Biology subjects

Turner, M.

Publications and source records attributed to Turner, M..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Regulation of IFNgamma production by ZFP36L2 in T cells is context-dependent

CD8+ T cells kill target cells by releasing cytotoxic molecules and pro-inflammatory cytokines, such as TNF and IFN{gamma}. The magnitude and duration of cytokine production is defined by post-transcriptional regulation, and a critical regulator herein are RNA-binding proteins (RBPs). Although the functional importance of RBPs in regulating cytokine production is established, the kinetics and mode of action through which RBPs control cytokine production is not well understood. Previously, we showed that the RBP ZFP36L2 blocks translation of pre-formed cytokine encoding mRNA in quiescent memory T cells. Here, we uncover that ZFP36L2 regulates cytokine production in a context-dependent manner. T cell-specific deletion of ZFP36L2 (CD4-cre) had no effect on T cell development, or on cytokine production during early time points (2-6h) of T cell activation. In contrast, ZFP36L2 specifically dampened the production of IFN{gamma} during prolonged T cell activation (20-48h). ZFP36L2 deficiency also resulted in increased production of IFN{gamma} production in tumour-infiltrating T cells that are chronically exposed to antigen. Mechanistically, ZFP36L2 regulates IFN{gamma} production at late time points of activation by destabilizing Ifng mRNA in an AU-rich element-dependent manner. Together, our results reveal that ZFP36L2 employs different regulatory nodules in effector and memory T cells to regulate cytokine production.

immunology↗

bioRxiv2023

MiR-155 epigenetically counteracts hypoxia for mitochondrial fitness during germinal center positive selection

To increase antibody affinity against pathogens, positively selected GC-B cells initiate cell division in the light zone (LZ) of germinal centres (GCs). Among those, higher-affinity clones migrate to the dark zone (DZ) and vigorously proliferate by relying on oxidative phosphorylation (OXPHOS). However, it remains unknown how positively selected GC-B cells adapt their metabolism for cell division in the glycolysis-dominant, cell cycle arrest-inducing, hypoxic LZ microenvironment. Here, we show that microRNA (miR)-155 mediates metabolic reprogramming during positive selection to protect high-affinity clones. Transcriptome examination and mass spectrometry analysis revealed that miR-155 regulates H3K36me2 levels by directly repressing hypoxia-induced histone lysine demethylase, Kdm2a. This is indispensable for enhancing OXPHOS through optimizing the expression of vital nuclear mitochondrial genes under hypoxia. The miR-155-Kdm2a interaction is crucial to prevent excessive production of reactive oxygen species and apoptosis. Thus, miR-155-mediated epigenetic regulation promotes mitochondrial fitness in high-affinity clones, ensuring their expansion and consequently affinity maturation.

immunology↗

bioRxiv2023

Zfp36l1 establishes the high affinity CD8 T cell response by directly linking TCR affinity to cytokine sensing

How individual T cells compete for and respond to IL2 at the molecular level, and, as a consequence, how this shapes population dynamics and the selection of high affinity clones is still poorly understood. Here we describe how the RNA binding protein ZFP36L1, acts as a sensor of TCR affinity to promote clonal expansion of high affinity CD8 T cells. As part of an incoherent feed forward loop ZFP36L1 has a non-redundant role in suppressing negative regulators of cytokine signalling and mediating a selection mechanism based on competition for IL2. We suggest that ZFP36L1 acts as a sensor of antigen affinity and establishes dominance of high affinity T cells by installing a hierarchical response to IL2.

immunology↗

bioRxiv2023

Inference of single cell profiles from histology stains with the Single-Cell omics from Histology Analysis Framework (SCHAF)

Tissue biology involves an intricate balance between cell-intrinsic processes and interactions between cells organized in specific spatial patterns, which can be respectively captured by single cell profiling methods, such as single cell RNA-seq (scRNA-seq) and spatial transcriptomics, and histology imaging data, such as Hematoxylin-and-Eosin (H&E) stains. While single cell profiles provide rich molecular information, they can be challenging to collect routinely in the clinic and either lack spatial resolution or high gene throughput. Conversely, histological H&E assays have been a cornerstone of tissue pathology for decades, but do not directly report on molecular details, although the observed structure they capture arises from molecules and cells. Here, we leverage vision transformers and adversarial deep learning to develop the Single Cell omics from Histology Analysis Framework (SCHAF), which generates a tissue samples spatially-resolved whole transcriptome single cell omics dataset from its H&E histology image. We demonstrate SCHAF on a variety of tissues-- including lung cancer, metastatic breast cancer, placentae, and whole mouse pups--training with matched samples analyzed by sc/snRNA-seq, H&E staining, and, when available, spatial transcriptomics. SCHAF generated appropriate single cell profiles from histology images in test data, related them spatially, and compared well to ground-truth scRNA-Seq, expert pathologist annotations, or direct spatial transcriptomic measurements, with some limitations. SCHAF opens the way to next-generation H&E analyses and an integrated understanding of cell and tissue biology in health and disease.

bioinformatics↗

bioRxiv2022

An integrated proteome and transcriptome of B cell maturation defines poised activation states of transitional and mature B cells

During B cell maturation, transitional and mature B cells acquire cell-intrinsic features that determine their ability to exit quiescence and mount effective immune responses. We used label-free mass spectrometry to quantify the proteome of B cell subsets from the mouse spleen and map the differential expression of environmental sensing, transcription- and translation initiation-factors that define cellular identity and function. By comparing the full-length transcriptome and proteome within the same sample, we identified mRNAs linked to B cell activation and antibody secretion that are expressed without detectable protein. These "poised" mRNAs might enable rapid protein production through increased translation or protein stability. In addition, through interrogation of our proteomic dataset, we found that the translational repressor PDCD4 restrains the response of marginal zone B cells to a T-independent antigen. Our molecular characterization of B cell maturation is a valuable resource to further explore the mechanisms underpinning the specialised functions of B cell subsets.

immunology↗

bioRxiv2022

Lysosomal swelling and lysis mediate delivery of C3 toxins to their cytoplasmic targets

Unlike other cholera-like toxins that contain separate binding/translocation and catalytic subunits, C3-like mono-ADP-ribosyltransferases consist of a single subunit that serves both functions. The manner whereby C3 toxins reach the host cell cytoplasm is poorly understood and was addressed in this study by monitoring the fate of fluorescently-labelled C3larvinA. Following binding to the macrophage membrane in a discontinuous, punctate pattern, the toxin was internalized, traversing the endocytic pathway to reach lysosomes. Strikingly, the lysosomes of C3larvinA-treated cells underwent massive swelling over the course of 1-4 hrs. Lysosomal swelling preceded the extensive rearrangement of the cellular F-actin caused by ADP-ribosylation of cytosolic Rho-GTPases. This suggested that lysosome swelling might be required for escape of the toxin into the cytoplasm where the GTPases reside. Accordingly, preventing swelling by osmotic manipulation or by arresting macropinocytosis precluded the F-actin rearrangement. Toxin-induced swelling was associated with leakage of sulforhodamine B and dextran from the lysosomes, implying membrane rupture or activation of mechano-sensitive pores, enabling the toxin itself to reach the cytosol. Finally, comparison of the cellular traffic and actin remodelling activities of C3larvinA with that of two related toxins, C3larvintrunc and Plx2A, highlighted the importance of the N-terminal 1-helix for lysosomal swelling and successful infection.

microbiology↗

bioRxiv2022

Genotyping, sequencing and analysis of 140,000 adults from the Mexico City Prospective Study

The Mexico City Prospective Study (MCPS) is a prospective cohort of over 150,000 adults recruited two decades ago from the urban districts of Coyoacan and Iztapalapa in Mexico City. We generated genotype and exome sequencing data for all individuals, and whole genome sequencing for 10,000 selected individuals. We uncovered high levels of relatedness and substantial heterogeneity in ancestry composition across individuals. Most sequenced individuals had admixed Native American, European and African ancestry, with extensive admixture from indigenous groups in Central, Southern and South Eastern Mexico. Native Mexican segments of the genome had lower levels of coding variation, but an excess of homozygous loss of function variants compared with segments of African and European origin. We estimated population specific allele frequencies at 142 million genomic variants, with an effective sample size of 91,856 for Native Mexico at exome variants, all available via a public browser. Using whole genome sequencing, we developed an imputation reference panel which outperforms existing panels at common variants in individuals with high proportions of Central, South and South Eastern Native Mexican ancestry. Our work illustrates the value of genetic studies in populations with diverse ancestry and provides foundational imputation and allele frequency resources for future genetic studies in Mexico and in the United States where the Hispanic/Latino population is predominantly of Mexican descent.

genetics↗

bioRxiv2022

The contribution of Neanderthal introgression and natural selection to neurodegenerative diseases

Humans are thought to be more susceptible to neurodegeneration than equivalently-aged primates. It is not known whether this vulnerability is specific to anatomically-modern humans or shared with other hominids. The contribution of introgressed Neanderthal DNA to neurodegenerative disorders remains uncertain. It is also unclear how common variants associated with neurodegenerative disease risk are maintained by natural selection in the population despite their deleterious effects. In this study, we aimed to quantify the genome-wide contribution of Neanderthal introgression and positive selection to the heritability of complex neurodegenerative disorders to address these questions. We used stratified-linkage disequilibrium score regression to investigate the relationship between five SNP-based signatures of natural selection, reflecting different timepoints of evolution, and genome-wide associated variants of the three most prevalent neurodegenerative disorders: Alzheimers disease, amyotrophic lateral sclerosis and Parkinsons disease. We found no evidence for enrichment of positively-selected SNPs in the heritability of Alzheimers disease, amyotrophic lateral sclerosis and Parkinsons disease,, suggesting that common deleterious disease variants are unlikely to be maintained by positive selection. There was no enrichment of Neanderthal introgression in the SNP-heritability of these disorders, suggesting that Neanderthal admixture is unlikely to have contributed to disease risk. These findings provide insight into the origins of neurodegenerative disorders within the evolution of Homo sapiens and addresses a long-standing debate, showing that Neanderthal admixture is unlikely to have contributed to common genetic risk of neurodegeneration in anatomically-modern humans.

neuroscience↗

bioRxiv2022

Does fatigue influence joint-specific work and ground force production during the first steps of maximal accelerative running?

The rate of initial acceleration during the first steps of a maximal-effort (sprint) run often determines success or failure in prey capture and predator evasion, and is a vital factor of success in many modern sports. However, accelerative events are commonly performed after having already run considerable distances, and the associated fatigue should impair muscle force production and thus reduce acceleration rate. Despite this, the effects of running-induced fatigue on our ability to accelerate as well as the running technique used to achieve it has been incompletely studied. We recorded 3-D kinematics and ground reaction forces during the first three steps of the acceleration phase from a standing start before and after performing a high-speed, multi-directional, fatiguing run-walk protocol in well-trained running athletes who were habituated to accelerative sprinting. We found that the athletes were able to maintain their rate of initial acceleration despite changing running technique, which was associated with use of a more upright posture, longer ground contact time, increased vertical ground reaction impulse, decreased hip flexion and extension velocities, and a shift in peak joint moments, power, and positive work from the hip to the knee joint; no changes were detected in ankle joint function. Thus, a compensatory increase in knee joint function alleviated the reduction in hip flexor-extensor capacity. These acute adaptations may indicate that the hip extensors (gluteal and hamstring muscle groups) were more susceptible to fatigue than the ankle and knee musculature, and may thus be a primary target for interventions promoting fatigue resistance.

biophysics↗

bioRxiv2022

Polypyrimidine Tract Binding Protein 1 regulates the activation of mouse CD8 T cells

We show that the RNA-binding protein Polypyrimidine Tract Binding Protein 1 (PTBP1) is dispensable for the development of naive mouse CD8 T cells, but is necessary for the optimal expansion and production of effector molecules by antigen-specific CD8 T cells in vivo. PTBP1 has an essential role in regulating the early events following activation of the naive CD8 T cell leading to IL-2 and TNF production. It is also required to protect activated CD8 T cells from apoptosis. PTBP1 controls alternative splicing of over 400 genes in naive CD8 T cells in addition to regulating the abundance of [~]200 mRNAs. PTBP1 is required for the nuclear accumulation of c-Fos, NFATc2 and NFATc3, but not NFATc1. This selective effect on NFAT proteins correlates with PTBP1-promoted expression of the shorter A{beta}1 isoform and exon 13 skipped A{beta}2 isoform of the catalytic A-subunit of calcineurin phosphatase. These findings reveal a crucial role for PTBP1 in regulating CD8 T cell activation.

immunology↗

bioRxiv2021

Maintenance of ankle power and decreased inter-limb asymmetry during sprint running in fatigued multi-speed athletes

The ability to shift from walking and jogging to sprinting gaits, even when fatigued after prolonged effort, would have been as useful to our hunter-gatherer ancestors as it is to modern athletes. During prolonged jogging, joint moment and work decrease in the distal (ankle) joint but increase at proximal (hip/knee) joints as fatigue progresses, and such adaptations might be expected to occur in sprinting. Fatigue is also thought to increase inter-limb kinematic and force production asymmetries, which are speculated to influence injury risk. However, the effects of running-related fatigue on sprint running gait have been incompletely studied, so these hypotheses remain untested. We studied 3-D kinematics and ground reaction force production in dominant (DL) and non-dominant (NDL) legs during both non-fatigued and fatigued sprinting in habitual but uncoached running athletes. Contrary to the tested hypotheses, relative between-leg differences were greater in non-fatigued than fatigued sprinting. When not fatigued, DL produced greater propulsive impulse through both greater positive and negative work being done at the ankle, whilst NDL produced more vertical impulse, possibly resulting from the greater hip flexion observed prior to the downwards acceleration of the foot towards the ground. Whilst few changes were detected in DL once fatigued, NDL shifted towards greater horizontal force production, largely resulting from an increase in plantarflexion (distal-joint) moments and power. After fatiguing running, therefore, inter-limb asymmetry was reduced during sprinting and no distal-to-proximal shift in work/power was detected. Speculatively, these adaptations may help to attenuate decreases in running speed whilst minimising injury risk. SignificanceThe ability to attain fast running speeds may critically determine success in tasks such as prey chase- and-capture in hunter-gatherer societies as well as success in modern sports competitions. At times, sprint running may have to be performed whilst fatigued from previous, longer-distance running, when speeds are reduced, and injury risk may be higher. Previous work indicated that fatigue prompts a proximal shift in joint work and power production and an increase in inter-limb asymmetry. On the contrary, we show that relative ankle positive and negative joint work was maintained in the face of fatigue and that inter-limb asymmetry was reduced in a group of runners experienced, but not formally instructed, in both long-distance and sprint running.

biophysics↗

bioRxiv2021

Ex Vivo and In Vivo CD46 Receptor Utilization by Species D Human Adenovirus Serotype 26 (HAdV26)

Human adenovirus serotype 26 (Ad26) is used as a gene-based vaccine against SARS-CoV-2 and HIV-1. Yet, its primary receptor portfolio remains controversial, potentially including sialic acid, CAR, integrins, and CD46. We and others have shown that Ad26 can use CD46, but these observations were questioned by the inability to co-crystallize Ad26 fiber with CD46. Recent work demonstrated that Ad26 binds CD46 with its hexon protein rather than its fiber. We examined the functional consequences of Ad26 for infection in vitro and in vivo. Ectopic expression of human CD46 on Chinese hamster ovary cells increased Ad26 infection significantly. Deletion of the complement control protein domains CCP1 or CCP2 or the serine-threonine-proline (STP) region of CD46 reduced infection. Comparing wild type and sialic acid-deficient CHO cells, we show that the usage of CD46 is independent of its sialylation status. Ad26 transduction was increased in CD46 transgenic mice after intramuscular (IM) injection, but not after intranasal (IN) administration. Ad26 transduction was 10-fold lower than Ad5 after intratumoral (IT) injection of CD46-expressing tumors. Ad26 transduction of liver was 1000-fold lower than Ad5 after intravenous (IV) injection. These data demonstrate the use of CD46 by Ad26 under certain situations, but also show that the receptor has little consequence by other routes of administration. Finally, IV injection of high doses of Ad26 into CD46 mice induced release of liver enzymes in the bloodstream and reduced white blood cell counts, but did not induce thrombocytopenia. This suggests that Ad26 virions do not induce direct clotting side effects seen during COVID-19 vaccination with this serotype of adenovirus. IMPORTANCEHuman species D Ad26 is being pursued as a low seroprevalence vector for oncolytic virotherapy and gene-based vaccination against HIV-1 and SARS-CoV-2. However, there is debate in the literature about its tropism and receptor utilization, which directly influence its efficiency for certain applications. This work was aimed at determining which receptor(s) this virus uses for infection, and its role in virus biology, vaccine efficacy, and importantly, in vaccine safety.

pharmacology and toxicology↗

bioRxiv2021

An improved iCLIP protocol

Crosslinking and Immunoprecipitation (CLIP) is a powerful technique to obtain transcriptome-wide maps of in vivo protein-RNA interactions, which are important to understand the post-transcriptional mechanisms mediated by RNA binding proteins (RBPs). Many variant CLIP protocols have been developed to improve the efficiency and convenience of cDNA library preparation. Here we describe an improved individual nucleotide resolution CLIP protocol (iiCLIP), which can be completed within 4 days from UV crosslinking to libraries for sequencing. For benchmarking, we directly compared PTBP1 iiCLIP libraries with the iCLIP2 protocol produced under standardised conditions, and with public eCLIP and iCLIP PTBP1 data. We visualised enriched motifs surrounding the identified crosslink positions and RNA maps of these crosslinks around the alternative exons regulated by PTBP1. Notably, motif enrichment was higher in iiCLIP and iCLIP2 in comparison to public eCLIP and iCLIP, and we show how this impacts the specificity of RNA maps. In conclusion, iiCLIP is technically convenient and efficient, and enables production of highly specific datasets for identifying RBP binding sites.

molecular biology↗

bioRxiv2021

The RNA m6A binding protein YTHDF2 promotes the B cell to plasma cell transition.

To identify roles of RNA binding proteins (RBPs) in the differentiation of B cells to antibody-secreting plasma cells we performed a CRISPR/Cas9 knockout screen of 1213 mouse RBPs for their ability to affect proliferation and/or survival, and the emergence of differentiated CD138+ cells in vitro. We identified RBPs that promoted the appearance of CD138+ cells including CSDE1 and STRAP, as well as RBPs that inhibited CD138+ cell appearance such as EIF3 subunits EIF3K and EIF3L. Furthermore, we identified RBPs that share the property of recruiting the CCR4-NOT complex to their target transcripts have the potential to mediate opposing outcomes on B cell differentiation. One such RBP, the m6A binding protein YTHDF2 promotes the appearance of CD138+ cells in vitro. In chimeric mouse models YTHDF2-deficient B cells formed germinal centers in a cell-autonomous manner, however plasma cells failed to accumulate.

immunology↗

bioRxiv2021

The timing of differentiation and potency of CD8 effector function is set by RNA binding proteins

CD8+ T cell differentiation into effector cells is initiated early after antigen encounter by signals from the T cell antigen receptor and costimulatory molecules. The molecular mechanisms that determine the timing and rate of differentiation however are not defined. Here we show that the RNA binding proteins (RBP) ZFP36 and ZFP36L1 limit the rate of differentiation of activated naive CD8+ T cells and the potency of the resulting cytotoxic lymphocytes. The RBP act in an early and short temporal window to enforce dependency on costimulation via CD28 for full T cell activation and effector differentiation by directly binding mRNA of NF-B, IRF8 and NOTCH1 transcription factors and cytokines, including IL2. Their absence in T cells, or the adoptive transfer of a small numbers of CD8+ T cells lacking the RBP, promotes resilience to influenza A virus infection without immunopathology. These findings highlight ZFP36 and ZFP36L1 as nodes for the integration of the early T cell activation signals determining the speed and quality of the CD8 response.

immunology↗

bioRxiv2021

Effects of vapor exposure to Δ9-tetrahydrocannabinol (THC) in the Maine Lobster (Homarus americanus)

RationaleDespite a long history of use in synaptic physiology, the lobster has been a neglected model for behavioral pharmacology. A restaurateur proposed that exposing lobster to cannabis smoke reduces anxiety and pain during the cooking process. It is unknown if lobster gill respiration in air would result in significant {Delta}9-tetrahydrocannabinol (THC) uptake and whether this would have any detectable behavioral effects. ObjectiveThe primary goal was to determine tissue THC levels in the lobster after exposure to THC vapor. Secondary goals were to determine if THC vapor altered locomotor behavior or nociception. MethodsTissue samples were collected (including muscle, brain and hemolymph) from Homarus americanus (N=3 per group) following 30 or 60 minutes of exposure to vapor generated by an e-cigarette device using THC (100 mg/mL in a propylene glycol vehicle). Separate experiments assessed locomotor behavior and hot water nociceptive responses following THC vapor exposure. ResultsTHC vapor produced duration-related THC levels in all tissues examined. Locomotor activity was decreased (distance, speed, time-mobile) by 30 min inhalation of THC. Lobsters exhibit a temperature-dependent withdrawal response to immersion of tail, antennae or claws in warm water; this is novel evidence of thermal nociception for this species. THC exposure for 60 minutes had only marginal effect on nociception under the conditions assessed. ConclusionsVapor exposure of lobsters, using an e-cigarette based model, produces dose-dependent THC levels in all tissues and reduces locomotor activity. Hot water nociception was temperature dependent, but only minimal anti-nociceptive effect of THC exposure was confirmed.

pharmacology and toxicology↗

bioRxiv2021

Modelling seeding and neuroanatomic spread of pathology in amyotrophic lateral sclerosis

The neurodegenerative disorder amyotrophic lateral sclerosis (ALS) is characterized by the progressive loss of upper and lower motor neurons, with pathological involvement of cerebral motor and, additionally, extra-motor areas, in a clinicopathological spectrum with frontotemporal dementia (FTD). A key unresolved question is whether the distribution of pathology in ALS is driven by molecular factors such as regional gene expression, by differential network vulnerability, or is a combination of both. A system of histopathological staging of ALS based on the regional burden of TDP-43 pathology observed in post mortem brains has been supported to some extent by analysis of distribution of in vivo structural MRI changes. In this paper, computational modelling using a Network Diffusion Model (NDM) was used to investigate whether a process of focal pathological seeding followed by structural network-based spread recapitulated post mortem histopathological staging and, secondly, whether this had any relationship to the pattern of expression of a panel of genes implicated in ALS across the healthy brain. Regionally parcellated T1-weighted MRI data from ALS patients (baseline n=79) was studied in relation to a healthy control structural connectome and a database of associated regional cerebral gene expression. The NDM provided strong support for a structural network-based basis for regional pathological spread in ALS, but no simple relationship to the spatial distribution of ALS-related genes in the healthy brain. Intriguingly, the critical seed regions for spread within the model were not within the primary motor cortex but basal ganglia, thalamus and insula, where NDM recapitulated aspects of the post mortem histopathological staging system. Within the ALS-FTD clinicopathological spectrum, non-primary motor structures may be among the earliest sites of cerebral pathology.

neuroscience↗

bioRxiv2021

ORFLine: a bioinformatic pipeline to prioritise small open reading frames identifies candidate secreted small proteins from lymphocytes

The annotation of small open reading frames (smORFs) of less than 100 codons (<300 nucleotides) is challenging due to the large number of such sequences in the genome. The recent development of next generation sequence and ribosome profiling enables identification of actively translated smORFs. In this study, we developed a computational pipeline, which we have named ORFLine, that stringently identifies smORFs and classifies them according to their position within transcripts. We identified a total of 5744 unique smORFs in datasets from mouse B and T lymphocytes and systematically characterized them using ORFLine. We further searched smORFs for the presence of a signal peptide, which predicted known secreted chemokines as well as novel micropeptides. Five novel micropeptides show evidence of secretion and are therefore candidate mediators of immunoregulatory functions.

bioinformatics↗