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Multiphasic architectures are found ubiquitously in biomolecular condensates and are thought to have important implications for the organisation of multiple chemical reactions within the same compartment. Many of these multiphasic condensates contain RNA in addition to proteins. Here, we investigate the importance of different interactions in multiphasic condensates comprising two different proteins and RNA using computer simulations with a residue-resolution coarse-grained model of proteins and RNA. We find that in multilayered condensates containing RNA in both phases, protein-RNA interactions dominate, with aromatic residues and arginine forming the key stabilising interactions. The total aromatic and arginine content of the two proteins must be appreciably different for distinct phases to form, and we show that this difference increases as the system is driven towards greater multiphasicity. Using the trends observed in the different interaction energies of this system, we demonstrate that we can also construct multilayered condensates with RNA preferentially concentrated in one phase. The 'rules' identified can thus enable the design of synthetic multiphasic condensates to facilitate further study of their organisation and function.

Purpose of Review Neuroblastoma, a paediatric malignancy of the sympathoadrenal lineage with a variable clinical course, is the most prevalent extra-cranial cancer in children. The majority of multi-modal therapeutics utilised for treating neuroblastoma may drive cells towards cell death or cellular senescence. Recent Findings Although cellular senescence has been historically regarded as a permanent state of non-proliferation, new evidence supports the notion that this process may indeed be much more dynamic than previously thought. Further, senescent tumour cells may escape treatment and further promote inflammation and migration through their repertoire of secreted molecules, leading to disease relapse. Summary Given this background, we review here the role of non-coding RNAs inclusive of long non-coding RNAs (lncRNAs) and miRNAs in therapy-induced senescence-related processes in neuroblastoma and discuss how these molecules may be manipulated for therapeutic gain.

Cellular senescence, a persistent form of cell cycle arrest, has been linked to the formation of heterochromatic foci, accompanied by additional concentric epigenetic layers. However, senescence is a highly heterogeneous phenotype, and the formation of these structures is context dependent. Recent developments in the understanding of the high-order chromatin organization have opened new avenues for contextualizing the nuclear and chromatin phenotypes of senescence. Oncogene-induced senescence displays prominent foci and typically exhibits increased chromatin compartmentalization, based on the chromosome conformation assays, as marked by increased transcompaction and segregation of the heterochromatin and euchromatin. However, other types of senescence (e.g., replicative senescence) exhibit comparatively lower levels of compartmentalization. Thus, a more integrative view of the global rearrangement of the chromatin architecture that occurs during senescence is emerging, with potential functional implications for the heterogeneity of the senescence phenotype.

In this issue of Blood1, Bamezai et al reveal an unexpected, moonlighting* function of the germ cell associated RNA binding protein, PIWIL4 in AML. The authors show a tumor-specific requirement for PIWIL4 in R-loop homeostasis. Loss of PIWIL4 in AML cells led to uncontrolled R-loop formation, transcriptional stalling, DNA damage and an enhanced sensitivity to ATR inhibition, findings that may inform future therapeutic approaches.

The RAD51 ATPase polymerises on single-stranded DNA to form nucleoprotein filaments (NPFs) that are critical intermediates in the reaction of Homologous Recombination. ATP binding maintains the NPF in a competent conformation for strand pairing and exchange. Once strand exchange is completed, ATP hydrolysis licenses the filament for disassembly. Here we show that the ATP-binding site of the RAD51 NPF contains a second metal ion. In the presence of ATP, the metal ion promotes the local folding of RAD51 into the conformation required for DNA binding. The metal ion is absent in the ADP-bound RAD51 filament, that rearranges in a conformation incompatible with DNA binding. The presence of the second metal ion explains how RAD51 couples the nucleotide state of the filament to DNA binding. We propose that loss of the second metal ion upon ATP hydrolysis drives RAD51 dissociation from the DNA and weakens filament stability, contributing to NPF disassembly.

Biomechanical cues are instrumental in guiding embryonic development and cell differentiation. Understanding how these physical stimuli translate into transcriptional programs will provide insight into mechanisms underlying mammalian pre-implantation development. Here, we explore this type of regulation by exerting microenvironmental control over mouse embryonic stem cells. Microfluidic encapsulation of mouse embryonic stem cells in agarose microgels stabilizes the naive pluripotency network and specifically induces expression of Plakoglobin (Jup), a vertebrate homolog of β-catenin. Overexpression of Plakoglobin is sufficient to fully re-establish the naive pluripotency gene regulatory network under metastable pluripotency conditions, as confirmed by single-cell transcriptome profiling. Finally, we find that, in the epiblast, Plakoglobin was exclusively expressed at the blastocyst stage in human and mouse embryos - further strengthening the link between Plakoglobin and naive pluripotency in vivo. Our work reveals Plakoglobin as a mechanosensitive regulator of naive pluripotency and provides a paradigm to interrogate the effects of volumetric confinement on cell-fate transitions.

Abstract. Emotion recognition ability of emotions expressed by other people (ERA-O) can be important for job performance, leadership, bargaining, and career success. Traditional personnel assessment tools of this ability, however, are contaminated by linguistic skills. In a time of global work migration, more and more people speak a language at work that is not their mother tongue. Consequently, we developed and validated the Face-Based Emotion Matching Test (FEMT), a nonlinguistic objective test of ERA-O in gainfully employed adults. We demonstrate the FEMT’s validity with psychological constructs (cognitive and emotional intelligence, Big Five personality traits) and its criterion validity and interethnic fit.

Extracellular vesicles (EV) and the microRNAs that they contain are increasingly recognised as a rich source of informative biomarkers, reflecting pathological processes and fundamental biological pathways and responses. Their presence in biofluids makes them particularly attractive for biomarker identification. However, a frequent caveat in relation to clinical studies is low abundance of EV RNA content. In this study, we used NanoString nCounter technology to assess the microRNA profiles of n = 64 EV low concentration RNA samples (180–49125 pg), isolated from serum and cell culture media using precipitation reagent or sequential ultracentrifugation. Data was subjected to robust quality control parameters based on three levels of limit of detection stringency, and differential microRNA expression analysis was performed between biological subgroups. We report that RNA concentrations > 100 times lower than the current NanoString recommendations can be successfully profiled using nCounter microRNA assays, demonstrating acceptable output ranges for imaging parameters, binding density, positive/negative controls, ligation controls and normalisation quality control. Furthermore, despite low levels of input RNA, high‐level differential expression analysis between biological subgroups identified microRNAs of biological relevance. Our results demonstrate that NanoString nCounter technology offers a sensitive approach for the detection and profiling of low abundance EV‐derived microRNA, and may provide a solution for research studies that focus on limited sample material.

Obesity is a global health epidemic. Excess white adipose tissue typifies obesity, but newly described beige adipose tissue burns calories through its thermogenic activity, leading to weight loss and metabolic improvements. There is a network of beneficial type 2 immune cells residing in healthy adipose tissue, including macrophages, eosinophils, innate lymphoid cells, and T lymphocyte subsets, which drives adipose beiging and thermogenesis. Thus, studying these cells may enable identification of new molecular targets for treating obesity. The work in this thesis aimed to study these immune regulators of beiging in the Krüppel-like Factor 3 (KLF3) transcription factor knockout (KO) mouse model. These KLF3 KO (Klf3−/−) mice are lean, protected from diet induced obesity and display elevated adipose beiging. We previously hypothesised that regulation of gene expression by KLF3 in adipose-resident eosinophils may contribute to the lean, beige phenotype in Klf3−/− mice. Here, we provided evidence supporting this hypothesis by demonstrating the enhanced ability of adipose eosinophils from Klf3−/− mice to increase thermogenic gene expression in beige adipocytes through in vitro co-culture. We further studied KLF3’s regulation of eosinophils by developing in vitro systems to identify ‘eosinokines’ – proteins that adipose eosinophils secrete to drive beiging. CRISPR-Cas9 genome editing was optimised for the EoL-1 human eosinophilic cell line to generate clonal cells lacking KLF3. To assess the potential of KLF3 KO EoL-1 secreted proteins to stimulate thermogenesis, a beiging assay was optimised with primary beige adipocytes. Next, we expanded this work to investigate if KLF3 regulates gene expression in other adipose-resident immune cells to contribute to beiging in Klf3−/− mice. Invariant natural killer T cells (iNKTs), regulatory T cells and natural killer cells were isolated from subcutaneous adipose tissue of WT and Klf3−/− mice and RNA-seq was performed to interrogate how KLF3 deficiency altered their transcriptomes. We observed the most altered gene expression in Klf3−/− iNKTs which, along with iNKTs being twice as abundant in Klf3−/− adipose tissue, suggested that KLF3 may regulate the function of adipose-resident iNKTs towards a less inflammatory phenotype. This work has contributed to a growing understanding of immune regulation of metabolism which will support the development of targeted obesity therapeutics for activating energy expenditure via beiging.

Distinct localisation of macromolecular structures relative to cell shape is a common feature across the domains of life. One mechanism for achieving spatiotemporal intracellular organisation is the Turing reaction-diffusion system (e.g. Min system in the bacterium Escherichia coli controlling in cell division). In this thesis, I explore potential Turing systems in archaea and eukaryotes as well as the effects of subdiffusion. Recently, a MinD homologue, MinD4, in the archaeon Haloferax volcanii was found to form a dynamic spatiotemporal pattern that is distinct from E. coli in its localisation and function. I investigate all four archaeal Min paralogue systems in H. volcanii by identifying four putative MinD activator proteins based on their genomic location and show that they alter motility but do not control MinD4 patterning. Additionally, one of these proteins shows remarkably fast dynamic motion with speeds comparable to eukaryotic molecular motors, while its function appears to be to control motility via interaction with the archaellum. In metazoa, neurons are highly specialised cells whose functions rely on the proper segregation of proteins to the axonal and somatodendritic compartments. These compartments are bounded by a structure called the axon initial segment (AIS) which is precisely positioned in the proximal axonal region during early neuronal development. How neurons control these self-organised localisations is poorly understood. Using a top-down analysis of developing neurons in vitro, I show that the AIS lies at the nodal plane of the first non-homogeneous spatial harmonic of the neuron shape while a key axonal protein, Tau, is distributed with a concentration that matches the same harmonic. These results are consistent with an underlying Turing patterning system which remains to be identified. The complex intracellular environment often gives rise to the subdiffusive dynamics of molecules that may affect patterning. To simulate the subdiffusive transport of biopolymers, I develop a stochastic simulation algorithm based on the continuous time random walk framework, which is then applied to a model of a dimeric molecular motor. This provides insight into the effects of subdiffusion on motor dynamics, where subdiffusion reduces motor speed while increasing the stall force. Overall, this thesis makes progress towards understanding intracellular patterning systems in different organisms, across the domains of life.

Most animal cells are surrounded by a cell membrane and an underlying actomyosin cortex. Both structures are linked, and they are under tension. In-plane membrane tension and cortical tension both influence many cellular processes, including cell migration, division, and endocytosis. However, while actomyosin tension is regulated by substrate stiffness, how membrane tension responds to mechanical substrate properties is currently poorly understood. Here, we probed the effective membrane tension of neurons and fibroblasts cultured on glass and polyacrylamide substrates of varying stiffness using optical tweezers. In contrast to actomyosin-based traction forces, both peak forces and steady-state tether forces of cells cultured on hydrogels were independent of substrate stiffness and did not change after blocking myosin II activity using blebbistatin, indicating that tether and traction forces are not directly linked. Peak forces in fibroblasts on hydrogels were about twice as high as those in neurons, indicating stronger membrane–cortex adhesion in fibroblasts. Steady-state tether forces were generally higher in cells cultured on hydrogels than on glass, which we explain by a mechanical model. Our results provide new insights into the complex regulation of effective membrane tension and pave the way for a deeper understanding of the biological processes it instructs.

Various physics- and data-driven sequence-dependent protein coarse-grained models have been developed to study biomolecular phase separation and elucidate the dominant physicochemical driving forces. Here we present Mpipi, a multiscale coarse-grained model that describes almost quantitatively the change in protein critical temperatures as a function of amino acid sequence. The model is parameterized from both atomistic simulations and bioinformatics data and accounts for the dominant role of π–π and hybrid cation–π/π–π interactions and the much stronger attractive contacts established by arginines than lysines. We provide a comprehensive set of benchmarks for Mpipi and seven other residue-level coarse-grained models against experimental radii of gyration and quantitative in vitro phase diagrams, demonstrating that Mpipi predictions agree well with experiments on both fronts. Moreover, Mpipi can account for protein–RNA interactions, correctly predicts the multiphase behavior of a charge-matched poly-arginine/poly-lysine/RNA system, and recapitulates experimental liquid–liquid phase separation trends for sequence mutations on FUS, DDX4 and LAF-1 proteins. Combining bioinformatics data and atomistic simulations, this study develops a sequence-dependent coarse-grained model for biomolecular phase separation. This model achieves a quantitative agreement with experimental observations. Extensive benchmarks exemplify its performance.

Aggregated α-synuclein (α-syn) accumulates in the neuronal Lewy body (LB) inclusions in Parkinson's disease (PD) and LB dementia. Yet, under nonpathological conditions, monomeric α-syn is hypothesized to exist in an equilibrium between disordered cytosolic- and partially α-helical lipid-bound states: a feature presumably important in synaptic vesicle release machinery. The exact underlying role of α-syn in these processes, and the mechanisms regulating membrane-binding of α-syn remains poorly understood. Herein we demonstrate that Protein kinase R (PKR) can phosphorylate α-syn at several Ser/Thr residues located in the membrane-binding region that is essential for α-syn's vesicle-interactions. α-Syn phosphorylated by PKR or α-syn isolated from PKR overexpressing cells, exhibit decreased binding to lipid membranes. Phosphorylation of Thr64 and Thr72 appears as the major contributor to this effect, as the phosphomimetic Thr64Glu/Thr72Glu-α-syn mutant displays reduced overall attachment to brain vesicles due to a decrease in vesicle-affinity of the last two thirds of α-syn's membrane binding region. This allows enhancement of the “double-anchor” vesicle-binding mechanism that tethers two vesicles and thus promote the clustering of presynaptic vesicles in vitro. Furthermore, phosphomimetic Thr64Glu/Thr72Glu-α-syn inhibits α-syn oligomerization and completely abolishes nucleation, elongation, and seeding of α-syn fibrillation in vitro and in cells, and prevents trans-synaptic spreading of aggregated α-syn pathology in organotypic hippocampal slice cultures. Overall, our findings demonstrate that normal and abnormal functions of α-syn, like membrane-binding, synaptic vesicle clustering and aggregation can be regulated by phosphorylation, e.g., via PKR. Mechanisms that could potentially be modulated for the benefit of patients suffering from α-syn aggregate-related diseases.

The oxidation of membrane lipids has been widely studied for several decades due to its significance in biological systems. However, despite its damaging physiological impact and its known role in many diseases, relatively little is understood about the specific structural consequences of oxidative action, particularly in vivo. In this work, a combination of sum-frequency generation (SFG) spectroscopy, surface tensiometry, and surface-selective infrared spectroscopies are used to gain deeper insight into the oxidation of phospholipids by reactive oxygen species (ROS) generated in situ. Oxidation is achieved by employing the Fenton reaction to convert physiological levels of H2O2 into OH and HO2 radicals in proximity to the head-groups of lipid monolayers at the air-water interface. By temporally monitoring the surface tension and spectroscopic changes at the interface as the oxidation proceeds, the impact of oxidation on the structure, conformation, and intermolecular interactions within the membrane has been revealed.

Fumarate hydratase (FH) is a mitochondrial enzyme that catalyzes the reversible hydration of fumarate to malate in the tricarboxylic acid (TCA) cycle. Germline mutations of FH lead to hereditary leiomyomatosis and renal cell carcinoma (HLRCC), a cancer syndrome characterized by a highly aggressive form of renal cancer. Although HLRCC tumors metastasize rapidly, FH-deficient mice develop premalignant cysts in the kidneys, rather than carcinomas. How Fh1-deficient cells overcome these tumor-suppressive events during transformation is unknown. Here, we perform a genome-wide CRISPR-Cas9 screen to identify genes that, when ablated, enhance the proliferation of Fh1-deficient cells. We found that the depletion of the histone cell cycle regulator (HIRA) enhances proliferation and invasion of Fh1-deficient cells in vitro and in vivo. Mechanistically, Hira loss activates MYC and its target genes, increasing nucleotide metabolism specifically in Fh1-deficient cells, independent of its histone chaperone activity. These results are instrumental for understanding mechanisms of tumorigenesis in HLRCC and the development of targeted treatments for patients.

Fibrillar protein aggregates are a hallmark of the pathology of a range of human disorders, from prion diseases to dementias. Yet, the same aggregated structures that are formed in disease are also encountered in several functional contexts. The fundamental properties that determine whether these protein assembly processes are functional or, by contrast, pathological, have remained elusive. Here, we address this question by analysing the aggregation kinetics of a large set of self-assembling proteins, from those associated with disease, over those whose aggregates fulfil functional roles in biology, to those that aggregate only under artificial conditions. Remarkably, we find that essentially all systems that assemble by a nucleated-growth mechanism are capable of significant self-replication on experimentally accessible timescales. However, comparing the intrinsic timescales of self-replication with the timescales over which the corresponding aggregates form in a biological context yields a clear distinction; for aggregates which have evolved to fulfil a structural role, the rate of self-replication is too low to be significant on the biologically relevant timescale. By contrast, all analysed proteins that aggregate in the context of disease are able to self-replicate quickly compared to the timescale of the associated disease. Our findings establish the ability to self-replicate as both a ubiquitous property of protein aggregates and one that has the potential to be a key process across aggregation-related disorders.

Nucleic acid and histone modifications critically depend on the tricarboxylic acid (TCA) cycle for substrates and cofactors. Although a few TCA cycle enzymes have been reported in the nucleus, the corresponding pathways are considered to operate in mitochondria. Here, we show that a part of the TCA cycle is operational also in the nucleus. Using13C-tracer analysis, we identified activity of glutamine-to-fumarate, citrate-to-succinate, and glutamine-to-aspartate routes in the nuclei ofHeLacells. Proximity labeling mass spectrometry revealed a spatial vicinity of the involved enzymes with core nuclear proteins. We further show nuclear localization of aconitase 2 and 2-oxoglutarate dehydrogenase in mouse embryonic stem cells. Nuclear localization of the latter enzyme, which produces succinyl-CoA, changed from pluripotency to a differentiated state with accompanying changes in the nuclear protein succinylation. Together, our results demonstrate operation of an extended metabolic pathway in the nucleus, warranting a revision of the canonical view on metabolic compartmentalization.

Points for accumulation in nanoscale topography (PAINT) allows the acquisition of practically unlimited measurements in localisation microscopy. However, PAINT is inherently limited by unwanted background fluorescence at high probe concentrations, especially in large depth-of-field volumetric imaging techniques. Here we present reservoir-PAINT (resPAINT), in which we combine PAINT with active control of probe photophysics. In resPAINT, a ‘reservoir’ of non-fluorescent activatable probes accumulate on the target, which makes it possible to drastically improve the localisation rate (by up to 50-fold) compared to conventional PAINT, without any compromise in contrast. By combining resPAINT with large depth-of-field microscopy, we demonstrate volumetric super-resolution imaging of entire cell surfaces. We then generalise the approach by implementing multiple switching strategies, including photoactivation and spontaneous blinking. We also implement alternative volumetric imaging modalities including the double-helix pointspread function, the tetrapod point-spread function and singlemolecule light field microscopy. Finally, we show that resPAINT can be used with a Fab to image membrane proteins, effectively extending the operating regime of conventional PAINT to encompass a larger range of biological interactions.

Funder: Funder: The Ministry of Science and Technology Grant Reference Number: 2018YFA0801100 Funder: The National Science Foundation of China Grant Reference Number: 31630091, Funder: Funder: The National Science Foundation of China Grant Reference Number:31871185, Exploring the functions of human-specific genes (HSGs) is challenging due to the lack of a tractable genetic model system. Testosterone is essential for maintaining human spermatogenesis and fertility, but the underlying mechanism is unclear. Here, we identified Cancer/Testis Antigen gene family 47 (CT47) as an essential regulator of human-specific spermatogenesis by stabilizing arginine methyltransferase 5 (PRMT5). A humanized mouse model revealed that CT47 functions to arrest spermatogenesis by interacting with and regulating CT47/PRMT5 accumulation in the nucleus during the leptotene/zygotene-to-pachytene transition of meiosis. We demonstrate that testosterone induces nuclear depletion of CT47/PRMT5 and rescues leptotene-arrested spermatocyte progression in humanized testes. Loss of CT47 in human embryonic stem cells (hESCs) by CRISPR/Cas9 led to an increase in haploid cells but blocked the testosterone-induced increase in haploid cells when hESCs were differentiated into haploid spermatogenic cells. Moreover, CT47 levels were decreased in nonobstructive azoospermia. Together, these results established CT47 as a crucial regulator of human spermatogenesis by preventing meiosis initiation before the testosterone surge.

ATP-competitive inhibitors are currently the largest class of clinically approved drugs for protein kinases. By targeting the ATP-binding pocket, these compounds block the catalytic activity, preventing substrate phosphorylation. A problem with these drugs, however, is that inhibited kinases may still recognize and bind downstream substrates, acting as scaffolds or binding hubs for signaling partners. Here, using protein kinase A as a model system, we show that chemically different ATP-competitive inhibitors modulate the substrate binding cooperativity by tuning the conformational entropy of the kinase and shifting the populations of its conformationally excited states. Since we found that binding cooperativity and conformational entropy of the enzyme are correlated, we propose a new paradigm for the discovery of ATP-competitive inhibitors, which is based on their ability to modulate the allosteric coupling between nucleotide and substrate-binding sites.

The development and implementation of a universal, evidence-based radiation protection policy is one of the greatest challenges that we face. While different contexts and unique situations make the implementation of a global policy complex, the idea that there might be a commonly accepted set of principles around which regulation, legislation and monitoring might be built has long been accepted as desirable, if not even essential. Recent developments in radiation protection and in radiobiology have started to suggest that the current, anthropocentric approach may underestimate the impact of radiation on the environment. This, through ecosystem perturbations, may prove deleterious to humans as well as other species in a way not captured by assessing human cancer risk as the main endpoint under consideration. Our evolving understanding of other human endpoints such as cardiovascular disease and the impact of multiple stressors such as pesticides and heavy metals have started to broaden our concept of an endpoint for human radiation protection and in doing so start to draw in the importance of the non-radioactive anthropogenic and natural environments. The questions raised by considering non-medical radiation protection as primarily an issue of environmental protection, stimulated a recent discussion and accompanying paper in IJRB (Mothersill et al. 2020) and were the stimulus for this special issue.

Clathrin-mediated endocytosis (CME) is the main mechanism by which mammalian cells control their cell surface proteome. Proper operation of the pivotal CME cargo-adaptor AP2 requires membrane-localised FCHO. Here, live-cell eTIRF-SIM shows that FCHO marks sites of clathrin-coated pit (CCP) initiation, which mature into uniform sized CCPs comprising a central patch of AP2 and clathrin corralled by an FCHO/Eps15 ring. We dissect the network of interactions between the FCHO interdomain-linker and AP2, which concentrates, orients, tethers and partially destabilizes closed AP2 at the plasma membrane. AP2’s subsequent membrane deposition drives its opening, which triggers FCHO displacement through steric competition with PtdIns4,5P2, clathrin, cargo and CME accessory factors. FCHO can now relocate toward a CCP's outer edge to engage and activate further AP2s to drive CCP growth/maturation.

Misfolding of secretory proteins in the endoplasmic reticulum (ER) features in many human diseases. In α1-antitrypsin deficiency, the pathogenic Z variant aberrantly assembles into polymers in the hepatocyte ER, leading to cirrhosis. We show that α1-antitrypsin polymers undergo a liquid:solid phase transition, forming a protein matrix that retards mobility of ER proteins by size-dependent molecular filtration. The Z-α1-antitrypsin phase transition is promoted during ER stress by an ATF6-mediated unfolded protein response. Furthermore, the ER chaperone calreticulin promotes Z-α1-antitrypsin solidification and increases protein matrix stiffness. Single-particle tracking reveals that solidification initiates in cells with normal ER morphology, previously assumed to represent a healthy pool. We show that Z-α1-antitrypsin-induced hypersensitivity to ER stress can be explained by immobilization of ER chaperones within the polymer matrix. This previously unidentified mechanism of ER dysfunction provides a template for understanding a diverse group of related proteinopathies and identifies ER chaperones as potential therapeutic targets., Alpha-1 Foundation Grifols Alpha-1-Antitrypsin Laurell’s Training Award National Biofilms Innovation Centre MINECO MedImmune Infinitus NIH The American Cancer Society The Pershing Square Sohn Cancer Research Award The Chan Zuckerberg Initiative UK Dementia Research Institute grant Canadian Institutes of Health Research US Alzheimer Society Integrated Biological Imaging Network

The composition of extracellular vesicles (EVs) is altered in many pathological conditions, and their molecular content provides essential information on features of parent cells and mechanisms of crosstalk between cells and organs. Metabolic Syndrome (MetS) is a cluster of clinical manifestations including obesity, insulin resistance, dyslipidemia and hypertension that increases the risk of cardiovascular disease and type 2 diabetes mellitus. Here, we investigated the crosstalk between liver and adipocytes by characterizing EVs secreted by primary hepatocytes isolated from Zucker rat model, and studied the effect they have on 3T3‐L1 adipocytes. We found that steatotic hepatocytes secrete EVs with significantly reduced exosomal markers in comparison with their lean counterpart. Moreover, proteomic analysis revealed that those EVs reflect the metabolic state of the parent cell in that the majority of proteins upregulated relate to fat metabolism, fatty acid synthesis, glycolysis, and pentose phosphate pathway. In addition, hepatocytes‐secreted EVs influenced lipolysis and insulin sensitivity in recipient 3T3‐L1 adipocytes. Untargeted metabolomic analysis detected alterations in different adipocyte metabolic pathways in cells treated with hepatic EVs. In summary, our work showed that steatosis has a significant impact in the amount and composition of EVs secreted by hepatocytes. Moreover, our data point to the involvement of hepatic‐EVs in the development of pathologies associated with MetS.

Description, Neuroserpin preferentially inhibits amyloid formation; a 14-mer region in neuroserpin is implicated in binding to amyloid., Neuroserpin is a secreted protease inhibitor known to inhibit amyloid formation by the Alzheimer’s beta peptide (Aβ). To test whether this effect was constrained to Aβ, we used a range of in vitro assays to demonstrate that neuroserpin inhibits amyloid formation by several different proteins and protects against the associated cytotoxicity but, unlike other known chaperones, has a poor ability to inhibit amorphous protein aggregation. Collectively, these results suggest that neuroserpin has an unusual chaperone selectivity for intermediates on the amyloid-forming pathway. Bioinformatics analyses identified a highly conserved 14-residue region containing an α helix shared between neuroserpin and the thyroxine-transport protein transthyretin, and we subsequently demonstrated that transthyretin also preferentially inhibits amyloid formation. Last, we used rationally designed neuroserpin mutants to demonstrate a direct involvement of the conserved 14-mer region in its chaperone activity. Identification of this conserved region may prove useful in the future design of anti-amyloid reagents.

The DREAM (DP, Retinoblastoma [Rb]-like, E2F, and MuvB) complex controls cellular quiescence by repressing cell cycle and other genes, but its mechanism of action is unclear. Here we demonstrate that two C. elegans THAP domain proteins, LIN-15B and LIN-36, co-localize with DREAM and function by different mechanisms for repression of distinct sets of targets. LIN-36 represses classical cell cycle targets by promoting DREAM binding and gene body enrichment of H2A.Z, and we find that DREAM subunit EFL-1/E2F is specific for LIN-36 targets. In contrast, LIN-15B represses germline specific targets in the soma by facilitating H3K9me2 promoter marking. We further find that LIN-36 and LIN-15B differently regulate DREAM binding. In humans, THAP proteins have been implicated in cell cycle regulation by poorly understood mechanisms. We propose that THAP domain proteins are key mediators of Rb/DREAM function.

T cells are critical in the body's defence against viruses and cancerous cells. They specifically recognise viral or tumour antigens presented on antigen presenting cells using their T cell receptor (TCR). Antigen binding triggers the TCR, transmitting signal intracellularly and resulting in the recruitment of a plethora of signalling proteins to the membrane. The signal is transmitted by post-translational modifications, such as the phosphorylation of tyrosine residues in intracellular tails of receptors, resulting in the recruitment of signalling proteins via their interaction domains. The spatial organisation of signalling proteins at the membrane determines the effector response of the T cell and is therefore critical for understanding the complex array of T cell responses. In this thesis, I develop a novel microscopy technique that reports on the nanoscale locations of signalling proteins and their binding kinetics to receptors at the T cell membrane. This technique utilizes the SH2 interaction domains of various signalling proteins which selectively and transiently bind to phosphorylated tyrosines on receptors. This transient binding results in the stochastic blinking necessary for super-resolution microscopy. Using this technique, termed protein point accumulation in nanoscale topography (pPAINT), I investigate the binding of multiple signalling proteins, achieving multiplexed imaging both simultaneously and sequentially with a combined microfluidic and microscopy approach. In the second half of this thesis, I apply pPAINT to study how chimeric antigen receptors (CARs) signal in T cells. In CAR-T therapy, patient cytotoxic T cells are isolated and transduced with a CAR construct that recognises tumour antigens, and are then reintroduced into the patient where they find and eliminate cells expressing the CAR target antigen. CAR constructs are made up of an antigen recognition domain fused to various intracellular signalling motifs from the TCR complex and co-stimulatory receptors, such as CD28, which are crucial for T cell activation. The first-generation of CARs contained an intracellular tail of the TCR, the CD3ζ chain, but it has been the second-generation CARs, with the addition of co-stimulatory receptor signalling domains, that have proven clinically effective. However, CAR therapy is not successful in all patients; limitations reducing their efficacy include inefficient recognition of low antigen densities, finite persistence in the body and off-target side effects in patients. It follows that a detailed knowledge and understanding of CAR activation and signalling is needed to optimise CAR design. In this thesis I use pPAINT to gain a unique perspective on how different generations of CARs signal upon activation, identifying key similarities and differences to signalling from the standard TCR. Signalling was investigated in CAR-T cells generated in a similar way to clinically used CAR-T therapies. In doing so, unique signalling mechanisms utilized by CARs were identified that will be valuable for the development of more effective chimeric antigen receptors. The results demonstrate that although CARs utilise the signalling domains of the TCR and co-stimulatory receptors, the pattern of adaptor protein recruitment is different from that of T cells stimulated through the TCR and co- stimulatory receptors. Specifically, I found that whilst hubs of signalling proteins spatially diverged from clusters of activated TCR, they were instead closely colocalised with activated CARs. The incorporation of the CD28 coreceptor in CAR design improves signalling protein recruitment patterns, however, the patterns of protein binding were still vastly different to co-stimulated T cells. Collectively, the results indicate that CARs utilize a signalling pathway unique to that of costimulated T cells, in a mechanism that may have ramifications in the functional responses exhibited by cells used in CAR-T therapy.

Pathological left ventricular hypertrophy (LVH) induced by mechanical pressure overload is the most powerful independent predictor of cardiac mortality in clinic. Current treatment methods focus on removing the pressure overload stimulus for LVH, which does not reverse adverse cardiac remodelling completely. Importantly, the molecular signalling pathways involved in pressure overload induced-LVH are potential targets for therapeutic intervention. Although numerous molecular signalling steps in the induction of LVH have been identified to date, the initial step when mechanical stretch associated with cardiac pressure overload is converted into a chemical signal that initiates hypertrophic signalling remains unresolved. As the primary molecular transducers of mechanical stimuli that function in the cardiovascular system, several ion channels, including mechanosensitive ones, have been considered as potential contributors to cardiac hypertrophy signalling pathways. The focus of this project is to investigate the role of two types of ion channels, including TRPM4 and Piezo1, in cardiac mechanotransduction underlying pressure overload-induced LVH. By employing wild-type and conditional cardiac gene knockout mouse LVH model based on aortic transverse constriction, this project demonstrates that TRPM4 channel is an important component of the Ca2+-dependent mechanosensory signalling pathway that contributes to LVH in response to pressure overload; selective deletion of TRPM4 channels in mouse cardiomyocytes results in an approximately 50% reduction in pressure overload-induced LVH. Also, this project provides methodological foundation of new in vitro approaches to investigate Piezo1-mediated cardiac mechanotransduction. Chemical agonist-evoked Piezo1 activation and likely isotropic stretch-induced Piezo1 activation were shown in both HL-1 atrial myocyte-like cell line and isolated mouse ventricular cardiomyocytes, visualised by Ca2+ imaging. In addition, the data from this project provide in vitro evidence of the functional interaction between Piezo1 and TRPM4 channels, reflected in altered action potential frequency in HL-1 cells. These findings contribute to better understanding of the mechano-electrochemical transduction which initiates the signalling pathway in pressure overload-induced LVH and thus provide the basis for development of potential novel therapeutic targets for prevention of pathological LVH.

The benign condition Hereditary Persistence of Foetal Haemoglobin (HPFH) alleviates -haemoglobinopathies, such as sickle cell disease and -thalassemia. HPFH can be associated with point mutations in the foetal globin promoters that disrupt the binding of the repressors BCL11A or LRF or create de novo binding sites for activators. HPFH is also associated with an extensive range of deletions within the -globin locus that reside downstream of the foetal HBG2 gene. Numerous mechanisms have been proposed to explain how downstream deletions can boost the expression of the foetal globin genes, including the deletion of silencer elements, deletion of genes encoding non-coding RNA, and bringing downstream enhancer elements into proximity with the foetal globin gene promoters. Here we systematically analysed the deletions associated with both HPFH and a related condition known as -thalassemia and propose a unifying mechanism. We found that in all cases where foetal globin is up-regulated the proximal adult -globin promoter is deleted. We used CRISPR gene editing to delete or disrupt transcription factor binding sites within the promoter and found that virtually all deletions that reduce HBB promoter activity, result in elevated foetal globin expression. These results fit with previous models where the foetal and adult globin genes compete for the distal locus control element. Under this model by disrupting the adult globin promoter, the foetal globin genes expression would be reactivated by allowing the foetal globin gene to better compete for the locus control element. We also found that a disruption of transcription factor binding sites by point mutations did not exert the same effect, suggesting that point mutations do not sufficiently disrupt competition for the locus control element. Our findings suggest that targeting the HBB promoter might be explored to elevate foetal globin and reduce sickle globin expression as a treatment for sickle cell disease.

Motivation The conventional methods to detect homologous protein pairs use the comparison of protein sequences. But the sequences of two homologous proteins may diverge significantly and consequently may be undetectable by standard approaches. The release of the AlphaFold 2.0 software enables the prediction of highly accurate protein structures and opens many opportunities to advance our understanding of protein functions, including the detection of homologous protein structure pairs. Results In this proof-of-concept work, we search for the closest homologous protein pairs using the structure models of five model organisms from the AlphaFold database. We compare the results with homologous protein pairs detected by their sequence similarity and show that the structural matching approach finds a similar set of results. In addition, we detect potential novel homologs solely with the structural matching approach, which can help to understand the function of uncharacterized proteins and make previously overlooked connections between well-characterized proteins. We also observe limitations of our implementation of the structure-based approach, particularly when handling highly disordered proteins or short protein structures. Our work shows that high accuracy protein structure models can be used to discover homologous protein pairs, and we expose areas for improvement of this structural matching approach. Availability and Implementation Information to the discovered homologous protein pairs can be found at the following URL: https://doi.org/10.17863/CAM.87873. The code can be accessed here: https://github.com/VivianMonzon/Reciprocal_Best_Structure_Hits. Supplementary information Supplementary data are available at Bioinformatics Advances online.

Despite being the target of extensive research efforts due to the COVID-19 (coronavirus disease 2019) pandemic, relatively little is known about the dynamics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication within cells. We investigate and characterize the tightly orchestrated virus assembly by visualizing the spatiotemporal dynamics of the four structural SARS-CoV-2 proteins at high resolution. The nucleoprotein is expressed first and accumulates around folded endoplasmic reticulum (ER) membranes in convoluted layers that contain viral RNA replication foci. We find that, of the three transmembrane proteins, the membrane protein appears at the Golgi apparatus/ER-to-Golgi intermediate compartment before the spike and envelope proteins. Relocation of a lysosome marker toward the assembly compartment and its detection in transport vesicles of viral proteins confirm an important role of lysosomes in SARS-CoV-2 egress. These data provide insights into the spatiotemporal regulation of SARS-CoV-2 assembly and refine the current understanding of SARS-CoV-2 replication.

Several software tools for the simulation and analysis of biochemical reaction networks have been developed in the last decades; however, assessing and comparing their computational performance in executing the typical tasks of computational systems biology can be limited by the lack of a standardized benchmarking approach. To overcome these limitations, we propose here a novel tool, named SMGen, designed to automatically generate synthetic models of reaction networks that, by construction, are characterized by relevant features (e.g., system connectivity and reaction discreteness) and non-trivial emergent dynamics of real biochemical networks. The generation of synthetic models in SMGen is based on the definition of an undirected graph consisting of a single connected component that, generally, results in a computationally demanding task; to speed up the overall process, SMGen exploits a main–worker paradigm. SMGen is also provided with a user-friendly graphical user interface, which allows the user to easily set up all the parameters required to generate a set of synthetic models with any number of reactions and species. We analysed the computational performance of SMGen by generating batches of symmetric and asymmetric reaction-based models (RBMs) of increasing size, showing how a different number of reactions and/or species affects the generation time. Our results show that when the number of reactions is higher than the number of species, SMGen has to identify and correct a large number of errors during the creation process of the RBMs, a circumstance that increases the running time. Still, SMGen can generate synthetic models with hundreds of species and reactions in less than 7 s.

The homeostatic regulation of amino acid concentrations is crucial for optimal brain function and development. Different amino acid transporters at cell membranes work together to facilitate the movement of amino acids into and out of the brain. Despite countless in vitro and in vivo research on these amino acids' activities, many fundamental concerns about their metabolic function in different brain areas and pathophysiological conditions remain unanswered. In the framework of this thesis, the effects of exogenous administration of several non-essential amino acids and the participation of their specific transporters in brain metabolism were investigated in Guinea pig cortical brain slices and mouse brain tissues using a targeted neuropharmacological and metabolomic strategy. Alterations in brain metabolism were analyzed using 1H and 13C nuclear magnetic resonance spectroscopy to evaluate changes in metabolite pools and 13C-enriched substrates. All the amino acid transporters mentioned in this study were addressed by the existing solute carrier (SLC) gene nomenclature system for amino acid transporters. The effect of exogenous L-aspartate, L-ornithine, and their salt, L-aspartate-L-ornithine (LOLA), on brain metabolism was investigated with or without an intact blood-brain barrier (BBB). The results indicated that neither L-aspartate, L-ornithine, nor LOLA, affected brain metabolism with an intact BBB. In cortical tissue slices L-aspartate increased brain metabolism concentration-dependently, L-ornithine significantly slowed it at higher concentrations (100 μmol/L), and the effects of LOLA was largely dependent on the balance of its two constituent amino acids. D-aspartate, another isoform of aspartate, produced a range of metabolic impacts, particularly on glutamatergic and GABAergic systems, with varying concentrations. In principal component analysis, the effects of D-aspartate were clearly distinguished from those of L-aspartate, indicating a metabolic pattern distinct from that of excitatory mechanisms. L-Proline administration significantly inhibited brain metabolism in Guinea pig cortical tissue slices, indicating a GABA-like effect; however, it was not a significant metabolic substrate. While it was actively taken up by cells in a concentration-dependent manner but was not completely metabolized. The metabolic pattern revealed that L-proline's effects clustered with 3-aminopropyl(methyl)phosphinic acid (SKF 97541), GABA, 1,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) and (5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a]thieno[2,3-f][1,4]diazepine-3-carboxylic acid) 1,1-dimethylethyl ester (RO194603) at lower concentrations (10 μmol/L) and with vigabatrin and RO194603 at higher concentrations (100 μmol/L); indicating that proline may act as a GABAB receptor agonist or GABAArho antagonist. Deletion of SLC6A17/NTT4 (neurotransmitter transporter 4) gene significantly impaired glutamate-glutamine cycle, reduced incorporation of 13C into Krebs cycle intermediates, and increased incorporation into lactate in the brain of mice lacking the gene. NTT4 knockout also altered several important metabolites in glutamatergic neurones, implying that it is a crucial transporter for maintaining brain amino acid homeostasis. Investigation of glutamine transport in cerebellum demonstrated that system A dominates glutamine transport in the cerebellum, with contributions from system N, which is inhibited by histidine and 2-(Methylamino)-2-methylpropionic acid (MeAIB) exerting the most metabolic influence. Inhibition of systems A and L by L-γ-Glutamyl-p-nitroanilide (GPNA) and 2-amino-4-bis(aryloxybenzyl)aminobutanoic acid (AABA) did not influence glutamine transport due to their low affinity for the transporters. Inhibition of systems L and B0 by 2-Aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH) showed little effect on fluxes from [1-13C]D-glucose but increased the flux of [1,2-13C]acetate into Glu C4,5 and Gln C4,5. Effects of cycloleucine were comparable to BCH but less powerful. This study provided new insight into the role of several non-essential amino acids in brain metabolism and also showed how brain metabolism is regulated in different brain regions.

The international Genetically Engineered Machine (iGEM) is an educational benchmark in synthetic biology. Eighteen years after its inception, it has also catalysed the infusion of synthetic biology with interdisciplinary fundamental and translational research as well as with inspired young scientists. Here, we communicate a quantitative analysis of compiled published work associated to iGEM projects, highlighting trends in their dissemination and versatility. As iGEM comes of age, we anticipate it will continue to revolutionise, alongside SynBio, several disciplines of science and industries through the development of synthetic biological systems towards a sustainable future.

Many bioconjugation strategies for DNA oligonucleotides and antibodies suffer limitations, such as site���specificity, stoichiometry and hydrolytic instability of the conjugates, which makes them unsuitable for biological applications. Here, we report a new platform for the preparation of DNA���antibody bioconjugates with a simple benzoylacrylic acid pentafluorophenyl ester reagent. Benzoylacrylic���labelled oligonucleotides prepared with this reagent can be site���specifically conjugated to a range of proteins and antibodies through accessible cysteine residues. The homogeneity of the prepared DNA���antibody bioconjugates was confirmed by a new LC���MS protocol and the bioconjugate probes were used in fluorescence or super���resolution microscopy cell imaging experiments. This work demonstrates the versatility and robustness of our bioconjugation protocol that gives site���specific, well���defined and plasma���stable DNA���antibody bioconjugates for biological applications.

[Figure: see text]., We acknowledge funding from Sidney Sussex College Cambridge (GM) and the European Research Council Grant Number 669237 (to D.K.) and the Royal Society (to D.K.). The Cambridge Brain Bank is supported by the NIHR Cambridge Biomedical Research Centre.

The solubility of proteins correlates with a variety of their properties, including function, production yield, pharmacokinetics, and formulation at high concentrations. High solubility is therefore a key requirement for the development of protein-based reagents for applications in life sciences, biotechnology, diagnostics, and therapeutics. Accurate solubility measurements, however, remain challenging and resource intensive, which limits their throughput and hence their applicability at the early stages of development pipelines, when long-lists of candidates are typically available in minute amounts. Here, we present an automated method based on the titration of a crowding agent (polyethylene glycol, PEG) to quantitatively assess relative solubility of proteins using about 200 µg of purified material. Our results demonstrate that this method is accurate and economical in material requirement and costs of reagents, which makes it suitable for high-throughput screening. This approach is freely-shared and based on a low cost, open-source liquid-handling robot. We anticipate that this method will facilitate the assessment of the developability of proteins and make it substantially more accessible.

Description, Microglia change their behavior when eating live neurons containing tau protein aggregates, a hallmark of Alzheimer’s disease., The microtubule-associated protein tau aggregates in multiple neurodegenerative diseases, causing inflammation and changing the inflammatory signature of microglia by unknown mechanisms. We have shown that microglia phagocytose live neurons containing tau aggregates cultured from P301S tau mice due to neuronal tau aggregate-induced exposure of the “eat me” signal phosphatidylserine. Here, we show that after phagocytosing tau aggregate-bearing neurons, microglia become hypophagocytic while releasing seed-competent insoluble tau aggregates. These microglia express a senescence-like phenotype, demonstrated by acidic β-galactosidase activity, secretion of paracrine senescence-associated cytokines, and maturation of matrix remodeling enzymes, results that are corroborated in P301S mouse brains and ex vivo brain slices. In particular, the nuclear factor κB–dependent activation of matrix metalloprotease 3 (MMP3/stromelysin1) was replicated in brains from patients with tauopathy. These data show that microglia that have been activated to ingest live tau aggregates-bearing neurons behave hormetically, becoming hypofunctional while acting as vectors of tau aggregate spreading.

Maintenance of protein homeostasis depends on cellular stress response pathways that mediate adaptive changes in gene expression. In the endoplasmic reticulum additional mechanisms adjust the availability of the abundant Hsp70-type chaperone, BiP, during short-term fluctuations in the unfolded protein load. Here, recent insights into the regulation of BiP by incorporation into inactive oligomers and reversible AMPylation are discussed.

In the present study we investigated the participation of hepatic peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) in the metabolic programming of newborn rats exposed in utero to dexamethasone (DEX). On the 21st day of life, fasted offspring born to DEX-treated mothers displayed increased conversion of pyruvate into glucose with simultaneous upregulation of PEPCK (phosphoenolpyruvate carboxykinase) and G6Pase (glucose-6-phosphatase). Increased oxidative phosphorylation, higher ATP/ADP ratio and mitochondrial biogenesis and lower pyruvate levels were also found in the progeny of DEX-treated mothers. On the other hand, the 21-day-old progeny of DEX-treated mothers had increased hepatic triglycerides (TAG) and lower CPT-1 activity when subjected to short-term fasting. At the mechanistic level, rats exposed in utero to DEX exhibited increased hepatic PGC-1α protein content with lower miR-29a-c expression. Increased PGC-1α content was concurrent with increased association to HNF-4α and NRF1 and reduced PPARα expression. The data presented herein reveal that changes in the transcription machinery in neonatal liver of rats born to DEX-treated mothers leads to an inflexible metabolic response to fasting. Such programming is hallmarked by increased oxidative phosphorylation of pyruvate with impaired FFA oxidation and hepatic TAG accumulation.