Translocation renal cell carcinoma (tRCC) is a rare, aggressive kidney cancer primarily occurring in children. They are genetically defined by translocations involving MiT/TFE gene family members TFE3 or TFEB. The biology underlying tRCC development remains poorly understood, partly due to the lack of representative experimental models. We utilized human kidney organoids, or tubuloids, to engineer a tRCC model by expressing one of the most common MiT/TFE fusions, SFPQ-TFE3. Fusion-expressing tubuloids adopt a tRCC-like phenotype and gene expression signature in vitro and grow as clear cell RCC upon xenotransplantation in mice. Genome-wide binding analysis suggests that SFPQ-TFE3 reprograms gene expression signatures by widespread, aberrant DNA binding. Combining these analyses with single-cell mRNA readouts reveals a derailed epithelial differentiation trajectory that is at the root of transformation toward tRCC. Our study demonstrates that SFPQ-TFE3 expression is sufficient to transform kidney epithelial cells into tRCC and defines the trajectories underlying malignant transformation.

Introduction:The COVID-19 Disease Map project is a large-scale community effort uniting 277 scientists from 130 Institutions around the globe. We use high-quality, mechanistic content describing SARS-CoV-2-host interactions and develop interoperable bioinformatic pipelines for novel target identification and drug repurposing. Methods:Extensive community work allowed an impressive step forward in building interfaces between Systems Biology tools and platforms. Our framework can link biomolecules from omics data analysis and computational modelling to dysregulated pathways in a cell-, tissue- or patient-specific manner. Drug repurposing using text mining and AI-assisted analysis identified potential drugs, chemicals and microRNAs that could target the identified key factors. Results:Results revealed drugs already tested for anti-COVID-19 efficacy, providing a mechanistic context for their mode of action, and drugs already in clinical trials for treating other diseases, never tested against COVID-19. Discussion:The key advance is that the proposed framework is versatile and expandable, offering a significant upgrade in the arsenal for virus-host interactions and other complex pathologies.

INTRODUCTION:A role for innate immune memory in protection during COVID-19 infection or vaccination has been recently reported. However, no study so far has shown whether the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can train innate immune cells. The aim of this study was to investigate whether this virus can induce trained immunity in human monocytes. METHODS:Monocytes were exposed to inactivated SARS-CoV-2 (iSARS-CoV-2) for 24 h, followed by a resting period in the medium only and a secondary stimulation on day 6 after which the cytokine/chemokine and transcriptomic profiles were determined. RESULTS:Compared to untrained cells, the iSARS-CoV-2-trained monocytes secreted significantly higher levels of IL-6, TNF-α, CXCL10, CXCL9, and CXCL11 upon restimulation. Transcriptome analysis of iSARS-CoV-2-trained monocytes revealed increased expression of several inflammatory genes. As epigenetic and metabolic modifications are hallmarks of trained immunity, we analyzed the expression of genes related to these processes. Findings indicate that indeed SARS-CoV-2-trained monocytes show changes in the expression of genes involved in metabolic pathways including the tricarboxylic acid cycle, amino acid metabolism, and the expression of several epigenetic regulator genes. Using epigenetic inhibitors that block histone methyl and acetyltransferases, we observed that the capacity of monocytes to be trained by iSARS-CoV-2 was abolished. CONCLUSION:Overall, our findings indicate that iSARS-CoV-2 can induce properties associated with trained immunity in human monocytes. These results contribute to the knowledge required for improving vaccination strategies to prevent infectious diseases.

Early and persistent defects in B cell subsets such as memory B cells were shown to be correlated with poor outcomes in COVID-19 patients. This research aimed to develop a molecular pathway model to understand the B cell development in COVID-19. A B cell transcriptomics dataset, obtained from COVID-19 patients, was analyzed on the resulting pathway model to study B cell activation. The pathway showed two distinct gene expression profiles between asymptomatic and symptomatic patients. In asymptomatic patients, there is an increase in transcript levels of antiviral interferon-stimulated genes such as ISG15, IFITM1, and NEAT1 and a driving gene for the extrafollicular pathway CXCR4 indicating a formation of plasmablast. In symptomatic patients, the results suggest an inhibition occurring at the germinal center hinting at a reduction in memory B cell production. Transcripts of driver gene CXCR5 involved in germinal center development is one of the most downregulated genes. This could contribute to the shortage in the formation of memory B cells in COVID-19. Concluding, in SARS-CoV-2 infection, B cells follow different activation routes in asymptomatic and symptomatic patients. In this study, we constructed a pathway that allowed us to analyze and interpret activation patterns of B cells in COVID-19 patients and their link to disease severity. Importantly, the pathway and approach can be reused for further research in COVID-19 or other diseases.

Break-Induced Replication (BIR) is a homologous recombination (HR) pathway that differentiates itself from all other HR pathways by involving extensive DNA synthesis of up to hundreds of kilobases. This DNA synthesis occurs in G2/M arrested cells by a mechanism distinct from regular DNA replication. BIR initiates by strand invasion of a single end of a DNA double-strand break (DSB) followed by extensive D-loop migration. The main replicative helicase Mcm2-7 is dispensable for BIR, however, Pif1 helicase and its PCNA interaction domain are required. Pif1 helicase was shown to be important for extensive repair-specific DNA synthesis at DSB in budding and fission yeasts, flies, and human cells, implicating conservation of the mechanism. Additionally, Mph1 helicase negatively regulates BIR by unwinding migrating D-loops, and Srs2 promotes BIR by eliminating the toxic joint molecules. Here, we describe the methods that address the following questions in studying BIR: (i) how to distinguish enzymes needed specifically for BIR from enzymes needed for other HR mechanisms that require short patch DNA synthesis, (ii) what are the phenotypes expected for mutants deficient in extensive synthesis during BIR, (iii) how to follow extensive DNA synthesis during BIR? Methods are described using yeast model organism and wild-type cells are compared side-by-side with Pif1 deficient cells.

The ACE2 receptors essential for SARS-CoV-2 infections are expressed not only in the lung but also in many other tissues in the human body. To better understand the disease mechanisms and progression, it is essential to understand how the virus affects and alters molecular pathways in the different affected tissues. In this study, we mapped the proteomics data obtained from Nie X. et al. (2021) to the pathway models of the COVID-19 Disease Map project and WikiPathways. The differences in pathway activities between COVID-19 and non-COVID-19 patients were calculated using the Wilcoxon test. As a result, 46% (5,235) of the detected proteins were found to be present in at least one pathway. Only a few pathways were altered in multiple tissues. As an example, the Kinin-Kallikrein pathway, an important inflammation regulatory pathway, was found to be less active in the lung, spleen, testis, and thyroid. We can confirm previously reported changes in COVID-19 patients such as the change in cholesterol, linolenic acid, and arachidonic acid metabolism, complement, and coagulation pathways in most tissues. Of all the tissues, we found the thyroid to be the organ with the most changed pathways. In this tissue, lipid pathways, energy pathways, and many COVID-19 specific pathways such as RAS and bradykinin pathways, thrombosis, and anticoagulation have altered activities in COVID-19 patients. Concluding, our results highlight the systemic nature of COVID-19 and the effect on other tissues besides the lung.

The COVID-19 Disease Map project is a large-scale community effort uniting 277 scientists from 130 Institutions around the globe. We use high-quality, mechanistic content describing SARS-CoV-2-host interactions and develop interoperable bioinformatic pipelines for novel target identification and drug repurposing. Community-driven and highly interdisciplinary, the project is collaborative and supports community standards, open access, and the FAIR data principles. The coordination of community work allowed for an impressive step forward in building interfaces between Systems Biology tools and platforms. Our framework links key molecules highlighted from broad omics data analysis and computational modeling to dysregulated pathways in a cell-, tissue- or patient-specific manner. We also employ text mining and AI-assisted analysis to identify potential drugs and drug targets and use topological analysis to reveal interesting structural features of the map. The proposed framework is versatile and expandable, offering a significant upgrade in the arsenal used to understand virus-host interactions and other complex pathologies.

DNA synthesis during homologous recombination is highly mutagenic and prone to template switches. Two-ended DNA double-strand breaks (DSBs) are usually repaired by gene conversion with a short patch of DNA synthesis, thus limiting the mutation load to the vicinity of the DSB. Single-ended DSBs are repaired by break-induced replication (BIR), which involves extensive and mutagenic DNA synthesis spanning up to hundreds of kilobases. It remains unknown how mutagenic BIR is suppressed at two-ended DSBs. Here, we demonstrate that BIR is suppressed at two-ended DSBs by proteins coordinating the usage of two ends of a DSB: (i) ssDNA annealing proteins Rad52 and Rad59 that promote second end capture, (ii) D-loop unwinding helicase Mph1, and (iii) Mre11-Rad50-Xrs2 complex that promotes synchronous resection of two ends of a DSB. Finally, BIR is also suppressed when Sir2 silences a normally heterochromatic repair template. All of these proteins are particularly important for limiting BIR when recombination occurs between short repetitive sequences, emphasizing the significance of these mechanisms for species carrying many repetitive elements such as humans.

Additional file 6. Biomass component at glucose and unlimited nitrogen condition.

Additional file 4. Condition-specific biomass generation.

BACKGROUND:Cutaneotrichosporon oleaginosus ATCC 20509 is a fast-growing oleaginous basidiomycete yeast that is able to grow in a wide range of low-cost carbon sources including crude glycerol, a byproduct of biodiesel production. When glycerol is used as a carbon source, this yeast can accumulate more than 50% lipids (w/w) with high concentrations of mono-unsaturated fatty acids.RESULTS:To increase our understanding of this yeast and to provide a knowledge base for further industrial use, a FAIR re-annotated genome was used to build a genome-scale, constraint-based metabolic model containing 1553 reactions involving 1373 metabolites in 11 compartments. A new description of the biomass synthesis reaction was introduced to account for massive lipid accumulation in conditions with high carbon-to-nitrogen (C/N) ratio in the media. This condition-specific biomass objective function is shown to better predict conditions with high lipid accumulation using glucose, fructose, sucrose, xylose, and glycerol as sole carbon source.CONCLUSION:Contributing to the economic viability of biodiesel as renewable fuel, C. oleaginosus ATCC 20509 can effectively convert crude glycerol waste streams in lipids as a potential bioenergy source. Performance simulations are essential to identify optimal production conditions and to develop and fine tune a cost-effective production process. Our model suggests ATP-citrate lyase as a possible target to further improve lipid production.

1 Institute of Data Science and Biotechnology, Gladstone Institutes, San Francisco, California, United States of America, 2 Department of Bioinformatics—BiGCaT, NUTRIM, Maastricht University, Maastricht, the Netherlands, 3 Maastricht Centre for Systems Biology (MaCSBio), Maastricht University, Maastricht, the Netherlands, 4 Department of Pharmaceutical Sciences, Division of Pharmaceutical Sciences, University of Vienna, Vienna, Austria, 5 Instituto de Ciencias Biologicas, Departamento de Bioquimica e Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil, 6 Micelio, Antwerp, Belgium

We need to effectively combine the knowledge from surging literature with complex datasets to propose mechanistic models of SARS-CoV-2 infection, improving data interpretation and predicting key targets of intervention. Here, we describe a large-scale community effort to build an open access, interoperable and computable repository of COVID-19 molecular mechanisms. The COVID-19 Disease Map (C19DMap) is a graphical, interactive representation of disease-relevant molecular mechanisms linking many knowledge sources. Notably, it is a computational resource for graph-based analyses and disease modelling. To this end, we established a framework of tools, platforms and guidelines necessary for a multifaceted community of biocurators, domain experts, bioinformaticians and computational biologists. The diagrams of the C19DMap, curated from the literature, are integrated with relevant interaction and text mining databases. We demonstrate the application of network analysis and modelling approaches by concrete examples to highlight new testable hypotheses. This framework helps to find signatures of SARS-CoV-2 predisposition, treatment response or prioritisation of drug candidates. Such an approach may help deal with new waves of COVID-19 or similar pandemics in the long-term perspective.

The Rad51/RecA family of recombinases perform a critical function in typical repair of double-strand breaks (DSBs): strand invasion of a resected DSB end into a homologous double-stranded DNA (dsDNA) template sequence to initiate repair. However, repair of a DSB using single stranded DNA (ssDNA) as a template, a common method of CRISPR/Cas9-mediated gene editing, is Rad51-independent. We have analyzed the genetic requirements for these Rad51-independent events in Saccharomyces cerevisiae by creating a DSB with the site-specific HO endonuclease and repairing the DSB with 80-nt single-stranded oligonucleotides (ssODNs), and confirmed these results by Cas9-mediated DSBs in combination with a bacterial retron system that produces ssDNA templates in vivo. We show that single strand template repair (SSTR), is dependent on Rad52, Rad59, Srs2 and the Mre11-Rad50-Xrs2 (MRX) complex, but unlike other Rad51-independent recombination events, independent of Rdh54. We show that Rad59 acts to alleviate the inhibition of Rad51 on Rad52's strand annealing activity both in SSTR and in single strand annealing (SSA). Gene editing is Rad51-dependent when double-stranded oligonucleotides of the same size and sequence are introduced as templates. The assimilation of mismatches during gene editing is dependent on the activity of Msh2, which acts very differently on the 3' side of the ssODN which can anneal directly to the resected DSB end compared to the 5' end. In addition DNA polymerase Polδ's 3' to 5' proofreading activity frequently excises a mismatch very close to the 3' end of the template. We further report that SSTR is accompanied by as much as a 600-fold increase in mutations in regions adjacent to the sequences directly undergoing repair. These DNA polymerase ζ-dependent mutations may compromise the accuracy of gene editing.

DNA synthesis during homologous recombination (HR) is highly mutagenic and prone to template switches. Two-ended DNA double strand breaks (DSBs) are usually repaired by gene conversion with a short patch of DNA synthesis, thus limiting the mutation load to the vicinity of the DSB. Single-ended DSBs are repaired by Break-Induced Replication (BIR) that involve extensive and mutagenic DNA synthesis spanning even hundreds of kilobases. It remains unknown how mutagenic BIR is suppressed at two-ended DSBs. Here we demonstrate that BIR is suppressed at two-ended DSBs by several proteins coordinating the usage of both DSB ends: ssDNA annealing protein Rad52 and Rad59, D-loop unwinding helicase Mph1, and DSB ends tethering Mre11-Rad50-Xrs2 complex. Finally, BIR is also suppressed when a normally heterochromatic repair template is silenced by Sir2. These findings suggest several mechanisms restricting mutagenic BIR during repair of two-ended DSBs.

Genome-scale metabolic models (GEMs) are manually curated repositories describing the metabolic capabilities of an organism. GEMs have been successfully used in different research areas, ranging from systems medicine to biotechnology. However, the different naming conventions (namespaces) of databases used to build GEMs limit model reusability and prevent the integration of existing models. This problem is known in the GEM community, but its extent has not been analyzed in depth. In this study, we investigate the name ambiguity and the multiplicity of non-systematic identifiers and we highlight the (in)consistency in their use in 11 biochemical databases of biochemical reactions and the problems that arise when mapping between different namespaces and databases. We found that such inconsistencies can be as high as 83.1%, thus emphasizing the need for strategies to deal with these issues. Currently, manual verification of the mappings appears to be the only solution to remove inconsistencies when combining models. Finally, we discuss several possible approaches to facilitate (future) unambiguous mapping.

Rad52 is a key factor for homologous recombination (HR) in yeast. Rad52 helps assemble Rad51-ssDNA nucleoprotein filaments that catalyze DNA strand exchange, and it mediates single-strand DNA annealing. We find that Rad52 has an even earlier function in HR in restricting DNA double-stranded break ends resection that generates 3' single-stranded DNA (ssDNA) tails. In fission yeast, Exo1 is the primary resection nuclease, with the helicase Rqh1 playing a minor role. We demonstrate that the choice of two extensive resection pathways is regulated by Rad52. In rad52 cells, the resection rate increases from ∼3-5 kb/h up to ∼10-20 kb/h in an Rqh1-dependent manner, while Exo1 becomes dispensable. Budding yeast Rad52 similarly inhibits Sgs1-dependent resection. Single-molecule analysis with purified budding yeast proteins shows that Rad52 competes with Sgs1 for DNA end binding and inhibits Sgs1 translocation along DNA. These results identify a role for Rad52 in limiting ssDNA generated by end resection.

Insertions of mobile elements1–4, mitochondrial DNA5 and fragments of nuclear chromosomes6 at DNA double-strand breaks (DSBs) threaten genome integrity and are common in cancer7–9. Insertions of chromosome fragments at V(D)J recombination loci can stimulate antibody diversification10. The origin of insertions of chromosomal fragments and the mechanisms that prevent such insertions remain unknown. Here we reveal a yeast mutant, lacking evolutionarily conserved Dna2 nuclease, that shows frequent insertions of sequences between approximately 0.1 and 1.5 kb in length into DSBs, with many insertions involving multiple joined DNA fragments. Sequencing of around 500 DNA inserts reveals that they originate from Ty retrotransposons (8%), ribosomal DNA (rDNA) (15%) and from throughout the genome, with preference for fragile regions such as origins of replication, R-loops, centromeres, telomeres or replication fork barriers. Inserted fragments are not lost from their original loci and therefore represent duplications. These duplications depend on nonhomologous end-joining (NHEJ) and Pol4. We propose a model in which alternative processing of DNA structures arising in Dna2-deficient cells can result in the release of DNA fragments and their capture at DSBs. Similar DNA insertions at DSBs are expected to occur in any cells with linear extrachromosomal DNA fragments. A deficiency of Dna2 causes duplication of DNA fragments and their insertion into the genome at double-stranded breaks in yeast.