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Researchers have found a way to identify multiple cell signalling proteins using a single cell rather than the billions of cells used previously.
The new measurement technology, developed by researchers at the ARC Centre of Excellence for Nanoscale Biophotonics, brings precision medicine a step closer.
“Cells secrete various messenger molecules, such as cytokines. They may indicate the presence of a disease or act as a driver of key therapeutic effects,” says Dr Guozhen Liu, lead author of paper detailing the technology.
The method, termed OnCELISA, uses antibodies attached on specially engineered cell surfaces to capture cytokine molecules before they have a chance to disperse away from the cell.
The secreted messenger proteins such as cytokines are reported, at the single cell level, by using fluorescent magnetic nanoparticles.
Cytokines secreted from cells play a critical role in controlling many physiological functions, including immunity, inflammation, response to cancer, and tissue repair.
The OnCELISA system can be used for ultrasensitive monitoring of cytokine release by individual cells, and it can also help discover cell populations with therapeutic value.
“The ability to identify and select cell populations based on their cytokine release is particularly valuable in commercial cell technologies and it can help develop unique products, such as future non-opioid pain relief” says Dr Liu.
“Importantly, our design uses commercially available reagents only, so it can be easily reproduced by others,” she adds.
While the published work focuses on specific proinflammatory cytokines IL-6 and IL-1β, the method is potentially suitable for a broad range of other secreted proteins and cell types.
The new technique represents an advance on traditional methods such as the enzyme-linked immunosorbent assays (ELISA) that detect average levels of secreted molecules from cell ensembles.
The OnCELISA takes the ELISA approach to its absolute extreme, by detecting cytokines on the surface of individual, single live cells.
The publication has been reported by prestigious iScience journal and can be found at https://www.sciencedirect.com/science/article/pii/S2589004219303578.
Publication Title: A Nanoparticle-Based Affinity Sensor that Identifies and Selects Highly Cytokine-Secreting Cells
Authors: Guozhen Liu; Christina Bursill; Siân P.Cartland; Ayad G.Anwer; Lindsay M.Parker; Kaixin Zhang; Shilun Feng; Meng He; David W.Inglis; Mary M.Kavurma; Mark R.Hutchinson; Ewa M.Goldys
Summary: We developed a universal method termed OnCELISA to detect cytokine secretion from individual cells by applying a capture technology on the cell membrane. OnCELISA uses fluorescent magnetic nanoparticles as assay reporters that enable detection on a single-cell level in microscopy and flow cytometry and fluorimetry in cell ensembles. This system is flexible and can be modified to detect different cytokines from a broad range of cytokine-secreting cells. Using OnCELISA we have been able to select and sort highly cytokine-secreting cells and identify cytokine-secreting expression profiles of different cell populations in vitro and ex vivo. We show that this system can be used for ultrasensitive monitoring of cytokines in the complex biological environment of atherosclerosis that contains multiple cell types. The ability to identify and select cell populations based on their cytokine expression characteristics is valuable in a host of applications that require the monitoring of disease progression.
CNBP scientists Chris Ashwood (pictured) and Prof Nicki Packer at Macquarie University have shown that sugars with exactly the same chemical composition but slightly different structure break apart differently in their latest publication in the area of mass spectrometry. This work is their first step in automating sugar analysis, to understand the role sugars play in human disease.
Journal: Journal of The American Society for Mass Spectrometry.
Authors: Christopher Ashwood, Chi-Hung Lin, Morten Thaysen-Andersen, Nicolle H. Packer.
Profiling cellular protein glycosylation is challenging due to the presence of highly similar glycan structures that play diverse roles in cellular physiology. As the anomericity and the exact linkage type of a single glycosidic bond can influence glycan function, there is a demand for improved and automated methods to confirm detailed structural features and to discriminate between structurally similar isomers, overcoming a significant bottleneck in the analysis of data generated by glycomics experiments. We used porous graphitized carbon-LC-ESI-MS/MS to separate and detect released N- and O-glycan isomers from mammalian model glycoproteins using negative mode resonance activation CID-MS/MS. By interrogating similar fragment spectra from closely related glycan isomers that differ only in arm position and sialyl linkage, product fragment ions for discrimination between these features were discovered. Using the Skyline software, at least two diagnostic fragment ions of high specificity were validated for automated discrimination of sialylation and arm position in N-glycan structures, and sialylation in O-glycan structures, complementing existing structural diagnostic ions. These diagnostic ions were shown to be useful for isomer discrimination using both linear and 3D ion trap mass spectrometers when analyzing complex glycan mixtures from cell lysates. Skyline was found to serve as a useful tool for automated assessment of glycan isomer discrimination. This platform-independent workflow can potentially be extended to automate the characterization and quantitation of other challenging glycan isomers.
A new paper from CNBP researchers reports on an improvement to deterministic lateral displacement arrays, which allows for higher particle concentration enhancement. The work has just been published in the journal ‘Biomicrofluidics’ and is accessible online.
Title: Maximizing particle concentration in deterministic lateral displacement arrays.
Authors: Shilun Feng, Alison M. Skelley, Ayad G. Anwer (pictured top left), Guozhen Liu and David W. Inglis.
Abstract: We present an improvement to deterministic lateral displacement arrays, which allows higher particle concentration enhancement. We correct and extend previous equations to a mirror-symmetric boundary. This approach allows particles to be concentrated into a central channel, no wider than the surrounding gaps, thereby maximizing the particle enrichment. The resulting flow patterns were, for the first time, experimentally measured. The performance of the device with hard micro-spheres and cells was investigated. The observed flow patterns show important differences from our model and from an ideal pattern. The 18 μm gap device showed 11-fold enrichment of 7 μm particles and nearly perfect enrichment—of more than 50-fold—for 10 μm particles and Jurkat cells. This work shows a clear path to achieve higher-than-ever particle concentration enhancement in a deterministic microfluidic separation system.
A new paper from CNBP researchers (lead author Wenjie Chen pictured) reports on the design of a new light-triggerable liposome. The work has just been published in the journal ‘Molecular Therapy: Nucleic Acid’ and is accessible online.
Journal: Molecular Therapy: Nucleic Acid.
Title: Light-triggerable liposomes for enhanced endo/lysosomal escape and gene silencing in PC12 cells.
Authors: Wenjie Chen, Wei Deng, Ewa M. Goldys.
Abstract: Liposomes are an effective gene/drug delivery system, widely used in biomedical applications including gene therapy and chemotherapy. Here we designed a photo-responsive liposome (lipVP) loaded with a photosensitizer verteporfin (VP). This photosensitizer is clinically approved for photodynamic therapy (PDT). LipVP was employed as a DNA carrier for pituitary adenylyl cyclase-activating polypeptide (PACAP) receptor 1 (PAC1R) gene knockdown in PC12 cells. This has been done by incorporating PAC1R antisense oligonucleotides inside the lipVP cavity. Cells which have taken up the lipVP were exposed to light from a UV light source. As a result of this exposure, reactive oxygen species (ROS) were generated from VP, destabilising the endo/lysosomal membranes and enhancing the liposomal release of antisense DNA into the cytoplasm. Endo/lysosomal escape of DNA was documented at different time points based on quantitative analysis of colocalization between fluorescently labelled DNA and endo/lysosomes. The released antisense oligonucleotides were found to silence PAC1R mRNA. The efficiency of this photo-induced gene silencing was demonstrated by a 74 ± 5% decrease in PAC1R fluorescence intensity. Following the light-induced DNA transfer into cells, cell differentiation with exposure to two kinds of PACAP peptides was observed to determine the cell phenotypic change after PAC1R gene knockdown.