Biocompatibility characterisation of CMOS-based Lab-on-Chip electrochemical sensors for in vitro cancer cell culture applications.

Lab-on-Chip electrochemical sensors, such as Ion-Sensitive Field-Effect Transistors (ISFETs), are being developed for use in point-of-care diagnostics, such as pH detection of tumour microenvironments, due to their integration with standard Complementary Metal Oxide Semiconductor (CMOS) technology. With this approach, the passivation of the CMOS process is used as a sensing layer to minimise post-processing, and Silicon Nitride (SiN) is the most common material at the microchip surface. ISFETs have the potential to be used for cell-based assays however, there is a poor understanding of the biocompatibility of microchip surfaces.

Insights from multi-omic modeling of neurodegeneration in xeroderma pigmentosum using an induced pluripotent stem cell system.

Xeroderma pigmentosum (XP) is caused by defective nucleotide excision repair of DNA damage. This results in hypersensitivity to ultraviolet light and increased skin cancer risk, as sunlight-induced photoproducts remain unrepaired. However, many XP patients also display early-onset neurodegeneration, which leads to premature death.

Environmentally dependent and independent control of 3D cell shape.

How cancer cells determine their shape in response to three-dimensional (3D) geometric and mechanical cues is unclear. We develop an approach to quantify the 3D cell shape of over 60,000 melanoma cells in collagen hydrogels using high-throughput stage-scanning oblique plane microscopy (ssOPM). We identify stereotypic and environmentally dependent changes in shape and protrusivity depending on whether a cell is proximal to a flat and rigid surface or is embedded in a soft environment.

Actin nucleators safeguard replication forks by limiting nascent strand degradation.

Accurate genome replication is essential for all life and a key mechanism of disease prevention, underpinned by the ability of cells to respond to replicative stress (RS) and protect replication forks. These responses rely on the formation of Replication Protein A (RPA)-single stranded (ss) DNA complexes, yet this process remains largely uncharacterized. Here, we establish that actin nucleation-promoting factors (NPFs) associate with replication forks, promote efficient DNA replication and facilitate association of RPA with ssDNA at sites of RS.

Actin nucleators safeguard replication forks by limiting nascent strand degradation.

Accurate genome replication is essential for all life and a key mechanism of disease prevention, underpinned by the ability of cells to respond to replicative stress (RS) and protect replication forks. These responses rely on the formation of Replication Protein A (RPA)-single stranded (ss) DNA complexes, yet this process remains largely uncharacterized. Here we establish that actin nucleation-promoting factors (NPFs) associate with replication forks, promote efficient DNA replication and facilitate association of RPA with ssDNA at sites of RS.

Quantitative imaging of single-cell phenotypes in cancer cells cultured on hydrogel surfaces.

Small interfering RNA (siRNA) screening approaches used with quantitative single-cell analysis can uncover the roles of genes in cell morphogenesis. Here, we present a high-throughput automated phenotypic screening technique to quantify a single cell shape in cancer cells cultured on top of soft 3D hydrogels. We describe reverse transfection of cells with siRNAs and seeding of these cells on top of collagen, followed by image analysis to quantify morphology of a single cell and population levels in low-elasticity matrices.

ARHGEF9 regulates melanoma morphogenesis in environments with diverse geometry and elasticity by promoting filopodial-driven adhesion.

Rho GTP Exchange Factors (RhoGEFs) and Rho GTPase Activating Proteins (RhoGAPs) are large families of molecules that regulate shape determination in all eukaryotes. In pathologies such as melanoma, RhoGEF and RhoGAP activity underpins the ability of cells to invade tissues of varying elasticity. To identify RhoGEFs and RhoGAPs that regulate melanoma cell shape on soft and/or stiff materials, we performed genetic screens, in tandem with single-cell quantitative morphological analysis.

Robust deep learning optical autofocus system applied to automated multiwell plate single molecule localization microscopy.

We presenta robust, long-range optical autofocus system for microscopy utilizing machine learning. This can be useful for experiments with long image data acquisition times that may be impacted by defocusing resulting from drift of components, for example due to changes in temperature or mechanical drift. It is also useful for automated slide scanning or multiwell plate imaging where the sample(s) to be imaged may not be in the same horizontal plane throughout the image data acquisition.

A high-content RNAi screen reveals multiple roles for long noncoding RNAs in cell division.

Genome stability relies on proper coordination of mitosis and cytokinesis, where dynamic microtubules capture and faithfully segregate chromosomes into daughter cells. With a high-content RNAi imaging screen targeting more than 2,000 human lncRNAs, we identify numerous lncRNAs involved in key steps of cell division such as chromosome segregation, mitotic duration and cytokinesis. Here, we provide evidence that the chromatin-associated lncRNA, linc00899, leads to robust mitotic delay upon its depletion in multiple cell types.

WDR5, BRCA1, and BARD1 Co-regulate the DNA Damage Response and Modulate the Mesenchymal-to-Epithelial Transition during Early Reprogramming.

Differentiated cells are epigenetically stable, but can be reprogrammed to pluripotency by expression of the OSKM transcription factors. Despite significant effort, relatively little is known about the cellular requirements for reprogramming and how they affect the properties of induced pluripotent stem cells. We have performed high-content screening with small interfering RNAs targeting 300 chromatin-associated factors and extracted colony-level quantitative features.

RNAi screens for Rho GTPase regulators of cell shape and YAP/TAZ localisation in triple negative breast cancer.

In order to metastasise, triple negative breast cancer (TNBC) must make dynamic changes in cell shape. The shape of all eukaryotic cells is regulated by Rho Guanine Nucleotide Exchange Factors (RhoGEFs), which activate Rho-family GTPases in response to mechanical and informational cues. In contrast, Rho GTPase-activating proteins (RhoGAPs) inhibit Rho GTPases.

Apolar and polar transitions drive the conversion between amoeboid and mesenchymal shapes in melanoma cells.

Melanoma cells can adopt two functionally distinct forms, amoeboid and mesenchymal, which facilitates their ability to invade and colonize diverse environments during the metastatic process. Using quantitative imaging of single living tumor cells invading three-dimensional collagen matrices, in tandem with unsupervised computational analysis, we found that melanoma cells can switch between amoeboid and mesenchymal forms via two different routes in shape space--an apolar and polar route. We show that whereas particular Rho-family GTPases are required for the morphogenesis of amoeboid and mesenchymal forms, others are required for transitions via the apolar or polar route and not amoeboid or mesenchymal morphogenesis per se.

Signaling networks converge on TORC1-SREBP activity to promote endoplasmic reticulum homeostasis.

The function and capacity of the endoplasmic reticulum (ER) is determined by multiple processes ranging from the local regulation of peptide translation, translocation, and folding, to global changes in lipid composition. ER homeostasis thus requires complex interactions amongst numerous cellular components. However, describing the networks that maintain ER function during changes in cell behavior and environmental fluctuations has, to date, proven difficult.

Cross-talk between Rho and Rac GTPases drives deterministic exploration of cellular shape space and morphological heterogeneity.

One goal of cell biology is to understand how cells adopt different shapes in response to varying environmental and cellular conditions. Achieving a comprehensive understanding of the relationship between cell shape and environment requires a systems-level understanding of the signalling networks that respond to external cues and regulate the cytoskeleton. Classical biochemical and genetic approaches have identified thousands of individual components that contribute to cell shape, but it remains difficult to predict how cell shape is generated by the activity of these components using bottom-up approaches because of the complex nature of their interactions in space and time.

Differential RNAi screening provides insights into the rewiring of signalling networks during oxidative stress.

Reactive Oxygen Species (ROS) are a natural by-product of cellular growth and proliferation, and are required for fundamental processes such as protein-folding and signal transduction. However, ROS accumulation, and the onset of oxidative stress, can negatively impact cellular and genomic integrity. Signalling networks have evolved to respond to oxidative stress by engaging diverse enzymatic and non-enzymatic antioxidant mechanisms to restore redox homeostasis.

The thiazolidinedione pioglitazone increases cholesterol biosynthetic gene expression in primary cortical neurons by a PPARgamma-independent mechanism.

In a recent clinical study, the thiazolidinedione (TZD) pioglitazone (Actos was reported to preserve cognitive function in patients with mild to moderate Alzheimer's disease and type II diabetes mellitus. TZDs are agonists of the nuclear hormone receptor peroxisome proliferator-activated receptor-gamma (PPARgamma), are peripheral insulin sensitizers, and have recently been reported to increase mitochondrial biogenesis in the central nervous system and dendritic spine density. We report a transcriptional profile of the TZD pioglitazone and the non-TZD PPARgamma agonist GW347845 in primary cortical culture.