supervised-machine-learning-for-semi-quantification-extracellular-dna

Supervised Machine Learning for Semi-Quantification of Extracellular DNA in Glomerulonephritis

Glomerular cell death is a pathological feature of myeloperoxidase anti neutrophil cytoplasmic antibody associated vasculitis (MPO-AAV). Extracellular ...

Monash University

For efficient metastatic dissemination, tumor cells form invadopodia to degrade and move through three-dimensional extracellular matrix. However, little is known about the conditions that favor invadopodia formation. Here, we investigated the effect of β-adrenoceptor signaling - which allows cells to respond to stress neurotransmitters - on the formation of invadopodia and examined the effect on tumor cell invasion.

β2-adrenoceptor signaling regulates invadopodia formation to enhance tumor cell invasion.

Chronic stress accelerates metastasis - the main cause of death in cancer patients - through the activation of β-adrenoceptors (βARs). We have previously shown that β2AR signaling in MDA-MB-231(HM) breast cancer cells, facilitates invadopodia formation and invasion in vitro. However, in the tumor microenvironment where many stromal cells also express βAR, the role of β2AR signaling in tumor cells in metastasis is unclear. Therefore, to investigate the contribution of β2AR signaling in tumor cells to metastasis in vivo, we used RNA interference to generate MDA-MB-231(HM) breast cancer cells that are deficient in β2AR. β2AR knockdown in tumor cells reduced the proportion of cells with a mesenchymal-like morphology and, as expected, reduced tumor cell invasion in vitro. Conversely, overexpression of β2AR in low metastatic MCF-7 breast cancer cells induced an invasive phenotype. Importantly, we found that knockdown of β2AR in tumor cells significantly reduced the impact of stress on metastasis in vivo. These findings highlight a crucial role for β2AR tumor cell signaling in the adverse effects of stress on metastasis, and indicate that it may be necessary to block β2AR on tumor cells to fully control metastatic progression. 

β2-Adrenoceptors on tumor cells play a critical role in stress-enhanced metastasis in a mouse model of breast cancer.

Mitochondria and mitochondrial dynamics play vital roles in health and disease. With the intricate nanometer-scale structure and rapid dynamics of mitochondria, super-resolution microscopy techniques possess great un-tapped potential to significantly contribute to understanding mitochondrial biology and kinetics. Here we present a novel mitochondrial probe (MitoRed AIE) suitable for live mitochondrial dynamics imaging and single particle tracking (SPT), together with a multi-dimensional data analysis approach to assess local mitochondrial (membrane) fluidity. The MitoRed AIE probe localizes primarily to mitochondrial membranes, with 95 ms fluorophore on-time delivering 106 photons/ms, characteristics which we exploit to demonstrate live cell 100 fps 3D time-lapse tracking of mitochondria. Combining our experimental and analytical approaches, we uncover mitochondrial dynamics at unprecedented time scales. This approach opens up a new regime into high spatio-temporal resolution dynamics in many areas of mitochondrial biology.

Novel super-resolution capable mitochondrial probe, MitoRed AIE, enables assessment of real-time molecular mitochondrial dynamics.

Helicobacter pylori (H. pylori) causes chronic inflammation which is a key precursor to gastric carcinogenesis. It has been suggested that H. pylori may limit this immunopathology by inducing the production of interleukin 33 (IL-33) in gastric epithelial cells, thus promoting T helper 2 immune responses. The molecular mechanism underlying IL-33 production in response to H. pylori infection, however, remains unknown. In this study, we demonstrate that H. pylori activates signalling via the pathogen recognition molecule Nucleotide-Binding Oligomerisation Domain-Containing Protein 1 (NOD1) and its adaptor protein receptor-interacting serine-threonine Kinase 2, to promote production of both full-length and processed IL-33 in gastric epithelial cells. Furthermore, IL-33 responses were dependent on the actions of the H. pylori Type IV secretion system, required for activation of the NOD1 pathway, as well as on the Type IV secretion system effector protein, CagA. Importantly, Nod1 mice with chronic H. pylori infection exhibited significantly increased gastric IL-33 and splenic IL-13 responses, but decreased IFN-γ responses, when compared with Nod1 animals. Collectively, our data identify NOD1 as an important regulator of mucosal IL-33 responses in H. pylori infection. We suggest that NOD1 may play a role in protection against excessive inflammation.

NOD1 is required for Helicobacter pylori induction of IL-33 responses in gastric epithelial cells.

Macrophage migration inhibitory factor (MIF) exerts multiple effects on immune cells, as well as having functions outside the immune system. MIF can promote inflammation through the induction of other cytokines, including TNF, IL-6, and IL-1 family cytokines. Here, we show that inhibition of MIF regulates the release of IL-1α, IL-1β, and IL-18, not by affecting transcription or translation of these cytokines, but via activation of the NLRP3 inflammasome. MIF is required for the interaction between NLRP3 and the intermediate filament protein vimentin, which is critical for NLRP3 activation. Further, we demonstrate that MIF interacts with NLRP3, indicating a role for MIF in inflammasome activation independent of its role as a cytokine. These data advance our understanding of how MIF regulates inflammation and identify it as a factor critical for NLRP3 inflammasome activation.

Macrophage migration inhibitory factor is required for NLRP3 inflammasome activation.

The mitochondrial membrane potential (Δψ) drives the generation of ATP by mitochondria. Interestingly, Δψ is higher in many cancer cells comparted to healthy noncancerous cell types, providing a unique metabolic marker. This feature has also been exploited for therapeutic use by utilizing drugs that specifically accumulate in the mitochondria of cancer cells with high Δψ. As such, the assessment of Δψ can provide very useful information as to the metabolic state of a cancer cell, as well as its potential for malignancy. In addition, the measurement of Δψ can also be used to test the ability of novel anticancer therapies to disrupt mitochondrial metabolism and cause cell death.Here, we outline two methods for assessing Δψ in cancer cells using confocal microscopy and the potentiometric fluorescent dye tetramethylrhodamine methyl ester (TMRM). In the first protocol, we describe a technique to quantitatively measure Δψ, which can be used to compare Δψ between different cell types. In the second protocol, we describe a technique for assessing changes to Δψ over time, which can be used to determine the effectiveness of different therapeutic compounds or drugs in modulating mitochondrial function.

Measurement of Mitochondrial Membrane Potential with the Fluorescent Dye Tetramethylrhodamine Methyl Ester (TMRM).

dsRNA is a common by-product of viral replication and acts as a potent trigger of antiviral immunity. SIDT1 and SIDT2 are closely related members of the SID-1 transmembrane family. SIDT2 functions as a dsRNA transporter and is required to traffic internalized dsRNA from endocytic compartments into the cytosol for innate immune activation, but the role of SIDT1 in dsRNA transport and in the innate immune response to viral infection is unclear. In this study, we show that  expression is upregulated in response to dsRNA and type I IFN exposure and that SIDT1 interacts with SIDT2. Moreover, similar to SIDT2, SIDT1 localizes to the endolysosomal compartment, interacts with the long dsRNA analog poly(I:C), and, when overexpressed, enhances endosomal escape of poly(I:C) in vitro. To elucidate the role of SIDT1 in vivo, we generated SIDT1-deficient mice. Similar to  mice, SIDT1-deficient mice produced significantly less type I IFN following infection with HSV type 1. In contrast to  mice, however, SIDT1-deficient animals showed no impairment in survival postinfection with either HSV type 1 or encephalomyocarditis virus. Consistent with this, we observed that, unlike SIDT2, tissue expression of SIDT1 was relatively restricted, suggesting that, whereas SIDT1 can transport extracellular dsRNA into the cytoplasm following endocytosis in vitro, the transport activity of SIDT2 is likely to be functionally dominant in vivo.

SIDT1 Localizes to Endolysosomes and Mediates Double-Stranded RNA Transport into the Cytoplasm.

Fluorescence microscopy has become a powerful tool to investigate proteins in their natural environment. Well-established techniques like widefield and confocal fluorescence microscopy have commonly been used for decades to visualize biomolecules in single cells and tissue sections. Live cell microscopy allows for the investigation of biomolecular trafficking, and other specialized techniques, such as proximity ligation assays (PLA) and fluorescence lifetime imaging microscopy (FLIM), can be used to study interactions between biomolecules of interest. Finally, with the most recent rise of optical super-resolution microscopy, we can investigate target biomolecules in situ with unprecedented detail on the nanometer scale. Here, we discuss various optical microscopy techniques that have successfully been used to image MIF. We highlight applications, advantages, and limitations of each technique. The techniques described here can easily be adapted to investigate other target proteins, their localization, interaction partners, and mechanisms of action.

Article	Year
β2-adrenoceptor signaling regulates invadopodia formation to enhance tumor cell invasion. Breast cancer research : BCR\| PubMed ID: 26607426	2015
β2-Adrenoceptors on tumor cells play a critical role in stress-enhanced metastasis in a mouse model of breast cancer. Brain, behavior, and immunity\| PubMed ID: 27321906	2016
Novel super-resolution capable mitochondrial probe, MitoRed AIE, enables assessment of real-time molecular mitochondrial dynamics. Scientific reports\| PubMed ID: 27492961	2016
NOD1 is required for Helicobacter pylori induction of IL-33 responses in gastric epithelial cells. Cellular microbiology\| PubMed ID: 29392836	2018
Macrophage migration inhibitory factor is required for NLRP3 inflammasome activation. Nature communications\| PubMed ID: 29884801	2018
Measurement of Mitochondrial Membrane Potential with the Fluorescent Dye Tetramethylrhodamine Methyl Ester (TMRM). Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 30725451	2019
SIDT1 Localizes to Endolysosomes and Mediates Double-Stranded RNA Transport into the Cytoplasm. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 31061008	2019
Microscopy Methods for Imaging MIF and Its Interaction Partners. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 31745874	2020

Sarah Creed

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