protein secretion in primary adipocytes. In mice, miR-29 abundance inversely correlated with leptin levels in two independent models of obesity. This work represents the only experimentally generated miRNA targetome in adipose tissue and identifies the first known post-transcriptional regulator of leptin. Future work aimed at manipulating miR-29:leptin binding may provide a therapeutic opportunity to treat obesity and its sequelae.

cell biology↗

bioRxiv2019

Isolation of an antimicrobial resistant, biofilm forming, Klebsiella grimontii isolate from a re-usable water bottle.

A re-usable water bottle was swabbed as part of the citizen science project Swab and Send and a Klebsiella grimontii isolate was recovered on chromogenic agar and designated SS141. Whole genome sequencing of SS141 showed it has the potential to be a human pathogen as it contains the biosynthetic gene cluster for the potent cytotoxin, kleboxymycin, and genes for other virulence factors. The genome also contains blaOXY-6-4 and fosA which is likely to explain the observed resistance to ampicillin, amoxicillin and fosfomycin. We have also shown that SS141 is a potent biofilm former, providing a reasonable explanation for its ability to colonise a re-usable water bottle. With the increasing use of re-usable water bottles as an alternative to disposables, and a strong forecast for growth in this industry over the next decade, this study highlights the need for cleanliness comparable to other re-usable culinary items.

microbiology↗

bioRxiv2019

Transcription factor NF-κB in a basal metazoan, the sponge, has conserved and unique sequences, activities, and regulation

Biological and biochemical functions of immunity transcription factor NF-{kappa}B in basal metazoans are largely unknown. Herein, we characterize transcription factor NF-{kappa}B from the demosponge Amphimedon queenslandica (Aq), in the phylum Porifera. Structurally and phylogenetically, the Aq-NF-{kappa}B protein is most similar to NF-{kappa}B p100 and p105 among vertebrate proteins, with an N-terminal DNA-binding/dimerization domain, a C-terminal Ankyrin (ANK) repeat domain, and a DNA binding-site profile more similar to human NF-{kappa}B proteins than Rel proteins. Aq-NF-{kappa}B also resembles the mammalian NF-{kappa}B protein p100 in that C-terminal truncation results in translocation of Aq-NF-{kappa}B to the nucleus and increases its transcriptional activation activity. Overexpression of a human or sea anemone I{kappa}B kinase (IKK) can induce C-terminal processing of Aq-NF-{kappa}B in vivo, and this processing requires C-terminal serine residues in Aq-NF-{kappa}B. Unlike human NF-{kappa}B p100, however, the C-terminal sequences of Aq-NF-{kappa}B do not effectively inhibit its DNA-binding activity when expressed in human cells. Tissue of another demosponge, a black encrusting sponge, contains NF-{kappa}B site DNA-binding activity and an NF-{kappa}B protein that appears mostly processed and in the nucleus of cells. NF-{kappa}B DNA-binding activity and processing is increased by treatment of sponge tissue with LPS. By transcriptomic analysis of A. queenslandica we identified likely homologs to many upstream NF-{kappa}B pathway components. These results present a functional characterization of the most ancient metazoan NF-{kappa}B protein to date, and show that many characteristics of mammalian NF-{kappa}B are conserved in sponge NF-{kappa}B, but the mechanism by which NF-{kappa}B functions and is regulated in the sponge may be somewhat different.

cell biology↗

bioRxiv2018

The expa1-1 mutant reveals a new biophysical lateral root organogenesis checkpoint

In plants, post-embryonic formation of new organs helps shape the adult organism. This requires the tight regulation of when and where a new organ is formed, and a coordination of the underlying cell divisions. To build a root system, new lateral roots are continuously developing, and this process requires asymmetric cell division in adjacent pericycle cells. Characterization of an expansin a1 (expa1) mutant has revealed a novel checkpoint during lateral root formation. Specifically, a minimal pericycle width was found to be necessary and sufficient to trigger asymmetric pericycle cell divisions during auxin-driven lateral root formation. We conclude that a localized radial expansion of adjacent pericycle cells is required to position the asymmetric cell divisions and generate a core of small daughter cells, which is a prerequisite for lateral root organogenesis.\n\nSIGNFICANCE STATEMENTOrgan formation is an essential process in plants and animals, driven by cell division and cell identity establishment. Root branching, where lateral roots form along the primary root axis, increases the root system and aids capture of water and nutrients. We have discovered that tight control of cell width is necessary to co-ordinate asymmetric cell divisions in cells that give rise to a new lateral root organ. While biomechanical processes have been shown to play a role in plant organogenesis, including lateral root formation, our data give new mechanistic insights into the cell size checkpoint during lateral root initiation.

plant biology↗

bioRxiv2018

Cell type-specific CLIP reveals that NOVA regulates cytoskeleton interactions in motoneurons

BackgroundAlternative RNA processing plays an essential role in shaping cell identity and connectivity in the central nervous system (CNS). This is believed to involve differential regulation of RNA processing in various cell types. However, in vivo study of cell-type specific post-transcriptional regulation has been a challenge. Here, we developed a sensitive and stringent method combining genetics and CLIP (crosslinking and immunoprecipitation) to globally identify regulatory interactions between NOVA and RNA in the mouse spinal cord motoneurons (MNs).\n\nResultsWe developed a means of undertaking MN-specific CLIP to explore MN-specific protein-RNA interactions relative to studies of the whole spinal cord. This allowed us to pinpoint differential RNA regulation specific to MNs, revealing major role for NOVA in regulating cytoskeleton interactions in MNs. In particular, NOVA specifically promotes the palmitoylated isoform of a cytoskeleton protein Septin 8 in MNs, which enhances dendritic arborization.\n\nConclusionsOur study demonstrates that cell type-specific RNA regulation is important for fine-tuning motoneuron physiology, and highlights the value of defining RNA processing regulation at single cell type resolution.

neuroscience↗

bioRxiv2017

Paleo-Oscillomics: Reconstructing Language-Relevant Computational Capacities in Neanderthals from the Molecular Basis of Neural Oscillations

Language seemingly evolved from changes in brain anatomy and wiring. We argue that language evolution can be better understood if particular changes in phasal and cross-frequency coupling properties of neural oscillations, resulting in core features of language, are considered. Because we cannot track the oscillatory activity of the brain from extinct hominins, we used our current understanding of the language oscillogenome (that is, the set of genes responsible for basic aspects of the oscillatory activity relevant for language) to infer some properties of the Neanderthal oscillome. We have found that several candidates for the language oscillogenome show differences in their methylation patterns between Neanderthals and humans. We argue that differences in their expression levels could be informative of differences in cognitive functions important for language.

neuroscience↗

bioRxiv2017

The language oscillogenome

Language has been argued to arise, both ontogenetically and phylogenetically, from specific patterns of brain wiring. We argue that it can further be shown that core features of language processing emerge from particular phasal and cross-frequency coupling properties of neural oscillations; what has been referred to as the language oscillome. It is expected that basic aspects of the language oscillome result from genetic guidance, what we will here call the language oscillogenome, for which we will put forward a list of candidate genes. We have considered genes for altered brain rhythmicity in conditions involving language deficits (autism spectrum disorders, schizophrenia, specific language impairment and dyslexia) for which we have confident genome-oscillome-phenome connections. These selected genes map on to aspects of brain function, particularly on to neurotransmitter function. Our aim is to propose a set of biologically robust genome-to-language linking hypotheses that, given testing, would grant causal and explanatory power to brain rhythms with respect to language processing.

neuroscience↗