No evidence for epitranscriptomic m5C modification of SARS-CoV-2, HIV and MLV viral RNA.

The addition of chemical groups to cellular RNA to modulate RNA fate and/or function is summarized under the term epitranscriptomic modification. More than 170 different modifications have been identified on cellular RNA, such as tRNA, rRNA and, to a lesser extent, on other RNA types. Recently, epitranscriptomic modification of viral RNA has received considerable attention as a possible additional mechanism regulating virus infection and replication. N6-methyladenosine (m6A) and C5-methylcytosine (m5C) have been most broadly studied in different RNA viruses. Various studies, however, reported varying results with regard to number and extent of the modification. Here we investigated the m5C methylome of SARS-CoV-2, and we reexamined reported m5C sites in HIV and MLV. Using a rigorous bisulfite-sequencing protocol and stringent data analysis, we found no evidence for the presence of m5C in these viruses. The data emphasize the necessity for optimizing experimental conditions and bioinformatic data analysis.

Next-generation computational tools for interrogating cancer immunity.

The remarkable success of cancer therapies with immune checkpoint blockers is revolutionizing oncology and has sparked intensive basic and translational research into the mechanisms of cancer-immune cell interactions. In parallel, numerous novel cutting-edge technologies for comprehensive molecular and cellular characterization of cancer immunity have been developed, including single-cell sequencing, mass cytometry and multiplexed spatial cellular phenotyping. In order to process, analyse and visualize multidimensional data sets generated by these technologies, computational methods and software tools are required. Here, we review computational tools for interrogating cancer immunity, discuss advantages and limitations of the various methods and provide guidelines to assist in method selection.

ExonSurfer: a web-tool to design primers at exon-exon junctions.

BACKGROUND: Reverse transcription quantitative PCR (RT-qPCR) with intercalating dyes is one of the main techniques to assess gene expression levels used in basic and applied research as well as in diagnostics. However, primer design for RT-qPCR can be complex due to the high demands on primer quality. Primers are best placed on exon junctions, should avoid polymorphic regions, be specific to the target transcripts and also prevent genomic amplification accurately, among others. Current software tools manage to meet all the necessary criteria only insufficiently. Here, we present ExonSurfer, a novel, user-friendly web-tool for qPCR primer design. RESULTS: ExonSurfer combines the different steps of the primer design process, encompassing target selection, specificity and self-complementarity assessment, and the avoidance of issues arising from polymorphisms. Amplification of potentially contaminating genomic DNA is avoided by designing primers on exon-exon junctions, moreover, a genomic alignment is performed to filter the primers accordingly and inform the user of any predicted interaction. In order to test the whole performance of the application, we designed primer pairs for 26 targets and checked both primer efficiency, amplicon melting temperature and length and confirmed the targeted amplicon by Sanger sequencing. Most of the tested primers accurately and selectively amplified the corresponding targets. CONCLUSION: ExonSurfer offers a comprehensive end-to-end primer design, guaranteeing transcript-specific amplification. The user interface is intuitive, providing essential specificity and amplicon details. The tool can also be used by command line and the source code is available. Overall, we expect ExonSurfer to facilitate RT-qPCR set-up for researchers in many fields.

Kinesin family member 12-related hepatopathy: A generally indolent disorder with elevated gamma-glutamyl-transferase activity.

Exome sequencing (ES) has identified biallelic kinesin family member 12 (KIF12) mutations as underlying neonatal cholestatic liver disease. We collected information on onset and progression of this entity. Among consecutively referred pediatric patients at our centers, diagnostic ES identified 4 patients with novel, biallelic KIF12 variants using the human GRCh38 reference sequence, as KIF12 remains incompletely annotated in the older reference sequence GRCh37. A review of these and of 21 reported patients with KIF12 variants found that presentation with elevated serum transaminase activity in the context of trivial respiratory infection, without clinical features of liver disease, was more common (n = 18) than manifest cholestatic disease progressing rapidly to liver transplantation (LT; n = 7). Onset of liver disease was at age <1 year in 15 patients; LT was more common in this group. Serum gamma-glutamyl transpeptidase activity (GGT) was elevated in all patients, and total bilirubin was elevated in 15 patients. Liver fibrosis or cirrhosis was present in 14 of 18 patients who were biopsied. The 16 different pathogenic variants and 11 different KIF12 genotypes found were not correlated with age of onset or progression to LT. Identification of biallelic pathogenic KIF12 variants distinguishes KIF12-related disease from other entities with elevated GGT.

Validation of RNA Extraction Methods and Suitable Reference Genes for Gene Expression Studies in Developing Fetal Human Inner Ear Tissue.

A comprehensive gene expression investigation requires high-quality RNA extraction, in sufficient amounts for real-time quantitative polymerase chain reaction and next-generation sequencing. In this work, we compared different RNA extraction methods and evaluated different reference genes for gene expression studies in the fetal human inner ear. We compared the RNA extracted from formalin-fixed paraffin-embedded tissue with fresh tissue stored at -80 °C in RNAlater solution and validated the expression stability of 12 reference genes (from gestational week 11 to 19). The RNA from fresh tissue in RNAlater resulted in higher amounts and a better quality of RNA than that from the paraffin-embedded tissue. The reference gene evaluation exhibited four stably expressed reference genes (B2M, HPRT1, GAPDH and GUSB). The selected reference genes were then used to examine the effect on the expression outcome of target genes (OTOF and TECTA), which are known to be regulated during inner ear development. The selected reference genes displayed no differences in the expression profile of OTOF and TECTA, which was confirmed by immunostaining. The results underline the importance of the choice of the RNA extraction method and reference genes used in gene expression studies.

MIO: microRNA target analysis system for immuno-oncology.

SUMMARY: MicroRNAs have been shown to be able to modulate the tumor microenvironment and the immune response and hence could be interesting biomarkers and therapeutic targets in immuno-oncology; however, dedicated analysis tools are missing. Here, we present a user-friendly web platform MIO and a Python toolkit miopy integrating various methods for visualization and analysis of provided or custom bulk microRNA and gene expression data. We include regularized regression and survival analysis and provide information of 40 microRNA target prediction tools as well as a collection of curated immune related gene and microRNA signatures and processed TCGA data including estimations of infiltrated immune cells and the immunophenoscore. The integration of several machine learning methods enables the selection of prognostic and predictive microRNAs and gene interaction network biomarkers. AVAILABILITY AND IMPLEMENTATION: https://mio.icbi.at, https://github.com/icbi-lab/mio and https://github.com/icbi-lab/miopy. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

nextNEOpi: a comprehensive pipeline for computational neoantigen prediction.

SUMMARY: Somatic mutations and gene fusions can produce immunogenic neoantigens mediating anticancer immune responses. However, their computational prediction from sequencing data requires complex computational workflows to identify tumor-specific aberrations, derive the resulting peptides, infer patients' Human Leukocyte Antigen types and predict neoepitopes binding to them, together with a set of features underlying their immunogenicity. Here, we present nextNEOpi (nextflow NEOantigen prediction pipeline) a comprehensive and fully automated bioinformatic pipeline to predict tumor neoantigens from raw DNA and RNA sequencing data. In addition, nextNEOpi quantifies neoepitope- and patient-specific features associated with tumor immunogenicity and response to immunotherapy. AVAILABILITY AND IMPLEMENTATION: nextNEOpi source code and documentation are available at https://github.com/icbi-lab/nextNEOpi. CONTACT: dietmar.rieder@i-med.ac.at or francesca.finotello@uibk.ac.at. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Cardiopulmonary recovery after COVID-19: an observational prospective multicentre trial.

BACKGROUND: After the 2002/2003 severe acute respiratory syndrome outbreak, 30% of survivors exhibited persisting structural pulmonary abnormalities. The long-term pulmonary sequelae of coronavirus disease 2019 (COVID-19) are yet unknown, and comprehensive clinical follow-up data are lacking. METHODS: In this prospective, multicentre, observational study, we systematically evaluated the cardiopulmonary damage in subjects recovering from COVID-19 at 60 and 100 days after confirmed diagnosis. We conducted a detailed questionnaire, clinical examination, laboratory testing, lung function analysis, echocardiography and thoracic low-dose computed tomography (CT). RESULTS: Data from 145 COVID-19 patients were evaluated, and 41% of all subjects exhibited persistent symptoms 100 days after COVID-19 onset, with dyspnoea being most frequent (36%). Accordingly, patients still displayed an impaired lung function, with a reduced diffusing capacity in 21% of the cohort being the most prominent finding. Cardiac impairment, including a reduced left ventricular function or signs of pulmonary hypertension, was only present in a minority of subjects. CT scans unveiled persisting lung pathologies in 63% of patients, mainly consisting of bilateral ground-glass opacities and/or reticulation in the lower lung lobes, without radiological signs of pulmonary fibrosis. Sequential follow-up evaluations at 60 and 100 days after COVID-19 onset demonstrated a vast improvement of symptoms and CT abnormalities over time. CONCLUSION: A relevant percentage of post-COVID-19 patients presented with persisting symptoms and lung function impairment along with radiological pulmonary abnormalities >100 days after the diagnosis of COVID-19. However, our results indicate a significant improvement in symptoms and cardiopulmonary status over time.

NeoFuse: predicting fusion neoantigens from RNA sequencing data.

SUMMARY: Gene fusions can generate immunogenic neoantigens that mediate anticancer immune responses. However, their computational prediction from RNA sequencing (RNA-seq) data requires deep bioinformatics expertise to assembly a computational workflow covering the prediction of: fusion transcripts, their translated proteins and peptides, Human Leukocyte Antigen (HLA) types, and peptide-HLA binding affinity. Here, we present NeoFuse, a computational pipeline for the prediction of fusion neoantigens from tumor RNA-seq data. NeoFuse can be applied to cancer patients' RNA-seq data to identify fusion neoantigens that might expand the repertoire of suitable targets for immunotherapy. AVAILABILITY AND IMPLEMENTATION: NeoFuse source code and documentation are available under GPLv3 license at https://icbi.i-med.ac.at/NeoFuse/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Scirpy: a Scanpy extension for analyzing single-cell T-cell receptor-sequencing data.

SUMMARY: Advances in single-cell technologies have enabled the investigation of T-cell phenotypes and repertoires at unprecedented resolution and scale. Bioinformatic methods for the efficient analysis of these large-scale datasets are instrumental for advancing our understanding of adaptive immune responses. However, while well-established solutions are accessible for the processing of single-cell transcriptomes, no streamlined pipelines are available for the comprehensive characterization of T-cell receptors. Here, we propose single-cell immune repertoires in Python (Scirpy), a scalable Python toolkit that provides simplified access to the analysis and visualization of immune repertoires from single cells and seamless integration with transcriptomic data. AVAILABILITY AND IMPLEMENTATION: Scirpy source code and documentation are available at https://github.com/icbi-lab/scirpy. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Adoptive T Cell Therapy: New Avenues Leading to Safe Targets and Powerful Allies.

Adoptive transfer of TCR-engineered T cells is a potent therapy, able to induce clinical responses in different human malignancies. Nevertheless, treatment toxicities may occur and, in particular for solid tumors, responses may be variable and often not durable. To address these challenges, it is imperative to carefully select target antigens and to immunologically interrogate the corresponding tumors when designing optimal T cell therapies. Here, we review recent advances, covering both omics- and laboratory tools that can enable the selection of optimal T cell epitopes and TCRs as well as the identification of dominant immune evasive mechanisms within tumor tissues. Furthermore, we discuss how these techniques may aid in a rational design of effective combinatorial adoptive T cell therapies.

Genes regulated by SATB2 during neurodevelopment contribute to schizophrenia and educational attainment.

SATB2 is associated with schizophrenia and is an important transcription factor regulating neocortical organization and circuitry. Rare mutations in SATB2 cause a syndrome that includes developmental delay, and mouse studies identify an important role for SATB2 in learning and memory. Interacting partners BCL11B and GATAD2A are also schizophrenia risk genes indicating that other genes interacting with or are regulated by SATB2 are making a contribution to schizophrenia and cognition. We used data from Satb2 mouse models to generate three gene-sets that contain genes either functionally related to SATB2 or targeted by SATB2 at different stages of development. Each was tested for enrichment using the largest available genome-wide association studies (GWAS) datasets for schizophrenia and educational attainment (EA) and enrichment analysis was also performed for schizophrenia and other neurodevelopmental disorders using data from rare variant sequencing studies. These SATB2 gene-sets were enriched for genes containing common variants associated with schizophrenia and EA, and were enriched for genes containing rare variants reported in studies of schizophrenia, autism and intellectual disability. In the developing cortex, genes targeted by SATB2 based on ChIP-seq data, and functionally affected when SATB2 is not expressed based on differential expression analysis using RNA-seq data, show strong enrichment for genes associated with EA. For genes expressed in the hippocampus or at the synapse, those targeted by SATB2 are more strongly enriched for genes associated EA than gene-sets not targeted by SATB2. This study demonstrates that single gene findings from GWAS can provide important insights to pathobiological processes. In this case we find evidence that genes influenced by SATB2 and involved in synaptic transmission, axon guidance and formation of the corpus callosum are contributing to schizophrenia and cognition.

FoxH1 represses miR-430 during early embryonic development of zebrafish via non-canonical regulation.

BACKGROUND: FoxH1 is a forkhead transcription factor with conserved key functions in vertebrate mesoderm induction and left-right patterning downstream of the TGF-beta/Nodal signaling pathway. Binding of the forkhead domain (FHD) of FoxH1 to a highly conserved proximal sequence motif was shown to regulate target gene expression. RESULTS: We identify the conserved microRNA-430 family (miR-430) as a novel target of FoxH1. miR-430 levels are increased in foxH1 mutants, resulting in a reduced expression of transcripts that are targeted by miR-430 for degradation. To determine the underlying mechanism of miR-430 repression, we performed chromatin immunoprecipitation studies and overexpression experiments with mutant as well as constitutive active and repressive forms of FoxH1. Our studies reveal a molecular interaction of FoxH1 with miR-430 loci independent of the FHD. Furthermore, we show that previously described mutant forms of FoxH1 that disrupt DNA binding or that lack the C-terminal Smad Interaction Domain (SID) dominantly interfere with miR-430 repression, but not with the regulation of previously described FoxH1 targets. CONCLUSIONS: We were able to identify the distinct roles of protein domains of FoxH1 in the regulation process of miR-430. We provide evidence that the indirect repression of miR-430 loci depends on the connection to a distal repressive chromosome environment via a non-canonical mode. The widespread distribution of such non-canonical binding sites of FoxH1, found not only in our study, argues against a function restricted to regulating miR-430 and for a more global role of FoxH1 in chromatin folding.

Thioguanosine Conversion Enables mRNA-Lifetime Evaluation by RNA Sequencing Using Double Metabolic Labeling (TUC-seq DUAL).

Temporal information about cellular RNA populations is essential to understand the functional roles of RNA. We have developed the hydrazine/NH4 Cl/OsO4 -based conversion of 6-thioguanosine (6sG) into A', where A' constitutes a 6-hydrazino purine derivative. A' retains the Watson-Crick base-pair mode and is efficiently decoded as adenosine in primer extension assays and in RNA sequencing. Because 6sG is applicable to metabolic labeling of freshly synthesized RNA and because the conversion chemistry is fully compatible with the conversion of the frequently used metabolic label 4-thiouridine (4sU) into C, the combination of both modified nucleosides in dual-labeling setups enables high accuracy measurements of RNA decay. This approach, termed TUC-seq DUAL, uses the two modified nucleosides in subsequent pulses and their simultaneous detection, enabling mRNA-lifetime evaluation with unprecedented precision.

TIminer: NGS data mining pipeline for cancer immunology and immunotherapy.

SUMMARY: Recently, a number of powerful computational tools for dissecting tumor-immune cell interactions from next-generation sequencing data have been developed. However, the assembly of analytical pipelines and execution of multi-step workflows are laborious and involve a large number of intermediate steps with many dependencies and parameter settings. Here we present TIminer, an easy-to-use computational pipeline for mining tumor-immune cell interactions from next-generation sequencing data. TIminer enables integrative immunogenomic analyses, including: human leukocyte antigens typing, neoantigen prediction, characterization of immune infiltrates and quantification of tumor immunogenicity. AVAILABILITY AND IMPLEMENTATION: TIminer is freely available at http://icbi.i-med.ac.at/software/timiner/timiner.shtml. CONTACT: zlatko.trajanoski@i-med.ac.at. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

meRanTK: methylated RNA analysis ToolKit.

UNLABELLED: The significance and function of posttranscriptional cytosine methylation in poly(A)RNA attracts great interest but is still poorly understood. High-throughput sequencing of RNA treated with bisulfite (RNA-BSseq) or subjected to enrichment techniques like Aza-IP or miCLIP enables transcriptome wide studies of this particular modification at single base pair resolution. However, to date, there are no specialized software tools available for the analysis of RNA-BSseq or Aza-IP data. Therefore, we developed meRanTK, the first publicly available tool kit which addresses the special demands of high-throughput RNA cytosine methylation data analysis. It provides fast and easy to use splice-aware bisulfite sequencing read mapping, comprehensive methylation calling and identification of differentially methylated cytosines by statistical analysis of single- and multi-replicate experiments. Application of meRanTK to RNA-BSseq or Aza-IP data produces accurate results in standard compliant formats. AVAILABILITY AND IMPLEMENTATION: meRanTK, source code and test data are released under the GNU GPLv3+ license and are available at http://icbi.at/software/meRanTK/ CONTACT: dietmar.rieder@i-med.ac.at.

Osmium-Mediated Transformation of 4-Thiouridine to Cytidine as Key To Study RNA Dynamics by Sequencing.

To understand the functional roles of RNA in the cell, it is essential to elucidate the dynamics of their production, processing and decay. A recent method for assessing mRNA dynamics is metabolic labeling with 4-thiouridine (4sU), followed by thio-selective attachment of affinity tags. Detection of labeled transcripts by affinity purification and hybridization to microarrays or by deep sequencing then reveals RNA expression levels. Here, we present a novel sequencing method (TUC-seq) that eliminates affinity purification and allows for direct assessment of 4sU-labeled RNA. It employs an OsO4 -mediated transformation to convert 4sU into cytosine. We exemplify the utility of the new method for verification of endogenous 4sU in tRNAs and for the detection of pulse-labeled mRNA of seven selected genes in mammalian cells to determine the relative abundance of the new transcripts. The results prove TUC-seq as a straight-forward and highly versatile method for studies of cellular RNA dynamics.

Co-expressed genes prepositioned in spatial neighborhoods stochastically associate with SC35 speckles and RNA polymerase II factories.

Chromosomally separated, co-expressed genes can be in spatial proximity, but there is still debate about how this nuclear organization is achieved. Proposed mechanisms include global genome organization, preferential positioning of chromosome territories, or gene-gene sharing of various nuclear bodies. To investigate this question, we selected a set of genes that were co-expressed upon differentiation of human multipotent stem cells. We applied a novel multi-dimensional analysis procedure which revealed that prior to gene expression, the relative position of these genes was conserved in nuclei. Upon stem cell differentiation and concomitant gene expression, we found that co-expressed genes were closer together. In addition, we found that genes in the same 1-µm-diameter neighborhood associated with either the same splicing speckle or to a lesser extent with the same transcription factory. Dispersal of speckles by overexpression of the serine-arginine (SR) protein kinase cdc2-like kinase Clk2 led to a significant drop in the number of genes in shared neighborhoods. We demonstrate quantitatively that the frequencies of speckle and factory sharing can be explained by assuming stochastic selection of a nuclear body within a restricted sub-volume defined by the original global gene positioning present prior to gene expression. We conclude that the spatial organization of these genes is a two-step process in which transcription-induced association with nuclear bodies enhances and refines a pre-existing global organization.

NAT8L (N-acetyltransferase 8-like) accelerates lipid turnover and increases energy expenditure in brown adipocytes.

NAT8L (N-acetyltransferase 8-like) catalyzes the formation of N-acetylaspartate (NAA) from acetyl-CoA and aspartate. In the brain, NAA delivers the acetate moiety for synthesis of acetyl-CoA that is further used for fatty acid generation. However, its function in other tissues remained elusive. Here, we show for the first time that Nat8l is highly expressed in adipose tissues and murine and human adipogenic cell lines and is localized in the mitochondria of brown adipocytes. Stable overexpression of Nat8l in immortalized brown adipogenic cells strongly increases glucose incorporation into neutral lipids, accompanied by increased lipolysis, indicating an accelerated lipid turnover. Additionally, mitochondrial mass and number as well as oxygen consumption are elevated upon Nat8l overexpression. Concordantly, expression levels of brown marker genes, such as Prdm16, Cidea, Pgc1α, Pparα, and particularly UCP1, are markedly elevated in these cells. Treatment with a PPARα antagonist indicates that the increase in UCP1 expression and oxygen consumption is PPARα-dependent. Nat8l knockdown in brown adipocytes has no impact on cellular triglyceride content, lipogenesis, or oxygen consumption, but lipolysis and brown marker gene expression are increased; the latter is also observed in BAT of Nat8l-KO mice. Interestingly, the expression of ATP-citrate lyase is increased in Nat8l-silenced adipocytes and BAT of Nat8l-KO mice, indicating a compensatory mechanism to sustain the acetyl-CoA pool once Nat8l levels are reduced. Taken together, our data show that Nat8l impacts on the brown adipogenic phenotype and suggests the existence of the NAT8L-driven NAA metabolism as a novel pathway to provide cytosolic acetyl-CoA for lipid synthesis in adipocytes.

Bi-allelic variants in CELSR3 are implicated in central nervous system and urinary tract anomalies.

CELSR3 codes for a planar cell polarity protein. We describe twelve affected individuals from eleven independent families with bi-allelic variants in CELSR3. Affected individuals presented with an overlapping phenotypic spectrum comprising central nervous system (CNS) anomalies (7/12), combined CNS anomalies and congenital anomalies of the kidneys and urinary tract (CAKUT) (3/12) and CAKUT only (2/12). Computational simulation of the 3D protein structure suggests the position of the identified variants to be implicated in penetrance and phenotype expression. CELSR3 immunolocalization in human embryonic urinary tract and transient suppression and rescue experiments of Celsr3 in fluorescent zebrafish reporter lines further support an embryonic role of CELSR3 in CNS and urinary tract formation.