Skeletal muscle fiber type distribution has implications for human health, muscle function, and performance. This knowledge has been gathered using labor-intensive and costly methodology that limited these studies. Here, we present a method based on muscle tissue RNA sequencing data (totRNAseq) to estimate the distribution of skeletal muscle fiber types from frozen human samples, allowing for a larger number of individuals to be tested. By using single-nuclei RNA sequencing (snRNAseq) data as a reference, cluster expression signatures were produced by averaging gene expression of cluster gene markers and then applying these to totRNAseq data and inferring muscle fiber nuclei type via linear matrix decomposition. This estimate was then compared with fiber type distribution measured by ATPase staining or myosin heavy chain protein isoform distribution of 62 muscle samples in two independent cohorts (n = 39 and 22). The correlation between the sequencing-based method and the other two were rATPas = 0.44 [0.13-0.67], [95% CI], and rmyosin = 0.83 [0.61-0.93], with p = 5.70 × 10-3 and 2.00 × 10-6, respectively. The deconvolution inference of fiber type composition was accurate even for very low totRNAseq sequencing depths, i.e., down to an average of ~ 10,000 paired-end reads. This new method ( https://github.com/OlaHanssonLab/PredictFiberType ) consequently allows for measurement of fiber type distribution of a larger number of samples using totRNAseq in a cost and labor-efficient way. It is now feasible to study the association between fiber type distribution and e.g. health outcomes in large well-powered studies.

Efficient and specific delivery of mRNA to target tissues is critical for maximising therapeutic benefits while minimising off-target effects and systemic toxicity. Systemic administration of mRNA using lipid nanoparticles (LNPs) or extracellular vesicles (EVs) typically leads to predominant accumulation in the liver. We hypothesised that cardiac-specific EVs could promote enhanced relative cardiac enrichment of delivered mRNA compared with non-cardiac EVs or LNPs. In mice, intravenous administration of cardiac progenitor cell-derived EVs (CPC-EVs) achieved the greatest relative cardiac selectivity of modified mRNA encoding vascular endothelial growth factor A (VEGF-A) to the heart, with reduced liver accumulation relative to non-cardiac EVs and LNPs. Cytokine profiling across seven organs revealed that LNP delivery triggered a widespread pro-inflammatory response, whereas CPC-EVs elicited only a localised and limited cytokine activation, suggesting a more favourable safety profile. Furthermore, direct intramyocardial injection of CPC-EVs not only led to efficient mRNA uptake by cardiac tissue and robust VEGF-A protein expression, but also minimal transcriptomic perturbation in the cardiac tissue, as confirmed by RNA-seq. In contrast, LNPs and non-cardiac EVs induced widespread perturbation in the transcriptome of cardiac tissue. Functionally, VEGF-A mRNA delivery via CPC-EVs markedly increased CD31 and α-SMA expression and vessel formation in ex vivo aortic ring assays, confirming enhanced angiogenic potential. Together, these findings support CPC-EVs as a promising platform for achieving enhanced cardiac delivery of mRNA, with reduced liver accumulation, limited off-target transcriptomic perturbation, a more selective cytokine response, and enhanced angiogenic activity in ex vivo assays.

The introduction of the CTLA-4 recombinant fusion protein has demonstrated therapeutic effects by selectively modulating T-cell activation in rheumatoid arthritis. Here we show, using a forward genetic approach, that a mutation in the SH3gl1 gene encoding the endocytic protein Endophilin A2 is associated with the development of arthritis in rodents. Defective expression of SH3gl1 affects T cell effector functions and alters the activation threshold of autoreactive T cells, thereby leading to complete protection from chronic autoimmune inflammatory disease in both mice and rats. We further show that SH3GL1 regulates human T cell signaling and T cell receptor internalization, and its expression is upregulated in rheumatoid arthritis patients. Collectively our data identify SH3GL1 as a key regulator of T cell activation, and as a potential target for treatment of autoimmune diseases.

Treatment-resistant glioblastoma stem and precursor cells (GPCs) drive glioblastoma (GBM) growth and recurrence. Thus, targeting the molecular machinery that sustains GPCs in an undifferentiated and self-renewing state is a promising therapeutic strategy. The transcription factor SOX21 effectively suppresses the tumorigenic capacity of GPCs, but the mechanism by which SOX21 impedes GPC features is unknown. By engineering patient-derived GPCs with a transgenic TetOn system we show that SOX21 expression induces an anti-tumorigenic transcriptional program, aligning with clinical data demonstrating a positive correlation between SOX21 levels and improved GBM patient survival. Induced SOX21 expression in GPCs within pre-established GBM reduces their capacity to sustain tumor growth and significantly extends the survival of the orthotopically transplanted mice. Mechanistically, SOX21 functions as a tumor suppressor by binding a large set of AP-1-targeted chromatin regions, leading to epigenetic repression of AP-1-activated genes. Consistently, the anti-tumorigenic activities of SOX21 are largely replicated by AP-1 inhibitors, which decrease GPC proliferation and survival, while overexpression of the AP-1 family member, c-JUN, counteracts these effects. Our findings identify SOX21 as a key regulator that prevents GPC malignancy by targeting and repressing an AP-1-driven, tumor-promoting gene expression program. These results highlight SOX21-regulated pathways as promising therapeutic targets for GBM.

RpoN, an alternative sigma factor commonly known as σ54, is implicated in persistent stages of Yersinia pseudotuberculosis infections in which genes associated with this regulator are upregulated. We here combined phenotypic and genomic assays to provide insight into its role and function in this pathogen. RpoN was found essential for Y. pseudotuberculosis virulence in mice, and in vitro functional assays showed that it controls biofilm formation and motility. Mapping genome-wide associations of Y. pseudotuberculosis RpoN using chromatin immunoprecipitation coupled with next-generation sequencing identified an RpoN binding motif located at 103 inter- and intragenic sites on both sense and antisense strands. Deletion of rpoN had a large impact on gene expression, including downregulation of genes encoding proteins involved in flagellar assembly, chemotaxis, and quorum sensing. There were also clear indications of cross talk with other sigma factors, together with indirect effects due to altered expression of other regulators. Matching differential gene expression with locations of the binding sites implicated around 130 genes or operons potentially activated or repressed by RpoN. Mutagenesis of selected intergenic binding sites confirmed both positive and negative regulatory effects of RpoN binding. Corresponding mutations of intragenic sense sites had less impact on associated gene expression. Surprisingly, mutating intragenic sites on the antisense strand commonly reduced expression of genes carried by the corresponding sense strand.IMPORTANCE The alternative sigma factor RpoN (σ54), which is widely distributed in eubacteria, has been implicated in controlling gene expression of importance for numerous functions including virulence. Proper responses to host environments are crucial for bacteria to establish infection, and regulatory mechanisms involved are therefore of high interest for development of future therapeutics. Little is known about the function of RpoN in the intestinal pathogen Y. pseudotuberculosis, and we therefore investigated its regulatory role in this pathogen. This regulator was indeed found to be critical for establishment of infection in mice, likely involving its requirement for motility and biofilm formation. The RpoN regulon involved both activating and suppressive effects on gene expression which could be confirmed with mutagenesis of identified binding sites. This is the first study of its kind of RpoN in Y. pseudotuberculosis, revealing complex regulation of gene expression involving both productive and silent effects of its binding to DNA, providing important information about RpoN regulation in enterobacteria.

The comma butterfly (Polygonia c-album, Nymphalidae, Lepidoptera) is a model insect species, most notably in the study of phenotypic plasticity and plant-insect coevolutionary interactions. In order to facilitate the integration of genomic tools with a diverse body of ecological and evolutionary research, we assembled the genome of a Swedish comma using 10X sequencing, scaffolding with matepair data, genome polishing, and assignment to linkage groups using a high-density linkage map. The resulting genome is 373 Mb in size, with a scaffold N50 of 11.7 Mb and contig N50 of 11,2Mb. The genome contained 90.1% of single-copy Lepidopteran orthologs in a BUSCO analysis of 5,286 genes. A total of 21,004 gene-models were annotated on the genome using RNA-Seq data from larval and adult tissue in combination with proteins from the Arthropoda database, resulting in a high-quality annotation for which functional annotations were generated. We further documented the quality of the chromosomal assembly via synteny assessment with Melitaea cinxia. The resulting annotated, chromosome-level genome will provide an important resource for investigating coevolutionary dynamics and comparative analyses in Lepidoptera.

Novel sequences (NSs), not present in the human reference genome, are abundant and remain largely unexplored. Here, we utilize de novo assembly to study NS in 1,000 Swedish individuals first sequenced as part of the SweGen project revealing a total of 46 Mb in 61,044 distinct contigs of sequences not present in GRCh38. The contigs were aligned to recently published catalogs of Icelandic and Pan-African NSs, as well as the chimpanzee genome, revealing a great diversity of shared sequences. Analyzing the positioning of NS across the chimpanzee genome, we find that 2,807 NS align confidently within 143 chimpanzee orthologs of human genes. Aligning the whole genome sequencing data to the chimpanzee genome, we discover ancestral NS common throughout the Swedish population. The NSs were searched for repeats and repeat elements: revealing a majority of repetitive sequence (56%), and enrichment of simple repeats (28%) and satellites (15%). Lastly, we align the unmappable reads of a subset of the thousand genomes data to our collection of NS, as well as the previously published Pan-African NS: revealing that both the Swedish and Pan-African NS are widespread, and that the Swedish NSs are largely a subset of the Pan-African NS. Overall, these results highlight the importance of creating a more diverse reference genome and illustrate that significant amounts of the NS may be of ancestral origin.