Publications

The Babraham Institute Publications database contains details of all publications resulting from our research groups and scientific services.

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Title / Authors / Details Open Access Download

CCR8 marks highly suppressive Treg cells within tumours but is dispensable for their accumulation and suppressive function.
Whiteside SK, Grant FM, Gyori DS, Conti AG, Imianowski CJ, Kuo P, Nasrallah R, Sadiyah F, Lira SA, Tacke F, Eil RL, Burton OT, Dooley J, Liston A, Okkenhaug K, Yang J, Roychoudhuri R

CD4 regulatory T (Treg) cells, dependent upon the transcription factor Foxp3, contribute to tumour immunosuppression but are also required for immune homeostasis. There is interest in developing therapies that selectively target the immunosuppressive function of Treg cells within tumours without disrupting their systemic anti-inflammatory function. High levels of expression of Chemokine (C-C motif) receptor 8 (CCR8) discriminate Treg cells within tumours from those found in systemic lymphoid tissues. It has recently been proposed that disruption of CCR8 function using blocking anti-CCR8 antibodies results in reduced accumulation of Treg cells within tumours and disruption of their immunosuppressive function. Here, using Ccr8 mice, we show that CCR8 function is not required for Treg cell accumulation or immunosuppression in the context of syngeneic MC38 colorectal adenocarcinoma and B16 melanoma tumours. We observed high levels of CCR8 expression on tumour-infiltrating Treg cells which was abolished in Ccr8 mice. High levels of CCR8 marked cells with high levels of suppressive function. However, whereas systemic ablation of Treg cells resulted in strikingly diminished tumour burden, growth of subcutaneously implanted tumours was unaffected by systemic CCR8 loss. Consistently, we observed minimal impact of systemic CCR8 ablation on the frequency, phenotype and function of tumour-infiltrating Treg cells and conventional T (Tconv) function. These findings suggest that CCR8 is not required for Treg cell accumulation and immunosuppressive function within tumours and that depletion of CCR8 Treg cells rather than blockade of CCR8 function is a more promising avenue for selective immunotherapy.

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Immunology , 1 , 1 ,

PMID: 33838058

Genetic deletion of Nox4 enhances cancerogen-induced formation of solid tumors.
Helfinger V, Freiherr von Gall F, Henke N, Kunze MM, Schmid T, Rezende F, Heidler J, Wittig I, Radeke HH, Marschall V, Anderson K, Shah AM, Fulda S, Brüne B, Brandes RP, Schröder K

Reactive oxygen species (ROS) can cause cellular damage and promote cancer development. Besides such harmful consequences of overproduction of ROS, all cells utilize ROS for signaling purposes and stabilization of cell homeostasis. In particular, the latter is supported by the NADPH oxidase 4 (Nox4) that constitutively produces low amounts of HO By that mechanism, Nox4 forces differentiation of cells and prevents inflammation. We hypothesize a constitutive low level of HO maintains basal activity of cellular surveillance systems and is unlikely to be cancerogenic. Utilizing two different murine models of cancerogen-induced solid tumors, we found that deletion of Nox4 promotes tumor formation and lowers recognition of DNA damage. Nox4 supports phosphorylation of H2AX (γH2AX), a prerequisite of DNA damage recognition, by retaining a sufficiently low abundance of the phosphatase PP2A in the nucleus. The underlying mechanism is continuous oxidation of AKT by Nox4. Interaction of oxidized AKT and PP2A captures the phosphatase in the cytosol. Absence of Nox4 facilitates nuclear PP2A translocation and dephosphorylation of γH2AX. Simultaneously AKT is left phosphorylated. Thus, in the absence of Nox4, DNA damage is not recognized and the increased activity of AKT supports proliferation. The combination of both events results in genomic instability and promotes tumor formation. By identifying Nox4 as a protective source of ROS in cancerogen-induced cancer, we provide a piece of knowledge for understanding the role of moderate production of ROS in preventing the initiation of malignancies.

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Proceedings of the National Academy of Sciences of the United States of America , 118 , 11 ,

PMID: 33836590

Widespread reorganisation of pluripotent factor binding and gene regulatory interactions between human pluripotent states.
Chovanec P, Collier AJ, Krueger C, Várnai C, Semprich CI, Schoenfelder S, Corcoran AE, Rugg-Gunn PJ

The transition from naive to primed pluripotency is accompanied by an extensive reorganisation of transcriptional and epigenetic programmes. However, the role of transcriptional enhancers and three-dimensional chromatin organisation in coordinating these developmental programmes remains incompletely understood. Here, we generate a high-resolution atlas of gene regulatory interactions, chromatin profiles and transcription factor occupancy in naive and primed human pluripotent stem cells, and develop a network-graph approach to examine the atlas at multiple spatial scales. We uncover highly connected promoter hubs that change substantially in interaction frequency and in transcriptional co-regulation between pluripotent states. Small hubs frequently merge to form larger networks in primed cells, often linked by newly-formed Polycomb-associated interactions. We identify widespread state-specific differences in enhancer activity and interactivity that correspond with an extensive reconfiguration of OCT4, SOX2 and NANOG binding and target gene expression. These findings provide multilayered insights into the chromatin-based gene regulatory control of human pluripotent states.

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Nature communications , 12 , 1 ,

PMID: 33828098

Open Access

Mutations in phospholipase C eta-1 () are associated with holoprosencephaly.
Drissi I, Fletcher E, Shaheen R, Nahorski M, Alhashem AM, Lisgo S, Fernández-Jaén A, Schon K, Tlili-Graiess K, Smithson SF, Lindsay S, J Sharpe H, Alkuraya FS, Woods G

Holoprosencephaly is a spectrum of developmental disorder of the embryonic forebrain in which there is failed or incomplete separation of the prosencephalon into two cerebral hemispheres. To date, dominant mutations in sonic hedgehog (SHH) pathway genes are the predominant Mendelian causes, and have marked interfamilial and intrafamilial phenotypical variabilities.

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Journal of medical genetics , ` , ` ,

PMID: 33820834

PI3Kδ Forms Distinct Multiprotein Complexes at the TCR Signalosome in Naïve and Differentiated CD4 T Cells.
Luff DH, Wojdyla K, Oxley D, Chessa T, Hudson K, Hawkins PT, Stephens LR, Barry ST, Okkenhaug K

Phosphoinositide 3-kinases (PI3Ks) play a central role in adaptive immunity by transducing signals from the T cell antigen receptor (TCR) via production of PIP. PI3Kδ is a heterodimer composed of a p110δ catalytic subunit associated with a p85α or p85β regulatory subunit and is preferentially engaged by the TCR upon T cell activation. The molecular mechanisms leading to PI3Kδ recruitment and activation at the TCR signalosome remain unclear. In this study, we have used quantitative mass spectrometry, biochemical approaches and CRISPR-Cas9 gene editing to uncover the p110δ interactome in primary CD4 T cells. Moreover, we have determined how the PI3Kδ interactome changes upon the differentiation of small naïve T cells into T cell blasts expanded in the presence of IL-2. Our interactomic analyses identified multiple constitutive and inducible PI3Kδ-interacting proteins, some of which were common to naïve and previously-activated T cells. Our data reveals that PI3Kδ rapidly interacts with as many as seven adaptor proteins upon TCR engagement, including the Gab-family proteins, GAB2 and GAB3, a CD5-CBL signalosome and the transmembrane proteins ICOS and TRIM. Our results also suggest that PI3Kδ pre-forms complexes with the adaptors SH3KBP1 and CRKL in resting cells that could facilitate the localization and activation of p110δ at the plasma membrane by forming ternary complexes during early TCR signalling. Furthermore, we identify interactions that were not previously known to occur in CD4 T cells, involving BCAP, GAB3, IQGAP3 and JAML. We used CRISPR-Cas9-mediated gene knockout in primary T cells to confirm that BCAP is a positive regulator of PI3K-AKT signalling in CD4 T cell blasts. Overall, our results provide evidence for a large protein network that regulates the recruitment and activation of PI3Kδ in T cells. Finally, this work shows how the PI3Kδ interactome is remodeled as CD4 T cells differentiate from naïve T cells to activated T cell blasts. These activated T cells upregulate additional PI3Kδ adaptor proteins, including BCAP, GAB2, IQGAP3 and ICOS. This rewiring of TCR-PI3K signalling that occurs upon T cell differentiation may serve to reduce the threshold of activation and diversify the inputs for the PI3K pathway in effector T cells.

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Frontiers in immunology , 12 , 1 ,

PMID: 33763075

Open Access

Genome-wide analysis of DNA replication and DNA double-strand breaks using TrAEL-seq.
Kara N, Krueger F, Rugg-Gunn P, Houseley J

Faithful replication of the entire genome requires replication forks to copy large contiguous tracts of DNA, and sites of persistent replication fork stalling present a major threat to genome stability. Understanding the distribution of sites at which replication forks stall, and the ensuing fork processing events, requires genome-wide methods that profile replication fork position and the formation of recombinogenic DNA ends. Here, we describe Transferase-Activated End Ligation sequencing (TrAEL-seq), a method that captures single-stranded DNA 3' ends genome-wide and with base pair resolution. TrAEL-seq labels both DNA breaks and replication forks, providing genome-wide maps of replication fork progression and fork stalling sites in yeast and mammalian cells. Replication maps are similar to those obtained by Okazaki fragment sequencing; however, TrAEL-seq is performed on asynchronous populations of wild-type cells without incorporation of labels, cell sorting, or biochemical purification of replication intermediates, rendering TrAEL-seq far simpler and more widely applicable than existing replication fork direction profiling methods. The specificity of TrAEL-seq for DNA 3' ends also allows accurate detection of double-strand break sites after the initiation of DNA end resection, which we demonstrate by genome-wide mapping of meiotic double-strand break hotspots in a dmc1Δ mutant that is competent for end resection but not strand invasion. Overall, TrAEL-seq provides a flexible and robust methodology with high sensitivity and resolution for studying DNA replication and repair, which will be of significant use in determining mechanisms of genome instability.

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PLoS biology , 19 , 3 ,

PMID: 33760805

Open Access

Publisher Correction: LifeTime and improving European healthcare through cell-based interceptive medicine.
Rajewsky N, Almouzni G, Gorski SA, Aerts S, Amit I, Bertero MG, Bock C, Bredenoord AL, Cavalli G, Chiocca S, Clevers H, De Strooper B, Eggert A, Ellenberg J, Fernández XM, Figlerowicz M, Gasser SM, Hubner N, Kjems J, Knoblich JA, Krabbe G, Lichter P, Linnarsson S, Marine JC, Marioni JC, Marti-Renom MA, Netea MG, Nickel D, Nollmann M, Novak HR, Parkinson H, Piccolo S, Pinheiro I, Pombo A, Popp C, Reik W, Roman-Roman S, Rosenstiel P, Schultze JL, Stegle O, Tanay A, Testa G, Thanos D, Theis FJ, Torres-Padilla ME, Valencia A, Vallot C, van Oudenaarden A, Vidal M, Voet T,

n/a

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Nature , 1 , 1 ,

PMID: 33731935

Follicular regulatory T cells produce neuritin to regulate B cells.
Gonzalez-Figueroa P, Roco JA, Papa I, Núñez Villacís L, Stanley M, Linterman MA, Dent A, Canete PF, Vinuesa CG

Regulatory T cells prevent the emergence of autoantibodies and excessive IgE, but the precise mechanisms are unclear. Here, we show that BCL6-expressing Tregs, known as follicular regulatory T (Tfr) cells, produce abundant neuritin protein that targets B cells. Mice lacking Tfr cells or neuritin in Foxp3-expressing cells accumulated early plasma cells in germinal centers (GCs) and developed autoantibodies against histones and tissue-specific self-antigens. Upon immunization, these mice also produced increased plasma IgE and IgG1. We show that neuritin is taken up by B cells, causes phosphorylation of numerous proteins, and dampens IgE class switching. Neuritin reduced differentiation of mouse and human GC B cells into plasma cells, downregulated BLIMP-1, and upregulated BCL6. Administration of neuritin to Tfr-deficient mice prevented the accumulation of early plasma cells in GCs. Production of neuritin by Tfr cells emerges as a central mechanism to suppress B cell-driven autoimmunity and IgE-mediated allergies.

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Cell , 1 , 1 ,

PMID: 33711260

Protein tyrosine phosphatases in cell adhesion.
Young KA, Biggins L, Sharpe HJ

Adhesive structures between cells and with the surrounding matrix are essential for the development of multicellular organisms. In addition to providing mechanical integrity, they are key signalling centres providing feedback on the extracellular environment to the cell interior, and vice versa. During development, mitosis and repair, cell adhesions must undergo extensive remodelling. Post-translational modifications of proteins within these complexes serve as switches for activity. Tyrosine phosphorylation is an important modification in cell adhesion that is dynamically regulated by the protein tyrosine phosphatases (PTPs) and protein tyrosine kinases. Several PTPs are implicated in the assembly and maintenance of cell adhesions, however, their signalling functions remain poorly defined. The PTPs can act by directly dephosphorylating adhesive complex components or function as scaffolds. In this review, we will focus on human PTPs and discuss their individual roles in major adhesion complexes, as well as Hippo signalling. We have collated PTP interactome and cell adhesome datasets, which reveal extensive connections between PTPs and cell adhesions that are relatively unexplored. Finally, we reflect on the dysregulation of PTPs and cell adhesions in disease.

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The Biochemical journal , 478 , 5 ,

PMID: 33710332

Phenotypic analysis of Pyrin-Associated Autoinflammation with Neutrophilic Dermatosis patients during treatment.
Van Nieuwenhove E, De Langhe E, Dooley J, Van Den Oord J, Shahrooei M, Parvaneh N, Ziaee V, Savic S, Kacar M, Bossuyt X, Humblet-Baron S, Liston A, Wouters C

In 2016 specific heterozygous gain-of-function mutations in MEFV were reported causal for a distinct autoinflammatory disease coined pyrin-associated autoinflammation with neutrophilic dermatosis (PAAND). We sought to provide an extended report on clinical manifestations in PAAND patients to date and evaluate the efficacy and safety of treatment with the IL-1-blocking agent anakinra.

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Rheumatology (Oxford, England) , 1 , 1 ,

PMID: 33693560

Dynamic 3D Locus Organization and Its Drivers Underpin Immunoglobulin Recombination.
Rogers CH, Mielczarek O, Corcoran AE

A functional adaptive immune system must generate enormously diverse antigen receptor (AgR) repertoires from a limited number of AgR genes, using a common mechanism, V(D)J recombination. The AgR loci are among the largest in the genome, and individual genes must overcome huge spatial and temporal challenges to co-localize with optimum variability. Our understanding of the complex mechanisms involved has increased enormously, due in part to new technologies for high resolution mapping of AgR structure and dynamic movement, underpinning mechanisms, and resulting repertoires. This review will examine these advances using the paradigm of the mouse immunoglobulin heavy chain (Igh) locus. We will discuss the key regulatory elements implicated in Igh locus structure. Recent next generation repertoire sequencing methods have shown that local chromatin state at V genes contribute to recombination efficiency. Next on the multidimensional scale, we will describe imaging studies that provided the first picture of the large-scale dynamic looping and contraction the Igh locus undergoes during recombination. We will discuss chromosome conformation capture (3C)-based technologies that have provided higher resolution pictures of Igh locus structure, including the different models that have evolved. We will consider the key transcription factors (PAX5, YY1, E2A, Ikaros), and architectural factors, CTCF and cohesin, that regulate these processes. Lastly, we will discuss a plethora of recent exciting mechanistic findings. These include Rag recombinase scanning for convergent RSS sequences within DNA loops; identification of Igh loop extrusion, and its putative role in Rag scanning; the roles of CTCF, cohesin and cohesin loading factor, WAPL therein; a new phase separation model for Igh locus compartmentalization. We will draw these together and conclude with some horizon-scanning and unresolved questions.

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Frontiers in immunology , 11 , 1 ,

PMID: 33679727

Open Access

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition).
Klionsky DJ, Ktistakis NT et al

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

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Autophagy , 1 , 1 ,

PMID: 33634751

Diagnosis of deficiency of adenosine deaminase type 2 in adulthood.
Betrains A, Staels F, Moens L, Delafontaine S, Hershfield MS, Blockmans D, Liston A, Humblet-Baron S, Meyts I, Schrijvers R, Vanderschueren S

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Scandinavian journal of rheumatology , 1 , 1 ,

PMID: 33627040

Therapeutic depletion of CCR8 tumor-infiltrating regulatory T cells elicits antitumor immunity and synergizes with anti-PD-1 therapy.
Van Damme H, Dombrecht B, Kiss M, Roose H, Allen E, Van Overmeire E, Kancheva D, Martens L, Murgaski A, Bardet PMR, Blancke G, Jans M, Bolli E, Martins MS, Elkrim Y, Dooley J, Boon L, Schwarze JK, Tacke F, Movahedi K, Vandamme N, Neyns B, Ocak S, Scheyltjens I, Vereecke L, Nana FA, Merchiers P, Laoui D, Van Ginderachter JA

Modulation and depletion strategies of regulatory T cells (Tregs) constitute valid approaches in antitumor immunotherapy but suffer from severe adverse effects due to their lack of selectivity for the tumor-infiltrating (ti-)Treg population, indicating the need for a ti-Treg specific biomarker.

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Journal for immunotherapy of cancer , 9 , 2 ,

PMID: 33589525

Open Access

BioPAN: a web-based tool to explore mammalian lipidome metabolic pathways on LIPID MAPS.
Gaud C, C Sousa B, Nguyen A, Fedorova M, Ni Z, O Donnell VB, Wakelam MJO, Andrews S, Lopez-Clavijo AF

Lipidomics increasingly describes the quantitation using mass spectrometry of all lipids present in a biological sample.  As the power of lipidomics protocols increase, thousands of lipid molecular species from multiple categories can now be profiled in a single experiment.  Observed changes due to biological differences often encompass large numbers of structurally-related lipids, with these being regulated by enzymes from well-known metabolic pathways.  As lipidomics datasets increase in complexity, the interpretation of their results becomes more challenging.  BioPAN addresses this by enabling the researcher to visualise quantitative lipidomics data in the context of known biosynthetic pathways.  BioPAN provides a list of genes, which could be involved in the activation or suppression of enzymes catalysing lipid metabolism in mammalian tissues.

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F1000Research , 10 , 1 ,

PMID: 33564392

Open Access

BioPAN: a web-based tool to explore mammalian lipidome metabolic pathways on LIPID MAPS.
Gaud C, C Sousa B, Nguyen A, Fedorova M, Ni Z, O'Donnell VB, Wakelam MJO, Andrews S, Lopez-Clavijo AF

Lipidomics increasingly describes the quantification using mass spectrometry of all lipids present in a biological sample.  As the power of lipidomics protocols increase, thousands of lipid molecular species from multiple categories can now be profiled in a single experiment.  Observed changes due to biological differences often encompass large numbers of structurally-related lipids, with these being regulated by enzymes from well-known metabolic pathways.  As lipidomics datasets increase in complexity, the interpretation of their results becomes more challenging.  BioPAN addresses this by enabling the researcher to visualise quantitative lipidomics data in the context of known biosynthetic pathways.  BioPAN provides a list of genes, which could be involved in the activation or suppression of enzymes catalysing lipid metabolism in mammalian tissues.

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F1000Research , 10 , 1 ,

PMID: 33564392

Open Access

Histone modifications form a cell-type-specific chromosomal bar code that persists through the cell cycle.
Halsall JA, Andrews S, Krueger F, Rutledge CE, Ficz G, Reik W, Turner BM

Chromatin configuration influences gene expression in eukaryotes at multiple levels, from individual nucleosomes to chromatin domains several Mb long. Post-translational modifications (PTM) of core histones seem to be involved in chromatin structural transitions, but how remains unclear. To explore this, we used ChIP-seq and two cell types, HeLa and lymphoblastoid (LCL), to define how changes in chromatin packaging through the cell cycle influence the distributions of three transcription-associated histone modifications, H3K9ac, H3K4me3 and H3K27me3. We show that chromosome regions (bands) of 10-50 Mb, detectable by immunofluorescence microscopy of metaphase (M) chromosomes, are also present in G and G. They comprise 1-5 Mb sub-bands that differ between HeLa and LCL but remain consistent through the cell cycle. The same sub-bands are defined by H3K9ac and H3K4me3, while H3K27me3 spreads more widely. We found little change between cell cycle phases, whether compared by 5 Kb rolling windows or when analysis was restricted to functional elements such as transcription start sites and topologically associating domains. Only a small number of genes showed cell-cycle related changes: at genes encoding proteins involved in mitosis, H3K9 became highly acetylated in GM, possibly because of ongoing transcription. In conclusion, modified histone isoforms H3K9ac, H3K4me3 and H3K27me3 exhibit a characteristic genomic distribution at resolutions of 1 Mb and below that differs between HeLa and lymphoblastoid cells but remains remarkably consistent through the cell cycle. We suggest that this cell-type-specific chromosomal bar-code is part of a homeostatic mechanism by which cells retain their characteristic gene expression patterns, and hence their identity, through multiple mitoses.

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Scientific reports , 11 , 1 ,

PMID: 33542322

Open Access

Genome-wide DNA methylation dynamics during epigenetic reprogramming in the porcine germline.
Gómez-Redondo I, Planells B, Cánovas S, Ivanova E, Kelsey G, Gutiérrez-Adán A

Prior work in mice has shown that some retrotransposed elements remain substantially methylated during DNA methylation reprogramming of germ cells. In the pig, however, information about this process is scarce. The present study was designed to examine the methylation profiles of porcine germ cells during the time course of epigenetic reprogramming.

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Clinical epigenetics , 13 , 1 ,

PMID: 33536045

Open Access

A Booster Dose Enhances Immunogenicity of the COVID-19 Vaccine Candidate ChAdOx1 nCoV-19 in Aged Mice.
Silva-Cayetano A, Foster WS, Innocentin S, Belij-Rammerstorfer S, Spencer AJ, Burton OT, Fra-Bidó S, Le Lee J, Thakur N, Conceicao C, Wright D, Barrett J, Evans-Bailey N, Noble C, Bailey D, Liston A, Gilbert SC, Lambe T, Linterman MA

The spread of SARS-CoV-2 has caused a worldwide pandemic that has affected almost every aspect of human life. The development of an effective COVID-19 vaccine could limit the morbidity and mortality caused by infection and may enable the relaxation of social-distancing measures. Age is one of the most significant risk factors for poor health outcomes after SARS-CoV-2 infection; therefore, it is desirable that any new vaccine candidates elicit a robust immune response in older adults.

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Med (New York, N.Y.) , 1 , 1 ,

PMID: 33521747

Open Access

Glyoxal fixation facilitates transcriptome analysis after antigen staining and cell sorting by flow cytometry.
Channathodiyil P, Houseley J

A simple method for extraction of high quality RNA from cells that have been fixed, stained and sorted by flow cytometry would allow routine transcriptome analysis of highly purified cell populations and single cells. However, formaldehyde fixation impairs RNA extraction and inhibits RNA amplification. Here we show that good quality RNA can be readily extracted from stained and sorted mammalian cells if formaldehyde is replaced by glyoxal-a well-characterised fixative that is widely compatible with immunofluorescent staining methods. Although both formaldehyde and glyoxal efficiently form protein-protein crosslinks, glyoxal does not crosslink RNA to proteins nor form stable RNA adducts, ensuring that RNA remains accessible and amenable to enzymatic manipulation after glyoxal fixation. We find that RNA integrity is maintained through glyoxal fixation, permeabilisation with methanol or saponin, indirect immunofluorescent staining and flow sorting. RNA can then be extracted by standard methods and processed into RNA-seq libraries using commercial kits; mRNA abundances measured by poly(A)+ RNA-seq correlate well between freshly harvested cells and fixed, stained and sorted cells. We validate the applicability of this approach to flow cytometry by staining MCF-7 cells for the intracellular G2/M-specific antigen cyclin B1 (CCNB1), and show strong enrichment for G2/M-phase cells based on transcriptomic data. Switching to glyoxal fixation with RNA-compatible staining methods requires only minor adjustments of most existing staining and sorting protocols, and should facilitate routine transcriptomic analysis of sorted cells.

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PloS one , 16 , 1 ,

PMID: 33481798

Open Access

Profiling of Phosphoinositide Molecular Species in Resting or Activated Human or Mouse Platelets by a LC-MS Method.
Chicanne G, Bertrand-Michel J, Viaud J, Hnia K, Clark J, Payrastre B

Our knowledge of the role and biology of the different phosphoinositides has greatly expanded over recent years. Reversible phosphorylation by specific kinases and phosphatases of positions 3, 4, and 5 on the inositol ring is a highly dynamic process playing a critical role in the regulation of the spatiotemporal recruitment and binding of effector proteins. The specific phosphoinositide kinases and phosphatases are key players in the control of many cellular functions, including proliferation, survival, intracellular trafficking, or cytoskeleton reorganization. Several of these enzymes are mutated in human diseases. The impact of the fatty acid composition of phosphoinositides in their function is much less understood. There is an important molecular diversity in the fatty acid side chains of PI. While stearic and arachidonic fatty acids are the major acyl species in PIP, PIP, and PIP, other fatty acid combinations are also found. The role of these different molecular species is still unknown, but it is important to quantify these different molecules and their potential changes during cell stimulation to better characterize this emerging field. Here, we describe a sensitive high-performance liquid chromatography-mass spectrometry method that we used for the first time to profile the changes in phosphoinositide molecular species (summed fatty acyl chain profiles) in human and mouse platelets under resting conditions and following stimulation. This method can be applied to other hematopoietic primary cells isolated from human or experimental animal models.

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Methods in molecular biology (Clifton, N.J.) , 2251 , 1 ,

PMID: 33481230

Protection against oxaliplatin-induced mechanical and thermal hypersensitivity in Sarm1 mice.
Gould SA, White M, Wilbrey AL, Pór E, Coleman MP, Adalbert R

Chemotherapy-induced peripheral neuropathy (CIPN) is a common dose-limiting side effect of cancer treatment, often associated with degeneration of sensory axons or their terminal regions. Presence of the slow Wallerian degeneration protein (WLD), or genetic deletion of sterile alpha and TIR motif containing protein 1 (SARM1), which strongly protect axons from degeneration after injury or axonal transport block, alleviate pain in several CIPN models. However, oxaliplatin can cause an acute pain response, suggesting a different mechanism of pain generation. Here, we tested whether the presence of WLD or absence of SARM1 protects against acute oxaliplatin-induced pain in mice after a single oxaliplatin injection. In BL/6 and Wld mice, oxaliplatin induced significant mechanical and cold hypersensitivities which were absent in Sarm1 mice. Despite the presence of hypersensitivity there was no significant loss of intraepidermal nerve fibers (IENFs) in the footpads of any mice after oxaliplatin treatment, suggesting that early stages of pain hypersensitivity could be independent of axon degeneration. To identify other changes that could underlie the pain response, RNA sequencing was carried out in DRGs from treated and control mice of each genotype. Sarm1 mice had fewer gene expression changes than either BL/6 or Wld mice. This is consistent with the pain measurements in demonstrating that Sarm1DRGs remain relatively unchanged after oxaliplatin treatment, unlike those in BL/6 and Wld mice. Changes in levels of four transcripts - Alas2, Hba-a1, Hba-a2, and Tfrc - correlated with oxaliplatin-induced pain, or absence thereof, across the three genotypes. Our findings suggest that targeting SARM1 could be a viable therapeutic approach to prevent oxaliplatin-induced acute neuropathic pain.

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Experimental neurology , 1 , 1 ,

PMID: 33460644

Cell-cell coupling and DNA methylation abnormal phenotypes in the after-hours mice.
Tinarelli F, Ivanova E, Colombi I, Barini E, Balzani E, Garcia CG, Gasparini L, Chiappalone M, Kelsey G, Tucci V

DNA methylation has emerged as an important epigenetic regulator of brain processes, including circadian rhythms. However, how DNA methylation intervenes between environmental signals, such as light entrainment, and the transcriptional and translational molecular mechanisms of the cellular clock is currently unknown. Here, we studied the after-hours mice, which have a point mutation in the Fbxl3 gene and a lengthened circadian period.

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Epigenetics & chromatin , 14 , 1 ,

PMID: 33407878

A RAC-GEF network critical for early intestinal tumourigenesis.
Pickering KA, Gilroy K, Cassidy JW, Fey SK, Najumudeen AK, Zeiger LB, Vincent DF, Gay DM, Johansson J, Fordham RP, Miller B, Clark W, Hedley A, Unal EB, Kiel C, McGhee E, Machesky LM, Nixon C, Johnsson AE, Bain M, Strathdee D, van Hoof SR, Medema JP, Anderson KI, Brachmann SM, Stucke VM, Malliri A, Drysdale M, Turner M, Serrano L, Myant K, Campbell AD, Sansom OJ

RAC1 activity is critical for intestinal homeostasis, and is required for hyperproliferation driven by loss of the tumour suppressor gene Apc in the murine intestine. To avoid the impact of direct targeting upon homeostasis, we reasoned that indirect targeting of RAC1 via RAC-GEFs might be effective. Transcriptional profiling of Apc deficient intestinal tissue identified Vav3 and Tiam1 as key targets. Deletion of these indicated that while TIAM1 deficiency could suppress Apc-driven hyperproliferation, it had no impact upon tumourigenesis, while VAV3 deficiency had no effect. Intriguingly, deletion of either gene resulted in upregulation of Vav2, with subsequent targeting of all three (Vav2 Vav3 Tiam1), profoundly suppressing hyperproliferation, tumourigenesis and RAC1 activity, without impacting normal homeostasis. Critically, the observed RAC-GEF dependency was negated by oncogenic KRAS mutation. Together, these data demonstrate that while targeting RAC-GEF molecules may have therapeutic impact at early stages, this benefit may be lost in late stage disease.

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Nature communications , 12 , 1 ,

PMID: 33397922

Open Access