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In the last century, we have seen a dramatic rise in the number of older persons globally, a trend known as the grey (or silver) tsunami. People live markedly longer than their predecessors worldwide, due to remarkable changes in their lifestyle and in progresses made by modern medicine. However, the older we become, the more susceptible we are to a series of age-related pathologies, including infections, cancers, autoimmune diseases, and multi-morbidities. Therefore, a key challenge for our modern societies is how to cope with this fragile portion of the population, so that everybody could have the opportunity to live a long and healthy life. From a holistic point of view, aging results from the progressive decline of various systems. Among them, the distinctive age-dependent changes in the immune system contribute to the enhanced frailty of the elderly. One of these affects a population of lymphocytes, known as regulatory T cells (Tregs), as accumulating evidence suggest that there is a significant increase in the frequency of these cells in secondary lymphoid organs (SLOs) of aged animals. Although there are still discrepancies in the literature about modifications to their functional properties during aging, mounting evidence suggests a detrimental role for Tregs in the elderly in the context of bacterial and viral infections by suppressing immune responses against non-self-antigens. Interestingly, Tregs seem to also contribute to the reduced effectiveness of immunizations against many pathogens by limiting the production of vaccine-induced protective antibodies. In this review, we will analyze the current state of understandings about the role of Tregs in acute and chronic infections as well as in vaccination response in both humans and mice. Lastly, we provide an overview of current strategies for Treg modulation with potential future applications to improve the effectiveness of vaccines in older individuals.
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Quiescent, differentiated adult vascular smooth muscle cells (VSMCs) can be induced to proliferate and switch phenotype. Such plasticity underlies blood vessel homeostasis and contributes to vascular disease development. Oligoclonal VSMC contribution is a hallmark of end-stage vascular disease. Here we aim to understand cellular mechanisms underpinning generation of this VSMC oligoclonality.
Vaccines typically protect against (re)infections by generating pathogen-neutralising antibodies. However, as we age, antibody-secreting cell formation and vaccine-induced antibody titres are reduced. Antibody-secreting plasma cells differentiate from B cells either early post-vaccination through the extrafollicular response or from the germinal centre (GC) reaction, which generates long-lived antibody-secreting cells. As the formation of both the extrafollicular antibody response and the GC requires the interaction of multiple cell types, the impaired antibody response in ageing could be caused by B cell intrinsic or extrinsic factors, or a combination of the two. Here, we show that B cells from older people do not have intrinsic defects in their proliferation and differentiation into antibody-secreting cells in vitro compared to those from the younger donors. However, adoptive transfer of B cells from aged mice to young recipient mice showed that differentiation into extrafollicular plasma cells was favoured at the expense of B cells entering the GC during the early stages of GC formation. In contrast, by the peak of the GC response, GC B cells derived from the donor cells of aged mice had expanded to the same extent as those from the younger donors. This indicates that age-related intrinsic B cell changes delay the GC response but are not responsible for the impaired antibody-secreting response or smaller peak GC response in ageing. Collectively, this study shows that B cells from aged individuals are not intrinsically defective in responding to stimulation and becoming antibody-secreting cells, implicating B cell-extrinsic factors as the primary cause of age-associated impairment in the humoral immunity.
Cellular RNAs are heterogeneous with respect to their alternative processing and secondary structures, but the functional importance of this complexity is still poorly understood. A set of alternatively processed antisense non-coding transcripts, which are collectively called COOLAIR, are generated at the Arabidopsis floral-repressor locus FLOWERING LOCUS C (FLC). Different isoforms of COOLAIR influence FLC transcriptional output in warm and cold conditions. Here, to further investigate the function of COOLAIR, we developed an RNA structure-profiling method to determine the in vivo structure of single RNA molecules rather than the RNA population average. This revealed that individual isoforms of the COOLAIR transcript adopt multiple structures with different conformational dynamics. The major distally polyadenylated COOLAIR isoform in warm conditions adopts three predominant structural conformations, the proportions and conformations of which change after cold exposure. An alternatively spliced, strongly cold-upregulated distal COOLAIR isoform shows high structural diversity, in contrast to proximally polyadenylated COOLAIR. A hyper-variable COOLAIR structural element was identified that was complementary to the FLC transcription start site. Mutations altering the structure of this region changed FLC expression and flowering time, consistent with an important regulatory role of the COOLAIR structure in FLC transcription. Our work demonstrates that isoforms of non-coding RNA transcripts adopt multiple distinct and functionally relevant structural conformations, which change in abundance and shape in response to external conditions.
The pre-conceptual, intrauterine, and early life environments can have a profound and long-lasting impact on the developmental trajectories and health outcomes of the offspring. Given the relatively low success rates of Assisted Reproductive Technologies (ART; ~25%), additives and adjuvants, such as glucocorticoids, are utilized to improve the success rate. Considering the dynamic developmental events that occur during this window, these exposures may alter blastocyst formation at a molecular level, and as such, affect not only the viability of the embryo and ability of the blastocyst to implant, but also the developmental trajectory of the first three cell lineages, ultimately influencing the physiology of the embryo. In this study we present a comprehensive single-cell transcriptome, methylome and small RNA atlas in the day 7 human embryo. We demonstrate that, despite no change in morphology and developmental features, preimplantation glucocorticoid exposure reprograms the molecular profile of the TE lineage and these changes are associated with an altered metabolic and inflammatory response. Our data also suggest that glucocorticoids can precociously mature the TE sub-lineages, supported by the presence of extravillous trophoblast markers in the polar sub-lineage and presence of X Chromosome dosage compensation. Further, we have elucidated that epigenetic regulation (DNA methylation and microRNAs (miRNAs)) likely underlie the transcriptional changes observed. This study suggests that exposures to exogenous compounds during preimplantation may unintentionally reprogram the human embryo, possibly leading to suboptimal development and longer-term health outcomes.
Li et al present the results of a proximity-interaction screen in mammalian cells for the effector proteins of 25 members of the Arf family of small GTPases. This study has generated an important resource for those working in several areas of cell biology and provided an initial characterisation of two new cellular roles for some of the least well studied members of this family, the regulation of PLD1 by ARL11/14 in phagocytosis, and the regulation of PI4KB by ARL5A/5B in the Golgi.
The advent of technologies that can characterize the phenotypes, functions and fates of individual cells has revealed extensive and often unexpected levels of diversity between cells that are nominally of the same subset. CD8 T cells, also known as cytotoxic T lymphocytes (CTLs), are no exception. Investigations of individual CD8 T cells both and have highlighted the heterogeneity of cellular responses at the levels of activation, differentiation and function. This review takes a broad perspective on the topic of heterogeneity, outlining different forms of variation that arise during a CD8 T cell response. Specific attention is paid to the impact of T cell receptor (TCR) stimulation strength on heterogeneity. In particular, this review endeavors to highlight connections between variation at different cellular stages, presenting known mechanisms and key open questions about how variation between cells can arise and propagate.
The RAS-regulated RAF-MEK1/2-ERK1/2 signalling pathway is frequently de-regulated in human cancer. Melanoma in particular exhibits a high incidence of activating BRAF and NRAS mutations and such cells are addicted to the activity of these mutant oncoproteins. As a result three different BRAF inhibitors (BRAFi) have now been approved for BRAFV600E/K- mutant melanoma and have transformed the treatment of this disease. Despite this, clinical responses are typically transient as tumour cells develop resistance. These resistance mechanisms frequently involve reinstatement of ERK1/2 signalling and BRAFi are now deployed in combination with one of three approved MEK1/2 inhibitors (MEKi) to provide more durable, but still transient, clinical responses. Furthermore, inhibitors to ERK1/2 (ERK1/2i) have also been developed to counteract ERK1/2 signalling. However, recent studies have suggested that BRAFi/MEKi and ERK1/2i resistance can arise through activation of a parallel signalling pathway leading to activation of ERK5, an unusual protein kinase that contains both a kinase domain and a transcriptional transactivation domain. Here we review the evidence supporting ERK5 as a mediator of BRAFi/MEKi and ERK1/2i resistance. We also review the challenges in targeting ERK5 signalling with small molecules, including paradoxical activation of the transcriptional transactivation domain, and discuss new therapeutic modalities that could be employed to target ERK5.
Upon antigen binding, the B cell receptor (BCR) undergoes clustering to form a signalosome that propagates downstream signaling required for normal B cell development and physiology. BCR clustering is dependent on remodeling of the cortical actin network, but the mechanisms that regulate actin remodeling in this context remain poorly defined. In this study, we identify the inositol 5-phosphatase INPP5B as a key regulator of actin remodeling, BCR clustering, and downstream signaling in antigen-stimulated B cells. INPP5B acts via dephosphorylation of the inositol lipid PI(4,5)P2 that in turn is necessary for actin disassembly, BCR mobilization, and cell spreading on immobilized surface antigen. These effects can be explained by increased actin severing by cofilin and loss of actin linking to the plasma membrane by ezrin, both of which are sensitive to INPP5B-dependent PI(4,5)P2 hydrolysis. INPP5B is therefore a new player in BCR signaling and may represent an attractive target for treatment of B cell malignancies caused by aberrant BCR signaling.
Mast cells and basophils have long been implicated in the pathogenesis of IgE-mediated hypersensitivity reactions. They express the high-affinity IgE receptor, FcϵRI, on their surface. Antigen-induced crosslinking of IgE antibodies bound to that receptor triggers a signaling cascade that results in activation, leading to the release of an array of preformed vasoactive mediators and rapidly synthesized lipids, as well as the production of inflammatory cytokines. In addition to bearing activating receptors like FcεRI, these effector cells of allergy express inhibitory ones including FcγR2b, an IgG Fc receptor with a cytosolic inhibitory motif that activates protein tyrosine phosphatases that suppress IgE-mediated activation. We and others have shown that food allergen-specific IgG antibodies strongly induced during the course of oral immunotherapy (OIT), signal FcγR2b to suppress IgE-mediated mast cell and basophil activation triggered by food allergen challenge. However, the potential inhibitory effects of IgA antibodies, which are also produced in response to OIT and are present at high levels at mucosal sites, including the intestine where food allergens are encountered, have not been well studied. Here we uncover an inhibitory function for IgA. We observe that IgA binds mouse bone marrow-derived mast cells (BMMCs) and peritoneal mast cells. Binding to BMMCs is dependent on calcium and sialic acid. We also found that IgA antibodies inhibit IgE-mediated mast cell degranulation in an allergen-specific fashion. Antigen-specific IgA inhibits IgE-mediated mast cell activation early in the signaling cascade, suppressing the phosphorylation of Syk, the proximal protein kinase mediating FcεRI signaling, and suppresses mast cell production of cytokines. Furthermore, using basophils from a peanut allergic donor we found that IgA binds to basophils and that activation by exposure to peanuts is effectively suppressed by IgA. We conclude that IgA serves as a regulator of mast cell and basophil degranulation, suggesting a physiologic role for IgA in the maintenance of immune homeostasis at mucosal sites.
The integrated stress response (ISR) plays a pivotal role in adaptation of translation machinery to cellular stress. Here, we demonstrate an ISR-independent osmoadaptation mechanism involving reprogramming of translation via coordinated but independent actions of mTOR and plasma membrane amino acid transporter SNAT2. This biphasic response entails reduced global protein synthesis and mTOR signaling followed by translation of SNAT2. Induction of SNAT2 leads to accumulation of amino acids and reactivation of mTOR and global protein synthesis, paralleled by partial reversal of the early-phase, stress-induced translatome. We propose SNAT2 functions as a molecular switch between inhibition of protein synthesis and establishment of an osmoadaptive translation program involving the formation of cytoplasmic condensates of SNAT2-regulated RNA-binding proteins DDX3X and FUS. In summary, we define key roles of SNAT2 in osmotolerance.
High-risk neuroblastoma, a pediatric tumor originating from the sympathetic nervous system, has a low mutation load but highly recurrent somatic DNA copy number variants. Previously, segmental gains and/or amplifications allowed identification of drivers for neuroblastoma development. Using this approach, combined with gene dosage impact on expression and survival, we identified ribonucleotide reductase subunit M2 (RRM2) as a candidate dependency factor further supported by growth inhibition upon in vitro knockdown and accelerated tumor formation in a neuroblastoma zebrafish model coexpressing human RRM2 with MYCN. Forced RRM2 induction alleviates excessive replicative stress induced by CHK1 inhibition, while high RRM2 expression in human neuroblastomas correlates with high CHK1 activity. MYCN-driven zebrafish tumors with RRM2 co-overexpression exhibit differentially expressed DNA repair genes in keeping with enhanced ATR-CHK1 signaling activity. In vitro, RRM2 inhibition enhances intrinsic replication stress checkpoint addiction. Last, combinatorial RRM2-CHK1 inhibition acts synergistic in high-risk neuroblastoma cell lines and patient-derived xenograft models, illustrating the therapeutic potential.
Complete, reproducible extraction of protein material is essential for comprehensive and unbiased proteome analyses. A current gold standard is single-pot, solid-phase-enhanced sample preparation (SP3), in which organic solvent and magnetic beads are used to denature and capture protein aggregates, with subsequent washes removing contaminants. However, SP3 is dependent on effective protein immobilization onto beads, risks losses during wash steps, and exhibits losses and greater costs at higher protein inputs. Here, we propose solvent precipitation SP3 (SP4) as an alternative to SP3 protein cleanup, capturing acetonitrile-induced protein aggregates by brief centrifugation rather than magnetism─with optional low-cost inert glass beads to simplify handling. SP4 recovered equivalent or greater protein yields for 1-5000 μg preparations and improved reproducibility (median protein 0.99 (SP4) 0.97 (SP3)). Deep proteome profiling revealed that SP4 yielded a greater recovery of low-solubility and transmembrane proteins than SP3, benefits to aggregating protein using 80 50% organic solvent, and equivalent recovery by SP4 and S-Trap. SP4 was verified in three other labs across eight sample types and five lysis buffers─all confirming equivalent or improved proteome characterization SP3. With near-identical recovery, this work further illustrates protein precipitation as the primary mechanism of SP3 protein cleanup and identifies that magnetic capture risks losses, especially at higher protein concentrations and among more hydrophobic proteins. SP4 offers a minimalistic approach to protein cleanup that provides cost-effective input scalability, the option to omit beads entirely, and suggests important considerations for SP3 applications─all while retaining the speed and compatibility of SP3.
Mendelian susceptibility to mycobacterial disease (MSMD) is caused by inborn errors of IFN-γ immunity. The most frequent genetic defects are found in IL12 or a subunit of its receptor. IL23R deficiency in MSMD has only been reported once, in two pediatric patients from the same kindred with isolated disseminated Bacille Calmette-Guérin disease. We evaluated the impact of a homozygous stop mutation in IL23R (R381X), identified by whole exome sequencing, in an adult patient with disseminated non-tuberculous mycobacterial disease.
The identification of CD4 T cells localizing to B cell follicles has revolutionized the knowledge of how humoral immunity is generated. Follicular helper T (T) cells support germinal center (GC) formation and regulate clonal selection and differentiation of memory and antibody-secreting B cells, thus controlling antibody affinity maturation and memory. T cells are essential in sustaining protective antibody responses necessary for pathogen clearance in infection and vaccine-mediated protection. Conversely, aberrant and excessive T cell responses mediate and sustain pathogenic antibodies to autoantigens, alloantigens, and allergens, facilitate lymphomagenesis, and even harbor viral reservoirs. T cell generation and function are determined by T cell antigen receptor (TCR), costimulation, and cytokine signals, together with specific metabolic and survival mechanisms. Such regulation is crucial to understanding disease pathogenesis and informing the development of emerging therapies for disease or novel approaches to boost vaccine efficacy.
Mammalian pre-implantation embryos accumulate substantial lipids, which are stored in lipid droplets (LDs). Despite the fundamental roles of lipids in many cellular functions, the significance of building-up LDs for the developing embryo remains unclear. Here we report that the accumulation and mobilization of LDs upon implantation are causal in the morphogenesis of the pluripotent epiblast and generation of the pro-amniotic cavity in mouse embryos, a critical step for all subsequent development. We show that the CIDEA protein, found abundantly in adipocytes, enhances lipid storage in blastocysts and pluripotent stem cells by promoting LD enlargement through fusion. The LD-stored lipids are mobilized into lysosomes at the onset of lumenogenesis, but without CIDEA are prematurely degraded by cytosolic lipases. Loss of lipid storage or inactivation of lipophagy leads to the aberrant formation of multiple cavities within disorganised epithelial structures. Thus, our study reveals an unexpected role for LDs in orchestrating tissue remodelling and uncovers underappreciated facets of lipid metabolism in peri-implantation development.
Phosphoinositides (PIPn) in mammalian tissues are enriched in the stearoyl/arachidonoyl acyl chain species ("C38:4"), but its functional significance is unclear. We have used metabolic tracers (isotopologues of inositol, glucose and water) to study PIPn synthesis in cell lines in which this enrichment is preserved to differing relative extents. We show that PIs synthesised from glucose are initially enriched in shorter/more saturated acyl chains, but then rapidly remodelled towards the C38:4 species. PIs are also synthesised by a distinct 're-cycling pathway', which utilises existing precursors and exhibits substantial selectivity for the synthesis of C38:4-PA and -PI. This re-cycling pathway is rapidly stimulated during receptor activation of phospholipase-C, both allowing the retention of the C38:4 backbone and the close coupling of PIPn consumption to its resynthesis, thus maintaining pool sizes. These results suggest that one property of the specific acyl chain composition of PIPn is that of a molecular code, to facilitate 'metabolic channelling' from PIP2 to PI via pools of intermediates (DG, PA and CDP-DG) common to other lipid metabolic pathways.
The maintenance of transcriptional states regulated by histone modifications and controlled switching between these states are fundamental concepts in our understanding of nucleosome-mediated epigenetic memory. Any approach relying on genome-wide bioinformatic analyses alone offers limited scope for dissecting the molecular mechanisms involved in maintenance and switching. Mechanistic mathematical models-describing the dynamics of histone modifications at individual genomic loci-offer an alternative way to investigate these mechanisms. These models, in conjunction with quantitative experimental data-ChIP data, quantification of mRNA levels, and single-cell fluorescence tracking in clonal lineages-can generate predictions that drive more targeted experiments, allowing us to understand mechanisms that would be challenging to unravel by a purely experimental approach. In this chapter, we describe a generic stochastic modeling framework that can be used to capture histone modification dynamics and associated molecular processes-including transcription and read-write feedback by chromatin modifying complexes-at individual genomic loci. Using a specific example-transcriptional silencing by Polycomb-mediated H3K27 methylation-we demonstrate how to construct and simulate a stochastic histone modification model. We provide a step-by-step guide to programming simulations for such a model and discuss how to analyze the simulation output.
Histone methyltransferases (HMTs) catalyze the methylation of lysine and arginine residues in histone as well as nonhistone substrates. In vitro histone methyltransferase assays have been instrumental in identifying HMTs, and they continue to be invaluable tools for the study of these important enzymes, revealing novel substrates and modes of regulation.Here we describe a universal protocol to examine HMT activity in vitro that can be adapted to a range of HMTs, substrates, and experimental objectives. We provide protocols for the detection of activity based on incorporation of H-labeled methyl groups from S-adenosylmethionine (SAM), methylation-specific antibodies, and quantification of the reaction product S-adenosylhomocysteine (SAH).
Heterochromatin maintains genome integrity and function, and is organised into distinct nuclear domains. Some of these domains are proposed to form by phase separation through the accumulation of HP1ɑ. Mouse heterochromatin contains noncoding major satellite repeats (MSR), which are highly transcribed in mouse embryonic stem cells (ESCs). Here, we report that MSR transcripts can drive the formation of HP1ɑ droplets in vitro, and modulate heterochromatin into dynamic condensates in ESCs, contributing to the formation of large nuclear domains that are characteristic of pluripotent cells. Depleting MSR transcripts causes heterochromatin to transition into a more compact and static state. Unexpectedly, changing heterochromatin's biophysical properties has severe consequences for ESCs, including chromosome instability and mitotic defects. These findings uncover an essential role for MSR transcripts in modulating the organisation and properties of heterochromatin to preserve genome stability. They also provide insights into the processes that could regulate phase separation and the functional consequences of disrupting the properties of heterochromatin condensates.
Gastrulation controls the emergence of cellular diversity and axis patterning in the early embryo. In mammals, this transformation is orchestrated by dynamic signalling centres at the interface of embryonic and extraembryonic tissues. Elucidating the molecular framework of axis formation in vivo is fundamental for our understanding of human development and to advance stem-cell-based regenerative approaches. Here, we illuminate early gastrulation of marmoset embryos in utero by spatial transcriptomics and stem cell-based embryo models. Gaussian process regression-based 3D-transcriptomes delineate the emergence of the anterior visceral endoderm, which is hallmarked by conserved (HHEX, LEFTY2, LHX1) and primate-specific (POSTN, SDC4, FZD5) factors. WNT signalling spatially coordinates primitive streak formation in the embryonic disc and is counteracted by SFRP1/2 to sustain pluripotency in the anterior domain. Amnion specification occurs at the boundaries of the embryonic disc through ID1/2/3 in response to BMP-signalling, providing a developmental rationale for amnion differentiation of primate pluripotent stem cells (PSCs). Spatial identity mapping demonstrates that primed marmoset PSCs exhibit highest similarity to the anterior embryonic disc, while naïve PSCs resemble the preimplantation epiblast. Our 3D-transcriptome models reveal the molecular code of lineage specification in the primate embryo and provide an in vivo reference to decipher human development.
Gastrulation controls the emergence of cellular diversity and axis patterning in the early embryo. In mammals, this transformation is orchestrated by dynamic signalling centres at the interface of embryonic and extraembryonic tissues. Elucidating the molecular framework of axis formation in vivo is fundamental for our understanding of human development and to advance stem-cell-based regenerative approaches. Here we illuminate early gastrulation of marmoset embryos in utero using spatial transcriptomics and stem-cell-based embryo models. Gaussian process regression-based 3D transcriptomes delineate the emergence of the anterior visceral endoderm, which is hallmarked by conserved (HHEX, LEFTY2, LHX1) and primate-specific (POSTN, SDC4, FZD5) factors. WNT signalling spatially coordinates the formation of the primitive streak in the embryonic disc and is counteracted by SFRP1 and SFRP2 to sustain pluripotency in the anterior domain. Amnion specification occurs at the boundaries of the embryonic disc through ID1, ID2 and ID3 in response to BMP signalling, providing a developmental rationale for amnion differentiation of primate pluripotent stem cells (PSCs). Spatial identity mapping demonstrates that primed marmoset PSCs exhibit the highest similarity to the anterior embryonic disc, whereas naive PSCs resemble the preimplantation epiblast. Our 3D transcriptome models reveal the molecular code of lineage specification in the primate embryo and provide an in vivo reference to decipher human development.
The post-translational modification of proteins expands the regulatory scope of the proteome far beyond what is achievable through genome regulation. The field of protein citrullination has seen significant progress in the last two decades. The small family of peptidylarginine deiminase (PADI or PAD) enzymes, which catalyse citrullination, have been implicated in virtually all facets of molecular and cell biology, from gene transcription and epigenetics to cell signalling and metabolism. We have learned about their association with a remarkable array of disease states and we are beginning to understand how they mediate normal physiological functions. However, while the biochemistry of PADI activation has been worked out in exquisite detail , we still lack a clear mechanistic understanding of the processes that regulate PADIs within cells, under physiological and pathophysiological conditions. This review summarizes and discusses the current knowledge, highlights some of the unanswered questions of immediate importance and gives a perspective on the outlook of the citrullination field.