Biology subjects

Turner, R.

Publications and source records attributed to Turner, R..

17 recordsLinked to original sources

bioRxiv2026

Human cytomegalovirus microRNAs target the Wnt signaling protein Dishevelled 3 to promote efficient reactivation in CD34+ hematopoietic progenitor cells

Human cytomegalovirus (HCMV) establishes latency in CD34+ hematopoietic progenitor cells (HPCs), yet differentiation of these cells promotes viral gene expression and replication. HCMV must therefore precisely regulate the signaling networks that govern HPC fate to maintain latency while preserving the capacity for reactivation. Virally encoded microRNAs (miRNAs) represent powerful tools used by HCMV to manipulate cellular pathways that are involved in HPC fate decisions. Wnt (Wingless/Integrated)/b-catenin signaling represents one such pathway that plays a critical role in regulating both hematopoiesis and virus replication. Previous work determined that HCMV stimulates b-catenin activity during reactivation in CD34+ HPCs. In contrast, here we show that HCMV miRNAs miR-US5-2 and miR-US25-2 downregulate the Wnt pathway adaptor protein Dvl3 during latency and that this downregulation is required for subsequent virus reactivation. These findings support a model of temporally distinct regulation of Wnt pathway components during latency and reactivation.

microbiology↗

bioRxiv2026

Evaluation of an Oxford Nanopore sequencing workflow for mycobacteria from primary MGIT culture

Illumina sequencing of primary MGIT cultures is an established workflow in several reference mycobacteriology laboratories. Oxford Nanopore Technologies (ONT) provides real-time genetic sequencing yielding long reads which help resolve repetitive genomes and is being explored for in-house implementation within diagnostic laboratories. However, low DNA yields from primary MGIT cultures frequently limit the application of ONT workflows, due to high minimum DNA input requirements for library preparation. We validated a modified ONT workflow combining rapid, semi-automated DNA extraction from MGIT cultures with Rapid PCR-Barcoding for whole-genome amplification, and compared its performance with Illumina sequencing for species identification and Mycobacterium tuberculosis complex (MTBC) single-nucleotide polymorphism (SNP) detection. A platform-agnostic analysis pipeline enabled consistent human read removal, taxonomic assignment, and MTBC genomic characterisation. ONT sequencing data was subsampled at 1, 6, and 72 hours to determine the earliest time point for reliable species identification. The concordance between sequencing platforms on species classification was 98.3% (95.8-99.5%) with all differences arising from potential mixed infections. SNP agreement was high, with a mean of 0.3 and a median of 0 SNP differences between sequencing platforms after masking. These findings demonstrate the feasibility of PCR-amplified ONT sequencing as a reliable alternative for routine genomic characterisation of MGIT cultures. IMPORTANCERapid identification of mycobacterial infections is essential for timely patient care and infection control. Many clinical laboratories currently rely on outsourcing sequencing to external reference centres, which adds time and delays the return of clinically actionable results. These services commonly use Illumina sequencing, which, while highly accurate, involves complex workflows and longer turnaround times. Newer technologies offer the potential to generate results in real time, but their use has been limited by the low amount of DNA available from routine culture samples. In this study, we developed an improved workflow that increases the amount of usable DNA and enables reliable, rapid sequencing directly from these samples. This approach allows multiple samples to be processed together and reduces the time needed to obtain results. Importantly, it could enable clinical laboratories to perform sequencing in-house, reducing reliance on external services and improving turnaround times.

molecular biology↗

bioRxiv2025

Mecp2 deficiency impairs microscale cortical network topology and dynamics in a Rett syndrome mouse model

Rett syndrome is a debilitating neurodevelopmental disorder with cerebral processing impairments caused by MECP2 loss-of-function mutations. Mecp2-deficient mouse models reveal disruptions of microscale cortical circuits. Yet how cellular-scale information processing is altered in Mecp2-deficient microscale functional networks is unknown. We investigated the development of functional connectivity, network topology, and dynamics in microelectrode array (MEA) recordings of primary cortical cultures from Mecp2-deficient and wild-type mice. Mecp2-deficient cortical networks developed more slowly and showed decreased functional connectivity compared to wild-type, leading to smaller network size, density, and strength of connectivity. Altered network topological features in Mecp2-deficient microscale circuits predicted decreased efficiency and information-sharing capacity. This reveals developmental deficits in microscale functional networks, which may in turn underlie the cortical decline and severe cognitive disability in Rett syndrome. These findings also offer circuit-level targets and an in-vitro approach for evaluating new therapeutic products for restoring microscale network function.

neuroscience↗

bioRxiv2025

The human cytomegalovirus chemokine binding protein UL22A is necessary for efficient reactivation from latency in CD34+ hematopoietic progenitor cells.

Herpesviruses and Poxviruses encode secreted chemokine binding proteins that prevent the interaction between chemokines and their cognate G protein coupled receptors to alter chemotactic gradients and intracellular signaling pathways. Human cytomegalovirus (HCMV) encodes the secreted protein UL22A (formerly UL21.5), which is described as a CCL5 (RANTES) binding protein and requires sulfation at two tyrosine residues (Y65 and Y69) for efficient RANTES interaction. In this report, we show that the UL22A protein, and the UL22A Y65 and Y69 residues are necessary for efficient HCMV reactivation from latency in CD34+ hematopoietic progenitor cells and that UL22A expression is essential for reactivation in a humanized mouse model of latency. However, RANTES neutralization is not sufficient to complement the in vitro reactivation defect of UL22A mutant viruses. These data suggest that UL22A plays an important role in latency, possibly through interactions with additional chemokines or other types of ligands via its tyrosine residues, in order to mediate efficient HCMV reactivation. IMPORTANCEHCMV is a ubiquitous herpesvirus that infects 60-90% of the population worldwide. In immunocompetent individuals, primary infection is asymptomatic and results in lifelong latent infection in CD34+ hematopoietic progenitor cells (HPCs). Viral reactivation remains a major complication for immunosuppressed individuals, but current therapeutics targeting HCMV replication show significant toxicity. Thus, a better understanding of the mechanisms controlling latency and reactivation is necessary to develop new therapeutics targeting these stages of the HCMV lifecycle. We show that virus lacking the HCMV chemokine binding protein UL22A is incapable of efficient reactivation in CD34+ HPCs and in vivo. UL22A tyrosine residues important for interaction with the chemokine RANTES are necessary for reactivation. However, neutralizing RANTES does not complement the reactivation defect of UL22A mutant viruses, demonstrating that UL22A has functions other than RANTES binding. Together, our results reveal a novel role for UL22A in HPCs and a new understanding of UL22A-chemokine interactions.

microbiology↗

bioRxiv2025

HCMV promotes viral reactivation through the coordinated regulation of Notch signaling by UL8 and miR-UL36

Human cytomegalovirus (HCMV) establishes latency in CD34+ hematopoietic progenitor cells (HPCs), where reactivation is intimately linked to cellular differentiation. We demonstrate that the Notch signaling pathway, a key regulator of stem cell maintenance and differentiation, functions as a barrier to HCMV reactivation. Two viral gene products, UL8 and miR-UL36, modulate this pathway during reactivation. UL8 promotes degradation of the Notch3 receptor via the endosomal/lysosomal pathway, dependent on two tyrosine-based motifs (Y305/314) in its cytoplasmic tail. A UL8 mutant lacking these motifs fails to degrade Notch3, resulting in sustained Notch signaling and impaired reactivation in vitro and in humanized mice. Similarly, miR-UL36 reduces expression of Notch3 and the Notch transcription factor Recombination Signal Binding Protein For Immunoglobulin Kappa J Region (RBPJ), suppressing Notch signaling. Deletion of miR-UL36 inhibits reactivation, but this defect, like that of the UL8 mutant, can be rescued by pharmacologic Notch inhibition. Thus, HCMV employs multiple gene products to suppress Notch signaling and promote conditions conducive to reactivation. These findings reveal how HCMV manipulates host differentiation pathways to control latency and suggest therapeutic strategies to prevent viral recurrence in immunocompromised patients. ImportanceHuman cytomegalovirus (HCMV) establishes lifelong latency, posing significant risks to transplant recipients and other immunocompromised individuals. Reactivation depends on progenitor cell differentiation, yet the viral mechanisms governing this process remain unclear. We identify Notch signaling as a major inhibitory pathway to reactivation and show that HCMV uses UL8 and miR-UL36 to suppress this pathway. UL8 degrades Notch3, while miR-UL36 downregulates Notch3 and RBPJ, together reducing Notch signaling and enabling reactivation. Mutant viruses lacking these regulators fail to reactivate efficiently, but this can be reversed by pharmacological inhibition of Notch. These findings establish Notch pathway suppression as a critical viral strategy for reactivation and highlight potential therapeutic targets for preventing HCMV disease.

microbiology↗

bioRxiv2025

Amplicon sequencing detects, identifies, and quantifies minority variants in mixed-species infections of Cryptosporidium parasites

Cryptosporidium is a globally endemic parasite genus with over 40 recognized species. While C. hominis and C. parvum are responsible for most human infections, human cases involving other species have also been reported. Furthermore, there is increasing evidence of simultaneous infections with multiple species. Therefore, we devised a new means to identify various species of Cryptosporidium in mixed infections by sequencing a 431 bp amplicon of the 18S rRNA gene encompassing two variable regions. Using the DADA2 pipeline, amplicons were first identified to a genus using the SILVA 132 reference database; then Cryptosporidium amplicons to a species using a custom database. This approach demonstrated sensitivity, successfully detecting and accurately identifying as little as 0.001 ng of C. parvum DNA in a complex stool background. Notably, we differentiated mixed infections and demonstrated the ability to identify potentially novel species of Cryptosporidium both in situ and in vitro. Using this method, we identified Cryptosporidium parvum in Egyptian rabbits with three samples showing minor mixed infections. By contrast, no mixed infections were detected in Egyptian children, who were primarily infected with C. hominis. Thus, this pipeline provides a sensitive tool for Cryptosporidium species-level identification, allowing for the detection and accurate identification of minor variants and mixed infections.

microbiology↗

bioRxiv2024

Enhancement and validation of the antibiotic resistance prediction performance of a cloud-based genetics processing platform for Mycobacteria

Tuberculosis remains a global health problem. Making it easier and quicker to identify which antibiotics an infection is likely to be susceptible to will be a key part of the solution. Whilst whole-genome sequencing offers many advantages, the processing of the genetic reads to produce the relevant public health and clinical information is, surprisingly, often the responsibility of the end user which inhibits uptake. Here we characterise how well a freely-available tool we have developed, gnomonicus, predicts the antibiotic resistance profile of a sample (given its variant call file) using our implementation of the second edition of the WHO catalogue of resistance-associated variants (WHOv2). To facilitate this, we have constructed a Diverse Testset of 2,663 publicly-available M. tuberculosis samples which have both genetic and drug susceptibility testing (DST) data. We have chosen to apply the catalogue such that our tool will return a result of (i) Fail if there are insufficient reads at a genetic locus associated with resistance, (ii) Unknown if a genetic variant in a resistance gene not listed in the catalogue is encountered and (iii) Resistant if three or more short-reads support the presence of a resistance-associated variant. The last step increases the sensitivity for all 15 antibiotics but only reaches significance in a few in our testset. Comparing our results to those of TB-Profiler, an existing tool, highlights the different design choices and demonstrates the performance of both tools on our Diverse Testset is comparable. By only considering high confidence DST results we show that gnomonicus, in combination with our translation of WHOv2, achieves sensitivities and specificities in excess of 95% for both isoniazid and rifampicin. Impact StatementWhole genome sequencing clinical samples taken from patients with tuberculosis is a potentially fast and accurate method for determining to which antibiotics the infection will be susceptible. Two barriers need to be overcome; the first, which is knowing which mutations are associated with resistance (or not) to a range of antibiotics is well on the way to be solved thanks to the efforts of the World Health Organization (WHO) who have published extensive catalogues containing lists of such mutations. The second barrier is that the processing of the raw genetic files remains a largely manual process overseen by bioinformaticians. Here we describe gnomonicus, our open-source AMR prediction tool, and report the performance of our translation of the second edition of the WHO catalogue using a carefully designed publicly-available dataset of 2,663 M. tuberculosis samples. We hope that not only will this tool be useful but also that this dataset will be used by other researchers to facilitate comparisons between pipelines, approaches and tools. Data SummaryThe attendant GitHub repository1 allows gnomonicus to be rerun on all 2,663 samples in the Diverse Testset; it therefore includes instructions, the necessary input files (all the variant call files, the version of the WHOv2 catalogue and a link to the H37Rv GenBank file used in this study) and also the output JSON files. The ENA accession numbers for all 2,663 samples (including a bash script to download them) and their corresponding phenotypic drug susceptibility testing results are included with the intention that people can either reproduce our results, or use the same dataset for other analyses. The JSON files output by TB-Profiler have also been added for comparison. The repository contains a series of Juypter notebooks containing Python3 code that allows the user to discover and parse the output JSON files from either tool and save the results as data tables. Other notebooks allow the user to reproduce all the analysis underlying this work, including reproducing the figures and many of the tables.

microbiology↗

bioRxiv2024

Viral microRNA regulation of Akt is necessary for reactivation of Human Cytomegalovirus from latency in CD34+ hematopoietic progenitor cells and humanized mice

Human cytomegalovirus (HCMV) actively manipulates cellular signaling pathways to benefit viral replication. Phosphatidyl-inositol 3-kinase (PI3K)/Akt signaling is an important negative regulator of HCMV replication, and during lytic infection the virus utilizes pUL38 to limit Akt phosphorylation and activation. During latency, PI3K/Akt signaling also limits virus replication, but how this is overcome at the time of reactivation is unknown. Virally encoded microRNAs (miRNAs) are a key component of the virus arsenal used to alter signaling during latency and reactivation. In the present study we show that three HCMV miRNAs (miR-UL36, miR-UL112 and miR-UL148D) downregulate Akt expression and attenuate downstream signaling, resulting in the activation of FOXO3a and enhanced internal promoter-driven IE transcription. A virus lacking expression of all three miRNAs is unable to reactivate from latency both in CD34+ hematopoietic progenitor cells and in a humanized mouse model of HCMV infection, however downregulating Akt restores the ability of the mutant virus to replicate. These findings highlight the negative role Akt signaling plays in HCMV replication in lytic and latent infection and how the virus has evolved miRNA-mediated countermeasures to promote successful reactivation. AUTHOR SUMMARYHuman cytomegalovirus (HCMV) infection results in lifelong persistence of the virus through the establishment of latency, and viral reactivation is a significant cause of morbidity and mortality in solid organ and stem cell transplant patients. HCMV latency is established in CD34+ hematopoietic progenitor cells (HPCs) where the virus manipulates cell signaling pathways to maintain the viral genome and remain poised to reinitiate gene expression under the appropriate conditions, although the molecular mechanisms surrounding these processes are poorly understood. HCMV encodes microRNAs (miRNAs) that modulate expression of hundreds of cellular and viral genes and play important roles in regulating signaling in HPCs. In this study, we show that HCMV miR-UL36, miR-UL112, and miR-UL148D coordinately inhibit Akt expression, activation, and downstream signaling through nonconventional mechanisms. A mutant lacking these miRNAs is unable to reactivate from latency, yet complementing Akt regulation restores the ability of the mutant virus to reactivate, pointing to an important role for miRNA-mediated inhibition of Akt to promote HCMV reactivation.

microbiology↗

bioRxiv2024

Evaluating the Benefits and Limits of Multiple Displacement Amplification with Whole-Genome Oxford Nanopore Sequencing

Multiple Displacement Amplification (MDA) outperforms conventional PCR in long fragment and whole genome amplification which makes it attractive to couple with long-read sequencing of samples with limited quantities of DNA to obtain improved genome assemblies. Here, we explore the efficacy and limits of MDA for genome sequence assembly using Oxford Nanopore Technologies (ONT) rapid library preparations and minION sequencing. We successfully generated almost complete genome sequences for all organisms examined, including Cryptosporidium meleagridis, Staphylococcus aureus, Enterococcus faecium, and Escherichia coli, with the ability to generate high-quality data from samples starting with only 0.025 ng of total DNA. Controlled sheared DNA samples exhibited a distinct pattern of size-increase after MDA, which may be associated with the amplification of long, low-abundance fragments present in the assay, as well as generating concatemeric sequences during amplification. To address concatemers, we developed a computational pipeline (CADECT: Concatemer Detection Tool) to identify and remove putative concatemeric sequences. This study highlights the efficacy of MDA in generating high-quality genome assemblies from limited amounts of input DNA. Also, the CADECT pipeline effectively mitigated the impact of concatemeric sequences, enabling the assembly of contiguous sequences even in cases where the input genomic DNA was degraded. These results have significant implications for the study of organisms that are challenging to culture in vitro, such as Cryptosporidium, and for expediting critical results in clinical settings with limited quantities of available genomic DNA.

bioinformatics↗

bioRxiv2023

The BCKDK inhibitor BT2 is a chemical uncoupler that lowers mitochondrial ROS production and de novo lipogenesis

Elevated levels of branched chain amino acids (BCAAs) and branched-chain -ketoacids (BCKAs) are associated with cardiovascular and metabolic disease, but the molecular mechanisms underlying a putative causal relationship remain unclear. The branched-chain ketoacid dehydrogenase kinase (BCKDK) inhibitor BT2 is often used in preclinical models to increase BCAA oxidation and restore steady-state BCAA and BCKA levels. BT2 administration is protective in various rodent models of heart failure and metabolic disease, but confoundingly, targeted ablation of Bckdk in specific tissues does not reproduce the beneficial effects conferred by pharmacologic inhibition. Here we demonstrate that BT2, a lipophilic weak acid, can act as a mitochondrial uncoupler. Measurements of oxygen consumption, mitochondrial membrane potential, and patch-clamp electrophysiology show BT2 increases proton conductance across the mitochondrial inner membrane independently of its inhibitory effect on BCKDK. BT2 is roughly five-fold less potent than the prototypical uncoupler 2,4-dinitrophenol (DNP), and phenocopies DNP in lowering de novo lipogenesis and mitochondrial superoxide production. The data suggest the therapeutic efficacy of BT2 may be attributable to the well-documented effects of mitochondrial uncoupling in alleviating cardiovascular and metabolic disease.

biochemistry↗

bioRxiv2022

Adipocytes reprogram carbon and nitrogen metabolism to maintain lipogenic flux in the absence of Bckdha

Dysregulated branched chain amino acid (BCAA) metabolism has emerged as a key metabolic feature associated with the obese insulin resistant state, and adipose BCAA catabolism is decreased in this context. BCAA catabolism is upregulated early in adipogenesis, but the impact of suppressing this pathway on the broader metabolic functions of the resultant adipocyte remain unclear. Here, we use CRISPR/Cas9 to target Bckdha and Acad8 in pre-adipocytes and induce a deficiency in BCAA or valine catabolism through differentiation. We characterise the transcriptional and metabolic phenotype of these cells using RNAseq and 13C metabolic flux analysis within a network spanning glycolysis, tricarboxylic (TCA) acid metabolism, BCAA catabolism, and fatty acid synthesis. While lipid droplet accumulation is maintained in Bckdha-deficient adipocytes, they display a more fibroblast-like transcriptional signature. In contrast, Acad8 deficiency minimally impacts gene expression. Decreased glycolytic flux emerges as the most distinct metabolic feature of Bckdha-deficient cells, accompanied by a [~]40% decrease in lactate secretion, yet pyruvate oxidation and utilization for de novo lipogenesis are increased to compensate for loss of BCAA carbon. Glutamine anaplerosis was also increased, though we observed a general decrease in levels of most non-essential amino acids consistent with an impact on nitrogen homeostasis. Overall, our data suggest that both metabolic and regulatory cross-talk exists between BCAA catabolism, glycolysis, and nitrogen metabolism in differentiated adipocytes. Suppression of BCAA catabolism associated with metabolic syndrome may result in a metabolically compromised adipocyte.

cell biology↗

bioRxiv2022

Fewer non-native insects in freshwater than in terrestrial habitats across continents

AimBiological invasions are a major threat to biodiversity in both aquatic and terrestrial habitats. Insects represent an important group of species in freshwater and terrestrial habitats, and they constitute a large proportion of non-native species. However, while many non-native insects are known from terrestrial ecosystems, it remains unclear how they are represented in freshwater habitats. Comparisons of the richness of invaders relative to the richness of native species between freshwater and terrestrial habitats are scarce, which hinders syntheses of invasion processes. Here, we used data from three regions on different continents to determine whether non-native insects are under- or overrepresented in freshwater compared to terrestrial assemblages. LocationEurope, North America, New Zealand MethodsWe compiled a comprehensive inventory of the native and non-native insect species established in freshwater and terrestrial habitats of the three study regions. We then contrasted the richness of non-native and native species among freshwater and terrestrial insects for all insect orders in each region. Using binomial regression, we analysed the proportions of non-native species in freshwater and terrestrial habitats across the three regions. ResultsIn most insect orders living in freshwater, non-native species were under-represented, while they were over-represented in a number of terrestrial orders. This pattern occurred in purely aquatic orders as well as in orders with both freshwater and terrestrial species. Overall, the proportion of non-native species was significantly lower in freshwater than in terrestrial species. Main conclusionsDespite the numerical and ecological importance of insects among all non-native species, non-native insect species are surprisingly rare in freshwater habitats, and this pattern is consistent across the three investigated study regions. We briefly review hypotheses concerning species traits and invasion pathways that are most likely to explain these patterns. Our findings contribute to a growing appreciation of drivers and impacts of biological invasions.

ecology↗

bioRxiv2021

Fatigue, Alertness and Risk Prediction for Shift Workers

Executive summaryO_LIThis report describes the principal outcomes of an Impact Acceleration Account project (grant number EP/I000992/1) between the University of Surrey and Transport for London carried out between Oct. 2019 and Mar. 2020. C_LIO_LIThe aim of the project was to compare the Health and Safety Executive (HSE) Fatigue Risk tool with SAFTE and other more recent models of fatigue, where fatigue here primarily means a reduced ability to function effectively and efficiently as a result of inadequate sleep. C_LIO_LIWe have not sought to discuss the useability of the HSE Fatigue Risk tool or SAFTE since this has been discussed comprehensively elsewhere (e.g. [1, 2]). We have instead focussed on the fundamental principles underlying the models. C_LIO_LIAll current biomathematical models have limitations and make asumptions that are not always evident from the accompanying documentation. Since full details of the HSE Fatigue Risk tool and the SAFTE model are not publicly available, Sections 1 and 2 give a mathematical description of the equations that we believe underlie each of these models. C_LIO_LIA comparison of predictions made by our versions of the HSE and SAFTE equations for one particular shift schedule of relevance to the UK and global tunnelling and construction industries is shown in Section 3. In this comparison, we use data collected durings TfLs Crossrail project by Dragados1. Essentially, both models give broadly the same message for the schedule we looked at, but the ability to display fatigue as it develops within a shift is a strength of SAFTE. C_LIO_LIA summary of the strengths and limitations of the use of these kind of scheduling tools is given in the final Section 4. Limitations include: O_LIModels do not describe fatigue during times when people are not in shift (e.g. driving home). However, they could readily be extended to do so. C_LIO_LIModels assume people start well-rested. This is not always a good assumption and can lead to an under-estimate of fatigue. C_LIO_LIMost models are currently based on population averages, but there are large individual different. It would be possible to further develop models to include uncertainty in fatigue predictions associated with individual differences. C_LIO_LIFew mdels include the light environment, which is important both to promote short-term alertness and facilitate circadian alignment. C_LIO_LIModels are not transparent, which makes them hard to independently validate. C_LIO_LIIt is hard to relate the outputs of current models to measureable outcomes in the field. C_LI C_LIO_LIWe also discuss briefly recent developments in mathematical modelling of fatigue and possible future directions. These include O_LIGuidance on scheduling and education on sleep and fatigue should be considered at least as important as current biomathematical models. C_LIO_LIOnly by analysing and integrating high quality individual data on sleep, fatigue, performance, near misses, accidents, actual shift patterns with models can we develop better models and management systems to reduce fatigue and associated risks. Wearables combined with apps present a great opportunity to collect data at scale but need to be used appropriately. C_LIO_LIThe importance of making time for sleep is not always recognised. Education, early diagnosis of sleep disorders such as sleep apnea, and self-monitoring all have a role to play in reducing fatigue-related risk in the work-place. C_LI C_LIO_LISection 3 and Section 4 may be understood without reading the intermediate more mathematical sections. C_LI

neuroscience↗

bioRxiv2020

Genetic and pharmacological evidence for kinetic competition between alternative poly(A) sites in yeast

Most eukaryotic mRNAs accommodate alternative sites of poly(A) addition in the 3 untranslated region in order to regulate mRNA function. Here we present a systematic analysis of 3 end formation factors, which revealed 3UTR lengthening in response to a loss of the core machinery, whereas a loss of the Sen1 helicase resulted in shorter 3UTRs. We show that the anti-cancer drug cordycepin, 3 deoxyadenosine, caused nucleotide accumulation and the usage of distal poly(A) sites. Mycophenolic acid, a drug which reduces GTP levels and impairs RNA polymerase II (RNAP II) transcription elongation, promoted the usage of proximal sites and reversed the effects of cordycepin on alternative polyadenylation. Moreover, cordycepin mediated usage of distal sites was associated with a permissive chromatin template and was suppressed in the presence of an rpb1 mutation, which slows RNAP II elongation rate. We propose that alternative polyadenylation is governed by temporal coordination of RNAP II transcription and 3 end processing and controlled by the availability of 3 end factors, nucleotide levels and chromatin landscape.

cell biology↗

bioRxiv2020

Validating layer-specific VASO across species

Cerebral blood volume (CBV) has been shown to be a robust and important physiological parameter for quantitative interpretation of functional (f)MRI, capable of delivering highly localized mapping of neural activity. Indeed, with recent advances in ultra-high-field (>=7T) MRI hardware and associated sequence libraries, it has become possible to capture non-invasive CBV weighted fMRI signals across cortical layers. One of the most widely used approaches to achieve this (in humans) is through vascular-space-occupancy (VASO) fMRI. Unfortunately, the exact contrast mechanisms of layer-dependent VASO fMRI have not been validated and thus interpretation of such data is confounded. Here we cross-validate layer-dependent VASO fMRI contrast in a preclinical rat model using well established (but invasive) imaging methods in response to neuronal activation (somatosensory cortex) and respiratory challenge (hypercapnia). In particular VASO derived CBV measures are directly compared to concurrent measures of total haemoglobin changes from high resolution intrinsic optical imaging spectroscopy (OIS). Through direct comparison of response magnitude, across time, negligible changes in hematocrit ratio during activation (neuronal or vascular) are inferred. Quantified cortical layer profiling is demonstrated and in agreement between both VASO and contrast enhanced fMRI (using monocrystalline iron oxide nanoparticles, MION). Responses show high spatial localisation to layers of cortical excitatory and inhibitory processing independent of confounding large draining veins which hamper BOLD fMRI studies. While we find increased VASO based CBV reactivity (3.1 {+/-} 1.2 fold increase) in humans compared to rats it is demonstrated that this reflects differences in stimulus design rather than confounds of the VASO signal source. Together, our findings confirm that the VASO contrast is indeed a reliable estimate of layer-specific CBV changes. This validation study increases the neuronal interpretability of human layer-dependent fMRI results and should supersede BOLD fMRI as the method of choice in neuroscience application studies. HighlightsO_LIOur goal is to validate layer-specific VASO fMRI with gold standard methods C_LIO_LILayer-specific VASO sequences are implemented for 7T imaging in humans and rats C_LIO_LIComparisons of VASO, optical imaging, and MION confirm the expected contrast origin C_LIO_LISomatosensory stimulation in humans and rats reveal the same layer-fMRI signatures C_LIO_LIWe confirm that VASO is a valid measure to estimate layer-specific neural activity C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/219378v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@4f9733org.highwire.dtl.DTLVardef@14fccd2org.highwire.dtl.DTLVardef@101edaorg.highwire.dtl.DTLVardef@598bae_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2019

Resolving multisensory and attentional influences across cortical depth in sensory cortices

In our environment our senses are bombarded with a myriad of signals, only a subset of which is relevant for our goals. Using sub-millimeter-resolution fMRI at 7T we resolved BOLD-response and activation patterns across cortical depth in early sensory cortices to auditory, visual and audiovisual stimuli under auditory or visual attention. In visual cortices, auditory stimulation induced widespread inhibition irrespective of attention, whereas auditory relative to visual attention suppressed mainly central visual field representations. In auditory cortices, visual stimulation suppressed activations, but amplified responses to concurrent auditory stimuli, in a patchy topography. Critically, multisensory interactions in auditory cortices were stronger in deeper laminae, while attentional influences were greatest at the surface. These distinct depth-dependent profiles suggest that multisensory and attentional mechanisms regulate sensory processing via partly distinct circuitries. Our findings are crucial for understanding how the brain regulates information flow across senses to interact with our complex multisensory world.

neuroscience↗

bioRxiv2018

Sensorimotor cortical-subthalamic network dynamics during force generation

The subthalamic nucleus (STN) is proposed to participate in pausing, or alternately, in dynamic scaling of behavioral responses, roles that have conflicting implications for understanding STN function in the context of deep brain stimulation (DBS) therapy. To examine the nature of event-related STN activity and subthalamic-cortical dynamics, we performed primary motor and somatosensory electrocorticography while subjects (n=10) performed a grip force task during DBS implantation surgery. The results provide the first evidence from humans that STN gamma activity can predict activity in the cortex both prior to and during movement, consistent with the idea that the STN participates in both motor planning and execution. We observed that STN activity appeared to facilitate movement: while both movement onset and termination both coincided with STN-cortical phase-locking, narrow-band gamma power was positively correlated with grip force, and event-related causality measures demonstrated that STN gamma activity predicted cortical gamma activity during movement. STN participation in somatosensory integration also was demonstrated by casual analysis. Information flow from the STN to somatosensory cortex was observed for both beta and gamma range frequencies, specific to particular movement periods and kinematics. Interactions in beta activity between the STN and somatosensory cortex, rather than motor cortex, predicted PD symptom severity. Thus, the STN contributes to multiple aspects of sensorimotor behavior dynamically across time.

neuroscience↗