3D Printing of Noncytotoxic High-Resolution Microchannels in Bisphenol-A Ethoxylate Dimethacrylate Tissue-Mimicking Materials.

The ability to create cell-laden fluidic models that mimic the geometries and physical properties of vascularized tissue would be extremely beneficial to the study of disease etiologies and future therapies, including in the case of cancer where there is increasing interest in studying alterations to the microvasculature. Engineered systems can present significant advantages over animal studies, alleviating challenges associated with variable complexity and control. Three-dimensional (3D)-printable tissue-mimicking hydrogels can offer an alternative, where control of the biophysical properties of the materials can be achieved. Hydrogel-based systems that can recreate complex 3D structures and channels with diameters <500 µm are challenging to produce. We present a noncytotoxic photo-responsive hydrogel that supports 3D printing of complex 3D structures with microchannels down to 150 µm in diameter. Fine tuning of the 3D-printing process has allowed the production of complex structures, where for demonstration purposes we present a helical channel with diameters between 250 and 370 µm around a central channel of 150 µm in diameter in materials with mechanical and acoustic properties that closely replicate those of tissue. The ability to control and accurately reproduce the complex features of the microvasculature has value across a wide range of biomedical applications, especially when the materials involved accurately mimic the physical properties of tissue. An approach that is additionally cell compatible provides a unique setup that can be exploited to study aspects of biomedical research with an unprecedented level of accuracy.

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.

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

KEY POINTS: Smooth muscle cell (SMC) phenotypic conversion from a contractile to a migratory phenotype is proposed to underlie cardiovascular disease but its contribution to vascular remodelling and even its existence have recently been questioned. Tracking the fate of individual SMCs is difficult as no specific markers of migratory SMCs exist. This study used a novel, prolonged time-lapse imaging approach to continuously track the behaviour of unambiguously identified, fully differentiated SMCs. In response to serum, highly-elongated, contractile SMCs initially rounded up, before spreading and migrating and these migratory cells displayed clear phagocytic activity. This study provides a direct demonstration of the transition of fully contractile SMCs to a non-contractile, migratory phenotype with phagocytic capacity that may act as a macrophage-like cell. ABSTRACT: Atherosclerotic plaques are populated with smooth muscle cells (SMCs) and macrophages. SMCs are thought to accumulate in plaques because fully differentiated, contractile SMCs reprogramme 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 1 µm 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.

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.

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.

From structure to function: mitochondrial morphology, motion and shaping in vascular smooth muscle.

The diversity of mitochondrial arrangements, which arise from the organelle being static or moving, or fusing and dividing in a dynamically reshaping network, is only beginning to be appreciated. While significant progress has been made in understanding the proteins that reorganise mitochondria, the physiological significance of the various arrangements is poorly understood. The lack of understanding may occur partly because mitochondrial morphology is studied most often in cultured cells. The simple anatomy of cultured cells presents an attractive model for visualizing mitochondrial behaviour but contrasts with the complexity of native cells in which elaborate mitochondrial movements and morphologies may not occur. Mitochondrial changes may take place in native cells (in response to stress and proliferation), but over a slow time-course and the cellular function contributed is unclear. To determine the role mitochondrial arrangements play in cell function, a crucial first step is characterisation of the interactions among mitochondrial components. Three aspects of mitochondrial behaviour are described in this review: (1) morphology, (2) motion and (3) rapid shape changes. The proposed physiological roles to which various mitochondrial arrangements contribute and difficulties in interpreting some of the physiological conclusions are also outlined.

Examining the role of mitochondria in Ca²âº signaling in native vascular smooth muscle.

Mitochondrial Ca²âº uptake contributes important feedback controls to limit the time course of Ca²âº signals. Mitochondria regulate cytosolic [Ca²âº] over an exceptional breath of concentrations (~200 nM to >10 µM) to provide a wide dynamic range in the control of Ca²âº signals. Ca²âº uptake is achieved by passing the ion down the electrochemical gradient, across the inner mitochondria membrane, which itself arises from the export of protons. The proton export process is efficient and on average there are less than three protons free within the mitochondrial matrix. To study mitochondrial function, the most common approaches are to alter the proton gradient and to measure the electrochemical gradient. However, drugs which alter the mitochondrial proton gradient may have substantial off target effects that necessitate careful consideration when interpreting their effect on Ca²âº signals. Measurement of the mitochondrial electrochemical gradient is most often performed using membrane potential sensitive fluorophores. However, the signals arising from these fluorophores have a complex relationship with the electrochemical gradient and are altered by changes in plasma membrane potential. Care is again needed in interpreting results. This review provides a brief description of some of the methods commonly used to alter and measure mitochondrial contribution to Ca²âº signaling in native smooth muscle.

Microdomains of muscarinic acetylcholine and Ins(1,4,5)P3 receptors create 'Ins(1,4,5)P3 junctions' and sites of Ca²+ wave initiation in smooth muscle.

Increases in cytosolic Ca(2+) concentration ([Ca(2+)](c)) mediated by inositol (1,4,5)-trisphosphate [Ins(1,4,5)P(3), hereafter InsP(3)] regulate activities that include division, contraction and cell death. InsP(3)-evoked Ca(2+) release often begins at a single site, then regeneratively propagates through the cell as a Ca(2+) wave. The Ca(2+) wave consistently begins at the same site on successive activations. Here, we address the mechanisms that determine the Ca(2+) wave initiation site in intestinal smooth muscle cells. Neither an increased sensitivity of InsP(3) receptors (InsP(3)R) to InsP(3) nor regional clustering of muscarinic receptors (mAChR3) or InsP(3)R1 explained the selection of an initiation site. However, examination of the overlap of mAChR3 and InsP(3)R1 localisation, by centre of mass analysis, revealed that there was a small percentage (∼10%) of sites that showed colocalisation. Indeed, the extent of colocalisation was greatest at the Ca(2+) wave initiation site. The initiation site might arise from a selective delivery of InsP(3) from mAChR3 activity to particular InsP(3)Rs to generate faster local [Ca(2+)](c) increases at sites of colocalisation. In support of this hypothesis, a localised subthreshold 'priming' InsP(3) concentration applied rapidly, but at regions distant from the initiation site, shifted the wave to the site of the priming. Conversely, when the Ca(2+) rise at the initiation site was rapidly and selectively attenuated, the Ca(2+) wave again shifted and initiated at a new site. These results indicate that Ca(2+) waves initiate where there is a structural and functional coupling of mAChR3 and InsP(3)R1, which generates junctions in which InsP(3) acts as a highly localised signal by being rapidly and selectively delivered to InsP(3)R1.

Intracellular protein determination using droplet-based immunoassays.

This paper describes the implementation of a sensitive, on-chip immunoassay for the analysis of intracellular proteins, developed using microdroplet technology. The system offers a number of analytical functionalities, enabling the lysis of low cell numbers, as well as protein detection and quantification, integrated within a single process flow. Cells were introduced into the device in suspension and were electrically lysed in situ. The cell lysate was subsequently encapsulated together with antibody-functionalized beads into stable, water-in-oil droplets, which were stored on-chip. The binding of intracellular proteins to the beads was monitored fluorescently. By analyzing many individual droplets and quantifying the data obtained against standard additions, we measured the level of two intracellular proteins, namely, HRas-mCitrine, expressed within HEK-293 cells, and actin-EGFP, expressed within MCF-7 cells. We determined the concentrations of these proteins over 5 orders of magnitude, from ~50 pM to 1 µM. The results from this semiautomated method were compared to those for determinations made using Western blots, and were found not only to be faster, but required a smaller number of cells.

On-chip immunoprecipitation for protein purification.

Immunoprecipitation (IP) is one of the most widely used and selective techniques for protein purification. Here, a miniaturised, polymer-supported immunoprecipitation (µIP) method for the on-chip purification of proteins from complex mixtures is described. A 4 µl PDMS column functionalised with covalently bound antibodies was created and all critical aspects of the µIP protocol (antibody immobilisation, blocking of potential non-specific adsorption sites, sample incubation and washing conditions) were assessed and optimised. The optimised µIP method was used to obtain purified fractions of affinity-tagged protein from a bacterial lysate.

Bilayer lipid membranes from falling droplets.

We describe a system that provides a rapid and simple way of forming suspended lipid bilayers within a microfluidic platform from an aqueous droplet. Bilayer lipid membranes are created in a polymeric device by contacting monolayers formed at a two-phase liquid-liquid interface. Microdroplets, containing membrane proteins, are injected onto an electrode positioned above an aperture machined through a conical cavity that is filled with a lipid-alkane solution. The formation of the BLM depends solely on the device geometry and leads to spontaneous formation of lipid bilayers simply by dispensing droplets of buffer. When an aqueous droplet containing transmembrane proteins or proteoliposomes is injected, straightforward electrophysiology measurements are possible. This method is suitable for incorporation into lab-on-a-chip devices and allows for buffer exchange and electrical measurements.

Formation of artificial lipid bilayers using droplet dielectrophoresis.

We describe the formation of artificial bilayer lipid membranes (BLMs) by the controlled, electrical manipulation of aqueous droplets immersed in a lipid-alkane solution. Droplet movement was generated using dielectrophoresis on planar microelectrodes covered in a thin insulator. Droplets, surrounded by lipid monolayers, were brought into contact and spontaneously formed a BLM. The method produced BLMs suitable for single-channel recording of membrane protein activity and the technique can be extended to create programmable BLM arrays and networks.

Binding of anionic lipids to at least three nonannular sites on the potassium channel KcsA is required for channel opening.

In addition to the annular or boundary lipids that surround the transmembrane surface of the potassium channel KcsA from Streptomyces lividans, x-ray crystallographic studies have detected one anionic lipid molecule bound at each protein-protein interface in the homotetrameric structure, at sites referred to as nonannular sites. The binding constant for phosphatidylglycerol at the nonannular sites has been determined using fluorescence quenching methods with a mutant of KcsA lacking the normal three lipid-exposed Trp residues. Binding is weak, with a binding constant of 0.42 +/- 0.06 in units of mol fraction, implying that the nonannular sites will only be approximately 70% occupied in bilayers of 100% phosphatidylglycerol. However, the nonannular sites show high selectivity for anionic lipids over zwitterionic lipids, and it is suggested that a change in packing at the protein-protein interface leads to a closing of the nonannular binding site in the unbound state. Increasing the anionic lipid content of the membrane leads to a large increase in open channel probability, from approximately 2.5% in the presence of 25 mol % phosphatidylglycerol to approximately 62% in 100 mol % phosphatidylglycerol. The relationship between open channel probability and phosphatidylglycerol content shows cooperativity. The data are consistent with a model in which three or four of the four nonannular sites in the KcsA homotetramer have to be occupied by anionic lipid for the channel to open. The conductance of the open channel increases with increasing concentration of anionic lipid, an effect possibly due to effects of anionic lipid on the concentration of K(+) close to the membrane surface.

Controlled delivery of proteins into bilayer lipid membranes on chip.

The study and the exploitation of membrane proteins for drug screening applications requires a controllable and reliable method for their delivery into an artificial suspended membrane platform based on lab-on-a-chip technology. In this work, a polymeric device for forming lipid bilayers suitable for electrophysiology studies and biosensor applications is presented. The chip supports a single bilayer and is configured for controlled protein delivery through on-chip microfluidics. In order to demonstrate the principle of protein delivery, the potassium channel KcsA was reconstituted into proteoliposomes, which were then fused with the suspended bilayer on-chip. Fusion of single proteoliposomes with the membrane was identified electrically. Single channel conductance measurements of KcsA in the on-chip bilayer were recorded and these were compared to previously published data obtained with a conventional planar bilayer system.

Air-exposure technique for the formation of artificial lipid bilayers in microsystems.

To develop a reliable method for on-chip bilayer lipid membrane (BLM) formation, which could be employed for use in a biosensor array platform, a polymer microfluidic device has been constructed, and the formation of suspended BLMs within it has been investigated. A simple, yet reproducible BLM formation protocol has been developed, in which a brief air-exposure period is employed to induce the rapid thinning of an initially thick lipid-solvent layer. The technique is rapid, reproducible, and amenable to the simple injection of proteins or analytes, as well as to buffer exchange on both sides of the membrane. Scaling up the technique for use in an array platform is also straightforward, the simultaneous formation of three individually addressable BLMs being demonstrated.

Nanofabrication of electrode arrays by electron-beam and nanoimprint lithographies.

The fabrication of ordered nanoelectrode arrays using both electron-beam lithography and nanoimprint lithography is described. Arrays of nanoelectrodes with varying individual electrode diameters were produced and characterised electrochemically. Whilst both methods are highly reproducibile, nanoimprint lithography has the potential to produce devices rapidly and at low-cost. To our knowledge, this is the first report where nanoimprint lithography is employed for the production of nanoelectrode arrays for electroanalytical sensors.

Optimization of the geometry and porosity of microelectrode arrays for sensor design.

This paper describes the systematic investigation of a range of microelectrode arrays with varying dimensions fabricated by standard photolithographic and reactive-ion etching techniques. As expected from theory, the electrochemical behavior of microelectrode arrays with a constant individual diameter varied strongly with center-to-center spacing, the larger the spacing the more sigmoidal the recorded voltammogram. Furthermore, the behavior of arrays with a constant relative center-to-center spacing is shown to vary with individual electrode diameter, the arrays with the smallest electrodes producing strongly peaked voltammograms. Peak current densities and signal-to-noise ratios were also obtained for a variety of array geometries, and the use of electrodeposited platinum black electrodes was investigated. To demonstrate one advantage of using a loosely packed microelectrode array in electroanalysis, a ferrocene-mediated enzyme-linked assay involving the biocatalytic reduction of H2O2 was investigated. Results showed an improved temporal response, with current-time transients reaching a steady-state response more quickly using arrays with increased center-center spacings.