Papers by Mirren Charnley
ABSTRACTCOVID-19 is primarily known as a respiratory disease caused by the virus SARS-CoV-2. Howe... more ABSTRACTCOVID-19 is primarily known as a respiratory disease caused by the virus SARS-CoV-2. However, neurological symptoms such as memory loss, sensory confusion, cognitive and psychiatric issues, severe headaches, and even stroke are reported in as many as 30% of cases and can persist even after the infection is over (so-called ‘long COVID’). These neurological symptoms are thought to be caused by brain inflammation, triggered by the virus infecting the central nervous system of COVID-19 patients, however we still don’t fully understand the mechanisms for these symptoms. The neurological effects of COVID-19 share many similarities to neurodegenerative diseases such as Alzheimer’s and Parkinson’s in which the presence of cytotoxic protein-based amyloid aggregates is a common etiological feature. Following the hypothesis that some neurological symptoms of COVID-19 may also follow an amyloid etiology we performed a bioinformatic scan of the SARS-CoV-2 proteome, detecting peptide frag...
BIO-PROTOCOL, 2018
Dense networks of amyloid nanofibrils fabricated from common globular proteins adsorbed to solid ... more Dense networks of amyloid nanofibrils fabricated from common globular proteins adsorbed to solid supports can improve cell adhesion, spreading and differentiation compared to traditional flat, stiff 2D cell culture substrates like Tissue Culture Polystyrene (TCPS). This is due to the fibrous, nanotopographic nature of the amyloid fibril networks and the fact that they closely mimic the mechanical properties and architecture of the extracellular matrix (ECM). However, precise cell responses are strongly dependent on the nanostructure of the network at the cell culture interface, thus accurate characterization of the immobilized network is important. Due to its exquisite lateral resolution and simple sample preparation techniques, Atomic Force Microscopy (AFM) is an ideal technique to characterize the fibril network morphology. Thus, here we describe a detailed protocol, for the characterization of amyloid fibril networks by tapping mode AFM.
BIO-PROTOCOL, 2018
Networks of amyloid nanofibrils fabricated from common globular proteins such as lysozyme and β-l... more Networks of amyloid nanofibrils fabricated from common globular proteins such as lysozyme and β-lactoglobulin have material properties that mimic the extracellular microenvironment of many cell types. Cells cultured on such amyloid fibril networks show improved attachment, spreading and in the case of mesenchymal stem cells improved differentiation. Here we describe a detailed protocol for fabricating amyloid fibril networks suitable for eukaryotic cell culture applications.
The β-selection checkpoint of T cell development tests whether the cell has recombined its genomi... more The β-selection checkpoint of T cell development tests whether the cell has recombined its genomic DNA to produce a functional T Cell Receptor β (TCRβ) receptor. Passage through the β-selection checkpoint requires the nascent TCRβ protein to mediate signaling through a pre-TCR complex. In this study, we show that developing T cells at the β-selection checkpoint establish an immunological synapse in in vitro & in situ, resembling that of the mature T cell. The immunological synapse is dependent on two key signaling pathways known to be critical for the transition beyond the β-selection checkpoint, Notch and CXCR4 signaling. In vitro and in situ analyses indicate that the immunological synapse promotes passage through the β-selection checkpoint. Collectively, these data indicate that developing T cells regulate pre-TCR signaling through the formation of an immunological synapse. This signaling platform integrates cues from Notch, CXCR4, and MHC on the thymic stromal cell, to allow tra...
Scientific reports, Jan 20, 2018
Microsample analysis is highly beneficial in blood-based testing where cutting-edge bioanalytical... more Microsample analysis is highly beneficial in blood-based testing where cutting-edge bioanalytical technologies enable the analysis of volumes down to a few tens of microliters. Despite the availability of analytical methods, the difficulty in obtaining high-quality and standardized microsamples at the point of collection remains a major limitation of the process. Here, we detail and model a blood separation principle which exploits discrete viscosity differences caused by blood particle sedimentation in a laminar flow. Based on this phenomenon, we developed a portable capillary-driven microfluidic device that separates blood microsamples collected from finger-pricks and delivers 2 µL of metered serum for bench-top analysis. Flow cytometric analysis demonstrated the high purity of generated microsamples. Proteomic and metabolomic analyses of the microsamples of 283 proteins and 1351 metabolite features was consistent with samples generated via a conventional centrifugation method. Th...
Journal of molecular biology, Jan 8, 2018
Cell polarity is an essential process shared by almost all animal tissues. Moreover, cell polarit... more Cell polarity is an essential process shared by almost all animal tissues. Moreover, cell polarity enables cells to sense and respond to the cues provided by the neighboring cells and the surrounding microenvironment. These responses play a critical role in regulating key physiological processes, including cell migration, proliferation, differentiation, vesicle trafficking and immune responses. The polarity protein complexes regulating these interactions are highly evolutionarily conserved between vertebrates and invertebrates. Interestingly, these polarity complexes interact with each other and key signaling pathways in a cell-polarity context-dependent manner. However, the exact mechanisms by which these interactions take place are poorly understood. In this review, we will focus on the roles of the key polarity complexes SCRIB, PAR and Crumbs in regulating different forms of cell polarity, including epithelial cell polarity, cell migration, asymmetric cell division and the T-cell...
Biointerphases, Jan 19, 2017
Force spectroscopy is a means of obtaining mechanical information of individual nanometer-scale s... more Force spectroscopy is a means of obtaining mechanical information of individual nanometer-scale structures in composite materials, such as protein assemblies for use in consumer films or gels. As a recently developed force spectroscopy technique, bimodal force spectroscopy relates frequency shifts in cantilevers simultaneously excited at multiple frequencies to the elastic properties of the contacted material, yet its utility for quantitative characterization of biopolymer assemblies has been limited. In this study, a linear correlation between experimental frequency shift and Young's modulus of polymer films was used to calibrate bimodal force spectroscopy and quantify Young's modulus of two protein nanostructures: β-lactoglobulin fibrils and zein nanoparticles. Cross-sectional Young's modulus of protein fibrils was determined to be 1.6 GPa while the modulus of zein nanoparticles was determined as 854 MPa. Parallel measurement of β-lactoglobulin fibril by a competing pu...
Pharmacognosy Research, 2017
Background: Conventional therapeutic agents used for treatment of Acne are associated with variou... more Background: Conventional therapeutic agents used for treatment of Acne are associated with various adverse effects necessitating development of safe and effective alternative therapeutic agents. In this context, a polyherbal formulation AHPL/AYCAP/0413 was developed for treatment of Acne. Objectives: To evaluate Anti-inflammatory and antimicrobial activity of AHPL/AYCAP/0413. Material and Methods: 1) Anti-inflammatory activity: Anti-inflammatory activity of AHPL/AYCAP/0413 in comparison with Diclofenac was assessed in carrageenan induced rat Paw edema model. 2) Anti-microbial activity for P. acne: Propionibacterium acnes were incubated under anaerobic conditions. Aliquots of molten BHI with glucose agar were used as the agar base. Formulation and clindamycin (10 μg/ml) were introduced in to the Agar wells randomly. 3) Anti-microbial activity for Staphylococcus epidermidis and Staphylococcus aureus: Staphylococcus epidermidis and Staphylococcus aureus were incubated under aerobic conditions at 37°C. TSB with glucose agar was used as the agar base. 0.5ml of formulation and clindamycin (10 μg/ml) were introduced in to the wells randomly. The antibacterial activity was evaluated by measuring zones of inhibition (in mm). Result: Significant reduction in rat paw edema (51% inhibition) was observed with formulation AHPL/AYCAP/0413 which was also comparable to that of Diclofenac (58% inhibition). Zone of inhibition for formulation was 18.33 mm, 19.20 mm and 26.30 mm for P. acnes, S. epidermidis and S. aureus respectively. This activity was also comparable to that of Clindamycin. Conclusion: AHPL/AYCAP/0413 capsule possesses significant Anti-inflammatory and Anti-microbial activities which further justifies its role in the management of Acne vulgaris.
Methods in molecular biology (Clifton, N.J.), 2017
Asymmetric cell division (ACD) controls cell fate decisions in model organisms such as Drosophila... more Asymmetric cell division (ACD) controls cell fate decisions in model organisms such as Drosophila and C. elegans and has recently emerged as a mediator of T cell fate and hematopoiesis. The most appropriate methods for assessing ACD in T cells are still evolving. Here we describe the methods currently applied to monitor and measure ACD of developing and activated T cells. We provide an overview of approaches for capturing cells in the process of cytokinesis in vivo, ex vivo, or during in vitro culture. We provide methods for in vitro fixed immunofluorescent staining and for time-lapse analysis. We provide an overview of the different approaches for quantification of ACD of lymphocytes, discuss the pitfalls and concerns in interpretation of these analyses, and provide detailed methods for the quantification of ACD in our group.
European Cells and Materials, 2010
Biomacromolecules, Jan 14, 2015
Networks of nanoscale fibrous coatings made from self-assembled peptides are promising candidates... more Networks of nanoscale fibrous coatings made from self-assembled peptides are promising candidates for biomaterials that can promote the growth of mammalian cells. One particularly attractive feature is the possibility of adding bio-functional sequences to peptides to promote cell attachment. We deconvolute the topographic and chemical effects of nanoscale fibrils on cells by depositing a plasma polymer film on TTR1-based fibrils decorated with a range of cell adhesive chemistries (RGD and cycloRGDfK), producing a surface that retains the nanoscale fibrous topography of surface bound fibrils but lacks the fibril surface chemistry. The surface topography was found to influence cell toxicity and spreading and the fibril surface chemistry influenced cell attachment and spreading. This study highlights the importance of considering both the chemical and physical features of novel biomaterials and illustrates the use of plasma polymer deposition as a tool for examining the relationship be...
Integrative Biology, 2009
Microwell arrays have emerged as robust and versatile alternatives to conventional mammalian cell... more Microwell arrays have emerged as robust and versatile alternatives to conventional mammalian cell culture substrates. Using standard microfabrication processes, biomaterials surfaces can be topographically patterned to comprise high-density arrays of micron-sized cavities with desirable geometry. Hundreds to thousands of individual cells or cell colonies with controlled size and shape can be trapped in these cavities by simple gravitational sedimentation. Efficient long-term cell confinement allows for parallel analyses and manipulation of cell fate during in vitro culture. These live-cell arrays have already found applications in cell biology, for example to probe the effect of cell colony size on embryonic stem cell differentiation, to dissect the heterogeneity in single cell proliferation kinetics of neural or hematopoietic stem/progenitor cell populations, or to elucidate the role of cell shape on cell function. Here, we highlight the key applications of these platforms, hopefully inspiring biologists to apply these systems for their own studies. Insight, innovation, integration Using straightforward microfabrication processes, biomaterials surfaces can be topographically structured to comprise high-density arrays of micron-sized cavities, termed 'microwell arrays'. These substrates are, for example, ideally suited to culture and probe the fate of large numbers of trapped single cells, such as rare and heterogeneous stem cells. Here we highlight the key applications of these platforms, hopefully inspiring biologists to apply these systems for their own studies.
INTRODUCTION: There is an increase in the use of implantable medical devices for the repair of so... more INTRODUCTION: There is an increase in the use of implantable medical devices for the repair of soft and hard tissue. Many such devices can initiate acute inflammation, resulting in device failure. The co-delivery of anti-inflammatories together with the device is proposed as a therapeutic strategy to reduce excessive inflammation. α-Melanocyte-stimulating hormone (MSH) is a natural and potent anti-inflammatory hormone produced in the body with very short peptide sequences that make it amenable for easy laboratory synthesis. The aim of this work is to immobilise short MSH peptides onto medical device surfaces using molecules called calixarenes, which are known to attach to a wide variety of material surfaces. This is being approached by synthesizing MSH-calixarene molecules with the aim of being able to 'dip and dry' treat medical devices with an anti-inflammatory 'coating'.
ACS Applied Materials & Interfaces, 2015
The success of synthetic bone implants requires good interface between the material and the host ... more The success of synthetic bone implants requires good interface between the material and the host tissue. To study the biological relevance of fibronectin (FN) density on the osteogenic commitment of human bone marrow mesenchymal stem cells (hBM-MSCs), human FN was adsorbed in a linear density gradient on the surface of PCL. The evolution of the osteogenic markers alkaline phosphatase and collagen 1 alpha 1 was monitored by immunohistochemistry, and the cytoskeletal organization and the cell-derived FN were assessed. The functional analysis of the gradient revealed that the lower FN-density elicited stronger osteogenic expression and higher cytoskeleton spreading, hallmarks of the stem cell commitment to the osteoblastic lineage. The identification of the optimal FN density regime for the osteogenic commitment of hBM-MSCs presents a simple and versatile strategy to significantly enhance the surface properties of polycaprolactone as a paradigm for other synthetic polymers intended for bone-related applications.
Biomaterials, 2014
Tissue engineering using scaffold-cell constructs holds the potential to develop functional strat... more Tissue engineering using scaffold-cell constructs holds the potential to develop functional strategies to regenerate bone. The interface of orthopedic implants with the host tissues is of great importance for its later performance. Thus, the optimization of the implant surface in a way that could stimulate osteogenic differentiation of mesenchymal stem cells (MSCs) is of significant therapeutic interest. The effect of surface roughness of polycaprolactone (PCL) on the osteogenic differentiation of human bone-marrow MSCs was investigated. We prepared surface roughness gradients of average roughness (Ra) varying from the sub-micron to the micrometer range (~0.5e4.7 mm), and mean distance between peaks (RS m) gradually varying from~214 mm to 33 mm. We analyzed the degree of cytoskeleton spreading, expression of alkaline phosphatase, collagen type 1 and mineralization. The response of cells to roughness divided the gradient into three groups of elicited stem cell behavior: 1) faster osteogenic commitment and strongest osteogenic expression; 2) slower osteogenic commitment but strong osteogenic expression, and 3) similar or inferior osteogenic potential in comparison to the control material. The stem-cell modulation by specific PCL roughness surfaces highlights the potential for creating effective solutions for orthopedic applications featuring a clinically relevant biodegradable material.
Proceedings of the National Academy of Sciences, 2008
This work explores time-resolved emission imaging microscopy (TREM) for noninvasive imaging and m... more This work explores time-resolved emission imaging microscopy (TREM) for noninvasive imaging and mapping of live cells on a hitherto uncharted microsecond time scale. Simple robust molecules for this purpose have long been sought. We have developed highly emissive, synthetically versatile, and photostable platinum(II) complexes that make TREM a practicable reality. [PtLCl], {HL = 1,3-di(2-pyridyl)benzene and derivatives}, are charge-neutral, small molecules that have low cytotoxicity and accumulate intracellularly within a remarkably short incubation time of 5 min, apparently under diffusion control. Their microsecond lifetimes and emission quantum yields of up to 70% are exceptionally high for transition metal complexes and permit the application of TREM to be demonstrated in a range of live cell types—normal human dermal fibroblast, neoplastic C8161 and CHO cells. [PtLCl] are thus likely to be suitable emission labels for any eukaryotic cell types. The high photostability of [PtLCl...
PLoS ONE, 2012
Background: Increasing evidence shows that the cancer microenvironment affects both tumorigenesis... more Background: Increasing evidence shows that the cancer microenvironment affects both tumorigenesis and the response of cancer to drug treatment. Therefore in vitro models that selectively reflect characteristics of the in vivo environment are greatly needed. Current methods allow us to screen the effect of extrinsic parameters such as matrix composition and to model the complex and three-dimensional (3D) cancer environment. However, 3D models that reflect characteristics of the in vivo environment are typically too complex and do not allow the separation of discrete extrinsic parameters. Methodology/Principal Findings: In this study we used a poly(ethylene glycol) (PEG) hydrogel-based microwell array to model breast cancer cell behavior in multilayer cell clusters that allows a rigorous control of the environment. The innovative array fabrication enables different matrix proteins to be integrated into the bottom surface of microwells. Thereby, extrinsic parameters including dimensionality, type of matrix coating and the extent of cell-cell adhesion could be independently studied. Our results suggest that cell to matrix interactions and increased cell-cell adhesion, at high cell density, induce independent effects on the response to Taxol in multilayer breast cancer cell clusters. In addition, comparing the levels of apoptosis and proliferation revealed that drug resistance mediated by cell-cell adhesion can be related to altered cell cycle regulation. Conversely, the matrix-dependent response to Taxol did not correlate with proliferation changes suggesting that cell death inhibition may be responsible for this effect. Conclusions/Significance: The application of the PEG hydrogel platform provided novel insight into the independent role of extrinsic parameters controlling drug response. The presented platform may not only become a useful tool for basic research related to the role of the cancer microenvironment but could also serve as a complementary platform for in vitro drug development.
PLoS ONE, 2011
The bone-degrading activity of osteoclasts depends on the formation of a cytoskeletal-adhesive su... more The bone-degrading activity of osteoclasts depends on the formation of a cytoskeletal-adhesive superstructure known as the sealing zone (SZ). The SZ is a dynamic structure, consisting of a condensed array of podosomes, the elementary adhesion-mediating structures of osteoclasts, interconnected by F-actin filaments. The molecular composition and structure of the SZ were extensively investigated, yet despite its major importance for bone formation and remodelling, the mechanisms underlying its assembly and dynamics are still poorly understood. Here we determine the relations between matrix adhesiveness and the formation, stability and expansion of the SZ. By growing differentiated osteoclasts on micropatterned glass substrates, where adhesive areas are separated by non-adhesive PLL-g-PEG barriers, we show that SZ growth and fusion strictly depend on the continuity of substrate adhesiveness, at the micrometer scale. We present a possible model for the role of mechanical forces in SZ formation and reorganization, inspired by the current data.
PLoS ONE, 2013
The formation and orientation of the mitotic spindle is a critical feature of mitosis. The morpho... more The formation and orientation of the mitotic spindle is a critical feature of mitosis. The morphology of the cell and the spatial distribution and composition of the cells' adhesive microenvironment all contribute to dictate the position of the spindle. However, the impact of the dimensionality of the cells' microenvironment has rarely been studied. In this study we present the use of a microwell platform, where the internal surfaces of the individual wells are coated with fibronectin, enabling the three-dimensional presentation of adhesive ligands to single cells cultured within the microwells. This platform was used to assess the effect of dimensionality and cell shape in a controlled microenvironment. Single HeLa cells cultured in circular microwells exhibited greater tilting of the mitotic spindle, in comparison to cells cultured in square microwells. This correlated with an increase in the time required to align the chromosomes at the metaphase plate due to prolonged activation of the spindle checkpoint in an actin dependent process. The comparison to 2D square patterns revealed that the dimensionality of cell adhesions alone affected both mitotic timings and spindle orientation; in particular the role of actin varied according to the dimensionality of the cells' microenvironment. Together, our data revealed that cell shape and the dimensionality of the cells' adhesive environment impacted on both the orientation of the mitotic spindle and progression through mitosis.
Uploads
Papers by Mirren Charnley