Publications
184 results found
Anand U, Yiangou Y, Sinisi M, et al., 2015, Mechanisms underlying clinical efficacy of Angiotensin II type 2 receptor (AT(2)R) antagonist EMA401 in neuropathic pain: clinical tissue and in vitro studies, Molecular Pain, Vol: 11, ISSN: 1744-8069
Background: The clinical efficacy of the Angiotensin II (AngII) receptor AT2R antagonist EMA401, a novel peripherallyrestrictedanalgesic, was reported recently in post-herpetic neuralgia. While previous studies have shown that AT2Ris expressed by nociceptors in human DRG (hDRG), and that EMA401 inhibits capsaicin responses in cultured hDRGneurons, the expression and levels of its endogenous ligands AngII and AngIII in clinical neuropathic pain tissues, andtheir signalling pathways, require investigation. We have immunostained AngII, AT2R and the capsaicin receptor TRPV1in control post-mortem and avulsion injured hDRG, control and injured human nerves, and in cultured hDRG neurons.AngII, AngIII, and Ang-(1-7) levels were quantified by ELISA. The in vitro effects of AngII, AT2R agonist C21, and Nervegrowth factor (NGF) were measured on neurite lengths; AngII, NGF and EMA401 effects on expression of p38 andp42/44 MAPK were measured using quantitative immunofluorescence, and on capsaicin responses using calciumimaging.Results: AngII immunostaining was observed in approximately 75% of small/medium diameter neurons in control(n = 5) and avulsion injured (n = 8) hDRG, but not large neurons i.e. similar to TRPV1. AngII was co-localised withAT2R and TRPV1 in hDRG and in vitro. AngII staining by image analysis showed no significant difference betweencontrol (n = 12) and injured (n = 13) human nerves. AngII levels by ELISA were also similar in control human nerves(4.09 ± 0.36 pmol/g, n = 31), injured nerves (3.99 ± 0.79 pmol/g, n = 7), and painful neuromas (3.43 ± 0.73 pmol/g,n = 12); AngIII and Ang-(1-7) levels were undetectable (<0.03 and 0.05 pmol/g respectively). Neurite lengths weresignificantly increased in the presence of NGF, AngII and C21 in cultured DRG neurons. AngII and, as expected, NGFsignificantly increased signal intensity of p38 and p42/44 MAPK, which was reversed by EMA401. AngII mediated sensitizationof capsaicin responses was not obs
Seifert J, Rheinlaender J, Novak P, et al., 2015, Comparison of Atomic Force Microscopy and Scanning Ion Conductance Microscopy for Live Cell Imaging, Langmuir, Vol: 31, Pages: 6807-6813, ISSN: 1520-5827
tomic force microscopy (AFM) and scanning ion conductance microscopy (SICM) are excellent and commonly used techniques for imaging the topography of living cells with high resolution. We present a direct comparison of AFM and SICM for imaging microvilli, which are small features on the surface of living cells, and for imaging the shape of whole cells. The imaging quality on microvilli increased significantly after cell fixation for AFM, whereas for SICM it remained constant. The apparent shape of whole cells in the case of AFM depended on the imaging force, which deformed the cell. In the case of SICM, cell deformations were avoided, owing to the contact-free imaging mechanism. We estimated that the lateral resolution on living cells is limited by the cell’s elastic modulus for AFM, while it is not for SICM. By long-term, time-lapse imaging of microvilli dynamics, we showed that the imaging quality decreased with time for AFM, while it remained constant for SICM.
Ivanov AP, Actis P, Jönsson P, et al., 2015, On-demand delivery of single DNA molecules using nanopipettes, ACS Nano, Vol: 9, Pages: 3587-3595, ISSN: 1936-086X
Understanding the behavioral properties of single molecules or larger scale populations interacting with single molecules is currently a hotly pursued topic in nanotechnology. This arises from the potential such techniques have in relation to applications such as targeted drug delivery, early stage detection of disease, and drug screening. Although label and label-free single molecule detection strategies have existed for a number of years, currently lacking are efficient methods for the controllable delivery of single molecules in aqueous environments. In this article we show both experimentally and from simulations that nanopipets in conjunction with asymmetric voltage pulses can be used for label-free detection and delivery of single molecules through the tip of a nanopipet with “on-demand” timing resolution. This was demonstrated by controllable delivery of 5 kbp and 10 kbp DNA molecules from solutions with concentrations as low as 3 pM.
Lopez-Cordoba A, Joensson P, Babakinejad B, et al., 2015, SICM-Based Nanodelivery System for Local TRPV1 Stimulation, 59th Annual Meeting of the Biophysical-Society, Publisher: CELL PRESS, Pages: 332A-332A, ISSN: 0006-3495
Takahashi Y, Kumatani A, Munakata H, et al., 2014, Nanoscale visualization of redox activity at lithium-ion battery cathodes, Nature Communications, Vol: 5, ISSN: 2041-1723
Intercalation and deintercalation of lithium ions at electrode surfaces are central to the operation of lithium-ion batteries. Yet, on the most important composite cathode surfaces, this is a rather complex process involving spatially heterogeneous reactions that have proved difficult to resolve with existing techniques. Here we report a scanning electrochemical cell microscope based approach to define a mobile electrochemical cell that is used to quantitatively visualize electrochemical phenomena at the battery cathode material LiFePO4, with resolution of ~100 nm. The technique measures electrode topography and different electrochemical properties simultaneously, and the information can be combined with complementary microscopic techniques to reveal new perspectives on structure and activity. These electrodes exhibit highly spatially heterogeneous electrochemistry at the nanoscale, both within secondary particles and at individual primary nanoparticles, which is highly dependent on the local structure and composition.
Jose Taberner F, Lopez-Cordoba A, Fernandez-Ballester G, et al., 2014, The Region Adjacent to the C-end of the Inner Gate in Transient Receptor Potential Melastatin 8 (TRPM8) Channels Plays a Central Role in Allosteric Channel Activation, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 289, Pages: 28579-28594
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- Citations: 27
Takahashi Y, Shevchuk AI, Novak P, et al., 2014, Erratum: Multifunctional nanoprobes for nanoscale chemical imaging and localized chemical delivery at surfaces and interfaces (Angewandte Chemie - International Edition (2011) (50) DOI: 10.1002/anie.201102796), Angewandte Chemie - International Edition, Vol: 53, ISSN: 1433-7851
Miragoli M, Yacoub MH, El-Hamamsy I, et al., 2014, Side-specific mechanical properties of valve endothelial cells, AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY, Vol: 307, Pages: H15-H24, ISSN: 0363-6135
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- Citations: 15
Novak P, Shevchuk A, Ruenraroengsak P, et al., 2014, Imaging single nanoparticle interactions with human lung cells using fast ion conductance microscopy, Nano Letters: a journal dedicated to nanoscience and nanotechnology, Vol: 14, Pages: 1202-1207, ISSN: 1530-6984
Experimental data on dynamic interactions between individual nanoparticles and membrane processes at nanoscale, essential for biomedical applications of nanoparticles, remain scarce due to limitations of imaging techniques. We were able to follow single 200 nm carboxyl-modified particles interacting with identified membrane structures at the rate of 15 s/frame using a scanning ion conductance microscope modified for simultaneous high-speed topographical and fluorescence imaging. The imaging approach demonstrated here opens a new window into the complexity of nanoparticle–cell interactions.
Clausmeyer J, Actis P, Cordoba AL, et al., 2014, Nanosensors for the detection of hydrogen peroxide, ELECTROCHEMISTRY COMMUNICATIONS, Vol: 40, Pages: 28-30, ISSN: 1388-2481
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- Citations: 57
Velez-Ortega AC, Belov O, Novak P, et al., 2014, High-Speed Hopping Probe Scanning Ion Conductance Microscopy, 58th Annual Meeting of the Biophysical-Society, Publisher: CELL PRESS, Pages: 797A-798A, ISSN: 0006-3495
Actis P, Tokar S, Klenerman D, et al., 2014, Tailoring Nanoprobes for Single-Cell Surgery, 58th Annual Meeting of the Biophysical-Society, Publisher: CELL PRESS, Pages: 414A-414A, ISSN: 0006-3495
Actis P, Tokar S, Clausmeyer J, et al., 2014, Electrochemical nanoprobes for single-cell analysis, ACS Nano, Vol: 8, Pages: 875-884, ISSN: 1936-0851
The measurement of key molecules in individual cells with minimal disruption to the biological milieu is the next frontier in single-cell analyses. Nanoscale devices are ideal analytical tools because of their small size and their potential for high spatial and temporal resolution recordings. Here, we report the fabrication of disk-shaped carbon nanoelectrodes whose radius can be precisely tuned within the range 5–200 nm. The functionalization of the nanoelectrode with platinum allowed the monitoring of oxygen consumption outside and inside a brain slice. Furthermore, we show that nanoelectrodes of this type can be used to impale individual cells to perform electrochemical measurements within the cell with minimal disruption to cell function. These nanoelectrodes can be fabricated combined with scanning ion conductance microscopy probes, which should allow high resolution electrochemical mapping of species on or in living cells.
Korchev YE, Actis P, Tokar S, et al., 2014, Nanopipet Based Nanoprobes for Single-Cell Analysis, 58th Annual Meeting of the Biophysical-Society, Publisher: CELL PRESS, Pages: 798A-799A, ISSN: 0006-3495
Babakinejad B, Joensson P, Lopez Cordoba A, et al., 2013, Local Delivery of Molecules from a Nanopipette for Quantitative Receptor Mapping on Live Cells, ANALYTICAL CHEMISTRY, Vol: 85, Pages: 9333-9342, ISSN: 0003-2700
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- Citations: 59
Novak P, Gorelik J, Vivekananda U, et al., 2013, Nanoscale-targeted patch-clamp recordings of functional presynaptic ion channels, Neuron, Vol: 79, Pages: 1067-1077, ISSN: 0896-6273
Direct electrical access to presynaptic ion channels has hitherto been limited to large specialized terminals such as the calyx of Held or hippocampal mossy fiber bouton. The electrophysiology and ion-channel complement of far more abundant small synaptic terminals (≤1 μm) remain poorly understood. Here we report a method based on superresolution scanning ion conductance imaging of small synapses in culture at approximately 100–150 nm 3D resolution, which allows presynaptic patch-clamp recordings in all four configurations (cell-attached, inside-out, outside-out, and whole-cell). Using this technique, we report presynaptic recordings of K+, Na+, Cl−, and Ca2+ channels. This semiautomated approach allows direct investigation of the distribution and properties of presynaptic ion channels at small central synapses.
Shevchuk AI, Novak P, Velazquez MA, et al., 2013, Combined ion conductance and fluorescence confocal microscopy for biological cell membrane transport studies, JOURNAL OF OPTICS, Vol: 15, ISSN: 2040-8978
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- Citations: 5
McKelvey K, Nadappuram BP, Actis P, et al., 2013, Fabrication, Characterization, and Functionalization of Dual Carbon Electrodes as Probes for Scanning Electrochemical Microscopy (SECM), ANALYTICAL CHEMISTRY, Vol: 85, Pages: 7519-7526, ISSN: 0003-2700
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- Citations: 48
Anand U, Facer P, Yiangou Y, et al., 2013, Angiotensin II type 2 receptor (AT(2)R) localization and antagonist-mediated inhibition of capsaicin responses and neurite outgrowth in human and rat sensory neurons, European Journal of Pain, Vol: 17, Pages: 1012-1026, ISSN: 1532-2149
BackgroundThe angiotensin II (AngII) receptor subtype 2 (AT2R) is expressed in sensory neurons and may play a role in nociception and neuronal regeneration.MethodsWe used immunostaining with characterized antibodies to study the localization of AT2R in cultured human and rat dorsal root ganglion (DRG) neurons and a range of human tissues. The effects of AngII and AT2R antagonist EMA401 on capsaicin responses in cultured human and rat (DRG) neurons were measured with calcium imaging, on neurite length and density with Gap43 immunostaining, and on cyclic adenosine monophosphate (cAMP) expression using immunofluorescence.ResultsAT2R expression was localized in small-/medium-sized cultured neurons of human and rat DRG. Treatment with the AT2R antagonist EMA401 resulted in dose-related functional inhibition of capsaicin responses (IC50 = 10 nmol/L), which was reversed by 8-bromo-cAMP, and reduced neurite length and density; AngII treatment significantly enhanced capsaicin responses, cAMP levels and neurite outgrowth. The AT1R antagonist losartan had no effect on capsaicin responses. AT2R was localized in sensory neurons of human DRG, and nerve fibres in peripheral nerves, skin, urinary bladder and bowel. A majority sub-population (60%) of small-/medium-diameter neuronal cells were immunopositive in both control post-mortem and avulsion-injured human DRG; some very small neurons appeared to be intensely immunoreactive, with TRPV1 co-localization. While AT2R levels were reduced in human limb peripheral nerve segments proximal to injury, they were preserved in painful neuromas.ConclusionsAT2R antagonists could be particularly useful in the treatment of chronic pain and hypersensitivity associated with abnormal nerve sprouting.
Joensson P, Cordoba AL, Babakinejad B, et al., 2013, Molecular nanomechanics and local stimulus of individual biomolecules on the surface of cells, 9th European-Biophysical-Societies-Association Congress, Publisher: SPRINGER, Pages: S108-S108, ISSN: 0175-7571
Miragoli M, Novak P, Ruenraroengsak P, et al., 2013, Functional interaction between charged nanoparticles and cardiac tissue: a new paradigm for cardiac arrhythmia?, NANOMEDICINE, Vol: 8, Pages: 725-737, ISSN: 1743-5889
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- Citations: 40
Babakinejad B, Yasufumi T, Yasufumi T, et al., 2013, Development of Carbon Nano-Heater: Stimulation of Sensory Neurons for Functional Study of Heat Sensitive Channels (vol 104, pg 519a, 2013), BIOPHYSICAL JOURNAL, Vol: 104, Pages: 1834-1834, ISSN: 0006-3495
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- Citations: 1
Klenerman D, Shevchuk A, Novak P, et al., 2013, Imaging the cell surface and its organization down to the level of single molecules, PHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY B-BIOLOGICAL SCIENCES, Vol: 368, ISSN: 0962-8436
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- Citations: 15
Taberner FJ, Lopez-Cordoba A, Fernandez-Ballester G, et al., 2013, Mutations in the TRP Domain Differentially affect the Function of TRPM8, 57th Annual Meeting of the Biophysical-Society, Publisher: CELL PRESS, Pages: 456A-456A, ISSN: 0006-3495
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- Citations: 1
Bhargava A, Lin X, Novak P, et al., 2013, Super-Resolution Scanning Patch-Clamp Reveals Clustering of Functional Ion Channels in the Adult Ventricular Myocyte, Circ Res, ISSN: 1524-4571
Rationale: Compartmentation of ion channels on the cardiomyocyte surface is important for electrical propagation and electromechanical coupling. The specialized T-tubule and costameric structures facilitate spatial coupling of various ion channels and receptors. Existing methods like immunofluorescence and patch-clamp techniques are limited in their ability to localize functional ion channels. As such, a correlation between channel protein location and channel function remains incomplete. Objective: To validate a method that permits to routinely image the topography of a live cardiomyocyte, and then study clustering of functional ion channels from a specific microdomain. Methods and Results: We used scanning ion conductance microscopy and conventional cell-attached patch-clamp with a software modification that allows controlled increase of pipette tip diameter. The sharp nanopipette used for topography scan was modified into a larger patch pipette which can be positioned with nanoscale precision to a specific site of interest (crest, groove or T-tubules of cardiomyocytes), and sealed to the membrane for cell-attached recording of ion channels. Using this method, we significantly increased the probability of detecting activity of L-type calcium channels in the T-tubules of ventricular cardiomyocytes. We also demonstrated that active sodium channels do not distribute homogenously on the sarcolemma but rather, they segregate into clusters of various densities -most crowded in the crest region- that are surrounded by areas virtually free of functional sodium channels. Conclusions: Our new method substantially increases the throughput of recording location-specific functional ion channels on the cardiomyocyte sarcolemma, thus allowing characterization of ion channels in relation to the microdomain in which they reside.
Zhukov A, Richards O, Ostanin V, et al., 2012, A hybrid scanning mode for fast scanning ion conductance microscopy (SICM) imaging, ULTRAMICROSCOPY, Vol: 121, Pages: 1-7, ISSN: 0304-3991
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- Citations: 32
Takahashi Y, Shevchuk AI, Novak P, et al., 2012, Topographical and electrochemical nanoscale imaging of living cells using voltage-switching mode scanning electrochemical microscopy, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 109, Pages: 11540-11545, ISSN: 0027-8424
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- Citations: 169
Shevchuk AI, Novak P, Taylor M, et al., 2012, An alternative mechanism of clathrin-coated pit closure revealed by ion conductance microscopy, JOURNAL OF CELL BIOLOGY, Vol: 197, Pages: 499-508, ISSN: 0021-9525
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- Citations: 65
Babakinejad B, Yasufumi T, Joensson P, et al., 2012, Quantitative Characterization of Local Chemical Delivery through Nanopipette, BIOPHYSICAL JOURNAL, Vol: 102, Pages: 313A-313A, ISSN: 0006-3495
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- Citations: 1
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