Biology subjects

Steele, R.

Publications and source records attributed to Steele, R..

10 recordsLinked to original sources

bioRxiv2025

Hydra in Flow: Investigating the Flow Response of Hydra via Microfluidic Systems

Hydra, a simple freshwater cnidarian, occurs in both moving and still bodies of water. Flow and corresponding forces are ubiquitous factors in Hydras environment. Even in still water, brief exposure to a burst of flow (e.g., due to wind) can influence the surface attachment and dispersal of polyps. Additionally, alignment with flow may play a crucial role in minimizing forces and regulating feeding behaviors in Hydra. However, the response to flow (particularly in the presence of gravity) has remained underexplored. Using vertically oriented microfluidic chambers, we investigated the biomechanical response of Hydra vulgaris and two additional Hydra species to fluid flow. For Hydra vulgaris, strong surface attachment was observed for all flow rates (0 - 100 mL/hr, corresponding to average velocities of 0 - 2.2 mm/s for the chambers). The experiments indicated alignment of the body column with the flow direction at high flow rates ([≥] 50 mL/hr). Alignment with flow was examined by quantifying the angle between the organisms (using a vector connecting head-to-foot) and the flow direction for multiple individuals (N = 9). Most individuals of H. vulgaris exhibited alignment under high flow. While preliminary, comparisons with H. hymanae and H. oxycnida suggested species-specific differences H. hymanae showed tentacle deformation but lacked clear alignment. H. oxycnida did not remain attached under flow. Additional experiments explored the combined effects of flow and osmolarity. These results support the use of microfluidic tools to examine flow-related behaviors and highlight the potential for comparative biomechanics across Hydra species.

biophysics↗

bioRxiv2023

A new look at the architecture and dynamics of the Hydra nerve net

The Hydra nervous system is the paradigm of a "simple nerve net". Nerve cells in Hydra, as in many cnidarian polyps, are organized in a nerve net extending throughout the body column. This nerve net is required for control of spontaneous behavior: elimination of nerve cells leads to polyps that do not move and are incapable of capturing and ingesting prey (Campbell, 1976). We have re-examined the structure of the Hydra nerve net by immunostaining fixed polyps with a novel antibody that stains all nerve cells in Hydra. Confocal imaging shows that there are two distinct nerve nets, one in the ectoderm and one in the endoderm, with the unexpected absence of nerve cells in the endoderm of the tentacles. The nerve nets in the ectoderm and endoderm do not contact each other. High-resolution TEM and serial block face SEM show that the nerve nets consist of bundles of parallel overlapping neurites. Results from transgenic lines show that neurite bundles include different neural circuits and hence that neurites in bundles require circuit-specific recognition. Nerve cell-specific innexins indicate that gap junctions can provide this specificity. The occurrence of bundles of neurites supports a model for continuous growth and differentiation of the nerve net by lateral addition of new nerve cells to the existing net. This model was confirmed by tracking newly differentiated nerve cells.

neuroscience↗

bioRxiv2022

IRF7 impacts on prostate cancer cell survival in response to radiation

Understanding the impact of radiotherapy on the evolution of treatment resistant prostate cancer is critical for selecting effective treatment combinations. Whilst activation of Type 1 interferon signalling is a hallmark of how cells respond to viral infection, in cancer cells, multiple stresses are known to activate this same response. In this study we have evaluated for the first time the changes in the interferon response induced by culturing prostate cancer cells under sphere- forming conditions and following irradiation. We report a conserved upregulated transcript profile for both conditions that is strongly associated with therapeutic resistance and cell survival in vitro and in vivo. The profile includes and is regulated by the Type 1 interferon master regulator IRF7 which, when depleted, delays tumour re-growth following irradiation. We immuno-stained two independent prostate cohorts for IRF7 and found that increased expression, particularly in cases with low PTEN expression, correlated with poor prognosis. To more comprehensively characterise the impact of IRF7 and radiation on cells, RNA-Seq was performed on IRF7 knockdown cells at different radiation doses. We identified a number of biological processes that were IRF7-dependent, including the formation of stem-like cell populations and also therapeutic vulnerabilities. For example, irradiation sensitised surviving cells to either a combination of an IKK{varepsilon}/TBK1 and a MEK inhibitor or treatment with an inhibitor of IDO1, an IRF7- dependent gene. Translationally our work suggests that IRF7 expression can be used to stratify patients who may not benefit from receiving radiotherapy alone but rather may benefit from treatment combinations. In two cohorts treated with radical intent, strong IRF7 staining was associated with disease-specific death implicating this pathway as a convergence point for therapeutic resistance in prostate and potentially other cancer types.

cancer biology↗

bioRxiv2022

Structures of human Malic Enzyme 3

Malic enzymes (ME1, ME2, and ME3) are involved in cellular energy regulation, redox homeostasis, and biosynthetic processes, through the production of pyruvate and reducing agent NAD(P)H. Recent studies have implicated the third and least well-characterized isoform, mitochondrial NADP+-dependent malic enzyme 3 (ME3), as a therapeutic target for pancreatic cancers. Here, we utilized an integrated structure approach to capture the structures of ME3 in various ligand binding states. ME3 exists as a stable tetramer and its dynamic Domain C is critical for activity. Catalytic assay results reveal that ME3 is a non-allosteric enzyme and does not require modulators for activity while structural analysis suggests that the inner stability of ME3 domain A relative to ME2 disables allostery in ME3. With structural information available for all three malic enzymes, the foundation has been laid to understand the structural and biochemical differences of these enzymes and could aid in the development of specific malic enzyme small molecule drugs.

molecular biology↗

bioRxiv2022

An ancient role for the Hippo pathway in axis formation and morphogenesis

How did cells of early metazoan organisms first organize themselves to form a body axis? The canonical Wnt pathway has been shown to be sufficient for induction of axis in Cnidaria, a sister group to Bilateria, and is important in bilaterian axis formation. Here, we provide experimental evidence that in cnidarian Hydra the Hippo pathway regulates the formation of a new axis during budding upstream of the Wnt pathway. The target of Hippo pathway, the transcriptional co-activator YAP, inhibits the initiation of budding in Hydra, and is regulated by Hydra LATS. In addition, we show functions of Hippo pathway in regulation of actin organization and cell proliferation in Hydra. We hypothesize that Hippo pathway served as a link between continuous cell division, cell density and axis formation early in metazoan evolution.

developmental biology↗

bioRxiv2021

Exosomes from COVID-19 patients carry tenascin-C and fibrinogen-β in triggering inflammatory signals in distant organ cells

SARS-CoV-2 infection causes cytokine storm and overshoot immunity in humans; however, it remains to be determined whether genetic material of SARS-CoV-2 and/or virus induced soluble mediators from lung epithelial cells as natural host are carried out by macrophages or other vehicles at distant organs causing tissue damage. We speculated that exosomes as extracellular vesicles are secreted from SARS-CoV-2 infected cells may transport messages to other cells of distant organs leading to pathogenic consequences. For this, we took an unbiased proteomic approach for analyses of exosomes isolated from plasma of healthy volunteers and SARS-CoV-2 infected patients. Our results revealed that tenascin-C (TNC) and fibrinogen-{beta} (FGB) are highly abundant in exosomes from SARS-CoV-2 infected patients plasma as compared to that of healthy normal controls. Since TNC and FGB stimulate pro-inflammatory cytokines via NF-{kappa}B pathway, we examined the status of TNF-, IL-6 and CCL5 expression upon exposure of hepatocytes to exosomes from COVID-19 patients and observed significant increase when compared with that from healthy subjects. Together, our results demonstrated that soluble mediators, like TNC and FGB, are transported through plasma exosomes in SARS-CoV-2 infected patients and trigger pro-inflammatory cytokine expression in cells of distant organs in COVID-19 patients. ImportanceExosomes play an important role in intercellular communication by inducing physiological changes in recipient cells by transferring bioactive proteins. Little is known about exosomes from SARS-CoV-2 infected cells and their role in pathogenesis. Here, we have carefully examined and analyzed this aspect of SARS-CoV-2 infection. Our results uncovered the potential mechanisms by which SARS-CoV-2 communicates with other cells of distant organs and promotes pathogenesis. We expect to detect whether other factors are modulated in the presence of COVID-19 exosomes. Our exosomes related proteomic experiments prioritize after initial verification to further examine their role in SARS-CoV-2 associated other pathogenic mechanisms to target for therapeutic modalities.

physiology↗

bioRxiv2020

Development of a novel allograft model of prostate cancer: a new tool to inform clinical translation

BackgroundAllograft models enable characterisation of genomic drivers and treatment responses by modelling immune and micro-environmental changes more accurately than xenografts. Despite this, few models are available to the prostate cancer (PCa) community. This study presents a novel allograft model of high-risk, localised PCa. In characterising this model we have focused on its response to radiotherapy (RT). MethodsThe DVL3 cell line was derived from a transgenic trp53-/-/Pten-/- mouse model and characterised both in vitro and in vivo. The DVL3 cells were allografted and response to RT was investigated and compared to the TRAMP-C1 model. Extensive tumour profiling in the DVL3 model was performed using flow cytometry, immunohistochemistry and RNA-seq. ResultsIn vitro the DVL3 cells expressed basal and luminal markers. DVL3 cells formed tumours with distinct glandular morphology which expressed androgen receptor, similar to human localised PC. DVL3 tumour growth was delayed following administration of fractionated RT, with infiltration of myeloid derived suppressor cells (MDSC). ConclusionsThe DVL3 allograft model represents substantial progress in PCa modelling, displaying; luminal differentiation, strong AR expression, and an immunosuppressive microenvironment, similar to observations in high-risk PCa patients. This model is ideally suited for development and validation of novel therapeutics, in particular immune-modulatory agents in combination with RT.

cancer biology↗

bioRxiv2020

Wnt signaling determines body axis polarity in regenerating Hydra tissue

How an animal establishes its body axis is a fundamental question in developmental biology. The freshwater cnidarian Hydra is an attractive model for studying axis formation because it is radially symmetric, with a single oral-aboral axis. It was recently proposed that the orientation of the new body axis in a regenerating Hydra is determined by the oral-aboral orientation of the actin-myosin contractile processes (myonemes) in the parent animals outer epithelial layer. However, because the myonemes are not known to possess polarity, it remained unclear how the oral-aboral polarity of the axis would be defined. As Wnt signaling is known to maintain axis polarity in Hydra and bilaterians, we hypothesized that it plays a role in axis specification in excised Hydra tissue pieces. We tested this hypothesis using pharmacological perturbations and novel grafting experiments to set Wnt-derived signals and myoneme orientation perpendicular to each other to determine which controls axis formation. Our results demonstrate that Wnt signaling is the dominant encoder of axis information, in line with its highly conserved role in anterior-posterior patterning.

developmental biology↗

bioRxiv2019

Modulating the unfolded protein response: Impacts of radiation on the response of prostate cancer cells to ONC201

Prostate cancer (PCa) is the most common non-cutaneous cancer in men and a notable cause of cancer mortality when it metastasises. Localised disease is mostly treated with surgery or radiotherapy. As PCa develops and treatment resistance emerges, the unfolded protein response (UPR) arises as an important adaptive biology co-amplifying with key cancer drivers [1]. The UPR can be cytoprotective but when acutely activated can lead to cell death. In this study we sought to enhance the acute activation of the UPR using radiation and ONC201, previously reported to be an UPR activator [2]. We found that treating PCa cells with ONC201 quickly increases the expression of components in all arms of the UPR - ATF4, ATF6 and IRE1-XBP1 - culminating in the subsequent cell death. During this time window between UPR activation and cell death we tested the priming effect of short-term administration of ONC201 on radiation responses. Pre-treatment with ONC201 for 24 hours prior to irradiation led to enhanced cytotoxicity compared to radiation alone assessed by cell viability and clonogenic assays. With priming, RNA-Seq analysis showed a sustained suppression of transcripts encoding cell cycle regulators as well as components of the DNA damage response pathways. Phenotypically this was reflected in enhanced cell cycle arrest and induction of necrosis and apoptosis. Furthermore, we demonstrated that short-term administration of inhibitors of cell cycle regulators (Dinaciclib and BI2536), could replicate this priming effect. Thus, we propose future studies to assess the impact of the short-term administration of drugs targeting the UPR and cell cycle regulation to enhance radiotherapy response.

cancer biology↗

bioRxiv2019

High-throughput propagation of human prostate tissue from induced-pluripotent stem cells

Primary culture of human prostate organoids is slow, inefficient and laborious. To overcome this, we demonstrate a new high-throughput model where rapidly proliferating and easily handled induced pluripotent stem cells, for the first time, enable generation of human prostate tissue in vivo and in vitro. Using a co-culture technique with urogenital sinus mesenchyme, we recapitulated the in situ prostate histology, including the stromal compartment and the full spectrum of epithelial differentiation. This approach overcomes major limitations in primary cultures of human prostate stem, luminal and neuroendocrine cells, as well as the stromal microenvironment. These models provide new opportunities to study prostate development, homeostasis and disease.

developmental biology↗