Biology subjects

Leary, S.

Publications and source records attributed to Leary, S..

3 recordsLinked to original sources

bioRxiv2020

Crosstalk between microglia and patient-derived glioblastoma cells inhibit invasion in a three-dimensional gelatin hydrogel model

BackgroundGlioblastoma is the most common and deadly form of primary brain cancer, accounting for more than thirteen thousand new diagnoses annually in the United States alone. Microglia are the innate immune cells within the central nervous system, acting as a front-line defense against injuries and inflammation via a process that involves transformation from a quiescent to an activated phenotype. Crosstalk between GBM cells and microglia represents an important axis to consider in the development of tissue engineering platforms to examine pathophysiological processes underlying GBM progression and therapy. MethodsThis work used a brain-mimetic hydrogel system to study patient-derived glioblastoma specimens and their interactions with microglia. Here, glioblastoma cells were either cultured alone in 3D hydrogels or in co-culture with microglia in a manner that allowed secretome-based signaling but prevented direct GBM-microglia contact. Patterns of GBM cell invasion were quantified using a three-dimensional spheroid assay. Secretome and transcriptome (via RNAseq) were used to profile the consequences of GBM-microglia interactions. ResultsMicroglia displayed an activated phenotype as a result of GBM crosstalk. Three-dimensional migration patterns of patient derived glioblastoma cells showed invasion was significantly decreased in response to microglia paracrine signaling. Potential molecular mechanisms underlying with this phenotype were identified from bioinformatic analysis of secretome and RNAseq data. ConclusionThe data demonstrate a tissue engineered hydrogel platform can be used to investigate crosstalk between immune cells of the tumor microenvironment related to GBM progression. Such multidimensional models may provide valuable insight to inform therapeutic innovations to improve GBM treatment.

cancer biology↗

bioRxiv2020

periscope: sub-genomic RNA identification in SARS-CoV-2 ARTIC Network Nanopore Sequencing Data

We have developed periscope, a tool for the detection and quantification of sub-genomic RNA (sgRNA) in SARS-CoV-2 genomic sequence data. The translation of the SARS-CoV-2 RNA genome for most open reading frames (ORFs) occurs via RNA intermediates termed "sub-genomic RNAs". sgRNAs are produced through discontinuous transcription which relies on homology between transcription regulatory sequences (TRS-B) upstream of the ORF start codons and that of the TRS-L which is located in the 5 UTR. TRS-L is immediately preceded by a leader sequence. This leader sequence is therefore found at the 5 end of all sgRNA. We applied periscope to 1,155 SARS-CoV-2 genomes from Sheffield, UK and validated our findings using orthogonal datasets and in vitro cell systems. Using a simple local alignment to detect reads which contain the leader sequence we were able to identify and quantify reads arising from canonical and non-canonical sgRNA. We were able to detect all canonical sgRNAs at expected abundances, with the exception of ORF10. A number of recurrent non-canonical sgRNAs are detected. We show that the results are reproducible using technical replicates and determine the optimum number of reads for sgRNA analysis. In VeroE6 ACE2+/- cell lines, periscope can detect the changes in the kinetics of sgRNA in orthogonal sequencing datasets. Finally, variants found in genomic RNA are transmitted to sgRNAs with high fidelity in most cases. This tool can be applied to all sequenced COVID-19 samples worldwide to provide comprehensive analysis of SARS-CoV-2 sgRNA.

bioinformatics↗

bioRxiv2020

Three adjacent nucleotide changes spanning two residues in SARS-CoV-2 nucleoprotein: possible homologous recombination from the transcription-regulating sequence

BackgroundGenetic variations across the SARS-CoV-2 genome may influence transmissibility of the virus and the hosts anti-viral immune response, in turn affecting the frequency of variants over-time. In this study, we examined the adjacent amino acid polymorphisms in the nucleocapsid (R203K/G204R) of SARS-CoV-2 that arose on the background of the spike D614G change and describe how strains harboring these changes became dominant circulating strains globally. MethodsDeep sequencing data of SARS-CoV-2 from public databases and from clinical samples were analyzed to identify and map genetic variants and sub-genomic RNA transcripts across the genome. ResultsSequence analysis suggests that the three adjacent nucleotide changes that result in the K203/R204 variant have arisen by homologous recombination from the core sequence (CS) of the leader transcription-regulating sequence (TRS) rather than by stepwise mutation. The resulting sequence changes generate a novel sub-genomic RNA transcript for the C-terminal dimerization domain of nucleocapsid. Deep sequencing data from 981 clinical samples confirmed the presence of the novel TRS-CS-dimerization domain RNA in individuals with the K203/R204 variant. Quantification of sub-genomic RNA indicates that viruses with the K203/R204 variant may also have increased expression of sub-genomic RNA from other open reading frames. ConclusionsThe finding that homologous recombination from the TRS may have occurred since the introduction of SARS-CoV-2 in humans resulting in both coding changes and novel sub-genomic RNA transcripts suggests this as a mechanism for diversification and adaptation within its new host.

microbiology↗