Collateral sensitivity (CS)-based antibiotic treatments, where increased resistance to one antibiotic leads to increased sensitivity to a second antibiotic, may have the potential to limit the... Show moreCollateral sensitivity (CS)-based antibiotic treatments, where increased resistance to one antibiotic leads to increased sensitivity to a second antibiotic, may have the potential to limit the emergence of antimicrobial resistance. However, it remains unclear how to best design CS-based treatment schedules. To address this problem, we use mathematical modelling to study the effects of pathogen- and drug-specific characteristics for different treatment designs on bacterial population dynamics and resistance evolution. We confirm that simultaneous and one-day cycling treatments could supress resistance in the presence of CS. We show that the efficacy of CS-based cycling therapies depends critically on the order of drug administration. Finally, we find that reciprocal CS is not essential to suppress resistance, a result that significantly broadens treatment options given the ubiquity of one-way CS in pathogens. Overall, our analyses identify key design principles of CS-based treatment strategies and provide guidance to develop treatment schedules to suppress resistance. Show less
Dongen, J. van; Gordon, S.D.; McRae, A.F.; Odintsova, V.V.; Mbarek, H.; Breeze, C.E.; ... ; Genetics DNA Methylation Consortiu 2021
The mechanisms underlying how monozygotic (or identical) twins arise are yet to be determined. Here, the authors investigate this in an epigenome-wide association study, showing that monozygotic... Show moreThe mechanisms underlying how monozygotic (or identical) twins arise are yet to be determined. Here, the authors investigate this in an epigenome-wide association study, showing that monozygotic twinning has a characteristic DNA methylation signature in adult somatic tissues.Monozygotic (MZ) twins and higher-order multiples arise when a zygote splits during pre-implantation stages of development. The mechanisms underpinning this event have remained a mystery. Because MZ twinning rarely runs in families, the leading hypothesis is that it occurs at random. Here, we show that MZ twinning is strongly associated with a stable DNA methylation signature in adult somatic tissues. This signature spans regions near telomeres and centromeres, Polycomb-repressed regions and heterochromatin, genes involved in cell-adhesion, WNT signaling, cell fate, and putative human metastable epialleles. Our study also demonstrates a never-anticipated corollary: because identical twins keep a lifelong molecular signature, we can retrospectively diagnose if a person was conceived as monozygotic twin. Show less
Mutations within subunits of the tRNA splicing endonuclease complex (TSEN) are associated with pontocerebellar hypoplasia (PCH). Here the authors show that tRNA intron excision is catalyzed by... Show moreMutations within subunits of the tRNA splicing endonuclease complex (TSEN) are associated with pontocerebellar hypoplasia (PCH). Here the authors show that tRNA intron excision is catalyzed by tetrameric TSEN assembled from inactive heterodimers, and provide evidence that modulation of TSEN stability may contribute to PCH phenotypes.Introns of human transfer RNA precursors (pre-tRNAs) are excised by the tRNA splicing endonuclease TSEN in complex with the RNA kinase CLP1. Mutations in TSEN/CLP1 occur in patients with pontocerebellar hypoplasia (PCH), however, their role in the disease is unclear. Here, we show that intron excision is catalyzed by tetrameric TSEN assembled from inactive heterodimers independently of CLP1. Splice site recognition involves the mature domain and the anticodon-intron base pair of pre-tRNAs. The 2.1-angstrom resolution X-ray crystal structure of a TSEN15-34 heterodimer and differential scanning fluorimetry analyses show that PCH mutations cause thermal destabilization. While endonuclease activity in recombinant mutant TSEN is unaltered, we observe assembly defects and reduced pre-tRNA cleavage activity resulting in an imbalanced pre-tRNA pool in PCH patient-derived fibroblasts. Our work defines the molecular principles of intron excision in humans and provides evidence that modulation of TSEN stability may contribute to PCH phenotypes. Show less
Schmidt, A.F.; Hunt, N.B.; Gordillo-Maranon, M.; Charoen, P.; Drenos, F.; Kivimaki, M.; ... ; Finan, C. 2021
Despite being studied in clinical trials, CETP inhibitors are not yet an approved treatment for coronary heart disease. Here, by analyzing results from clinical trials and drug target mendelian... Show moreDespite being studied in clinical trials, CETP inhibitors are not yet an approved treatment for coronary heart disease. Here, by analyzing results from clinical trials and drug target mendelian randomization studies, the authors demonstrate that previous failure of CETP inhibitors are likely compound and not drug target-related.Development of cholesteryl ester transfer protein (CETP) inhibitors for coronary heart disease (CHD) has yet to deliver licensed medicines. To distinguish compound from drug target failure, we compared evidence from clinical trials and drug target Mendelian randomization of CETP protein concentration, comparing this to Mendelian randomization of proprotein convertase subtilisin/kexin type 9 (PCSK9). We show that previous failures of CETP inhibitors are likely compound related, as illustrated by significant degrees of between-compound heterogeneity in effects on lipids, blood pressure, and clinical outcomes observed in trials. On-target CETP inhibition, assessed through Mendelian randomization, is expected to reduce the risk of CHD, heart failure, diabetes, and chronic kidney disease, while increasing the risk of age-related macular degeneration. In contrast, lower PCSK9 concentration is anticipated to decrease the risk of CHD, heart failure, atrial fibrillation, chronic kidney disease, multiple sclerosis, and stroke, while potentially increasing the risk of Alzheimer's disease and asthma. Due to distinct effects on lipoprotein metabolite profiles, joint inhibition of CETP and PCSK9 may provide added benefit. In conclusion, we provide genetic evidence that CETP is an effective target for CHD prevention but with a potential on-target adverse effect on age-related macular degeneration. Show less
The electrochemical reduction of CO2 to CO is a promising technology for replacing production processes employing fossil fuels. Still, low energy efficiencies hinder the production of CO at... Show moreThe electrochemical reduction of CO2 to CO is a promising technology for replacing production processes employing fossil fuels. Still, low energy efficiencies hinder the production of CO at commercial scale. CO2 electrolysis has mainly been performed in neutral or alkaline media, but recent fundamental work shows that high selectivities for CO can also be achieved in acidic media. Therefore, we investigate the feasibility of CO2 electrolysis at pH 2–4 at indrustrially relevant conditions, using 10 cm2 gold gas diffusion electrodes. Operating at current densities up to 200 mA cm−2, we obtain CO faradaic efficiencies between 80–90% in sulfate electrolyte, with a 30% improvement of the overall process energy efficiency, in comparison with neutral media. Additionally, we find that weakly hydrated cations are crucial for accomplishing high reaction rates and enabling CO2 electrolysis in acidic media. This study represents a step towards the application of acidic electrolyzers for CO2 electroreduction. Show less
Schimmel, J.; Muñoz-Subirana, N.; Kool, H.; Schendel, R. van; Tijsterman, M. 2021
Error-prone repair of DNA double-strand breaks have been implied to cause cancer-associated genome alterations, but the mechanism of their formation remains unclear. Here the authors find that DNA... Show moreError-prone repair of DNA double-strand breaks have been implied to cause cancer-associated genome alterations, but the mechanism of their formation remains unclear. Here the authors find that DNA polymerase alpha primase plays part in tandem duplication formation at CRISPR/Cas9-induced complementary 3 ' ssDNA protrusions.Small tandem duplications of DNA occur frequently in the human genome and are implicated in the aetiology of certain human cancers. Recent studies have suggested that DNA double-strand breaks are causal to this mutational class, but the underlying mechanism remains elusive. Here, we identify a crucial role for DNA polymerase alpha (Pol alpha)-primase in tandem duplication formation at breaks having complementary 3 ' ssDNA protrusions. By including so-called primase deserts in CRISPR/Cas9-induced DNA break configurations, we reveal that fill-in synthesis preferentially starts at the 3 ' tip, and find this activity to be dependent on 53BP1, and the CTC1-STN1-TEN1 (CST) and Shieldin complexes. This axis generates near-blunt ends specifically at DNA breaks with 3 ' overhangs, which are subsequently repaired by non-homologous end-joining. Our study provides a mechanistic explanation for a mutational signature abundantly observed in the genomes of species and cancer cells. Show less
Poly(ADP-ribosyl)ation (PAR) is a versatile and complex posttranslational modification composed of repeating units of ADP-ribose arranged into linear or branched polymers. This scaffold is linked... Show morePoly(ADP-ribosyl)ation (PAR) is a versatile and complex posttranslational modification composed of repeating units of ADP-ribose arranged into linear or branched polymers. This scaffold is linked to the regulation of many of cellular processes including the DNA damage response, alteration of chromatin structure and Wnt signalling. Despite decades of research, the principles and mechanisms underlying all steps of PAR removal remain actively studied. In this work, we synthesise well-defined PAR branch point molecules and demonstrate that PARG, but not ARH3, can resolve this distinct PAR architecture. Structural analysis of ARH3 in complex with dimeric ADP-ribose as well as an ADP-ribosylated peptide reveal the molecular basis for the hydrolysis of linear and terminal ADP-ribose linkages. We find that ARH3-dependent hydrolysis requires both rearrangement of a catalytic glutamate and induction of an unusual, square-pyramidal magnesium coordination geometry. Show less
Romano, A.; Sutter, M.; Liu, J.H.; Yamagishi, T.; Balliet, D. 2021
Cooperation within and across borders is of paramount importance for the provision of public goods. Parochialism – the tendency to cooperate more with ingroup than outgroup members – limits... Show moreCooperation within and across borders is of paramount importance for the provision of public goods. Parochialism – the tendency to cooperate more with ingroup than outgroup members – limits contributions to global public goods. National parochialism (i.e., greater cooperation among members of the same nation) could vary across nations and has been hypothesized to be associated with rule of law, exposure to world religions, relational mobility and pathogen stress. We conduct an experiment in participants from 42 nations (N = 18,411), and observe cooperation in a prisoner’s dilemma with ingroup, outgroup, and unidentified partners. We observe that national parochialism is a ubiquitous phenomenon: it is present to a similar degree across the nations studied here, is independent of cultural distance, and occurs both when decisions are private or public. These findings inform existing theories of parochialism and suggest it may be an obstacle to the provision of global public goods. Show less
Tet3 is the main alpha -ketoglutarate (alpha KG)-dependent dioxygenase in neurons that converts 5-methyl-dC into 5-hydroxymethyl-dC and further on to 5-formyl- and 5-carboxy-dC. Neurons possess... Show moreTet3 is the main alpha -ketoglutarate (alpha KG)-dependent dioxygenase in neurons that converts 5-methyl-dC into 5-hydroxymethyl-dC and further on to 5-formyl- and 5-carboxy-dC. Neurons possess high levels of 5-hydroxymethyl-dC that further increase during neural activity to establish transcriptional plasticity required for learning and memory functions. How alpha KG, which is mainly generated in mitochondria as an intermediate of the tricarboxylic acid cycle, is made available in the nucleus has remained an unresolved question in the connection between metabolism and epigenetics. We show that in neurons the mitochondrial enzyme glutamate dehydrogenase, which converts glutamate into alpha KG in an NAD(+)-dependent manner, is redirected to the nucleus by the alpha KG-consumer protein Tet3, suggesting on-site production of alpha KG. Further, glutamate dehydrogenase has a stimulatory effect on Tet3 demethylation activity in neurons, and neuronal activation increases the levels of alpha KG. Overall, the glutamate dehydrogenase-Tet3 interaction might have a role in epigenetic changes during neural plasticity. alpha -ketoglutarate (alpha KG) is an intermediate in the tricarboxylic acid cycle that is required in the nucleus for genomic DNA demethylation by Tet3. Here, the authors show that the enzyme glutamate dehydrogenase, which converts glutamate to alpha KG, is redirected from the mitochondria to the nucleus. Show less
To identify approaches to target DNA repair vulnerabilities in cancer, we discovered nanomolar potent, selective, low molecular weight (MW), allosteric inhibitors of the polymerase function of DNA... Show moreTo identify approaches to target DNA repair vulnerabilities in cancer, we discovered nanomolar potent, selective, low molecular weight (MW), allosteric inhibitors of the polymerase function of DNA polymerase Pol theta, including ART558. ART558 inhibits the major Pol theta -mediated DNA repair process, Theta-Mediated End Joining, without targeting Non-Homologous End Joining. In addition, ART558 elicits DNA damage and synthetic lethality in BRCA1- or BRCA2-mutant tumour cells and enhances the effects of a PARP inhibitor. Genetic perturbation screening revealed that defects in the 53BP1/Shieldin complex, which cause PARP inhibitor resistance, result in in vitro and in vivo sensitivity to small molecule Pol theta polymerase inhibitors. Mechanistically, ART558 increases biomarkers of single-stranded DNA and synthetic lethality in 53BP1-defective cells whilst the inhibition of DNA nucleases that promote end-resection reversed these effects, implicating these in the synthetic lethal mechanism-of-action. Taken together, these observations describe a drug class that elicits BRCA-gene synthetic lethality and PARP inhibitor synergy, as well as targeting a biomarker-defined mechanism of PARPi-resistance. Pol theta has been recently identified as a therapeutic target in cancer but specific inhibitors are currently unavailable. Here, the authors identify small molecule inhibitors of Pol theta 's polymerase activity which elicit BRCA1/2 synthetic lethality, enhance the effect of PARP inhibitors and target PARP inhibitor resistance caused by 53BP1/Shieldin pathway defects. Show less
Allahyar, A.; Pieterse, M.; Swennenhuis, J.; Los-de Vries, G.T.; Yilmaz, M.; Leguit, R.; ... ; Laat, W. de 2021
Preservation of cancer biopsies by FFPE introduces DNA fragmentation, hindering analysis of rearrangements. Here the authors introduce FFPE Targeted Locus Capture for identification of... Show morePreservation of cancer biopsies by FFPE introduces DNA fragmentation, hindering analysis of rearrangements. Here the authors introduce FFPE Targeted Locus Capture for identification of translocations in preserved samples.In routine diagnostic pathology, cancer biopsies are preserved by formalin-fixed, paraffin-embedding (FFPE) procedures for examination of (intra-) cellular morphology. Such procedures inadvertently induce DNA fragmentation, which compromises sequencing-based analyses of chromosomal rearrangements. Yet, rearrangements drive many types of hematolymphoid malignancies and solid tumors, and their manifestation is instructive for diagnosis, prognosis, and treatment. Here, we present FFPE-targeted locus capture (FFPE-TLC) for targeted sequencing of proximity-ligation products formed in FFPE tissue blocks, and PLIER, a computational framework that allows automated identification and characterization of rearrangements involving selected, clinically relevant, loci. FFPE-TLC, blindly applied to 149 lymphoma and control FFPE samples, identifies the known and previously uncharacterized rearrangement partners. It outperforms fluorescence in situ hybridization (FISH) in sensitivity and specificity, and shows clear advantages over standard capture-NGS methods, finding rearrangements involving repetitive sequences which they typically miss. FFPE-TLC is therefore a powerful clinical diagnostics tool for accurate targeted rearrangement detection in FFPE specimens. Show less
Krishnan-Schmieden M.; Konold P.E.; Kennis J.T.M.; Pandit A. 2021
Plants need to protect themselves from excess light, which causes photo-oxidative damage and lowers the efficiency of photosynthesis. Photosystem II subunit S (PsbS) is a pH sensor protein that... Show morePlants need to protect themselves from excess light, which causes photo-oxidative damage and lowers the efficiency of photosynthesis. Photosystem II subunit S (PsbS) is a pH sensor protein that plays a crucial role in plant photoprotection by detecting thylakoid lumen acidification in excess light conditions via two lumen-faced glutamates. However, how PsbS is activated under low-pH conditions is unknown. To reveal the molecular response of PsbS to low pH, here we perform an NMR, FTIR and 2DIR spectroscopic analysis of Physcomitrella patens PsbS and of the E176Q mutant in which an active glutamate has been replaced. The PsbS response mechanism at low pH involves the concerted action of repositioning of a short amphipathic helix containing E176 facing the lumen and folding of the luminal loop fragment adjacent to E71 to a 310-helix, providing clear evidence of a conformational pH switch. We propose that this concerted mechanism is a shared motif of proteins of the light-harvesting family that may control thylakoid inter-protein interactions driving photoregulatory responses. Show less
Michielsen, L.; Reinders, M.J.T.; Mahfouz, A. 2021
Supervised methods are increasingly used to identify cell populations in single-cell data. Yet, current methods are limited in their ability to learn from multiple datasets simultaneously, are... Show moreSupervised methods are increasingly used to identify cell populations in single-cell data. Yet, current methods are limited in their ability to learn from multiple datasets simultaneously, are hampered by the annotation of datasets at different resolutions, and do not preserve annotations when retrained on new datasets. The latter point is especially important as researchers cannot rely on downstream analysis performed using earlier versions of the dataset. Here, we present scHPL, a hierarchical progressive learning method which allows continuous learning from single-cell data by leveraging the different resolutions of annotations across multiple datasets to learn and continuously update a classification tree. We evaluate the classification and tree learning performance using simulated as well as real datasets and show that scHPL can successfully learn known cellular hierarchies from multiple datasets while preserving the original annotations. scHPL is available at https://github.com/lcmmichielsen/scHPL. Classification methods for scRNA-seq data are limited in their ability to learn from multiple datasets simultaneously. Here the authors present scHPL, a hierarchical progressive learning method that automatically finds relationships between cell populations across multiple datasets and constructs a classification tree. Show less
Determining divergent metabolic requirements of T cells, and the viruses and tumours they fail to combat, could provide new therapeutic checkpoints. Inhibition of acyl-CoA:cholesterol... Show moreDetermining divergent metabolic requirements of T cells, and the viruses and tumours they fail to combat, could provide new therapeutic checkpoints. Inhibition of acyl-CoA:cholesterol acyltransferase (ACAT) has direct anti-carcinogenic activity. Here, we show that ACAT inhibition has antiviral activity against hepatitis B (HBV), as well as boosting protective anti-HBV and anti-hepatocellular carcinoma (HCC) T cells. ACAT inhibition reduces CD8(+) T cell neutral lipid droplets and promotes lipid microdomains, enhancing TCR signalling and TCR-independent bioenergetics. Dysfunctional HBV- and HCC-specific T cells are rescued by ACAT inhibitors directly ex vivo from human liver and tumour tissue respectively, including tissue-resident responses. ACAT inhibition enhances in vitro responsiveness of HBV-specific CD8(+) T cells to PD-1 blockade and increases the functional avidity of TCR-gene-modified T cells. Finally, ACAT regulates HBV particle genesis in vitro, with inhibitors reducing both virions and subviral particles. Thus, ACAT inhibition provides a paradigm of a metabolic checkpoint able to constrain tumours and viruses but rescue exhausted T cells, rendering it an attractive therapeutic target for the functional cure of HBV and HBV-related HCC. Shared metabolic pathways could allow simultaneous manipulation of T cells, viruses and tumours. Here the authors show targeting cholesterol esterification restrains hepatitis B in vitro, whilst bolstering exhausted antigen-specific T cell responses from human liver and hepatocellular carcinoma. Show less
Many viral antigens have been identified in patients with COVID-19 patients, but which of these result in meaningful immune responses is unclear. Here the authors identify a range of SARS-CoV-2 CD8... Show moreMany viral antigens have been identified in patients with COVID-19 patients, but which of these result in meaningful immune responses is unclear. Here the authors identify a range of SARS-CoV-2 CD8(+) T cell responses across patients including a response targeting an epitope of ORF1ab with immunodominant properties.The COVID-19 pandemic caused by SARS-CoV-2 is a continuous challenge worldwide, and there is an urgent need to map the landscape of immunogenic and immunodominant epitopes recognized by CD8(+) T cells. Here, we analyze samples from 31 patients with COVID-19 for CD8(+) T cell recognition of 500 peptide-HLA class I complexes, restricted by 10 common HLA alleles. We identify 18 CD8(+) T cell recognized SARS-CoV-2 epitopes, including an epitope with immunodominant features derived from ORF1ab and restricted by HLA-A*01:01. In-depth characterization of SARS-CoV-2-specific CD8(+) T cell responses of patients with acute critical and severe disease reveals high expression of NKG2A, lack of cytokine production and a gene expression profile inhibiting T cell re-activation and migration while sustaining survival. SARS-CoV-2-specific CD8(+) T cell responses are detectable up to 5 months after recovery from critical and severe disease, and these responses convert from dysfunctional effector to functional memory CD8(+) T cells during convalescence. Show less
Karami, O.; Rahimi, A.; Mak, P.; Horstman, A.; Boutilier, K.; Compier, M.; ... ; Offringa, R. 2021
Plant somatic cells can be reprogrammed into totipotent embryonic cells that are able to form differentiated embryos in a process called somatic embryogenesis (SE), by hormone treatment or through... Show morePlant somatic cells can be reprogrammed into totipotent embryonic cells that are able to form differentiated embryos in a process called somatic embryogenesis (SE), by hormone treatment or through overexpression of certain transcription factor genes, such as BABY BOOM (BBM). Here we show that overexpression of the AT-HOOK MOTIF CONTAINING NUCLEAR LOCALIZED 15 (AHL15) gene induces formation of somatic embryos on Arabidopsis thaliana seedlings in the absence of hormone treatment. During zygotic embryogenesis, AHL15 expression starts early in embryo development, and AH15 and other AHL genes are required for proper embryo patterning and development beyond the globular stage. Moreover, AHL15 and several of its homologs are upregulated and required for SE induction upon hormone treatment, and they are required for efficient BBM-induced SE as downstream targets of BBM. A significant number of plants derived from AHL15 overexpression-induced somatic embryos are polyploid. Polyploidisation occurs by endomitosis specifically during the initiation of SE, and is caused by strong heterochromatin decondensation induced by AHL15 overexpression. Plant somatic embryogenesis (SE) can be triggered by hormone application or overexpression of certain transcription factors such as BBM. Here Karami et al. show that AHL15 is required for induction of downstream BBM targets and promotes heterochromatin decondensation and endomitosis during the induction of SE. Show less
Climate change poses a significant threat to global biodiversity, but freshwater fishes have been largely ignored in climate change assessments. Here, we assess threats of future flow and water... Show moreClimate change poses a significant threat to global biodiversity, but freshwater fishes have been largely ignored in climate change assessments. Here, we assess threats of future flow and water temperature extremes to similar to 11,500 riverine fish species. In a 3.2 degrees C warmer world (no further emission cuts after current governments' pledges for 2030), 36% of the species have over half of their present-day geographic range exposed to climatic extremes beyond current levels. Threats are largest in tropical and sub-arid regions and increases in maximum water temperature are more threatening than changes in flow extremes. In comparison, 9% of the species are projected to have more than half of their present-day geographic range threatened in a 2 degrees C warmer world, which further reduces to 4% of the species if warming is limited to 1.5 degrees C. Our results highlight the need to intensify (inter)national commitments to limit global warming if freshwater biodiversity is to be safeguarded. Show less
Computational flattening algorithms have been successfully applied to X-ray microtomography scans of damaged historical documents, but have so far been limited to scrolls, books, and documents with... Show moreComputational flattening algorithms have been successfully applied to X-ray microtomography scans of damaged historical documents, but have so far been limited to scrolls, books, and documents with one or two folds. The challenge tackled here is to reconstruct the intricate folds, tucks, and slits of unopened letters secured shut with “letterlocking,” a practice—systematized in this paper—which underpinned global communications security for centuries before modern envelopes. We present a fully automatic computational approach for reconstructing and virtually unfolding volumetric scans of a locked letter with complex internal folding, producing legible images of the letter’s contents and crease pattern while preserving letterlocking evidence. We demonstrate our method on four letterpackets from Renaissance Europe, reading the contents of one unopened letter for the first time. Using the results of virtual unfolding, we situate our findings within a novel letterlocking categorization chart based on our study of 250,000 historical letters Show less
