Exertional heat illness (EHI) is an occupational health hazard for athletes and military personnel–characterised by the inability to thermoregulate during exercise. The ability to thermoregulate can ...be studied using a standardised heat tolerance test (HTT) developed by The Institute of Naval Medicine. In this study, we investigated whole blood gene expression (at baseline, 2 h post-HTT and 24 h post-HTT) in male subjects with either a history of EHI or known susceptibility to malignant hyperthermia (MHS): a pharmacogenetic condition with similar clinical phenotype. Compared to healthy controls at baseline, 291 genes were differentially expressed in the EHI cohort, with functional enrichment in inflammatory response genes (up to a four-fold increase). In contrast, the MHS cohort featured 1019 differentially expressed genes with significant down-regulation of genes associated with oxidative phosphorylation (OXPHOS). A number of differentially expressed genes in the inflammation and OXPHOS pathways overlapped between the EHI and MHS subjects, indicating a common underlying pathophysiology. Transcriptome profiles between subjects who passed and failed the HTT (based on whether they achieved a plateau in core temperature or not, respectively) were not discernable at baseline, and HTT was shown to elevate inflammatory response gene expression across all clinical phenotypes.
Exertional heat illness (EHI) and malignant hyperthermia (MH) are life threatening conditions associated with muscle breakdown in the setting of triggering factors including volatile anesthetics, ...exercise, and high environmental temperature. To identify new genetic variants that predispose to EHI and/or MH, we performed genomic sequencing on a cohort with EHI/MH and/or abnormal caffeine-halothane contracture test. In five individuals, we identified rare, pathogenic heterozygous variants in ASPH, a gene encoding junctin, a regulator of excitation-contraction coupling. We validated the pathogenicity of these variants using orthogonal pre-clinical models, CRISPR-edited C2C12 myotubes and transgenic zebrafish. In total, we demonstrate that ASPH variants represent a new cause of EHI and MH susceptibility.
Over the past decade, the development of 'simple' blood tests that enable cancer screening, diagnosis or monitoring and facilitate the design of personalized therapies without the need for invasive ...tumour biopsy sampling has been a core ambition in cancer research. Data emerging from ongoing biomarker development efforts indicate that multiple markers, used individually or as part of a multimodal panel, are required to enhance the sensitivity and specificity of assays for early stage cancer detection. The discovery of cancer-associated molecular alterations that are reflected in blood at multiple dimensions (genome, epigenome, transcriptome, proteome and metabolome) and integration of the resultant multi-omics data have the potential to uncover novel biomarkers as well as to further elucidate the underlying molecular pathways. Herein, we review key advances in multi-omics liquid biopsy approaches and introduce the 'nano-omics' paradigm: the development and utilization of nanotechnology tools for the enrichment and subsequent omics analysis of the blood-circulating cancerome.
Abstract
Despite the tremendous potential of liquid biopsies to revolutionise cancer care, there has been limited success translating blood-circulating proteomic and genomic biomarkers into the ...clinic. This is fundamentally due to the extremely low concentration of tumour-derived biomolecules in blood circulation, particularly at an early disease stage, which makes the discovery phase of the biomarker pipeline extremely challenging. Nanotechnology offers a promising solution, with a nanoparticle-biomolecule enrichment tool recently developed to enrich low-abundant, low molecular weight proteins in the blood of ovarian cancer patients.1 Proteomic analysis followed by immunoassay-based validation of selected proteins demonstrated the potential of the nanoparticle-platform proposed to discover novel biomarkers with greater specificity and sensitivity than the clinically used biomarkers. In addition, we recently confirmed the presence of cell-free DNA (cfDNA) captured onto the surface lipid nanoparticles incubated ex vivo with human plasma.2 A significantly higher abundance of cfDNA was detected in the nanoparticle-enriched plasma samples of late-stage ovarian cancer patients compared to age-matched female controls. Proteomic analysis of the same samples also revealed tumour-specific elevations in histone proteins, which are commonly found in circulation complexed with cfDNA. These findings have highlighted the opportunity for the development of a nano-proteogenomics platform able to simultaneously purify both proteins and cell-free nucleic acids from human plasma, an important step in the discovery of novel multi-omic biomarker panels. Utilising the above patented nanotechnology, we have compared proteomic and genomic profiles derived from nanoparticle-biomolecule samples of cancer patients with age- and sex-matched controls to uncover new potential blood-based biomarkers in a proof-of-principle study. In brief, ex-vivo plasma samples were incubated with lipid-based nanoparticles and purified using a two-step size-based purification protocol. The purified samples were then analysed by label-free proteomics (LC-MS/MS) and next-generation sequencing to uncover both proteomic and genomic tumour-specific signatures, including differentially abundant proteins, genomic copy number alterations and tumour-specific mutations. This work highlights the potential of our nanotechnology-based enrichment platform to enhance the discovery of cancer-specific proteogenomic biomarker panels, a vital step in developing sensitive and specific liquid biopsies for the early detection of cancer.
References: 1 M. Hadjidemetriou, L. Papafilippou, R. D. Unwin, J. Rogan, A. Clamp, K. Kostarelos, Nano Today 2020, 34, 100901. 2 L. Gardner, J. Warrington, J. Rogan, D. G. Rothwell, G. Brady, C. Dive, K. Kostarelos, M. Hadjidemetriou, Nanoscale Horizons 2020, 5, 1476.
Citation Format: Lois Gardner, Dominic G. Rothwell, Caroline Dive, Kostas Kostarelos, Marilena Hadjidemetriou. Nanonets for multiomics blood analysis and cancer biomarker discovery abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 568.
The spontaneous adsorption of biomolecules onto the surface of nanoparticles (NPs) in complex physiological biofluids has been widely investigated over the last decade. Characterisation of the ...protein composition of the 'biomolecule corona' has dominated research efforts, whereas other classes of biomolecules, such as nucleic acids, have received no interest. Scarce, speculative statements exist in the literature about the presence of nucleic acids in the biomolecule corona, with no previous studies attempting to describe the contribution of genomic content to the blood-derived NP corona. Herein, we provide the first experimental evidence of the interaction of circulating cell-free DNA (cfDNA) with lipid-based NPs upon their incubation with human plasma samples, obtained from healthy volunteers and ovarian carcinoma patients. Our results also demonstrate an increased amount of detectable cfDNA in patients with cancer. Proteomic analysis of the same biomolecule coronas revealed the presence of histone proteins, suggesting an indirect, nucleosome-mediated NP-cfDNA interaction. The finding of cfDNA as part of the NP corona, offers a previously unreported new scope regarding the chemical composition of the 'biomolecule corona' and opens up new possibilities for the potential exploitation of the biomolecule corona for the enrichment and analysis of blood-circulating nucleic acids.
The biomolecule corona spontaneously adsorbed onto lipid-based nanoparticles (NPs), upon incubation with human plasma, contains circulating cell-free DNA (cfDNA).
Exertional heat illness (EHI) is a clinically important disorder, notifiable in military personnel, and is characterised by an inability to thermoregulate. Research investigating the genetic risk ...factors contributing to this potentially fatal condition is limited and the pathophysiology of EHI remains poorly understood. EHI shares a similar clinical manifestation to malignant hyperthermia (MH), a pharmacogenetic disorder associated with calcium dysregulation in skeletal muscle. Interestingly, 34% of the EHI patients in this study developed muscle contractures during an in vitro contracture test (IVCT), the gold-standard diagnostic test for MH susceptibility. The coding regions of fifty genes relating to calcium homeostasis and energy metabolism were sequenced in sixty-four EHI patients using a next-generation sequencing (NGS) approach. Many of these genes have been previously implicated in MH, congenital myopathies and metabolic disorders. Seventy-nine rare (minor allele frequency ≤1%) and potentially pathogenic (CADD-score ≥15) non-synonymous variants were identified across twenty-four genes, potentially conferring susceptibility to EHI. Around 75% of MH susceptible individuals in the UK carry a diagnostic ryanodine receptor type-1 (RYR1) variant. Uncharacterised RYR1 variants were identified in 38% of EHI patients in this study, 16% of which were annotated as rare and potentially pathogenic. Global gene expression profiles were examined in a heterozygous RyR1 R163C mutant mouse model associated with EHI and MH to investigate the acute heat stress response. These mice demonstrated elevated basal O2 consumption and increased expression of heat shock proteins (HSPs) after heat exposure. RNA-seq was also used to explore the exertional heat stress response in a cohort of EHI, MH and healthy control volunteers. Elevated HSPs were detected in the blood of MH individuals along with a basal reduction of key oxidative phosphorylation enzymes, both suggestive of oxidative stress. In contrast, increased expression of metabolic enzymes required for acetyl-coA synthesis were detected in both EHI and MH susceptible patients relative to controls. This thesis highlights the likely role of calcium dysregulation and energy metabolism in the pathophysiology of this complex disorder.
Exertional heat illness (EHI) is a clinically important disorder, notifiable in military personnel, and is characterised by an inability to thermoregulate. Research investigating the genetic risk ...factors contributing to this potentially fatal condition is limited and the pathophysiology of EHI remains poorly understood. EHI shares a similar clinical manifestation to malignant hyperthermia (MH), a pharmacogenetic disorder associated with calcium dysregulation in skeletal muscle. Interestingly, 34% of the EHI patients in this study developed muscle contractures during an in vitro contracture test (IVCT), the gold-standard diagnostic test for MH susceptibility. The coding regions of fifty genes relating to calcium homeostasis and energy metabolism were sequenced in sixty-four EHI patients using a next-generation sequencing (NGS) approach. Many of these genes have been previously implicated in MH, congenital myopathies and metabolic disorders. Seventy-nine rare (minor allele frequency ≤1%) and potentially pathogenic (CADD-score ≥15) non-synonymous variants were identified across twenty-four genes, potentially conferring susceptibility to EHI. Around 75% of MH susceptible individuals in the UK carry a diagnostic ryanodine receptor type-1 (RYR1) variant. Uncharacterised RYR1 variants were identified in 38% of EHI patients in this study, 16% of which were annotated as rare and potentially pathogenic. Global gene expression profiles were examined in a heterozygous RyR1 R163C mutant mouse model associated with EHI and MH to investigate the acute heat stress response. These mice demonstrated elevated basal O2 consumption and increased expression of heat shock proteins (HSPs) after heat exposure. RNA-seq was also used to explore the exertional heat stress response in a cohort of EHI, MH and healthy control volunteers. Elevated HSPs were detected in the blood of MH individuals along with a basal reduction of key oxidative phosphorylation enzymes, both suggestive of oxidative stress. In contrast, increased expression of metabolic enzymes required for acetyl-coA synthesis were detected in both EHI and MH susceptible patients relative to controls. This thesis highlights the likely role of calcium dysregulation and energy metabolism in the pathophysiology of this complex disorder.
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