Smith, R. H. | Search bioRxiv

bioRxiv2024

Inhibition of the RNA Regulator HuR mitigates spinal cord injury by potently suppressing post-injury neuroinflammation

BackgroundNeuroinflammation plays a significant role in promoting secondary tissue injury after spinal cord trauma. Within minutes after spinal cord injury (SCI), microglia and astrocytes become activated and produce inflammatory mediators such as TNF-, IL-6, iNOS and COX-2 which induce tissue injury through cytotoxicity, vascular hyperpermeability, and secondary ischemia. The inflammatory cascade is amplified by chemokines such as CCL2 and CXCL1 that promote recruitment of peripheral inflammatory cells into the injured spinal cord. HuR is a key post-transcriptional RNA regulator that controls glial expression of many pro-inflammatory factors by binding to adenylate- and uridylate-rich elements in 3 untranslated regions of the mRNA. SRI-42127 is a small molecule inhibitor that blocks HuR nucleocytoplasmic translocation, a process critical for its regulatory function. The goal of this study was to assess the potential of SRI-42127 for suppressing neuroinflammation after SCI and improving functional outcome. MethodsAdult female mice underwent a contusion injury at the T10 level. SRI-42127 or vehicle was administered intraperitoneally starting 1 h after injury and up to 5 days. Locomotor function was assessed by open field testing, balance beam and rotarod. Immunohistochemistry was used to assess lesion size, neuronal loss, myelin sparing, microglial activation and HuR localization. Molecular analyses of spinal cord and peripheral tissues for expression of inflammatory mediators included qPCR, immunohistochemistry, ELISA, or western blot. Post-SCI pain was assessed by the mouse grimace scale. ResultsSRI-42127 significantly attenuated loss of locomotor function and post-SCI pain. Histologic correlates to these beneficial effects included reduced lesion size, neuronal loss, and an increase in myelin sparing. There was reduced microglial activation at the epicenter with concomitant attenuation of HuR nucleocytoplasmic translocation. Molecular analysis revealed a striking reduction of pro-inflammatory mediators at the epicenter including IL-6, MMP-12, IL-1{beta}, TNF-, iNOS, COX-2, and chemokines CCL2, CXCL1, and CXCL2. Suppression of inflammatory responses extended peripherally including serum, liver, and spleen. ConclusionTargeting HuR after SCI is a viable therapeutic approach for suppressing neuroinflammatory responses after tissue injury and improving functional outcome.

neuroscience↗

bioRxiv2022

Investigating the impact of database choice on the accuracy of metagenomic read classification for the rumen microbiome

Microbiome analysis is quickly moving towards high-throughput methods such as metagenomic sequencing. Accurate taxonomic classification of metagenomic data relies on reference sequence databases, and their associated taxonomy. However, for understudied environments such as the rumen microbiome many sequences will be derived from novel or uncultured microbes that are not present in reference databases. As a result, taxonomic classification of metagenomic data from understudied environments may be inaccurate. To assess the accuracy of taxonomic read classification, this study classified metagenomic data that had been simulated from cultured rumen microbial genomes from the Hungate collection. To assess the impact of reference databases on the accuracy of taxonomic classification, the data was classified with Kraken 2 using several reference databases. We found that the choice and composition of reference database significantly impacted on taxonomic classification results, and accuracy. In particular, NCBI RefSeq proved to be a poor choice of database. Our results indicate that inaccurate read classification is likely to be a significant problem, affecting all studies that use insufficient reference databases. We observe that adding cultured reference genomes from the rumen to the reference database greatly improves classification rate and accuracy. We also demonstrate that metagenome-assembled genomes (MAGs) have the potential to further enhance classification accuracy by representing uncultivated microbes, sequences of which would otherwise be unclassified or incorrectly classified. However, classification accuracy was strongly dependent on the taxonomic labels assigned to these MAGs. We therefore highlight the importance of accurate reference taxonomic information and suggest that, with formal taxonomic lineages, MAGs have the potential to improve classification rate and accuracy, particularly in environments such as the rumen that are understudied or contain many novel genomes.

genomics↗