Droop, A. P. | Search bioRxiv

bioRxiv2026

Adapting the OpenFlexure Microscope for Affordable Live-Cell Imaging

Live-cell imaging (LCI) captures dynamic cellular behaviours missed in fixed samples. Researchers in resource-limited settings are underrepresented in the adoption of LCI due to the prohibitive cost of dedicated systems. We adapted the 3D-printed OpenFlexure Microscope (OFM) for LCI in humid tissue culture incubators. We relocated electronics to prevent corrosion, and printed in acrylonitrile styrene acrylate (ASA) to improve image stability in prolonged experiments. Current-limiting resistors reduced heat buildup inside sealed incubators, while our simple graphical user interface (GUI)-based application enabled rapid setup of timelapse experiments. Using a 48-hour docetaxel treatment of breast cancer cells, we validated how the LCI-OFM seamlessly fits into established bioimaging pipelines to generate biologically meaningful data. We further demonstrated broad applicability of the LCI-OFM by imaging Leishmania-infected macrophages in a containment level 3 laboratory and gut fungi in an anaerobic environment. This accessible platform expands opportunities for resource-limited researchers to study locally-relevant health challenges.

cell biology↗

bioRxiv2020

Oncolytic virus treatment differentially affects the CD56dim and CD56bright NK cell subsets in vivo and regulates a spectrum of human NK cell activity

Natural killer (NK) cells protect against intracellular infection and cancer. These properties are exploited in oncolytic virus (OV) therapy, where anti-viral responses enhance anti-tumour immunity. We have analysed the mechanism by which reovirus, an oncolytic dsRNA virus, modulates human NK cell activity. Reovirus activates NK cells in a type I interferon (IFN-I) dependent manner, resulting in STAT1 and STAT4 signalling in both CD56dim and CD56bright NK cell subsets. Gene expression profiling revealed the dominance of IFN-I responses and identified induction of genes associated with NK cell cytotoxicity and cell cycle progression, with distinct responses in the CD56dim and CD56bright subsets. However, reovirus treatment, acting via IFN-I, inhibited NK cell proliferative responses to IL-15 and was associated with reduced AKT signalling. In vivo, human CD56dim and CD56bright NK cells responded with similar kinetics to reovirus treatment, but CD56bright NK cells were transiently lost from the peripheral circulation at the peak of the IFN-I response, suggestive of their redistribution to secondary lymphoid tissue. These results show that reovirus modulates a spectrum of NK cell activity in vivo, encompassing direct action on tumour cells and the regulation of adaptive immunity. Such activity is likely to mirror NK cell responses to natural viral infection.

immunology↗

bioRxiv2019

A high-content screen profiles cytotoxic microRNAs in pediatric and adult glioblastoma cells and identifies miR-1300 as a potent inducer of cytokinesis failure

BackgroundMicroRNAs play an important role in the regulation of mRNA translation, and have therapeutic potential in cancer and other diseases.\n\nMethodsTo profile the landscape of microRNAs with significant cytotoxicity in the context of glioblastoma (GBM), we performed a high-throughput screen using a synthetic oligonucleotide library representing all known human microRNAs in adult and pediatric GBM cells. Bio-informatics analysis were used to refine this list and the top seven microRNAs were validated in a larger panel of cells by flow-cytometry, and RTqPCR. The downstream mechanism of the strongest and most consistent candidate was investigated by siRNAs, 3UTR luciferase assays and Western Blotting.\n\nResultsOur screen identified [~]100 significantly cytotoxic microRNAs with 70% concordance between cell lines. MicroRNA-1300 (miR-1300) was the most potent and robust candidate. We observed a striking binucleated phenotype in miR-1300 expressing cells and characterized the mechanism of action as cytokinesis failure followed by apoptosis, which was observed in an extended GBM cell panel including two stem-like patient-derived cultures. We identified the physiological role of miR-1300 as a regulator of endomitosis in megakaryocyte differentiation where blockade of cytokinesis is an essential step. In glioblastoma cells, the oncogene Epithelial Cell Transforming 2 (ECT2) was validated as a direct key target of miR-1300. ECT2 siRNA phenocopied the effects of miR-1300, and its overexpression led to a significant rescue of miR-1300 induced binucleation.\n\nConclusionMiR-1300 was identified as a novel regulator of endomitosis with translatable potential for therapeutic application. The datatasets will be a resource for the neuro-oncology community.\n\nKey points (2 or 3 key points 85 characters plus spaces each)70% of cytotoxic microRNAs were shared between adult and pediatric glioblastoma cells\n\nMiR-1300 expression is restricted to endomitosis within megakaryocyte differentiation\n\nMiR-1300s ectopic expression is a potent and promising therapeutic tool in cancer\n\nImportance of StudyPrevious functional studies of microRNAs involved in the regulation of glioblastoma cell proliferation and/or survival have focused on adult glioblastoma alone and are restricted to only a few microRNAs at a time. Our study provides the first encompassing landscape of potent cytotoxic microRNAs in pediatric and adult glioblastoma.\n\nNot only, does our data provide an invaluable resource for the research community but it also revealed that 70% of microRNAs with significant cytotoxicity were shared by adult and pediatric cells. Finally, we identified and characterized the previously undescribed role of microRNA-1300 in the tight regulation of megakaryocyte differentiation into platelets and how, when expressed outside of this context, miR-1300 consistently causes cytokinesis failure followed by apoptosis, and thus represents a powerful cytotoxic tool with potential for translation towards therapeutic applications.

cancer biology↗