Molecular hallmarks of heterochronic parabiosis at single-cell resolution.

The ability to slow or reverse biological ageing would have major implications for mitigating disease risk and maintaining vitality1. Although an increasing number of interventions show promise for rejuvenation2, their effectiveness on disparate cell types across the body and the molecular pathways susceptible to rejuvenation remain largely unexplored. Here we performed single-cell RNA sequencing on 20 organs to reveal cell-type-specific responses to young and aged blood in heterochronic parabiosis. Adipose mesenchymal stromal cells, haematopoietic stem cells and hepatocytes are among those cell types that are especially responsive. On the pathway level, young blood invokes new gene sets in addition to reversing established ageing patterns, with the global rescue of genes encoding electron transport chain subunits pinpointing a prominent role of mitochondrial function in parabiosis-mediated rejuvenation. We observed an almost universal loss of gene expression with age that is largely mimicked by parabiosis: aged blood reduces global gene expression, and young blood restores it in select cell types. Together, these data lay the groundwork for a systemic understanding of the interplay between blood-borne factors and cellular integrity.

Dysregulation of brain and choroid plexus cell types in severe COVID-19.

Although SARS-CoV-2 primarily targets the respiratory system, patients with and survivors of COVID-19 can suffer neurological symptoms1-3. However, an unbiased understanding of the cellular and molecular processes that are affected in the brains of patients with COVID-19 is missing. Here we profile 65,309 single-nucleus transcriptomes from 30 frontal cortex and choroid plexus samples across 14 control individuals (including 1 patient with terminal influenza) and 8 patients with COVID-19. Although our systematic analysis yields no molecular traces of SARS-CoV-2 in the brain, we observe broad cellular perturbations indicating that barrier cells of the choroid plexus sense and relay peripheral inflammation into the brain and show that peripheral T cells infiltrate the parenchyma. We discover microglia and astrocyte subpopulations associated with COVID-19 that share features with pathological cell states that have previously been reported in human neurodegenerative disease4-6. Synaptic signalling of upper-layer excitatory neurons-which are evolutionarily expanded in humans7 and linked to cognitive function8-is preferentially affected in COVID-19. Across cell types, perturbations associated with COVID-19 overlap with those found in chronic brain disorders and reside in genetic variants associated with cognition, schizophrenia and depression. Our findings and public dataset provide a molecular framework to understand current observations of COVID-19-related neurological disease, and any such disease that may emerge at a later date.

Swarm Learning for decentralized and confidential clinical machine learning.

Fast and reliable detection of patients with severe and heterogeneous illnesses is a major goal of precision medicine1,2. Patients with leukaemia can be identified using machine learning on the basis of their blood transcriptomes3. However, there is an increasing divide between what is technically possible and what is allowed, because of privacy legislation4,5. Here, to facilitate the integration of any medical data from any data owner worldwide without violating privacy laws, we introduce Swarm Learning-a decentralized machine-learning approach that unites edge computing, blockchain-based peer-to-peer networking and coordination while maintaining confidentiality without the need for a central coordinator, thereby going beyond federated learning. To illustrate the feasibility of using Swarm Learning to develop disease classifiers using distributed data, we chose four use cases of heterogeneous diseases (COVID-19, tuberculosis, leukaemia and lung pathologies). With more than 16,400 blood transcriptomes derived from 127 clinical studies with non-uniform distributions of cases and controls and substantial study biases, as well as more than 95,000 chest X-ray images, we show that Swarm Learning classifiers outperform those developed at individual sites. In addition, Swarm Learning completely fulfils local confidentiality regulations by design. We believe that this approach will notably accelerate the introduction of precision medicine.

Ageing hallmarks exhibit organ-specific temporal signatures.

Ageing is the single greatest cause of disease and death worldwide, and understanding the associated processes could vastly improve quality of life. Although major categories of ageing damage have been identified-such as altered intercellular communication, loss of proteostasis and eroded mitochondrial function1-these deleterious processes interact with extraordinary complexity within and between organs, and a comprehensive, whole-organism analysis of ageing dynamics has been lacking. Here we performed bulk RNA sequencing of 17 organs and plasma proteomics at 10 ages across the lifespan of Mus musculus, and integrated these findings with data from the accompanying Tabula Muris Senis2-or 'Mouse Ageing Cell Atlas'-which follows on from the original Tabula Muris3. We reveal linear and nonlinear shifts in gene expression during ageing, with the associated genes clustered in consistent trajectory groups with coherent biological functions-including extracellular matrix regulation, unfolded protein binding, mitochondrial function, and inflammatory and immune response. Notably, these gene sets show similar expression across tissues, differing only in the amplitude and the age of onset of expression. Widespread activation of immune cells is especially pronounced, and is first detectable in white adipose depots during middle age. Single-cell RNA sequencing confirms the accumulation of T cells and B cells in adipose tissue-including plasma cells that express immunoglobulin J-which also accrue concurrently across diverse organs. Finally, we show how gene expression shifts in distinct tissues are highly correlated with corresponding protein levels in plasma, thus potentially contributing to the ageing of the systemic circulation. Together, these data demonstrate a similar yet asynchronous inter- and intra-organ progression of ageing, providing a foundation from which to track systemic sources of declining health at old age.

SingmiR: a single-cell miRNA alignment and analysis tool.

Single-cell RNA sequencing (RNA-seq) has revolutionized our understanding of cell biology, developmental and pathophysiological molecular processes, paving the way toward novel diagnostic and therapeutic approaches. However, most of the gene regulatory processes on the single-cell level are still unknown, including post-transcriptional control conferred by microRNAs (miRNAs). Like the established single-cell gene expression analysis, advanced computational expertise is required to comprehensively process newly emerging single-cell miRNA-seq datasets. A web server providing a workflow tailored for single-cell miRNA-seq data with a self-explanatory interface is currently not available. Here, we present SingmiR, enabling the rapid (pre-)processing and quantification of human miRNAs from noncoding single-cell samples. It performs read trimming for different library preparation protocols, generates automated quality control reports and provides feature-normalized count files. Numerous standard and advanced analyses such as dimension reduction, clustered feature heatmaps, sample correlation heatmaps and differential expression statistics are implemented. We aim to speed up the prototyping pipeline for biologists developing single-cell miRNA-seq protocols on small to medium-sized datasets. SingmiR is freely available to all users without the need for a login at https://www.ccb.uni-saarland.de/singmir.

miEAA 2023: updates, new functional microRNA sets and improved enrichment visualizations.

MicroRNAs (miRNAs) are small non-coding RNAs that play a critical role in regulating diverse biological processes. Extracting functional insights from a list of miRNAs is challenging, as each miRNA can potentially interact with hundreds of genes. To address this challenge, we developed miEAA, a flexible and comprehensive miRNA enrichment analysis tool based on direct and indirect miRNA annotation. The latest release of miEAA includes a data warehouse of 19 miRNA repositories, covering 10 different organisms and 139 399 functional categories. We have added information on the cellular context of miRNAs, isomiRs, and high-confidence miRNAs to improve the accuracy of the results. We have also improved the representation of aggregated results, including interactive Upset plots to aid users in understanding the interaction among enriched terms or categories. Finally, we demonstrate the functionality of miEAA in the context of ageing and highlight the importance of carefully considering the miRNA input list. MiEAA is free to use and publicly available at https://www.ccb.uni-saarland.de/mieaa/.

isomiRdb: microRNA expression at isoform resolution.

A significant fraction of mature miRNA transcripts carries sequence and/or length variations, termed isomiRs. IsomiRs are differentially abundant in cell types, tissues, body fluids or patients' samples. Not surprisingly, multiple studies describe a physiological and pathophysiological role. Despite their importance, systematically collected and annotated isomiR information available in databases remains limited. We thus developed isomiRdb, a comprehensive resource that compiles miRNA expression data at isomiR resolution from various sources. We processed 42 499 human miRNA-seq datasets (5.9 × 1011 sequencing reads) and consistently analyzed them using miRMaster and sRNAbench. Our database provides online access to the 90 483 most abundant isomiRs (>1 RPM in at least 1% of the samples) from 52 tissues and 188 cell types. Additionally, the full set of over 3 million detected isomiRs is available for download. Our resource can be queried at the sample, miRNA or isomiR level so users can quickly answer common questions about the presence/absence of a particular miRNA/isomiR in tissues of interest. Further, the database facilitates to identify whether a potentially interesting new isoform has been detected before and its frequency. In addition to expression tables, isomiRdb can generate multiple interactive visualisations including violin plots and heatmaps. isomiRdb is free to use and publicly available at: https://www.ccb.uni-saarland.de/isomirdb.

Skin treatment with non-thermal plasma modulates the immune system through miR-223-3p and its target genes.

Non-thermal plasma, a partially ionized gas, holds significant potential for clinical applications, including wound-healing support, oral therapies, and anti-tumour treatments. While its applications showed promising outcomes, the underlying molecular mechanisms remain incompletely understood. We thus apply non-thermal plasma to mouse auricular skin and conducted non-coding RNA sequencing, as well as single-cell blood sequencing. In a time-series analysis (five timepoints spanning 2 hours), we compare the expression of microRNAs in the plasma-treated left ears to the unexposed right ears of the same mice as well as to the ears of unexposed control mice. Our findings indicate specific effects in the treated ears for a set of five miRNAs: mmu-miR-144-5p, mmu-miR-144-3p, mmu-miR-142a-5p, mmu-miR-223-3p, and mmu-miR-451a. Interestingly, mmu-miR-223-3p also exhibits an increase over time in the right non-treated ear of the exposed mice, suggesting systemic effects. Notably, this miRNA, along with mmu-miR-142a-5p and mmu-miR-144-3p, regulates genes and pathways associated with wound healing and tissue regeneration (namely ErbB, FoxO, Hippo, and PI3K-Akt signalling). This co-regulation is particularly remarkable considering the significant seed dissimilarities among the miRNAs. Finally, single-cell sequencing of PBMCs reveals the downregulation of 12 from 15 target genes in B-cells, Cd4+ and Cd8+ T-cells. Collectively, our data provide evidence for a systemic effect of non-thermal plasma.

BusyBee Web: towards comprehensive and differential composition-based metagenomic binning.

Despite recent methodology and reference database improvements for taxonomic profiling tools, metagenomic assembly and genomic binning remain important pillars of metagenomic analysis workflows. In case reference information is lacking, genomic binning is considered to be a state-of-the-art method in mixed culture metagenomic data analysis. In this light, our previously published tool BusyBee Web implements a composition-based binning method efficient enough to function as a rapid online utility. Handling assembled contigs and long nanopore generated reads alike, the webserver provides a wide range of supplementary annotations and visualizations. Half a decade after the initial publication, we revisited existing functionality, added comprehensive visualizations, and increased the number of data analysis customization options for further experimentation. The webserver now allows for visualization-supported differential analysis of samples, which is computationally expensive and typically only performed in coverage-based binning methods. Further, users may now optionally check their uploaded samples for plasmid sequences using PLSDB as a reference database. Lastly, a new application programming interface with a supporting python package was implemented, to allow power users fully automated access to the resource and integration into existing workflows. The webserver is freely available under: https://www.ccb.uni-saarland.de/busybee.

PLSDB: advancing a comprehensive database of bacterial plasmids.

Plasmids are known to contain genes encoding for virulence factors and antibiotic resistance mechanisms. Their relevance in metagenomic data processing is steadily growing. However, with the increasing popularity and scale of metagenomics experiments, the number of reported plasmids is rapidly growing as well, amassing a considerable number of false positives due to undetected misassembles. Here, our previously published database PLSDB provides a reliable resource for researchers to quickly compare their sequences against selected and annotated previous findings. Within two years, the size of this resource has more than doubled from the initial 13,789 to now 34,513 entries over the course of eight regular data updates. For this update, we aggregated community feedback for major changes to the database featuring new analysis functionality as well as performance, quality, and accessibility improvements. New filtering steps, annotations, and preprocessing of existing records improve the quality of the provided data. Additionally, new features implemented in the web-server ease user interaction and allow for a deeper understanding of custom uploaded sequences, by visualizing similarity information. Lastly, an application programming interface was implemented along with a python library, to allow remote database queries in automated workflows. The latest release of PLSDB is freely accessible under https://www.ccb.uni-saarland.de/plsdb.

miRNATissueAtlas2: an update to the human miRNA tissue atlas.

Small non-coding RNAs (sncRNAs) are pervasive regulators of physiological and pathological processes. We previously developed the human miRNA Tissue Atlas, detailing the expression of miRNAs across organs in the human body. Here, we present an updated resource containing sequencing data of 188 tissue samples comprising 21 organ types retrieved from six humans. Sampling the organs from the same bodies minimizes intra-individual variability and facilitates the making of a precise high-resolution body map of the non-coding transcriptome. The data allow shedding light on the organ- and organ system-specificity of piwi-interacting RNAs (piRNAs), transfer RNAs (tRNAs), microRNAs (miRNAs) and other non-coding RNAs. As use case of our resource, we describe the identification of highly specific ncRNAs in different organs. The update also contains 58 samples from six tissues of the Tabula Muris collection, allowing to check if the tissue specificity is evolutionary conserved between Homo sapiens and Mus musculus. The updated resource of 87 252 non-coding RNAs from nine non-coding RNA classes for all organs and organ systems is available online without any restrictions (https://www.ccb.uni-saarland.de/tissueatlas2).

Encyclopedia of tools for the analysis of miRNA isoforms.

RNA sequencing data sets rapidly increase in quantity. For microRNAs (miRNAs), frequently dozens to hundreds of billion reads are generated per study. The quantification of annotated miRNAs and the prediction of new miRNAs are leading computational tasks. Now, the increased depth of coverage allows to gain deeper insights into the variability of miRNAs. The analysis of isoforms of miRNAs (isomiRs) is a trending topic, and a range of computational tools for the analysis of isomiRs has been developed. We provide an overview on 27 available computational solutions for the analysis of isomiRs. These include both stand-alone programs (17 tools) and web-based solutions (10 tools) and span a publication time range from 2010 to 2020. Seven of the tools were published in 2019 and 2020, confirming the rising importance of the topic. While most of the analyzed tools work for a broad range of organisms or are completely independent of a reference organism, several tools have been tailored for the analysis of human miRNA data or for plants. While 14 of the tools are general analysis tools of miRNAs, and isomiR analysis is one of their features, the remaining 13 tools have specifically been developed for isomiR analysis. A direct comparison on 20 deep sequencing data sets for selected tools provides insights into the heterogeneity of results. With our work, we provide users a comprehensive overview on the landscape of isomiR analysis tools and in that support the selection of the most appropriate tool for their respective research task.

Ageing-associated small RNA cargo of extracellular vesicles.

Previous work on murine models and humans demonstrated global as well as tissue-specific molecular ageing trajectories of RNAs. Extracellular vesicles (EVs) are membrane vesicles mediating the horizontal transfer of genetic information between different tissues. We sequenced small regulatory RNAs (sncRNAs) in two mouse plasma fractions at five time points across the lifespan from 2-18 months: (1) sncRNAs that are free-circulating (fc-RNA) and (2) sncRNAs bound outside or inside EVs (EV-RNA). Different sncRNA classes exhibit unique ageing patterns that vary between the fcRNA and EV-RNA fractions. While tRNAs showed the highest correlation with ageing in both fractions, rRNAs exhibited inverse correlation trajectories between the EV- and fc-fractions. For miRNAs, the EV-RNA fraction was exceptionally strongly associated with ageing, especially the miR-29 family in adipose tissues. Sequencing of sncRNAs and coding genes in fat tissue of an independent cohort of aged mice up to 27 months highlighted the pivotal role of miR-29a-3p and miR-29b-3p in ageing-related gene regulation that we validated in a third cohort by RT-qPCR.

Aviator: a web service for monitoring the availability of web services.

With Aviator, we present a web service and repository that facilitates surveillance of online tools. Aviator consists of a user-friendly website and two modules, a literature-mining based general and a manually curated module. The general module currently checks 9417 websites twice a day with respect to their availability and stores many features (frontend and backend response time, required RAM and size of the web page, security certificates, analytic tools and trackers embedded in the webpage and others) in a data warehouse. Aviator is also equipped with an analysis functionality, for example authors can check and evaluate the availability of their own tools or those of their peers. Likewise, users can check the availability of a certain tool they intend to use in research or teaching to avoid including unstable tools. The curated section of Aviator offers additional services. We provide API snippets for common programming languages (Perl, PHP, Python, JavaScript) as well as an OpenAPI documentation for embedding in the backend of own web services for an automatic test of their function. We query the respective APIs twice a day and send automated notifications in case of an unexpected result. Naturally, the same analysis functionality as for the literature-based module is available for the curated section. Aviator can freely be used at https://www.ccb.uni-saarland.de/aviator.

miRMaster 2.0: multi-species non-coding RNA sequencing analyses at scale.

Analyzing all features of small non-coding RNA sequencing data can be demanding and challenging. To facilitate this process, we developed miRMaster. After the analysis of over 125 000 human samples and 1.5 trillion human small RNA reads over 4 years, we present miRMaster 2 with a wide range of updates and new features. We extended our reference data sets so that miRMaster 2 now supports the analysis of eight species (e.g. human, mouse, chicken, dog, cow) and 10 non-coding RNA classes (e.g. microRNAs, piRNAs, tRNAs, rRNAs, circRNAs). We also incorporated new downstream analysis modules such as batch effect analysis or sample embeddings using UMAP, and updated annotation data bases included by default (miRBase, Ensembl, GtRNAdb). To accommodate the increasing popularity of single cell small-RNA sequencing data, we incorporated a module for unique molecular identifier (UMI) processing. Further, the output tables and graphics have been improved based on user feedback and new output formats that emerged in the community are now supported (e.g. miRGFF3). Finally, we integrated differential expression analysis with the miRNA enrichment analysis tool miEAA. miRMaster is freely available at https://www.ccb.uni-saarland.de/mirmaster2.

miRTargetLink 2.0-interactive miRNA target gene and target pathway networks.

Which genes, gene sets or pathways are regulated by certain miRNAs? Which miRNAs regulate a particular target gene or target pathway in a certain physiological context? Answering such common research questions can be time consuming and labor intensive. Especially for researchers without computational experience, the integration of different data sources, selection of the right parameters and concise visualization can be demanding. A comprehensive analysis should be central to present adequate answers to complex biological questions. With miRTargetLink 2.0, we develop an all-in-one solution for human, mouse and rat miRNA networks. Users input in the unidirectional search mode either a single gene, gene set or gene pathway, alternatively a single miRNA, a set of miRNAs or an miRNA pathway. Moreover, genes and miRNAs can jointly be provided to the tool in the bidirectional search mode. For the selected entities, interaction graphs are generated from different data sources and dynamically presented. Connected application programming interfaces (APIs) to the tailored enrichment tools miEAA and GeneTrail facilitate downstream analysis of pathways and context-annotated categories of network nodes. MiRTargetLink 2.0 is freely accessible at https://www.ccb.uni-saarland.de/mirtargetlink2.

CoolMPS: evaluation of antibody labeling based massively parallel non-coding RNA sequencing.

Results of massive parallel sequencing-by-synthesis vary depending on the sequencing approach. CoolMPS™ is a new sequencing chemistry that incorporates bases by labeled antibodies. To evaluate the performance, we sequenced 240 human non-coding RNA samples (dementia patients and controls) with and without CoolMPS. The Q30 value as indicator of the per base sequencing quality increased from 91.8 to 94%. The higher quality was reached across the whole read length. Likewise, the percentage of reads mapping to the human genome increased from 84.9 to 86.2%. For both technologies, we computed similar distributions between different RNA classes (miRNA, piRNA, tRNA, snoRNA and yRNA) and within the classes. While standard sequencing-by-synthesis allowed to recover more annotated miRNAs, CoolMPS yielded more novel miRNAs. The correlation between the two methods was 0.97. Evaluating the diagnostic performance, we observed lower minimal P-values for CoolMPS (adjusted P-value of 0.0006 versus 0.0004) and larger effect sizes (Cohen's d of 0.878 versus 0.9). Validating 19 miRNAs resulted in a correlation of 0.852 between CoolMPS and reverse transcriptase-quantitative polymerase chain reaction. Comparison to data generated with Illumina technology confirmed a known shift in the overall RNA composition. With CoolMPS we evaluated a novel sequencing-by-synthesis technology showing high performance for the analysis of non-coding RNAs.

CoolMPS for robust sequencing of single-nuclear RNAs captured by droplet-based method.

Massively-parallel single-cell and single-nucleus RNA sequencing (scRNA-seq, snRNA-seq) requires extensive sequencing to achieve proper per-cell coverage, making sequencing resources and availability of sequencers critical factors for conducting deep transcriptional profiling. CoolMPS is a novel sequencing-by-synthesis approach that relies on nucleotide labeling by re-usable antibodies, but whether it is applicable to snRNA-seq has not been tested. Here, we use a low-cost and off-the-shelf protocol to chemically convert libraries generated with the widely-used Chromium 10X technology to be sequenceable with CoolMPS technology. To assess the quality and performance of converted libraries sequenced with CoolMPS, we generated a snRNA-seq dataset from the hippocampus of young and old mice. Native libraries were sequenced on an Illumina Novaseq and libraries that were converted to be compatible with CoolMPS were sequenced on a DNBSEQ-400RS. CoolMPS-derived data faithfully replicated key characteristics of the native library dataset, including correct estimation of ambient RNA-contamination, detection of captured cells, cell clustering results, spatial marker gene expression, inter- and intra-replicate differences and gene expression changes during aging. In conclusion, our results show that CoolMPS provides a viable alternative to standard sequencing of RNA from droplet-based libraries.

Validation of human microRNA target pathways enables evaluation of target prediction tools.

MicroRNAs are regulators of gene expression. A wide-spread, yet not validated, assumption is that the targetome of miRNAs is non-randomly distributed across the transcriptome and that targets share functional pathways. We developed a computational and experimental strategy termed high-throughput miRNA interaction reporter assay (HiTmIR) to facilitate the validation of target pathways. First, targets and target pathways are predicted and prioritized by computational means to increase the specificity and positive predictive value. Second, the novel webtool miRTaH facilitates guided designs of reporter assay constructs at scale. Third, automated and standardized reporter assays are performed. We evaluated HiTmIR using miR-34a-5p, for which TNF- and TGFB-signaling, and Parkinson's Disease (PD)-related categories were identified and repeated the pipeline for miR-7-5p. HiTmIR validated 58.9% of the target genes for miR-34a-5p and 46.7% for miR-7-5p. We confirmed the targeting by measuring the endogenous protein levels of targets in a neuronal cell model. The standardized positive and negative targets are collected in the new miRATBase database, representing a resource for training, or benchmarking new target predictors. Applied to 88 target predictors with different confidence scores, TargetScan 7.2 and miRanda outperformed other tools. Our experiments demonstrate the efficiency of HiTmIR and provide evidence for an orchestrated miRNA-gene targeting.

A mouse tissue atlas of small noncoding RNA.

Small noncoding RNAs (ncRNAs) play a vital role in a broad range of biological processes both in health and disease. A comprehensive quantitative reference of small ncRNA expression would significantly advance our understanding of ncRNA roles in shaping tissue functions. Here, we systematically profiled the levels of five ncRNA classes (microRNA [miRNA], small nucleolar RNA [snoRNA], small nuclear RNA [snRNA], small Cajal body-specific RNA [scaRNA], and transfer RNA [tRNA] fragments) across 11 mouse tissues by deep sequencing. Using 14 biological replicates spanning both sexes, we identified that ∼30% of small ncRNAs are distributed across the body in a tissue-specific manner with some also being sexually dimorphic. We found that some miRNAs are subject to "arm switching" between healthy tissues and that tRNA fragments are retained within tissues in both a gene- and a tissue-specific manner. Out of 11 profiled tissues, we confirmed that brain contains the largest number of unique small ncRNA transcripts, some of which were previously annotated while others are identified in this study. Furthermore, by combining these findings with single-cell chromatin accessibility (scATAC-seq) data, we were able to connect identified brain-specific ncRNAs with their cell types of origin. These results yield the most comprehensive characterization of specific and ubiquitous small RNAs in individual murine tissues to date, and we expect that these data will be a resource for the further identification of ncRNAs involved in tissue function in health and dysfunction in disease.