Biology subjects

Sweeney, S. T.

Publications and source records attributed to Sweeney, S. T..

7 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↗

bioRxiv2023

Identification of GEFs and GAPS modulating phosphorylation and abundance of Rab10 by LRRK2-G2019S in neurons

The Parkinson s Disease associated kinase LRRK2 is proposed to act through phosphorylation of Rab proteins, most notably Rab10. All Rabs participate in a GTPase cycle, in which GEFs (Guanine nucleotide exchange factors) promote membrane binding, and GAPs (GTPase-activating proteins) promote release into the cytoplasm. LRRK2 is proposed to phosphorylate membrane bound Rab10. The hypothesis is that phosphoRab10 is less sensitive to GAP action and may remain membrane bound. To test how LRRK2 and Rab10 function in the GTPase cycle, we used an immunoblotting strategy in fly brains to show that a putative GEF Crag/DENND4C and three possible GAPs (pollux (plx), GAPcenA and Evi5, orthologs of AS160) interact with LRRK2 controlling the phosphorylation and abundance of Rab10. Crag behaves similarly to a Rab10 GEF and additionally modulates the level of panRab10. Only plx acts as a conventional GAP. GAPcenA seems to act as a GAP for phosphoRab10 more than panRab10. It is likely that Evi5 acts as a GAP for another Rab, possibly Rab11.

neuroscience↗

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

The Drosophila TMEM184B ortholog Tmep ensures proper locomotion by restraining ectopic firing at the neuromuscular junction

TMEM184B is a putative seven-pass membrane protein that promotes axon degeneration after injury. TMEM184B mutation causes aberrant neuromuscular architecture and sensory and motor behavioral defects in mice. The mechanism through which TMEM184B causes neuromuscular defects is unknown. We employed Drosophila melanogaster to investigate the function of the TMEM184B ortholog, Tmep (CG12004) at the neuromuscular junction. We show that Tmep is required for full adult viability and efficient larval locomotion. Tmep mutant larvae have a reduced body contraction rate compared to controls, with stronger deficits in females. Surviving adult Tmep mutant females show "bang sensitivity," a phenotype associated with epileptic seizures. In recordings from body wall muscles, Tmep mutants show substantial hyperexcitability, with many post-synaptic potentials fired in response to a single stimulation, consistent with a role for Tmep in restraining synaptic excitability. Neuromuscular junctions in Tmep mutants show modest structural defects and satellite boutons, which could also contribute to poor locomotor performance. Tmep is expressed in endosomes and synaptic vesicles within motor neurons, suggesting a possible role in synaptic membrane trafficking. Using RNAi knockdown, we show that Tmep is required in motor neurons for proper larval locomotion and excitability. Locomotor defects can be rescued by presynaptic knock-down of endoplasmic reticulum calcium channels or by reducing evoked release probability, suggesting that excess synaptic activity drives behavioral deficiencies. Our work establishes a critical function for the TMEM184B ortholog Tmep in the regulation of synaptic transmission and locomotor behavior.

neuroscience↗

bioRxiv2020

JNK signalling regulates antioxidant responses in neurons

Reactive oxygen species (ROS) are generated during physiological bouts of synaptic activity and as a consequence of pathological conditions in the central nervous system. How neurons respond to and distinguish between ROS in these different contexts is currently unknown. In Drosophila mutants with enhanced JNK activity, lower levels of ROS are observed and these animals are resistant to both changes in ROS and changes in synapse morphology induced by oxidative stress. In wild type flies, disrupting JNK-AP-1 signalling perturbs redox homeostasis suggesting JNK activity positively regulates neuronal antioxidant defense. We validated this hypothesis in mammalian neurons, finding that JNK activity regulates the expression of the antioxidant gene Srxn-1, in a c-Jun dependent manner. We describe a conserved adaptive role for neuronal JNK in the maintenance of redox homeostasis that is relevant to several neurodegenerative diseases.

neuroscience↗

bioRxiv2016

Reactive Oxygen Species Regulate Activity-Dependent Neuronal Structural Plasticity

Neurons are inherently plastic, adjusting their structure, connectivity and excitability in response to changes in activity. How neurons sense changes in their activity level and then transduce these to structural changes remains to be fully elucidated. Working with the Drosophila larval locomotor network, we show that neurons use reactive oxygen species (ROS), metabolic byproducts, to monitor their activity. ROS signals are both necessary and sufficient for activity-dependent structural adjustments of both pre- and postsynaptic terminals and for network output, as measured by larval crawling behavior. We find the highly conserved Parkinsons disease-linked protein DJ-1{beta} acts as a redox sensor in neurons where it regulates pre- and postsynaptic structural plasticity, in part via modulation of the PTEN-PI3Kinase pathway. Neuronal ROS thus play an important physiological role as second messengers required for neuronal and network tuning, whose dysregulation in the ageing brain and under neurodegenerative conditions may contribute to synaptic dysfunction.

neuroscience↗