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Biology subjects

Pritchard, A.

Publications and source records attributed to Pritchard, A..

3 recordsLinked to original sources

Small Extracellular Vesicle Signaling and Mitochondrial Transfer Reprograms T Helper Cell Function in Human Asthma

Small extracellular vesicles (sEVs) are known to orchestrate cell-cell communication, but the role of sEV signaling via mitochondria in perpetuating asthmatic airway inflammation is unknown. Myeloid-derived regulatory cells (MDRCs) are known to control CD4+ T cell responses in asthma. We demonstrate that airway MDRC-derived sEVs from asthmatics mediate T cell receptor engagement and transfer of mitochondria that induce antigen-specific activation and polarization of Th17 and Th2 cells; these cells are drivers of chronic airway inflammation in asthma. CD4+ T cells internalize sEVs containing mitochondria predominantly by membrane fusion, and blocking mitochondrial oxidant signaling in MDRC-derived sEVs mitigates T cell activation. Reactive oxygen species-mediated signaling that elicits T cell activation in asthmatics is sEV-dependent. Additionally, a Drp1-dependent mechanism in pro-inflammatory MDRCs promotes mitochondrial packaging within sEVs, which then co-localize with the polarized cytoskeleton and mitochondrial networks in recipient T cells. Importantly, intranasal transfer of mitochondria packaged sEVs enhances airway inflammation and Th polarization in vivo in a murine model of asthma. Thus, our studies indicate a previously unrecognized role for mitochondrial fission and sEV-mediated mitochondrial transfer-mediated signaling in dysregulated T cell activation and Th cell polarization in asthma which could constitute a novel therapeutic target.

immunology↗

Methods for the targeted sequencing and analysis of integrons and their gene cassettes from complex microbial communities

Integrons are bacterial genetic elements that can integrate mobile gene cassettes. They are mostly known for spreading antibiotic resistance cassettes among human pathogens. However, beyond clinical settings, gene cassettes encode an extraordinarily diverse range of functions important for bacterial adaptation. The recovery and sequencing of cassettes has promising applications, including: surveillance of clinically important genes, particularly antibiotic resistance determinants; investigating the functional diversity of integron-carrying bacteria; and novel enzyme discovery. Although gene cassettes can be directly recovered using PCR, there are no standardised methods for their amplification and, importantly, for validating sequences as genuine integron gene cassettes. Here, we present reproducible methods for the PCR amplification, sequence processing, and validation of gene cassette amplicons from complex communities. We describe two different PCR assays that either amplify cassettes together with integron integrases, or gene cassettes together within cassette arrays. We compare the use of Nanopore and Illumina sequencing, and present bioinformatic pipelines that filter sequences to ensure that they represent amplicons from genuine integrons. Using a diverse set of environmental DNAs, we show that our approach can consistently recover thousands of unique cassettes per sample and up to hundreds of different integron integrases. Recovered cassettes confer a wide range of functions, including antibiotic resistance, with as many as 300 resistance cassettes found in a single sample. In particular, we show that class 1 integrons appear to be collecting and concentrating antibiotic resistance genes out of the broader diversity of cassette functions. The methods described here can be applied to any environmental or clinical microbiome sample.

microbiology↗

A screen for combination therapies in BRAF/NRAS wild type melanoma identifies nilotinib plus MEK inhibitor as a synergistic combination

Despite recent therapeutic advances in the management of BRAFV600-mutant melanoma, there is still a compelling need for more effective treatments for patients who developed BRAF/NRAS wild type disease. Since the activity of single targeted agents is limited by innate and acquired resistance, we performed a high-throughput drug screen using 180 drug combinations to generate over 18,000 viability curves, with the aim of identifying agents that synergise to kill BRAF/NRAS wild type melanoma cells. From this screen we observed strong synergy between the tyrosine kinase inhibitor nilotinib and MEK inhibitors and validated this combination in an independent cell line collection. We found that AXL expression was associated with synergy to the nilotinib/MEK inhibitor combination, and that both drugs work in concert to suppress pERK. This finding was supported by genome-wide CRISPR screening which revealed that resistance mechanisms converge on regulators of the MAPK pathway. Finally, we validated the synergy of nilotinib/trametinib combination in vivo using patient-derived xenografts. Our results indicate that a nilotinib/MEK inhibitor combination may represent an effective therapy in BRAF/NRAS wild type melanoma patients.

cancer biology↗