Micro- and nano-electrode arrays for electroanalytical sensing

A systematic investigation of the electrochemical behaviour of two sets of microelectrode arrays, fabricated by standard photolithographic and reactive-ion etching techniques, is presented. The first set of microelectrode arrays had a constant relative centre-centre spacing of 10r (where r is the electrode radius). As a value of r was decreased, the cyclic voltammograms recorded from the array became increasingly peak-shaped, due to merging of the diffusion fields of the individual electrodes. Furthermore, it was shown that the peak current densities obtained were largest for the arrays with the smallest individual electrodes, as were the signal-to-noise ratios (SNRs). Electroplating the individuals electrodes with platinum black was also shown to increase the peak currents and the SNRs, due to an increase in the effective surface area. Sigmoidal voltammograms, which are indicative of radial diffusion, were obtained for an individual electrode radius of 25 mm but not for arrays with smaller electrodes. To obtain radial diffusion for an array of 2.5 mm electrodes, it was shown (using a second set of microelectrode arrays) that a minimum relative centre-centre spacing of 40r is required. Further enhancement of the peak current densities were obtained by decreasing the size of the individual electrodes. A series of nanoelectrode arrays were fabricated using electron-beam lithography (EBL). The voltammograms obtained from these arrays exhibited a continual increase in the recorded peak current as the individual electrodes radius was decreased to a value of 110 nm. Since EBL is a slow and costly technique, nanoimprint lithography (NIL) was investigated as an alternative method of fabricating nanoelectrode arrays and comparable results were obtained from arrays produced by EBL and NIL. A dissolved oxygen and temperature sensor incorporating a working microelectrode array was also designed and fabricated. The sector comprised a densely packed array of 2.5 mm radius electrodes and a micro-reference electrode, both of which were covered with an agarose electrolyte gel enclosed in an SU8 chamber. A thermal resistor was included for temperature compensation of the dissolved oxygen measurements. The Ag|AgCl micro-reference electrode was found to be stable for approximately 80 hours in 0.1 M KCl, with 100 nA of current passing through it. Linear calibration curves were obtained from both temperature and dissolved oxygen measurement

Heterogeneity in the proliferative capacity of smooth muscle cells (SMCs)

In cardiovascular disease, artery walls remodel through an increase in SMC numbers. The predominant hypothesis for this is that SMCs in the tunica media undergo phenotypic modulation into a proliferative cell type. However, direct evidence for SMC phenotypic modulation is scant. We therefore exploited time-lapse microscopy methods to track the fate of freshly isolated, contractile SMCs. As SMCs isolated from different smooth muscle (SM) tissues are heterogeneous in nature, we have used time lapse microscopy in combination with immunocytochemistry to investigate the proliferative capacity of SMCs from portal vein (PV), carotid artery (CA) and distal colon. The adventia/endothelium and mucosa/serosa were mechanically removed before isolating cells by enzymatic digestion and trituration. Highly elongated, contractile SMCs that stained for both SM α-actin (SMA) and SM myosin heavy chain (SM-MHC) were obtained from all tissues. Significantly, both colon and PV tissues also contained large numbers of spherical cells that did not stain for SMA or SM-MHC (nonSMCs). In standard culture conditions, the SMCs showed limited proliferative capacity: with the exception of one SMC that divided once (out of 15 tracked cells), colon SMCs did not proliferate. Of 11 PV SMCs tracked, 7 SMCs did divide, though none progressed beyond the 3rd generation (daughter of daughter) by confluency. Conversely, the nonSMCs proliferated rapidly (reaching the 5th generation in 5 days) and dominated the resulting cultures. In contrast, all CA cells stained for both SMA and SM-MHC i.e. no nonSMCs were present. Whilst most CA SMCs underwent apoptosis early in culture (27 of 31 cells), those that survived went on to proliferate at varying rates (up to the 6th generation in 5 days). These results illustrate the complexities involved in creating models of SMC proliferation

The transition of smooth muscle cells from a contractile to a migratory, phagocytic phenotype : direct demonstration of phenotypic modulation

Atherosclerotic plaques are populated with smooth muscle cells (SMCs) and macrophages. SMCs are thought to accumulate in plaques because fully-differentiated, contractile SMCs reprogram into a ‘synthetic’ migratory phenotype, so-called phenotypic modulation, whilst plaque macrophages are thought to derive from blood-borne myeloid cells. Recently, these views have been challenged, with reports that SMC phenotypic modulation may not occur during vascular remodelling and that plaque macrophages may not be of haematopoietic origin. Following the fate of SMCs is complicated by the lack of specific markers for the migratory phenotype and direct demonstrations of phenotypic modulation are lacking. Therefore, we employed long-term, high-resolution, time-lapse microscopy to track the fate of unambiguously identified, fully-differentiated, contractile SMCs in response to the growth factors present in serum. Phenotypic modulation was clearly observed. The highly-elongated, contractile SMCs initially rounded up, for 1-3 days, before spreading outwards. Once spread, the SMCs became motile and displayed dynamic cell-cell communication behaviours. Significantly, they also displayed clear evidence of phagocytic activity. This macrophage-like behaviour was confirmed by their internalisation of 1µm fluorescent latex beads. However, migratory SMCs did not uptake acetylated low-density lipoprotein or express the classic macrophage marker CD68. These results directly demonstrate that SMCs may rapidly undergo phenotypic modulation and develop phagocytic capabilities. Resident SMCs may provide a potential source of macrophages in vascular remodelling

Microwell arrays for monitoring phenotypic heterogeneity in vascular cell populations

Significant remodeling of the vascular wall underlies cardiovascular disease resulting in the formation of atherosclerotic plaques populated with macrophage and smooth muscle cells (SMCs). These SMCs are thought to arise from the vessel wall, as mature SMCs de-differentiate from a contractile to a migratory, proliferate phenotype. However, the remodeling process is not fully understood and uncertainties remain over plaque cell origins and the plasticity of cells within the vascular wall. Both drug development and regenerative medicine have been restricted by these uncertainties. Recently, through a combination of time-lapse, high-speed fluorescence and 3D reconstruction microscopy, we demonstrated unambiguously [1] that freshly isolated mature, contractile SMCs can rapidly transform into not only a migratory but a phagocytic phenotype, a characteristic behaviour of macrophage. Results also showed strong heterogeneity in the proliferative capacity of SMCs [2] and the presence of other highly proliferative cell types in vascular wall that readily interact with SMCs. To better understand vascular call fate, including characterizing the phenotype of cell subpopulations, we employed SU-8 microfabrication to create a series of addressable microwell arrays that enable screening at the single cell level of large numbers of freshly isolated vascular cells. By incorporating microwells of different areas (from 60x60 to 180x180) and seeding with a cell suspension of appropriate density (either a pure SMC population or a mixed vascular population), cells sedimented stochastically across the microwell arrays such that many wells contained single cells. These cells were characterized by imaging in situ prior to tracking them for >1 week as they were induced to de-differentiate in culture. To validate this approach, variation in the proliferation of individual cells was tracked and the expression of SMC markers (e.g. SMA) following phenotypic modulation quantified. This microwell array approach, which is amenable to drug screening applications, will enable detailed characterization of phenotypic changes in vascular cell sub-populations, providing new insights to inform tissue engineering applications

Investigating the response of skeletal muscle to prosthesis-related loading conditions : an ex vivo animal model

BACKGROUND The use of lower-limb prosthetics puts the soft tissue of the residuum, including the muscle envelop, under constant physical stress. To adapt to this unphysiological mechanical loading, the muscles need to maintain the balance between tissue damage and regenerative processes. However, in extreme cases of overload or with repeated impact, this balance may be disturbed [1], potentially leading to residual limb pain and Deep Tissue Injuries [2]. AIM A new ex vivo model of rat skeletal muscle tissue was developed to quantify cellular damage from prosthesis-related loading protocols (Fig. 1a). Preliminary exploration of different imaging procedures and the relevance of results for prosthetic research and practice are discussed. METHOD Freshly isolated soleus and extensor digitorum longus muscles dissected from male Sprague Dawley rats were subjected to transverse compressive loading (9-32kPa through a 2mm indenter). Control tissues were held in the same conditions for the same time without loading. Tissues were subsequently processed for imaging by standard histological procedures, using H&E staining for visualising cell and tissue morphology and Procion Yellow for fluorescent dead cell staining [3]. In addition, tissue clearing methods were investigated to enable full tissue depth imaging by confocal microscopy. Furthermore, biochemical changes caused by cellular damage were visualised via multiphoton Raman microscopy of unstained samples. RESULTS During the maximum experimental time frame of 3h, the control samples showed only minor loss in cell viability. By comparison, the extent of mechanical damage in loaded tissues was readily distinguishable by imaging (Fig. 1b), with partial loss of striations, disrupted muscle fibres, increased interstitial space, and loss of cell viability. With careful control of the experimental setup, detailed imaging of local cellular damage in response to loading conditions could be obtained. DISCUSSION AND CONCLUSION Our preliminary studies present an ex vivo model and experimental procedures that are suitable for quantifying cellular damage from prosthesis-related loading conditions on skeletal muscle. Looking at this microscale will provide important insights into the adaptive capabilities of skeletal muscle. This can provide the basis for further research into the role of soft tissue deformation in limb pain and ulcer formation and could inform future directions for socket design and fit

Quantification of functionalised gold nanoparticle-targeted knockdown of gene expression in HeLa cells

Introduction: Gene therapy continues to grow as an important area of research, primarily because of its potential in the treatment of disease. One significant area where there is a need for better understanding is in improving the efficiency of oligonucleotide delivery to the cell and indeed, following delivery, the characterization of the effects on the cell. Methods: In this report, we compare different transfection reagents as delivery vehicles for gold nanoparticles functionalized with DNA oligonucleotides, and quantify their relative transfection efficiencies. The inhibitory properties of small interfering RNA (siRNA), single-stranded RNA (ssRNA) and single-stranded DNA (ssDNA) sequences targeted to human metallothionein hMT-IIa are also quantified in HeLa cells. Techniques used in this study include fluorescence and confocal microscopy, qPCR and Western analysis. Findings: We show that the use of transfection reagents does significantly increase nanoparticle transfection efficiencies. Furthermore, siRNA, ssRNA and ssDNA sequences all have comparable inhibitory properties to ssDNA sequences immobilized onto gold nanoparticles. We also show that functionalized gold nanoparticles can co-localize with autophagosomes and illustrate other factors that can affect data collection and interpretation when performing studies with functionalized nanoparticles. Conclusions: The desired outcome for biological knockdown studies is the efficient reduction of a specific target; which we demonstrate by using ssDNA inhibitory sequences targeted to human metallothionein IIa gene transcripts that result in the knockdown of both the mRNA transcript and the target protein

Magnetite-doped polydimethylsiloxane (PDMS) for phosphopeptide enrichment

Reversible phosphorylation plays a key role in numerous biological processes. Mass spectrometry-based approaches are commonly used to analyze protein phosphorylation, but such analysis is challenging, largely due to the low phosphorylation stoichiometry. Hence, a number of phosphopeptide enrichment strategies have been developed, including metal oxide affinity chromatography (MOAC). Here, we describe a new material for performing MOAC that employs a magnetite-doped polydimethylsiloxane (PDMS), that is suitable for the creation of microwell array and microfluidic systems to enable low volume, high throughput analysis. Incubation time and sample loading were explored and optimized and demonstrate that the embedded magnetite is able to enrich phosphopeptides. This substrate-based approach is rapid, straightforward and suitable for simultaneously performing multiple, low volume enrichments

3D-printed high-resolution microchannels for contrast enhanced ultrasound research

Systemically circulating microbubbles are used as contrast agents to aid both drug targeting and delivery using ultrasound. Exploiting their acoustic behaviour in small diameter vessels is critical for both applications, but the highly controlled experiments required to support this are not possible in vivo and challenging in vitro. Experimental platforms with small diameter channels (below 200 microns) are not readily available nor able to represent vascular geometries, leaving the existence and extent of microbubble-microvessel interactions incompletely defined. In this work we present a 3D-printed microchannel platform using tissue-mimicking hydrogels featuring radii down to 75 microns. We demonstrate application to study microbubble behaviour via acoustic backscatter under controlled environments in physiologically-relevant conditions

MYC regulates fatty acid metabolism through a multigenic program in claudin-low triple negative breast cancer

Background: Recent studies have suggested that fatty acid oxidation (FAO) is a key metabolic pathway for the growth of triple negative breast cancers (TNBCs), particularly those that have high expression of MYC. However, the underlying mechanism by which MYC promotes FAO remains poorly understood. Methods: We used a combination of metabolomics, transcriptomics, bioinformatics, and microscopy to elucidate a potential mechanism by which MYC regulates FAO in TNBC. Results: We propose that MYC induces a multigenic program that involves changes in intracellular calcium signalling and fatty acid metabolism. We determined key roles for fatty acid transporters (CD36), lipases (LPL), and kinases (PDGFRB, CAMKK2, and AMPK) that each contribute to promoting FAO in human mammary epithelial cells that express oncogenic levels of MYC. Bioinformatic analysis further showed that this multigenic program is highly expressed and predicts poor survival in the claudin-low molecular subtype of TNBC, but not other subtypes of TNBCs, suggesting that efforts to target FAO in the clinic may best serve claudin-low TNBC patients. Conclusion: We identified critical pieces of the FAO machinery that have the potential to be targeted for improved treatment of patients with TNBC, especially the claudin-low molecular subtype

Abstract 1441 : MYC expression promotes lipid metabolism and metabolic plasticity in human mammary epithelial cell

MYC is one of the most commonly mutated and highly amplified oncogenes in human breast cancer. MYC amplifications occur most frequently in triple-negative breast cancers (TNBCs). TNBCs can be divided into two molecular subtypes: basal-like and claudin-low breast cancers. These cancers tend to be extremely aggressive and are strongly associated with disease recurrence, poor prognosis and high mortality. In particular, claudin-low tumors are classified by a loss of tight junctions and cell-to-cell contacts and an enrichment for genes associated with an epithelial-to-mesenchymal transition (EMT) and mammary stem cells (also known as tumor-initiating cells). Despite the high level of disease severity, there are no targeted therapies for claudin-low TNBCs. To address this unmet need, we utilized human mammary epithelial cells (HuMECs) that express oncogenic levels of MYC and a mutant MYC (T58A) to characterize the behavioral and metabolic changes that occur during the formation of MYC-driven breast cancers. We found that MYC regulates the expression of genes associated with cell stemness, EMT, lipid metabolism, and calcium (Ca2+) signaling and that the expression of this gene signature promotes cell growth, survival, migration, and metabolic plasticity. The gene signature of MYC-expressing HuMECs highly correlates with the gene signature of claudin-low breast cancers, therefore highlighting the relevance of our HuMEC model to human claudin-low breast cancer. We found the major drivers underlying the MYC-dependent changes in cell behavior to be stimulation of Ca2+ signaling and strong activation of lipid metabolism. Ca2+ signaling is stimulated through the MYC-dependent repression of Ca2+ efflux mechanisms; elevated cytosolic Ca2+ then consequently stimulates a Ca2+/calmodulin kinase kinase 2 (CAMKK2)/AMPK signaling axis that activates fatty acid scavenging and transport, as well as β-oxidation. Enhanced lipid metabolism thereby provides the necessary biomass (fatty acids) for phospholipid biosynthesis and energy (ATP) to support the metabolically demanding processes of cell growth, proliferation, and migration. In all, our findings provide a strong rationale for targeting lipid metabolism and the Ca2+/CAMKK2/AMPK signaling axis in MYC-driven, and potentially claudin-low, breast cancers