Biology subjects

Lambert, D. W.

Publications and source records attributed to Lambert, D. W..

3 recordsLinked to original sources

bioRxiv2023

Vault particles are common contaminants of extracellular vesicle preparations

Extracellular vesicles (EVs) may contain a variety of molecular cargo including proteins and nucleic acids. Vault particle components have been repeatedly reported in the literature as EV cargo. Here, we demonstrated by small RNA sequencing that vault RNA (vtRNA) were highly abundant in EV pellets enriched by differential centrifugation. EVs were prepared by commonly used enrichment methods and biochemical assays used to determine whether vault particle components were bona fide EV cargo. EVs were isolated by differential centrifugation, size exclusion chromatography (SEC) and Dynabead immunocapture. RNase and proteinase treatment of EV preparations demonstrated that most vtRNA and major vault protein (MVP) were not enclosed and protected within the EV membrane. Vault-like particles were visualised in differential centrifugation pellets by cryo-transmission electron microscopy. EVs enriched by size exclusion chromatography and those isolated by immunocapture post-ultracentrifugation showed co-purification of MVP, whereas EVs isolated by direct immunocapture from conditioned medium were MVP-negative. Taken together, commonly used isolation techniques, such as differential centrifugation and SEC, can lead to contamination of EVs with vault particles. The current study highlights the importance of determining the topology of putative EV-associated components to determine if they are EV cargo or contaminants that have been co-purified.

cell biology↗

bioRxiv2020

The role of icIL-1RA type 1 in oral keratinocyte senescence and the development of the senescence associated secretory phenotype

There is compelling evidence that senescent cells, through the senescence-associated secretory phenotype (SASP), can promote malignant transformation and invasion. IL-1 is a key mediator of this cytokine network, but the control of its activity in the senescence program has not been elucidated. IL-1 signalling is regulated by IL-1RA, which has four variants. Here, we show that expression of intracellular IL-1RA type 1 (icIL-1RA1), which competitively inhibits binding of IL-1 to its receptor, is progressively lost during oral carcinogenesis ex vivo and that the pattern of expression is associated with keratinocyte replicative fate in vitro. We demonstrate icIL-1RA1 is an important regulator of the SASP in mortal cells, as CRISPR-CAS9 mediated icIL-1RA1 knockdown in normal and mortal dysplastic oral keratinocytes is followed by increased IL-6 and IL-8 secretion, and rapid senescence following release from ROCK inhibition. Thus, we suggest that downregulation of icIL-1RA1 in early stages of the carcinogenesis process can enable the development of a premature and de-regulated SASP, creating a pro-inflammatory state in which cancer is more likely to arise.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cell biology↗

bioRxiv2018

Comprehensive functional profiling of long non-coding RNAs through a novel pan-cancer integration approach and modular analysis of their protein-coding gene association networks

Long non-coding RNAs (lncRNAs) are emerging as crucial regulators of cellular processes in diseases such as cancer, although the functions of most remain poorly understood. To address this, here we apply a novel strategy to integrate gene expression profiles across 32 cancer types, and cluster human lncRNAs based on their pan-cancer protein-coding gene associations. By doing so, we derive 16 lncRNA modules whose unique properties allow simultaneous inference of function, disease specificity and regulation for over 800 lncRNAs. Remarkably, modules could be grouped into just four functional themes: transcription regulation, immunological, extracellular, and neurological, with module generation frequently driven by lncRNA tissue specificity. Notably, three modules associated with the extracellular matrix represented potential networks of lncRNAs regulating key events in tumour progression. These included a tumour-specific signature of 33 lncRNAs that may play a role in inducing epithelialmesenchymal transition through modulation of TGF{beta} signalling, and two stromal-specific modules comprising 26 lncRNAs linked to a tumour suppressive microenvironment, and 12 lncRNAs related to cancer-associated fibroblasts. At least one member of the 12-lncRNA signature was experimentally supported by siRNA knockdown, which resulted in attenuated differentiation of quiescent fibroblasts to a cancer-associated phenotype. Overall, the study provides a unique pan-cancer perspective on the lncRNA functional landscape, acting as a global source of novel hypotheses on lncRNA contribution to tumour progression.\n\nAuthor SummaryThe established view of protein production is that genomic DNA is transcribed into RNA, which is then translated into protein. Proteins play a critical role in shaping the function of each individual cell in the human body yet they represent less than 2% of human genomic sequence whilst up to 90% of the genome is transcribed. To explain this disparity, the existence of thousands of long non-coding RNAs (lncRNAs) has emerged that do not encode proteins but perform function as an RNA molecule. Most lncRNAs have yet to be assigned a specific biological role, so to address this we apply a novel computational approach to characterise the function of >800 lncRNAs through consistent association with protein coding genes across multiple cancer types. By doing so, we discover 16 \"modules\" of closely related lncRNAs that share broad functional themes, the most compelling of which consists of 12 lncRNAs that could regulate activation of specific cells neighbouring the tumour, leading to accelerated tumour progression and invasion. Overall, the study provides the most robust view of the lncRNA-protein coding gene landscape to date, adding to growing evidence that lncRNAs are key regulators of cancer, and have therapeutic potential comparable to proteins.

bioinformatics↗