Biology subjects

Issitt, T.

Publications and source records attributed to Issitt, T..

6 recordsLinked to original sources

bioRxiv2026

Breast cancer cohort study identifies an effective, non-invasive, breath-based diagnostic linked to cellular environment-dependent, novel methylation metabolisms

Volatile organic compounds (VOCs) demonstrate promise as non-invasive diagnostic tools. However, lack of mechanistically linked VOCs with biomarker discovery platforms limit delivery to the clinic. In this cohort-based study, we observed significant alterations of chloride-containing volatile fluxes, inclusive of methyl chloride (MeCl), in the breath of cancer patients that are consistent with our newly described metabolic model, derived through in vitro cellular assays and in vivo mice models. The diagnostic accuracy of this cohort study (60 patients) is equivalent to mammogram approaches. This newly identified and novel metabolism along with associated diagnostic biomarkers were initially identified through headspace studies of breast cancer cell lines which were deprived of serum, glucose or oxygen, similar to cellular conditions in tumors. In these cellular assays MeCl was consistently informative of cellular stress. Under resource limited conditions cellular production of MeCl was significantly reduced, and in several cases, cellular metabolism shifted to consumption. We present a new "push-pull" model in which cellular production of MeCl is linked to cellular methylation potential and methyl-transferase activity while consumption of MeCl is associated with methionine generation. Neither consumption nor production metabolisms have been described or quantified in humans or human tissues previously. The cellular headspace-derived model was tested using xenograft tumour bearing mice, which demonstrated reduced MeCl production, consistent with this model This work therefore presents a potentially powerful breath biomarker for cancer that translates from cellular and mice models through to human subjects. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/726956v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@71fcdeorg.highwire.dtl.DTLVardef@1905726org.highwire.dtl.DTLVardef@149bb10org.highwire.dtl.DTLVardef@1981c33_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LI(Poly)chloromethane compounds, including methyl chloride and chloroform, are indicative of cancer status in breath sampled from breast cancer clinic patients C_LIO_LIGlucose, serum and oxygen starvation induce significant changes in cellular metabolic fluxes C_LIO_LIReduced methyl chloride production is indicative of cellular stress in vitro C_LIO_LIMethyl chloride production is linked to cellular methylation activity C_LIO_LIMethyl chloride consumption is linked to methionine synthesis, cellular enrichment in chloride concentration, and enhanced chloroform fluxes C_LIO_LIMethyl chloride fluxes in in vitro cell cultures under pathophysiologically relevant conditions translate to the breath of tumour bearing mice C_LI

molecular biology↗

bioRxiv2025

Chloroform release from ageing cells and Drosophila DJ-1 mutants

Volatile Organic Compounds (VOCs) offer potential for non-invasive diagnosis as biomarkers of disease and metabolism. In complex biological matrices, such as breath however, identifying useful biomarkers from hundreds, or even thousands of VOCs can be challenging. Models of disease, such as cellular or animal models, offer a means to elucidate VOC metabolisms, for accurate targeted studies in patient samples. Neurodegenerative conditions, such as parkinsons have been associated with changed VOCs, offering a potential for early diagnostics and interventions improving treatments and outcomes for patients. Here, three separate models including; human HEK-293t cells, isolated primary rat glial cells, Drosophila flies (wild type and a mutant of the parkinsons associated gene, DJ-1) were grown for an extended period and levels of the VOC chloroform (CHCl3) investigated using custom static headspace sampling chambers. Samples were analysed using targeted gas chromatography mass spectroscopy over time to generate metabolic flux values and chloroform shown to dramatically increase in all models as they aged. HEK-293t cells revealed a 60-fold increase after 10 weeks, glial cells revealed a 10-fold increase after 3 to 4 weeks and DJ-1 mutant flies revealed significant increases compared to control flies at 4 weeks. These results, taken together, indicate that chloroform release is related to ageing in these models and may provide a target for neurodegenerative studies moving forward. We present here the first evidence of chloroform being actively produced by human and rat cells and the first observation of volatile metabolisms in Drosophila.

biochemistry↗

bioRxiv2024

Asbestos Mineral Fibre Exposure Significantly Affects Volatile Organic Compound Profiles of Mesothelial Cell Lines In Vitro

Malignant mesothelioma (MM) is a rare cancer caused by exposure to asbestos, this condition continues to represent a significant diagnostic and therapeutic challenge. Due to the long disease latency, non-invasive diagnostic modalities such as breath analysis may enhance MM early detection by identifying the disease specific pattern of volatile organic compounds (VOCs) in exhaled breath. In this study, solid phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS) was used to extract VOCs from the headspace of two mesothelioma cell lines: MSTO-211H (biphasic mesothelioma) and NCI-H28 (epithelioid mesothelioma) in addition to MET-5a (non-malignant mesothelial cell line), following exposure to asbestos mineral fibers (actinolite, amosite, crocidolite and chrysotile) and a non-asbestos fiber control (wollastonite). Multivariate statistical analysis was applied to identify VOC-based biomarkers associated with asbestos exposure within the cell lines. Data confirms that exposure to mineral fibers (including the non-asbestos fiber control) induces significant alterations in the levels of as many as 24 VOCs in each cell line. This is the first study to investigate the impact of asbestos mineral fiber exposure on the VOC profile of MM cell lines. This may be relevant to studies involving mesothelioma patients, as these VOCs could also serve as indicators of asbestos exposure in individuals who have been exposed to asbestos.

cancer biology↗

bioRxiv2023

The hepcidin-ferroportin axis influences mitochondrial function, proliferation, and migration in pulmonary artery endothelial and smooth muscle cells

RationaleElevated circulating hepcidin levels have been reported in patients with pulmonary artery hypertension (PAH). Hepcidin has been shown to promote proliferation of human pulmonary artery smooth muscle cells (PASMCs) in vitro, suggesting a potential role in PAH pathogenesis. However, the role of human pulmonary artery endothelial cells (PAECs) as either a source of hepcidin, or the effect of hepcidin on PAECfunction has not previously been described. ObjectiveTo define the role of the hepcidin-ferroportin axis on the phenotype of pulmonary artery endothelial cells Methods and resultsPAECs treated with hepcidin, or IL-6 were investigated for both ferroportin and hepcidin release and regulation with immunofluorescence, mRNA levels and cellular release assays. Effects of hepcidin on PASMC and PAEC mitochondrial function was investigated using immunofluorescence and seahorse assay. Migration and proliferation of PASMC treated with conditioned media from hPAEC treated with hepcidin was investigated using the Xcelligence system and other tools. PAECs express ferroportin; hepcidin treatment of PAECs results in mitochondrial iron accumulation and intracellular hepcidin biosynthesis and release. Conditioned media from hepcidin treated PAECs causes PASMCs to down regulate ferroportin expression whilst promoting migration and proliferation. Inhibition of hepcidin in PAEC conditioned media limits these responses. PASMC cellular and mitochondrial iron retention are associated with migratory and proliferative responses. ConclusionsThe Hepcidin-ferroportin axis is present and operational in PAECs. Modulation of this axis shows distinct differences in responses seen between PAECS and PASMCs. Stimulation of this axis in PAECS with hepcidin may well institute proliferative and migratory responses in PASMCs of relevance to pathogenesis of PAH offering a potential therapeutic target.

cell biology↗

bioRxiv2023

Hypoxic volatile metabolic markers in the MDA-MB-231 breast cancer cell line

Hypoxia in disease describes persistent low oxygen conditions, observed in a range of pathologies, including cancer. In the discovery of biomarkers in biological models, pathophysiological traits present a source of translatable metabolic products for the diagnosis of disease in humans. Part of the metabolome is represented by its volatile, gaseous fraction; the volatilome. Human volatile profiles, such as those found in breath, are able to diagnose disease, however accurate volatile biomarker discovery is required to target reliable biomarkers to develop new diagnostic tools. Using custom chambers to control oxygen levels and facilitate headspace sampling, the MDA-MB-231 breast cancer cell line was exposed to hypoxia (1% oxygen) for 24 hours. The maintenance of hypoxic conditions in the system was successfully validated over this time period. Targeting and non-targeting gas chromatography mass spectrometry approaches revealed four significantly altered volatile organic compounds when compared to control cells. Three compounds were actively consumed by cells: methyl chloride, acetone and n-Hexane. Cells under hypoxia also produced significant amounts of styrene. This work presents a novel methodology for identification of volatile metabolisms under controlled gas conditions with novel observations of volatile metabolisms by breast cancer cells.

cell biology↗

bioRxiv2021

Human pulmonary artery endothelial cells upregulate ACE2 expression in response to iron-regulatory elements: potential implications for SARS-CoV-2 infection of vascular endothelial cells.

Emerging studies from the ongoing covid-19 pandemic have implicated vascular dysfunction and endotheliitis in many patients presenting with severe disease. However, there is limited evidence for the expression of ACE2 (the principal co-receptor for Sars-Cov-2 cellular attachment) in vascular endothelial cells which has prompted the suggestion that the virus does not infect these cell types. However, the studies presented here demonstrate enhanced expression of ACE2 at the level of both mRNA and protein, in human pulmonary artery endothelial cells (PAECs) challenged with either IL-6 or hepcidin. Notably elevated levels both these iron-regulatory elements have been described in Covid-19 patients with severe disease and are further associated with morbidity and mortality. Additionally, levels of both IL-6 and hepcidin are often elevated in the elderly and in chronic disease settings, these populations being at greater risk of adverse outcomes from Sars-Cov-2 infection. A role for IL-6 and hepcidin as modulators of ACE2 expression seems plausible, additional, studies are required to determine if viral infection can be demonstrated in PAECs challenged with either of these iron-regulatory elements.

cell biology↗