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Advances in a scientific discipline are often measured by small, incremental steps. In this review, we report on two intertwined disciplines in the protein structure prediction field, modeling of single chains and modeling of complexes, that have over decades emulated this pattern, as monitored by the community-wide blind prediction experiments CASP and CAPRI. However, over the past few years, dramatic advances were observed for the accurate prediction of single protein chains, driven by a surge of deep learning methodologies entering the prediction field. We review the main scientific developments that enabled these recent breakthroughs and feature the important role of blind prediction experiments in building up and nurturing the structure prediction field. We discuss how the new wave of artificial intelligence-based methods is impacting the fields of computational and experimental structural biology and highlight areas in which deep learning methods are likely to lead to future developments, provided that major challenges are overcome. Expected final online publication date for the Annual Review of Biophysics, Volume 52 is May 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

The structure of mRNA molecules plays an important role in its interactions with trans-acting factors, notably RNA binding proteins (RBPs), thus contributing to the functional consequences of this interplay. However, current transcriptome-wide experimental methods to chart these interactions are limited by their poor sensitivity. Here we extend the hiCLIP atlas of duplexes bound by Staufen1 (STAU1) ∼10-fold, through careful consideration of experimental assumptions, and the development of bespoke computational methods which we apply to existing data. We present Tosca, a Nextflow computational pipeline for the processing, analysis and visualisation of proximity ligation sequencing data generally. We use our extended duplex atlas to discover insights into the RNA selectivity of STAU1, revealing the importance of structural symmetry and duplex-span-dependent nucleotide composition. Furthermore, we identify heterogeneity in the relationship between STAU1-bound 3’ UTRs and metabolism of the associated RNAs that we relate to RNA structure: transcripts with short-range proximal 3’ UTR duplexes have high degradation rates, but those with long-range duplexes have low rates. Overall, our work enables the integrative analysis of proximity ligation data delivering insights into specific features and effects of RBP-RNA structure interactions.

Stress-induced tRNA fragmentation upon environmental insult is a conserved cellular process catalysed by endonucleolytic activities targeting mature tRNAs. The resulting tRNA-derived small RNAs (tsRNAs) have been implicated in various biological processes that impact cell-to-cell signalling, cell survival as well as gene expression regulation during embryonic development. However, how endonuclease-targeted tRNAs give rise to individual and potentially biologically active tsRNAs remains poorly understood. Here, we report on the in vivo identification of proteins associated with stress-induced tsRNAs-containing protein complexes, which, together with a ‘tracer tRNA’ assay, were used to uncover enzymatic activities that can bind and process specific endonuclease-targeted tRNAs in vitro. Among those, we identified conserved ATP-dependent RNA helicases which can robustly separate tRNAs with endonuclease-mediated ‘nicks’ in their anticodon loops. These findings shed light on the existence of cellular pathways dedicated to producing individual tsRNAs after stress-induced tRNA hydrolysis, which adds to our understanding as to how tRNA fragmentation and the resulting tsRNAs might exert physiological impact.

PURPOSE Failure to respond to induction chemotherapy portends a poor outcome in childhood acute lymphoblastic leukemia (ALL) and is more frequent in T-cell ALL (T-ALL) than B-cell ALL. We aimed to address the limited understanding of clinical and genetic factors that influence outcome in a cohort of patients with T-ALL induction failure (IF). METHODS We studied all cases of T-ALL IF on two consecutive multinational randomized trials, UKALL2003 and UKALL2011, to define risk factors, treatment, and outcomes. We performed multiomic profiling to characterize the genomic landscape. RESULTS IF occurred in 10.3% of cases and was significantly associated with increasing age, occurring in 20% of patients age 16 years and older. Five-year overall survival (OS) rates were 52.1% in IF and 90.2% in responsive patients ( P < .001). Despite increased use of nelarabine-based chemotherapy consolidated by hematopoietic stem-cell transplant in UKALL2011, there was no improvement in outcome. Persistent end-of-consolidation molecular residual disease resulted in a significantly worse outcome (5-year OS, 14.3% v 68.5%; HR, 4.10; 95% CI, 1.35 to 12.45; P = .0071). Genomic profiling revealed a heterogeneous picture with 25 different initiating lesions converging on 10 subtype-defining genes. There was a remarkable abundance of TAL1 noncoding lesions, associated with a dismal outcome (5-year OS, 12.5%). Combining TAL1 lesions with mutations in the MYC and RAS pathways produces a genetic stratifier that identifies patients highly likely to fail conventional therapy (5-year OS, 23.1% v 86.4%; HR, 6.84; 95% CI, 2.78 to 16.78; P < .0001) and who should therefore be considered for experimental agents. CONCLUSION The outcome of IF in T-ALL remains poor with current therapy. The lack of a unifying genetic driver suggests alternative approaches, particularly using immunotherapy, are urgently needed.

Genetic investigations of Upper Palaeolithic Europe have revealed a complex and transformative history of human population movements and ancestries, with evidence of several instances of genetic change across the European continent in the period following the Last Glacial Maximum (LGM). Concurrent with these genetic shifts, the post-LGM period is characterized by a series of significant climatic changes, population expansions and cultural diversification. Britain lies at the extreme northwest corner of post-LGM expansion and its earliest Late Glacial human occupation remains unclear. Here we present genetic data from Palaeolithic human individuals in the United Kingdom and the oldest human DNA thus far obtained from Britain or Ireland. We determine that a Late Upper Palaeolithic individual from Gough's Cave probably traced all its ancestry to Magdalenian-associated individuals closely related to those from sites such as El Mirón Cave, Spain, and Troisième Caverne in Goyet, Belgium. However, an individual from Kendrick's Cave shows no evidence of having ancestry related to the Gough’s Cave individual. Instead, the Kendrick’s Cave individual traces its ancestry to groups who expanded across Europe during the Late Glacial and are represented at sites such as Villabruna, Italy. Furthermore, the individuals differ not only in their genetic ancestry profiles but also in their mortuary practices and their diets and ecologies, as evidenced through stable isotope analyses. This finding mirrors patterns of dual genetic ancestry and admixture previously detected in Iberia but may suggest a more drastic genetic turnover in northwestern Europe than in the southwest.

Extrachromosomal DNA (ecDNA) is a common mode of oncogene amplification but is challenging to analyze. Here, we adapt CRISPR-CATCH, in vitro CRISPR-Cas9 treatment and pulsed field gel electrophoresis of agarose-entrapped genomic DNA, previously developed for bacterial chromosome segments, to isolate megabase-sized human ecDNAs. We demonstrate strong enrichment of ecDNA molecules containing EGFR, FGFR2 and MYC from human cancer cells and NRAS ecDNA from human metastatic melanoma with acquired therapeutic resistance. Targeted enrichment of ecDNA versus chromosomal DNA enabled phasing of genetic variants, identified the presence of an EGFRvIII mutation exclusively on ecDNAs and supported an excision model of ecDNA genesis in a glioblastoma model. CRISPR-CATCH followed by nanopore sequencing enabled single-molecule ecDNA methylation profiling and revealed hypomethylation of the EGFR promoter on ecDNAs. We distinguished heterogeneous ecDNA species within the same sample by size and sequence with base-pair resolution and discovered functionally specialized ecDNAs that amplify select enhancers or oncogene-coding sequences.

Objective To establish optimised diffusion weightings (‘b-values’) for acquisition of whole-body diffusion-weighted MRI (WB-DWI) for estimation of the apparent diffusion coefficient (ADC) in patients with metastatic melanoma (MM). Existing recommendations for WB-DWI have not been optimised for the tumour properties in MM; therefore, evaluation of acquisition parameters is essential before embarking on larger studies. Methods Retrospective clinical data and phantom experiments were used. Clinical data comprised 125 lesions from 14 examinations in 11 patients with multifocal MM, imaged before and/or after treatment with immunotherapy at a single institution. ADC estimates from these data were applied to a model to estimate the optimum b-value. A large non-diffusing phantom was used to assess eddy current–induced geometric distortion. Results Considering all tumour sites from pre- and post-treatment examinations together, metastases exhibited a large range of mean ADC values, [0.67–1.49] × 10−3 mm2/s, and the optimum high b-value (bhigh) for ADC estimation was 1100 (10th–90th percentile: 740–1790) s/mm2. At higher b-values, geometric distortion increased, and longer echo times were required, leading to reduced signal. Conclusions Theoretical optimisation gave an optimum bhigh of 1100 (10th–90th percentile: 740–1790) s/mm2 for ADC estimation in MM, with the large range of optimum b-values reflecting the wide range of ADC values in these tumours. Geometric distortion and minimum echo time increase at higher b-values and are not included in the theoretical optimisation; bhigh in the range 750–1100 s/mm2 should be adopted to maintain acceptable image quality but performance should be evaluated for a specific scanner. Key Points • Theoretical optimisation gave an optimum high b-value of 1100 (10th–90th percentile: 740–1790) s/mm2for ADC estimation in metastatic melanoma. • Considering geometric distortion and minimum echo time (TE), a b-value in the range 750–1100 s/mm2is recommended. • Sites should evaluate the performance of specific scanners to assess the effect of geometric distortion and minimum TE.

OBJECTIVE: Invasive brain-computer interfaces (BCIs) require neurosurgical implantation, which confers a range of risks. Despite this, no studies have assessed the acceptability of invasive BCIs amongst the neurosurgical team. This study aims to establish baseline knowledge of BCIs within the neurosurgical team and identify attitudes towards different applications of invasive BCI. METHOD: A two-stage cross-sectional international survey of the neurosurgical team (neurosurgeons, anaesthetists, and operating room nurses) was conducted. Results from the first, qualitative, survey were used to guide the second stage quantitative survey, which assessed acceptability of invasive BCI applications. 5-part Likert Scales were used to collect quantitative data. Surveys were distributed internationally via social media and collaborators. RESULTS: 108 qualitative responses were collected. Themes included the promise of BCIs positively impacting disease targets, concerns regarding stability, and an overall positive emotional reaction to BCI technology. The quantitative survey generated 538 responses from 32 countries. Baseline knowledge of BCI technology was poor, with 9% claiming to have a 'good' or 'expert' knowledge of BCIs. Acceptability of invasive BCI for rehabilitative purposes was >80%. Invasive BCI for augmentation in healthy populations divided opinion. CONCLUSION: The neurosurgical team's view of the acceptability of BCI was divided across a range of indications. Some applications (for example stroke rehabilitation) were viewed as more appropriate than other applications (such as augmentation for military use). This range in views highlights the need for stakeholder consultation on acceptable use cases along with regulation and guidance to govern initial BCI implantations if patients are to realise the potential benefits.

Mutations of theADAR1gene encoding an RNA deaminase cause severe diseases associated with chronic activation of type I interferon (IFN) responses, including Aicardi–Goutières syndrome and bilateral striatal necrosis1–3. The IFN-inducible p150 isoform of ADAR1 contains a Zα domain that recognizes RNA with an alternative left-handed double-helix structure, termed Z-RNA4,5. HemizygousADAR1mutations in the Zα domain cause type I IFN-mediated pathologies in humans2,3and mice6–8; however, it remains unclear how the interaction of ADAR1 with Z-RNA prevents IFN activation. Here we show that Z-DNA-binding protein 1 (ZBP1), the only other protein in mammals known to harbour Zα domains9, promotes type I IFN activation and fatal pathology in mice with impaired ADAR1 function. ZBP1 deficiency or mutation of its Zα domains reduced the expression of IFN-stimulated genes and largely prevented early postnatal lethality in mice with hemizygous expression of ADAR1 with mutated Zα domain (Adar1mZα/–mice).Adar1mZα/–mice showed upregulation and impaired editing of endogenous retroelement-derived complementary RNA reads, which represent a likely source of Z-RNAs activating ZBP1. Notably, ZBP1 promoted IFN activation and severe pathology inAdar1mZα/–mice in a manner independent of RIPK1, RIPK3, MLKL-mediated necroptosis and caspase-8-dependent apoptosis, suggesting a novel mechanism of action. Thus, ADAR1 prevents endogenous Z-RNA-dependent activation of pathogenic type I IFN responses by ZBP1, suggesting that ZBP1 could contribute to type I interferonopathies caused byADAR1mutations.

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

The chromosomal cohesin complex establishes sister chromatid cohesion during S phase, which forms the basis for faithful segregation of DNA replication products during cell divisions. Cohesion establishment is defective in the absence of either of three non-essential Saccharomyces cerevisiae replication fork components Tof1-Csm3 and Mrc1. Here, we investigate how these conserved factors contribute to cohesion establishment. Tof1-Csm3 and Mrc1 serve known roles during DNA replication, including replication checkpoint signaling, securing replication fork speed, as well as recruiting topoisomerase I and the histone chaperone FACT. By modulating each of these functions independently, we rule out that one of these known replication roles explains the contribution of Tof1-Csm3 and Mrc1 to cohesion establishment. Instead, using purified components, we reveal direct and multipronged protein interactions of Tof1-Csm3 and Mrc1 with the cohesin complex. Our findings open the possibility that a series of physical interactions between replication fork components and cohesin facilitate successful establishment of sister chromatid cohesion during DNA replication.

Recent years have seen exciting progress across human embryo research, including new methods for culturing embryos, transcriptional profiling of embryogenesis and gastrulation, mapping lineage trajectories, and experimenting on stem cell-based embryo models. These advances are beginning to define the dynamical principles of development across stages, tissues and organs, enabling a better understanding of human development before birth in health and disease, and potentially leading to improved treatments for infertility and developmental disorders. However, there are still significant roadblocks en route to this goal. Here, we highlight technical challenges to studying early human development and propose ways and means to overcome some of these constraints.

Cell migration is crucial for cancer dissemination. We find that AMP-activated protein kinase (AMPK) controls cell migration by acting as an adhesion sensing molecular hub. In 3-dimensional matrices, fast-migrating amoeboid cancer cells exert low adhesion/low traction linked to low ATP/AMP, leading to AMPK activation. In turn, AMPK plays a dual role controlling mitochondrial dynamics and cytoskeletal remodelling. High AMPK activity in low adhering migratory cells, induces mitochondrial fission, resulting in lower oxidative phosphorylation and lower mitochondrial ATP. Concurrently, AMPK inactivates Myosin Phosphatase, increasing Myosin II-dependent amoeboid migration. Reducing adhesion or mitochondrial fusion or activating AMPK induces efficient rounded-amoeboid migration. AMPK inhibition suppresses metastatic potential of amoeboid cancer cells in vivo, while a mitochondrial/AMPK-driven switch is observed in regions of human tumours where amoeboid cells are disseminating. We unveil how mitochondrial dynamics control cell migration and suggest that AMPK is a mechano-metabolic sensor linking energetics and the cytoskeleton.

Enhanced expression of the cold-shock protein RNA binding motif 3 (RBM3) is highly neuroprotective both in vitro and in vivo. Whilst upstream signalling pathways leading to RBM3 expression have been described, the precise molecular mechanism of RBM3 cold induction remains elusive. To identify temperature-dependent modulators of RBM3, we performed a genome-wide CRISPR-Cas9 knockout screen using RBM3-reporter human iPSC-derived neurons. We found that RBM3 mRNA and protein levels are robustly regulated by several splicing factors, with heterogeneous nuclear ribonucleoprotein H1 (HNRNPH1) being the strongest positive regulator. Splicing analysis revealed that moderate hypothermia significantly represses the inclusion of a poison exon, which, when retained, targets the mRNA for nonsense-mediated decay. Importantly, we show that HNRNPH1 mediates this cold-dependent exon skipping via its thermosensitive interaction with a G-rich motif within the poison exon. Our study provides novel mechanistic insights into the regulation of RBM3 and provides further targets for neuroprotective therapeutic strategies.

16 páginas, 6 figuras, 2 tablas, Lineage 7 (L7) emerged in the phylogeny of the Mycobacterium tuberculosis complex (MTBC) subsequent to the branching of 'ancient' lineage 1 and prior to the Eurasian dispersal of 'modern' lineages 2, 3 and 4. In contrast to the major MTBC lineages, the current epidemiology suggests that prevalence of L7 is highly confined to the Ethiopian population, or when identified outside of Ethiopia, it has mainly been in patients of Ethiopian origin. To search for microbiological factors that may contribute to its restricted distribution, we compared the genome of L7 to the genomes of globally dispersed MTBC lineages. The frequency of predicted functional mutations in L7 was similar to that documented in other lineages. These include mutations characteristic of modern lineages - such as constitutive expression of nitrate reductase - as well as mutations in the VirS locus that are commonly found in ancient lineages. We also identified and characterized multiple lineage-specific mutations in L7 in biosynthesis pathways of cell wall lipids, including confirmed deficiency of methoxy-mycolic acids due to a stop-gain mutation in the mmaA3 gene that encodes a methoxy-mycolic acid synthase. We show that the abolished biosynthesis of methoxy-mycolates of L7 alters the cell structure and colony morphology on selected growth media and impacts biofilm formation. The loss of these mycolic acid moieties may change the host-pathogen dynamic for L7 isolates, explaining the limited geographical distribution of L7 and contributing to further understanding the spread of MTBC lineages across the globe., This study was supported by the MRC Confidence in Concept Fund and the ISSF Wellcome Trust grants 105603/Z/14/Z (G.L-M) and 204833/Z/16/Z (S.J.W). E.H., A.A. and S.J.W. would like to acknowledge funding from the Association of Physicians of Great Britain and Ireland Links with Developing Countries Scheme, and the Human Hereditary and Health in Africa (H3Africa) ‘TBGEN: an integrated approach to unravelling susceptibility to tuberculosis in Africa’ [H3A/18/003]. H3Africa is managed by the Science for Africa (SFA) Foundation in partnership with the Wellcome Trust, the NIH (National Institutes of Health) and African Society of Human genetics (AfSHG). The views expressed herein are those of the authors and not necessarily those of the SFA Foundation and its partners.

Functional genomic strategies have become fundamental for annotating gene function and regulatory networks. Here, we combined functional genomics with proteomics by quantifying protein abundances in a genome-scale knockout library in Saccharomyces cerevisiae, using data-independent acquisition mass spectrometry. We find that global protein expression is driven by a complex interplay of (1) general biological properties, including translation rate, protein turnover, the formation of protein complexes, growth rate, and genome architecture, followed by (2) functional properties, such as the connectivity of a protein in genetic, metabolic, and physical interaction networks. Moreover, we show that functional proteomics complements current gene annotation strategies through the assessment of proteome profile similarity, protein covariation, and reverse proteome profiling. Thus, our study reveals principles that govern protein expression and provides a genome-spanning resource for functional annotation.

CD4+ T cells are typically considered as ‘helper’ or ‘regulatory’ populations that support and orchestrate the responses of other lymphocytes. However, they can also develop potent granzyme (Gzm)-mediated cytotoxic activity and CD4+ cytotoxic T cells (CTLs) have been amply documented both in humans and in mice, particularly in the context of human chronic infection and cancer. Despite the established description of CD4+ CTLs, as well as of the critical cytotoxic activity they exert against MHC class II-expressing targets, their developmental and memory maintenance requirements remain elusive. This is at least in part owing to the lack of a murine experimental system where CD4+ CTLs are stably induced. Here, we show that viral and bacterial vectors encoding the same epitope induce distinct CD4+ CTL responses in challenged mice, all of which are nevertheless transient in nature and lack recall properties. Consistent with prior reports, CD4+ CTL differentiation is accompanied by loss of TCF-1 expression, a transcription factor considered essential for memory T cell survival. Using genetic ablation of Tcf7, which encodes TCF-1, at the time of CD4+ T cell activation, we further show that, contrary to observations in CD8+ T cells, continued expression of TCF-1 is not required for CD4+ T cell memory survival. Whilst Tcf7-deficient CD4+ T cells persisted normally following retroviral infection, the CD4+ CTL subset still declined, precluding conclusive determination of the requirement for TCF-1 for murine CD4+ CTL survival. Using xenotransplantation of human CD4+ T cells into murine recipients, we demonstrate that human CD4+ CTLs develop and persist in the same experimental conditions where murine CD4+ CTLs fail to persist. These observations uncover a species-specific defect in murine CD4+ CTL persistence with implications for their use as a model system.

Gains and losses of DNA are prevalent in cancer and emerge as a consequence of inter-related processes of replication stress, mitotic errors, spindle multipolarity and breakage–fusion–bridge cycles, among others, which may lead to chromosomal instability and aneuploidy1,2. These copy number alterations contribute to cancer initiation, progression and therapeutic resistance3–5. Here we present a conceptual framework to examine the patterns of copy number alterations in human cancer that is widely applicable to diverse data types, including whole-genome sequencing, whole-exome sequencing, reduced representation bisulfite sequencing, single-cell DNA sequencing and SNP6 microarray data. Deploying this framework to 9,873 cancers representing 33 human cancer types from The Cancer Genome Atlas6 revealed a set of 21 copy number signatures that explain the copy number patterns of 97% of samples. Seventeen copy number signatures were attributed to biological phenomena of whole-genome doubling, aneuploidy, loss of heterozygosity, homologous recombination deficiency, chromothripsis and haploidization. The aetiologies of four copy number signatures remain unexplained. Some cancer types harbour amplicon signatures associated with extrachromosomal DNA, disease-specific survival and proto-oncogene gains such as MDM2. In contrast to base-scale mutational signatures, no copy number signature was associated with many known exogenous cancer risk factors. Our results synthesize the global landscape of copy number alterations in human cancer by revealing a diversity of mutational processes that give rise to these alterations.

How the production of biomass is controlled as cells increase in size and proceed through the cell cycle events is important for understanding the regulation of global cellular growth. This has been studied for decades but has not yielded consistent results, probably due to perturbations induced by the synchronisation methods used in most previous studies. To avoid this problem, we have developed a system to analyse unperturbed exponentially growing populations of fission yeast cells. We generated thousands of fixed single-cell measurements of cell size, cell cycle stage and the levels of global cellular translation and transcription. We show that translation scales with size, and additionally, increases at late S-phase/early G2 and early in mitosis and decreases later in mitosis, suggesting that cell cycle controls are also operative over global cellular translation. Transcription increases with both size and the amount of DNA, suggesting that the level of transcription of a cell may be the result of a dynamic equilibrium between the number of RNA polymerases associating and disassociating from DNA.

During early vertebrate development, signals from a special region of the embryo, the organizer, can redirect the fate of non-neural ectoderm cells to form a complete, patterned nervous system. This is called neural induction and has generally been imagined as a single signalling event, causing a switch of fate. Here, we undertake a comprehensive analysis, in very fine time course, of the events following exposure of competent ectoderm of the chick to the organizer (the tip of the primitive streak, Hensen’s node). Using transcriptomics and epigenomics we generate a gene regulatory network comprising 175 transcriptional regulators and 5614 predicted interactions between them, with fine temporal dynamics from initial exposure to the signals to expression of mature neural plate markers. Using in situ hybridization, single-cell RNA-sequencing, and reporter assays, we show that the gene regulatory hierarchy of responses to a grafted organizer closely resembles the events of normal neural plate development. The study is accompanied by an extensive resource, including information about conservation of the predicted enhancers in other vertebrates.

The Rho GTPase family proteins are key regulators of cytoskeletal dynamics. Deregulated activity of Rho GTPases is associated with cancers and neurodegenerative diseases, and their potential as drug targets has long been recognized. Using an economically effective drug screening workflow in fission yeast and human cells, we have identified a Rho GTPase inhibitor, O1. By a suppressor mutant screen in fission yeast, we find a point mutation in the rho1 gene that confers resistance to O1. Consistent with the idea that O1 is the direct inhibitor of Rho1, O1 reduced the cellular amount of activated, GTP-bound Rho1 in wild-type cells, but not in the O1-resistant mutant cells, in which the evolutionarily conserved Ala62 residue is mutated to Thr. Similarly, O1 inhibits activity of the human orthologue RhoA GTPase in tissue culture cells. Our studies illustrate the power of yeast phenotypic screens in the identification and characterization of drugs relevant to human cells and have identified a novel GTPase inhibitor for fission yeast and human cells.

Segmenting cells within cellular aggregates in 3D is a growing challenge in cell biology, due to improvements in capacity and accuracy of microscopy techniques. Here we describe a pipeline to segment images of cell aggregates in 3D. The pipeline combines neural network segmentations with active meshes. We apply our segmentation method to cultured mouse mammary duct organoids imaged over 24 hours with oblique plane microscopy, a high-throughput light-sheet fluorescence microscopy technique. We show that our method can also be applied to images of mouse embryonic stem cells imaged with a spinning disc microscope. We segment individual cells based on nuclei and cell membrane fluorescent markers, and track cells over time. We describe metrics to quantify the quality of the automated segmentation. Our segmentation pipeline involves a Fiji plugin which implement active meshes deformation and allows a user to create training data, automatically obtain segmentation meshes from original image data or neural network prediction, and manually curate segmentation data to identify and correct mistakes. Our active meshes-based approach facilitates segmentation postprocessing, correction, and integration with neural network prediction.Statement of significanceIn vitro culture of organ-like structures derived from stem cells, so-called organoids, allows to image tissue morphogenetic processes with high temporal and spatial resolution. Three-dimensional segmentation of cell shape in timelapse movies of these developing organoids is however a significant challenge. In this work, we propose an image analysis pipeline for cell aggregates that combines deep learning with active contour segmentations. This combination offers a flexible and efficient way to segment three-dimensional cell images, which we illustrate with by segmenting datasets of growing mammary gland organoids and mouse embryonic stem cells.

Effective secondary response to antigen is a hallmark of immunological memory. However, the extent of memory CD8 T cell response to secondary boost varies at different times after a primary response. Considering the central role of memory CD8 T cells in long-lived protection against viral infections and tumors, a better understanding of the molecular mechanisms underlying the changing responsiveness of these cells to antigenic challenge would be beneficial. We examined here primed CD8 T cell response to boost in a BALB/c mouse model of intramuscular vaccination by priming with HIV-1 gag-encoding Chimpanzee adenovector, and boosting with HIV-1 gag-encoding Modified Vaccinia virus Ankara. We found that boost was more effective at day(d)100 than at d30 post-prime, as evaluated at d45 post-boost by multi-lymphoid organ assessment of gag-specific CD8 T cell frequency, CD62L-expression (as a guide to memory status) and in vivo killing. RNA-sequencing of splenic gag-primed CD8 T cells at d100 revealed a quiescent, but highly responsive signature, that trended toward a central memory (CD62L+) phenotype. Interestingly, gag-specific CD8 T cell frequency selectively diminished in the blood at d100, relative to the spleen, lymph nodes and bone marrow. These results open the possibility to modify prime/boost intervals to achieve an improved memory CD8 T cell secondary response.

P110α is a member of the phosphoinositide 3-kinase (PI3K) enzyme family that functions downstream of RAS. RAS proteins contribute to the activation of p110α by interacting directly with its RAS binding domain (RBD), resulting in the promotion of many cellular functions such as cell growth, proliferation and survival. Previous work from our lab has highlighted the importance of the p110α/RAS interaction in tumour initiation and growth. Here we report the discovery and characterisation of a cyclic peptide inhibitor (cyclo-CRVLIR) that interacts with the p110α-RBD and blocks its interaction with KRAS. cyclo-CRVLIR was discovered by screening a “split-intein cyclisation of peptides and proteins” (SICLOPPS) cyclic peptide library. The primary cyclic peptide hit from the screen initially showed a weak affinity for the p110α-RBD (Kd about 360 µM). However, two rounds of amino acid substitution led to cyclo-CRVLIR, with an improved affinity for p110α-RBD in the low µM (Kd 3 µM). We show that cyclo-CRVLIR binds selectively to the p110α-RBD but not to KRAS or the structurally-related RAF-RBD. Further, using biophysical, biochemical and cellular assays, we show that cyclo-CRVLIR effectively blocks the p110α/KRAS interaction in a dose dependent manner and reduces phospho-AKT levels in several oncogenic KRAS cell lines.

Peer reviewed: True, Organoids have become a prominent model system in pulmonary research. The ability to establish organoid cultures directly from patient tissue has expanded the repertoire of physiologically relevant preclinical model systems. In addition to their derivation from adult lung stem/progenitor cells, lung organoids can be derived from fetal tissue or induced pluripotent stem cells to fill a critical gap in modelling pulmonary development in vitro. Recent years have seen important progress in the characterisation and refinement of organoid culture systems. Here, we address several open questions in the field, including how closely organoids recapitulate the tissue of origin, how well organoids recapitulate patient cohorts, and how well organoids capture diversity within a patient. We advocate deeper characterisation of models using single cell technologies, generation of more diverse organoid biobanks and further standardisation of culture media.

BACKGROUND: The crosstalk between cancer and the tumour immune microenvironment (TIME) has attracted significant interest in the latest years because of its impact on cancer evolution and response to treatment. Despite this, cancer-specific tumour-TIME interactions and their mechanistic insights are still poorly understood. METHODS: Here, we compute the significant interactions occurring between cancer-specific genetic drivers and five anti- and pro-tumour TIME features in 32 cancer types using Lasso regularised ordinal regression. Focusing on head and neck squamous cancer (HNSC), we rebuild the functional networks linking specific TIME driver alterations to the TIME state they associate with. RESULTS: The 477 TIME drivers that we identify are multifunctional genes whose alterations are selected early in cancer evolution and recur across and within cancer types. Tumour suppressors and oncogenes have an opposite effect on the TIME and the overall anti-tumour TIME driver burden is predictive of response to immunotherapy. TIME driver alterations predict the immune profiles of HNSC molecular subtypes, and perturbations in keratinization, apoptosis and interferon signalling underpin specific driver-TIME interactions. CONCLUSIONS: Overall, our study delivers a comprehensive resource of TIME drivers, gives mechanistic insights into their immune-regulatory role, and provides an additional framework for patient prioritisation to immunotherapy. The full list of TIME drivers and associated properties are available at http://www.network-cancer-genes.org .

Patient-derived xenograft (PDX) models involve the engraftment of tumour tissue in immunocompromised mice and represent an important pre-clinixtcal oncology research. A limitation of non-small cell lung cancer (NSCLC) PDX model derivation in NOD-scidIL2Rgammanull(NSG) mice is that a subset of initial engraftments are of lymphocytic, rather than tumour origin. In the lung TRACERx PDX pipeline, lymphoproliferations occurred in 17.8% of lung adenocarcinoma and 10% of lung squamous cell carcinoma transplantations, despite none of these patients having a prior or subsequent clinical history of lymphoproliferative disease. Lymphoproliferations were predominantly human CD20+ B cells and had the immunophenotype expected for post-transplantation diffuse large B cell lymphoma. All lymphoproliferations expressed Epstein-Barr-encoded RNAs (EBER). Analysis of immunoglobulin light chain gene rearrangements in three tumours where multiple tumour regions had resulted in lymphoproliferations suggested that each had independent clonal origins. Overall, these data suggest the presence of B cell clones with lymphoproliferative potential within primary NSCLC tumours that are under continuous immune surveillance. Since these cells can be expanded following transplantation into NSG mice, our data highlight the value of quality control measures to identify lymphoproliferations within xenograft pipelines and support the incorporation of strategies to minimise lymphoproliferations during the early stages of xenograft establishment pipelines. To present the histology data herein, we developed a Python-based tool for generating patient-level pathology overview figures from whole-slide image files; PATHOverview is available on GitHub (https://github.com/EpiCENTR-Lab/PATHOverview).

During embryonic development, mutually antagonistic signaling cascades determine gonadal fate toward a testicular or ovarian identity. Errors in this process result in disorders of sex development (DSDs), characterized by discordance between chromosomal, gonadal, and anatomical sex. The absence of an appropriate, accessible in vitro system is a major obstacle in understanding mechanisms of sex-determination/DSDs. Here, we describe protocols for differentiation of mouse and human pluripotent cells toward gonadal progenitors. Transcriptomic analysis reveals that the in vitro–derived murine gonadal cells are equivalent to embryonic day 11.5 in vivo progenitors. Using similar conditions, Sertoli-like cells derived from 46,XY human induced pluripotent stem cells (hiPSCs) exhibit sustained expression of testis-specific genes, secrete anti-Müllerian hormone, migrate, and form tubular structures. Cells derived from 46,XY DSD female hiPSCs, carrying an NR5A1 variant, show aberrant gene expression and absence of tubule formation. CRISPR-Cas9–mediated variant correction rescued the phenotype. This is a robust tool to understand mechanisms of sex determination and model DSDs.

Cloud labs, where experiments are executed remotely in robotic facilities, can improve the reproducibility, accessibility, and scalability of experimental biology. Funding and training programs will enable academics to overcome barriers to adopting such technology.

Monosynaptically restricted rabies viruses have been used for more than a decade for synaptic connectivity tracing. However, the verisimilitude of quantitative conclusions drawn from these experiments is largely unknown. The primary reason is the simple metrics commonly used, which generally disregard the effect of starter cell numbers. Here we present an experimental dataset with a broad range of starter cell numbers and explore their relationship with the number of input cells across the brain using descriptive statistics and modelling. We show that starter cell numbers strongly affect input fraction and convergence index measures, making quantitative comparisons unreliable. Furthermore, we suggest a principled way to analyse rabies derived connectivity data by taking advantage of the starter vs input cell relationship that we describe and validate across independent datasets.

The Hippo pathway was originally discovered to control tissue growth in Drosophila and includes the Hippo kinase (Hpo; MST1/2 in mammals), scaffold protein Salvador (Sav; SAV1 in mammals) and the Warts kinase (Wts; LATS1/2 in mammals). The Hpo kinase is activated by binding to Crumbs-Expanded (Crb-Ex) and/or Merlin-Kibra (Mer-Kib) proteins at the apical domain of epithelial cells. Here we show that activation of Hpo also involves the formation of supramolecular complexes with properties of a biomolecular condensate, including concentration dependence and sensitivity to starvation, macromolecular crowding, or 1,6-hexanediol treatment. Overexpressing Ex or Kib induces formation of micron-scale Hpo condensates in the cytoplasm, rather than at the apical membrane. Several Hippo pathway components contain unstructured low-complexity domains and purified Hpo-Sav complexes undergo phase separation in vitro. Formation of Hpo condensates is conserved in human cells. We propose that apical Hpo kinase activation occurs in phase separated "signalosomes" induced by clustering of upstream pathway components.

An intronic GGGGCC repeat expansion in C9orf72 is a common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. The repeats are transcribed in both sense and antisense directions to generate distinct dipeptide repeat proteins, of which poly(GA), poly(GR), and poly(PR) have been implicated in contributing to neurodegeneration. Poly(PR) binding to RNA may contribute to toxicity, but analysis of poly(PR)-RNA binding on a transcriptome-wide scale has not yet been carried out. We therefore performed crosslinking and immunoprecipitation (CLIP) analysis in human cells to identify the RNA binding sites of poly(PR). We found that poly(PR) binds to nearly 600 RNAs, with the sequence GAAGA enriched at the binding sites. In vitro experiments showed that poly(GAAGA) RNA binds poly(PR) with higher affinity than control RNA and induces the phase separation of poly(PR) into condensates. These data indicate that poly(PR) preferentially binds to poly(GAAGA)-containing RNAs, which may have physiological consequences.

Poster presented as part of the Crick BioImage Analysis Symposium. Atomic force microscopy (AFM) is unique in its ability to image single molecules in liquid with sub-molecular resolution, without the need for labelling or averaging. Enabling us to probe biomolecular structures in native-like states. However, the lack of automated analysis tools in AFM, and slow integration of machine learning (ML) pipelines limits the analysis of the powerful data produced. One limiting factor for the design and integration of these tools is AFM specific dataissues, including; varied image contents and small datasets (compared to e.g. Cryo EM). Raw AFM images undergomany cleaning steps where poorly generalisable threshold methods make identifying desired molecules of relative interest (MoRIs) difficult within a dataset. Initial trials using weakly-supervised random forests, and a recursive DBSCAN algorithm, can identify multiple MoRIs in one pass with higher accuracy and less user oversight than the gold-standard[1,2] software. Once MoIs are identified, we encounter a second problem - the inherent heterogeneity of many biomolecular structures. In molecules such as DNA, heterogeneity is driven by its inherent flexibility[3], but how can one distinguish between one conformation and another? Here, we guide ML instance segmentation labelling via surveys to empirically distinguishDNA minicircle conformations. Neural networks should then localise, classify and segment large AFM datasets to help unravel the role of structure in DNA interactions. Permission has been given by authors to upload to Crick Figshare. Copyright remains with the original holders.

Correlative multimodal imaging is a useful approach to investigate complex structural relations in life sciences across multiple scales. For these experiments, sample preparation workflows that are compatible with multiple imaging techniques must be established. In one such implementation, a fluorescently-labelled region of interest in a biological soft tissue sample can be imaged with light microscopy before staining the specimen with heavy metals, enabling follow-up higher resolution structural imaging at the targeted location, bringing context where it is required. Alternatively, or in addition to fluorescence imaging, other microscopy methods such as synchrotron X-ray computed tomography with propagation-based phase contrast (SXRT) or serial blockface scanning electron microscopy (SBF-SEM) might also be applied. When combining imaging techniques across scales, it is common that a volumetric region of interest (ROI) needs to be carved from the total sample volume before high resolution imaging with a subsequent technique can be performed. In these situations, the overall success of the correlative workflow depends on the precise targeting of the ROI and the trimming of the sample down to a suitable dimension and geometry for downstream imaging.Here we showcase the utility of a novel femtosecond laser device to prepare microscopic samples (1) of an optimised geometry for synchrotron X-ray microscopy as well as (2) for subsequent volume electron microscopy applications, embedded in a wider correlative multimodal imaging workflow (Fig. 1).

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Genetically identical cells are known to differ in many physiological parameters such as growth rate and drug tolerance. Metabolic specialization is believed to be a cause of such phenotypic heterogeneity, but detection of metabolically divergent subpopulations remains technically challenging. We developed a proteomics-based technology, termed differential isotope labelling by amino acids (DILAC), that can detect producer and consumer subpopulations of a particular amino acid within an isogenic cell population by monitoring peptides with multiple occurrences of the amino acid. We reveal that young, morphologically undifferentiated yeast colonies contain subpopulations of lysine producers and consumers that emerge due to nutrient gradients. Deconvoluting their proteomes using DILAC, we find evidence for in situ cross-feeding where rapidly growing cells ferment and provide the more slowly growing, respiring cells with ethanol. Finally, by combining DILAC with fluorescence-activated cell sorting, we show that the metabolic subpopulations diverge phenotypically, as exemplified by a different tolerance to the antifungal drug amphotericin B. Overall, DILAC captures previously unnoticed metabolic heterogeneity and provides experimental evidence for the role of metabolic specialization and cross-feeding interactions as a source of phenotypic heterogeneity in isogenic cell populations.

Coenzyme A (CoA) is an important cellular metabolite that is critical for metabolic processes and the regulation of gene expression. Recent discovery of the antioxidant function of CoA has highlighted its protective role that leads to the formation of a mixed disulfide bond with protein cysteines, which is termed protein CoAlation. To date, more than 2000 CoAlated bacterial and mammalian proteins have been identified in cellular responses to oxidative stress, with the majority being involved in metabolic pathways (60%). Studies have shown that protein CoAlation is a widespread post-translational modification which modulates the activity and conformation of the modified proteins. The induction of protein CoAlation by oxidative stress was found to be rapidly reversed after the removal of oxidizing agents from the medium of cultured cells. In this study, we developed an enzyme-linked immunosorbent assay (ELISA)-based deCoAlation assay to detect deCoAlation activity from Bacillus subtilis and Bacillus megaterium lysates. We then used a combination of ELISA-based assay and purification strategies to show that deCoAlation is an enzyme-driven mechanism. Using mass-spectrometry and deCoAlation assays, we identified B. subtilis YtpP (thioredoxin-like protein) and thioredoxin A (TrxA) as enzymes that can remove CoA from different substrates. With mutagenesis studies, we identified YtpP and TrxA catalytic cysteine residues and proposed a possible deCoAlation mechanism for CoAlated methionine sulfoxide reducatse A (MsrA) and peroxiredoxin 5 (PRDX5) proteins, which results in the release of both CoA and the reduced form of MsrA or PRDX5. Overall, this paper reveals the deCoAlation activity of YtpP and TrxA and opens doors to future studies on the CoA-mediated redox regulation of CoAlated proteins under various cellular stress conditions.

Poster presented as part of the Crick BioImage Analysis Symposium. Fluorescence microscopy is a well-developed imaging modality that is used in life sciences to study tissues, organoids, and cells. The inspection on the microscopic scale is limited by the resolving power of the used optical system, but more specialized techniques for Super-Resolution Microscopy (SRM) are able to render even nanoscopic details of biological samples. However, the accessibility to SRM has been historically hindered by the availability of high-tech, usually expensive, microscopy platforms. Unfortunately, scientific advances of nanoscopy in countries of Africa, Latin America, and Eastern Europe have historically lagged behind developed nations, due to the lack of geographically well-distributed infrastructures. Fortunately, the development of methods for Fluorescence Fluctuation-based Super-Resolution Microscopy (FF-SRM) has facilitated the access to nanoscopic information by assessing the statistical properties of fluorescence intermittency, hence, by analyzing a temporal stack of diffraction-limited images it is possible to study biological systems at the nanoscale. FF-SRM methods are applicable to data sets acquired with any fluorescence microscopy setup (reviewed in [1]), but have not been implemented yet in an integrated GUI: they are instead available as a multitude of scripts/software, based on Matlab [2,7], Python [3,7], and FIJI/ImageJ [2,4,5,6,7]. This project aims to unify the FF-SRM within the open-source “napari-superres” plugin, including at least: SOFI [3], 3B [5], ESI [4], MUSICAL [2], SRRF [6], and MSSR [7]. We believe that such effort will enrich the napari ecosystem, enabling the benchmarking of different approaches to SRM within the same software. Moreover, a suite of FF-SRM methods will foster the development of science, removing some economical inequalities, and granting global access to nanoscopic imaging, especially across countries lacking specialized hardware for SRM imaging. References 1. Alva, Alma, Eduardo Brito-Alarcón, Alejandro Linares, Esley Torres-García, Haydee O. Hernández, Raúl Pinto-Cámara, Damián Martínez, et al. 2022. “Fluorescence Fluctuation-Based Super-Resolution Microscopy: Basic Concepts for an Easy Start.” Journal of Microscopy, August. https://doi.org/10.1111/jmi.13135. 2. Agarwal, Krishna, and Radek Macháň. 2016. “Multiple Signal Classification Algorithm for Super-Resolution Fluorescence Microscopy.” Nature Communications 7 (1): 13752. https://doi.org/10.1038/ncomms13752. 3. Miao, Yuting, Shimon Weiss, and Xiyu Yi. 2022. “PySOFI: An Open Source Python Package for SOFI.” Biophysical Reports 2 (2): 100052. https://doi.org/10.1016/j.bpr.2022.100052. 4. Yahiatene, Idir, Simon Hennig, Marcel Müller, and Thomas Huser. 2015. “Entropy-Based Super-Resolution Imaging (ESI): From Disorder to Fine Detail.” ACS Photonics 2 (8): 1049–56. https://doi.org/10.1021/acsphotonics.5b00307. 5. Cox, Susan, Edward Rosten, James Monypenny, Tijana Jovanovic-Talisman, Dylan T. Burnette, Jennifer Lippincott-Schwartz, Gareth E. Jones, and Rainer Heintzmann. 2012. “Bayesian Localization Microscopy Reveals Nanoscale Podosome Dynamics.” Nature Methods 9 (2): 195–200. https://doi.org/10.1038/nmeth.1812. 6. Gustafsson, Nils, Siân Culley, George Ashdown, Dylan M. Owen, Pedro Matos Pereira, and Ricardo Henriques. 2016. "Fast live-cell conventional fluorophore nanoscopy with ImageJ through super-resolution radial fluctuations." Nature communications 7 (1): 1-9. https://doi.org/10.1038/ncomms12471 7. García, Esley Torres, Raúl Pinto Cámara, Alejandro Linares, Damián Martínez, Víctor Abonza, Eduardo Brito-Alarcón, Carlos Calcines-Cruz, et al. 2021. “Nanoscopic Resolution within a Single Imaging Frame.” BioRxiv, October, 2021.10.17.464398. https://doi.org/10.1101/2021.10.17.464398. Presenting author: Rocco D’Antuono

Transcription of protein-coding genes is regulated by dynamic association of co-factors with RNA polymerase II (RNAPII). The function of these factors and their relationship with RNAPII is often poorly understood. Here, we present an approach for elongation-factor-specific mNET capture (ELCAP) of RNAPII complexes for sequencing and mass spectrometry analysis aimed at investigating the function of such RNAPII regulatory proteins. As proof of principle, we apply ELCAP to the RNAPII-associated proteins SCAF4 and SCAF8, which share an essential role as mRNA anti-terminators but have individual roles at the 3′ end of genes. Mass spectrometry analysis shows that both SCAF4 and SCAF8 are part of RNAPII elongation complexes containing 3′ end processing factors but depleted of splicing components. Importantly, the ELCAP sequencing (ELCAP-seq) profiles of SCAF4- and SCAF8-RNAPII complexes nicely reflect their function as mRNA-anti-terminators and their competing functions at the end of genes, where they prevent or promote transcriptional readthrough.

Brain metastases (BM) are the most common brain neoplasm in adults. Current BM therapies still offer limited efficacy and reduced survival outcomes, emphasizing the need for a better understanding of the disease. Herein, we analyzed the transcriptional profile of brain metastasis initiating cells (BMICs) at two distinct stages of the brain metastatic cascade—the “premetastatic” or early stage when they first colonize the brain and the established macrometastatic stage. RNA sequencing was used to obtain the transcriptional profiles of premetastatic and macrometastatic (non-premetastatic) lung, breast, and melanoma BMICs. We identified that lung, breast, and melanoma premetastatic BMICs share a common transcriptomic signature that is distinct from their non-premetastatic counterparts. Importantly, we show that premetastatic BMICs exhibit increased expression of HLA-G, which we further demonstrate functions in an HLA-G/SPAG9/STAT3 axis to promote the establishment of brain metastatic lesions. Our findings suggest that unraveling the molecular landscape of premetastatic BMICs allows for the identification of clinically relevant targets that can possibly inform the development of preventive and/or more efficacious BM therapies.

Migration and adhesion play critical roles in B cells, regulating recirculation between lymphoid organs, migration within lymphoid tissue, and interaction with CD4+ T cells. However, there is limited knowledge of how B cells integrate chemokine receptor and integrin signaling with B cell activation to generate efficient humoral responses. Here, we show that the WNK1 kinase, a regulator of migration and adhesion, is essential in B cells for T-dependent and -independent antibody responses. We demonstrate that WNK1 transduces signals from the BCR, CXCR5, and CD40, and using intravital imaging, we show that WNK1 regulates migration of naive and activated B cells, and their interactions with T cells. Unexpectedly, we show that WNK1 is required for BCR- and CD40-induced proliferation, acting through the OXSR1 and STK39 kinases, and for efficient B cell-T cell collaboration in vivo. Thus, WNK1 is critical for humoral immune responses, by regulating B cell migration, adhesion, and T cell-dependent activation.

BACKGROUND: Most studies of immunity to SARS-CoV-2 focus on circulating antibody, giving limited insights into mucosal defences that prevent viral replication and onward transmission. We studied nasal and plasma antibody responses one year after hospitalisation for COVID-19, including a period when SARS-CoV-2 vaccination was introduced. METHODS: In this follow up study, plasma and nasosorption samples were prospectively collected from 446 adults hospitalised for COVID-19 between February 2020 and March 2021 via the ISARIC4C and PHOSP-COVID consortia. IgA and IgG responses to NP and S of ancestral SARS-CoV-2, Delta and Omicron (BA.1) variants were measured by electrochemiluminescence and compared with plasma neutralisation data. FINDINGS: Strong and consistent nasal anti-NP and anti-S IgA responses were demonstrated, which remained elevated for nine months (p < 0.0001). Nasal and plasma anti-S IgG remained elevated for at least 12 months (p < 0.0001) with plasma neutralising titres that were raised against all variants compared to controls (p < 0.0001). Of 323 with complete data, 307 were vaccinated between 6 and 12 months; coinciding with rises in nasal and plasma IgA and IgG anti-S titres for all SARS-CoV-2 variants, although the change in nasal IgA was minimal (1.46-fold change after 10 months, p = 0.011) and the median remained below the positive threshold determined by pre-pandemic controls. Samples 12 months after admission showed no association between nasal IgA and plasma IgG anti-S responses (R = 0.05, p = 0.18), indicating that nasal IgA responses are distinct from those in plasma and minimally boosted by vaccination. INTERPRETATION: The decline in nasal IgA responses 9 months after infection and minimal impact of subsequent vaccination may explain the lack of long-lasting nasal defence against reinfection and the limited effects of vaccination on transmission. These findings highlight the need to develop vaccines that enhance nasal immunity. FUNDING: This study has been supported by ISARIC4C and PHOSP-COVID consortia. ISARIC4C is supported by grants from the National Institute for Health and Care Research and the Medical Research Council. Liverpool Experimental Cancer Medicine Centre provided infrastructure support for this research. The PHOSP-COVD study is jointly funded by UK Research and Innovation and National Institute of Health and Care Research. The funders were not involved in the study design, interpretation of data or the writing of this manuscript.

Studying the development of neural circuits in rodent models requires surgical access to the neonatal brain. Since commercially available stereotaxic and anesthetic equipment is designed for use in adults, reliable targeting of brain structures in such young animals can be challenging. Hypothermic cooling (cryoanesthesia) has been used as a preferred anesthesia approach in neonates. This commonly involves submerging neonates in ice, an approach that is poorly controllable. We have developed an affordable, simple to construct device - CryoPup - that allows for fast and robust cryoanesthesia of rodent pups. CryoPup consists of a microcontroller controlling a Peltier element and a heat exchanger. It is capable of both cooling and heating, thereby also functioning as a heating pad during recovery. Importantly, it has been designed for size compatibility with common stereotaxic frames. We validate CryoPup in neonatal mice, demonstrating that it allows for rapid, reliable and safe cryoanesthesia and subsequent recovery. This open-source device will facilitate future studies into the development of neural circuits in the postnatal brain.

Pioneer transcription factors are thought to play pivotal roles in developmental processes by binding nucleosomal DNA to activate gene expression, though mechanisms through which pioneer transcription factors remodel chromatin remain unclear. Here, using single-cell transcriptomics, we show that endogenous expression of neurogenic transcription factor ASCL1, considered a classical pioneer factor, defines a transient population of progenitors in human neural differentiation. Testing ASCL1's pioneer function using a knockout model to define the unbound state, we found that endogenous expression of ASCL1 drives progenitor differentiation bycis-regulation both as a classical pioneer factor and as a nonpioneer remodeler, where ASCL1 binds permissive chromatin to induce chromatin conformation changes. ASCL1 interacts with BAF SWI/SNF chromatin remodeling complexes, primarily at targets where it acts as a nonpioneer factor, and we provide evidence for codependent DNA binding and remodeling at a subset of ASCL1 and SWI/SNF cotargets. Our findings provide new insights into ASCL1 function regulating activation of long-range regulatory elements in human neurogenesis and uncover a novel mechanism of its chromatin remodeling function codependent on partner ATPase activity.

Modular SCF (SKP1-CUL1-Fbox) ubiquitin E3 ligases orchestrate multiple cellular pathways in eukaryotes. Their variable SKP1-Fbox substrate receptor (SR) modules enable regulated substrate recruitment and subsequent proteasomal degradation. CAND proteins are essential for the efficient and timely exchange of SRs. To gain structural understanding of the underlying molecular mechanism, we reconstituted a human CAND1-driven exchange reaction of substrate-bound SCF alongside its co-E3 ligase DCNL1 and visualized it by cryo-EM. We describe high-resolution structural intermediates, including a ternary CAND1-SCF complex, as well as conformational and compositional intermediates representing SR- or CAND1-dissociation. We describe in molecular detail how CAND1-induced conformational changes in CUL1/RBX1 provide an optimized DCNL1-binding site and reveal an unexpected dual role for DCNL1 in CAND1-SCF dynamics. Moreover, a partially dissociated CAND1-SCF conformation accommodates cullin neddylation, leading to CAND1 displacement. Our structural findings, together with functional biochemical assays, help formulate a detailed model for CAND-SCF regulation.

Epithelial tissues provide front-line barriers shielding the organism from invading pathogens and harmful substances. In the airway epithelium, the combined action of multiciliated and secretory cells sustains the mucociliary escalator required for clearance of microbes and particles from the airways. Defects in components of mucociliary clearance or barrier integrity are associated with recurring infections and chronic inflammation. The timely and balanced differentiation of basal cells into mature epithelial cell subsets is therefore tightly controlled. While different growth factors regulating progenitor cell proliferation have been described, little is known about the role of metabolism in these regenerative processes. Here we show that basal cell differentiation correlates with a shift in cellular metabolism from glycolysis to fatty acid oxidation (FAO). We demonstrate both in vitro and in vivo that pharmacological and genetic impairment of FAO blocks the development of fully differentiated airway epithelial cells, compromising the repair of airway epithelia. Mechanistically, FAO links to the hexosamine biosynthesis pathway to support protein glycosylation in airway epithelial cells. Our findings unveil the metabolic network underpinning the differentiation of airway epithelia and identify novel targets for intervention to promote lung repair. ispartof: Nat Commun vol:14 issue:1 pages:721- ispartof: location:England status: Published online

Alanine racemase (Alr) is a pyridoxal 5′-phosphate-dependent enzyme that catalyzes the racemization of l-alanine to d-alanine. Alr is one of the two targets of the broad-spectrum antibiotic d-cycloserine (DCS), a structural analogue of d-alanine. Despite being an essential component of regimens used to treat multi- and extensively drug-resistant tuberculosis for almost seven decades, resistance to DCS has not been observed in patients. We previously demonstrated that DCS evades resistance due to an ultralow rate of emergence of mutations. Yet, we identified a single polymorphism (converting Asp322 to Asn) in the alr gene, which arose in 8 out of 11 independent variants identified and that confers resistance. Here, we present the crystal structure of the Alr variant D322N in both the free and DCS-inactivated forms and the characterization of its DCS inactivation mechanism by UV–visible and fluorescence spectroscopy. Comparison of these results with those obtained with wild-type Alr reveals the structural basis of the 240-fold reduced inhibition observed in Alr D322N.

Beyond tertiary lymphoid structures, a significant number of immune-rich areas without germinal center-like structures are observed in non–small cell lung cancer. Here, we integrated transcriptomic data and digital pathology images to study the prognostic implications, spatial locations, and constitution of immune rich areas (immune hotspots) in a cohort of 935 patients with lung cancer from The Cancer Genome Atlas. A high intratumoral immune hotspot score, which measures the proportion of immune hotspots interfacing with tumor islands, was correlated with poor overall survival in lung squamous cell carcinoma but not in lung adenocarcinoma. Lung squamous cell carcinomas with high intratumoral immune hotspot scores were characterized by consistent upregulation of B-cell signatures. Spatial statistical analyses conducted on serial multiplex IHC slides further revealed that only 4.87% of peritumoral immune hotspots and 0.26% of intratumoral immune hotspots were tertiary lymphoid structures. Significantly lower densities of CD20+CXCR5+ and CD79b+ B cells and less diverse immune cell interactions were found in intratumoral immune hotspots compared with peritumoral immune hotspots. Furthermore, there was a negative correlation between the percentages of CD8+ T cells and T regulatory cells in intratumoral but not in peritumoral immune hotspots, with tertiary lymphoid structures excluded. These findings suggest that the intratumoral immune hotspots reflect an immunosuppressive niche compared with peritumoral immune hotspots, independent of the distribution of tertiary lymphoid structures. A balance toward increased intratumoral immune hotspots is indicative of a compromised antitumor immune response and poor outcome in lung squamous cell carcinoma. Significance: Intratumoral immune hotspots beyond tertiary lymphoid structures reflect an immunosuppressive microenvironment, different from peritumoral immune hotspots, warranting further study in the context of immunotherapies.

Background: The risk factors for recovery from COVID-19 dyspnoea are poorly understood. We investigated determinants of recovery from dyspnoea in adults with COVID-19 and compared these to determinants of recovery from non-COVID-19 dyspnoea. Methods: We used data from two prospective cohort studies: PHOSP-COVID (patients hospitalised between March 2020 and April 2021 with COVID-19) and COVIDENCE UK (community cohort studied over the same time period). PHOSP-COVID data were collected during hospitalisation and at 5-month and 1-year follow-up visits. COVIDENCE UK data were obtained through baseline and monthly online questionnaires. Dyspnoea was measured in both cohorts with the Medical Research Council Dyspnoea Scale. We used multivariable logistic regression to identify determinants associated with a reduction in dyspnoea between 5-month and 1-year follow-up. Findings: We included 990 PHOSP-COVID and 3309 COVIDENCE UK participants. We observed higher odds of improvement between 5-month and 1-year follow-up among PHOSP-COVID participants who were younger (odds ratio 1.02 per year, 95% CI 1.01–1.03), male (1.54, 1.16–2.04), neither obese nor severely obese (1.82, 1.06–3.13 and 4.19, 2.14–8.19, respectively), had no pre-existing anxiety or depression (1.56, 1.09–2.22) or cardiovascular disease (1.33, 1.00–1.79), and shorter hospital admission (1.01 per day, 1.00–1.02). Similar associations were found in those recovering from non-COVID-19 dyspnoea, excluding age (and length of hospital admission). Interpretation: Factors associated with dyspnoea recovery at 1-year post-discharge among patients hospitalised with COVID-19 were similar to those among community controls without COVID-19. Funding: PHOSP-COVID is supported by a grant from the MRC-UK Research and Innovation and the Department of Health and Social Care through the National Institute for Health Research (NIHR) rapid response panel to tackle COVID-19. The views expressed in the publication are those of the author(s) and not necessarily those of the National Health Service (NHS), the NIHR or the Department of Health and Social Care. COVIDENCE UK is supported by the UK Research and Innovation, the National Institute for Health Research, and Barts Charity. The views expressed are those of the authors and not necessarily those of the funders.

IntroductionThe SARS-CoV-2 pandemic remains a threat to public health. Soon after its outbreak, it became apparent that children are less severely affected. Indeed, opposing clinical manifestations between children and adults are observed for other infections. The SARS-CoV-2 outbreak provides the unique opportunity to study the underlying mechanisms. This protocol describes the methods of an observational study that aims to characterise age dependent differences in immune responses to primary respiratory infections using SARS-CoV-2 as a model virus and to assess age differences in clinical outcomes including lung function.Methods and analysisThe study aims to recruit at least 120 children and 60 adults that are infected with SARS-CoV-2 and collect specimen for a multiomics analysis, including single cell RNA sequencing of nasal epithelial cells and peripheral blood mononuclear cells, mass cytometry of whole blood samples and nasal cells, mass spectrometry-based serum and plasma proteomics, nasal epithelial cultures with functional in vitro analyses, SARS-CoV-2 antibody testing, sequencing of the viral genome and lung function testing. Data obtained from this multiomics approach are correlated with medical history and clinical data. Recruitment started in October 2020 and is ongoing.Ethics and disseminationThe study was reviewed and approved by the Ethics Committee of Charité – Universitätsmedizin Berlin (EA2/066/20). All collected specimens are stored in the central biobank of Charité – Universitätsmedizin Berlin and are made available to all participating researchers and on request.Trial registration numberDRKS00025715, pre-results publication.