Publications
183 results found
Gorelkin P, Erofeev A, Shevchuk A, et al., 2019, Scanning Ion Conductance Microscope as a new tool for bionanotechnology, Publisher: WILEY, Pages: 169-169, ISSN: 2211-5463
Gopal S, Chiappini C, Penders J, et al., 2019, Porous silicon nanoneedles modulate endocytosis to deliver biological payloads, Advanced Materials, Vol: 31, ISSN: 0935-9648
Owing to their ability to efficiently deliver biological cargo and sense the intracellular milieu, vertical arrays of high aspect ratio nanostructures, known as nanoneedles, are being developed as minimally invasive tools for cell manipulation. However, little is known of the mechanisms of cargo transfer across the cell membrane‐nanoneedle interface. In particular, the contributions of membrane piercing, modulation of membrane permeability and endocytosis to cargo transfer remain largely unexplored. Here, combining state‐of‐the‐art electron and scanning ion conductance microscopy with molecular biology techniques, it is shown that porous silicon nanoneedle arrays concurrently stimulate independent endocytic pathways which contribute to enhanced biomolecule delivery into human mesenchymal stem cells. Electron microscopy of the cell membrane at nanoneedle sites shows an intact lipid bilayer, accompanied by an accumulation of clathrin‐coated pits and caveolae. Nanoneedles enhance the internalization of biomolecular markers of endocytosis, highlighting the concurrent activation of caveolae‐ and clathrin‐mediated endocytosis, alongside macropinocytosis. These events contribute to the nanoneedle‐mediated delivery (nanoinjection) of nucleic acids into human stem cells, which distribute across the cytosol and the endolysosomal system. This data extends the understanding of how nanoneedles modulate biological processes to mediate interaction with the intracellular space, providing indications for the rational design of improved cell‐manipulation technologies.
Xue L, Cadinu P, Paulose Nadappuram B, et al., 2018, Gated single-molecule transport in double-barreled nanopores, ACS Applied Materials and Interfaces, Vol: 10, Pages: 38621-38629, ISSN: 1944-8244
Single-molecule methods have been rapidly developing with the appealing prospect of transforming conventional ensemble-averaged analytical techniques. However, challenges remain especially in improving detection sensitivity and controlling molecular transport. In this article, we present a direct method for the fabrication of analytical sensors that combine the advantages of nanopores and field-effect transistors for simultaneous label-free single-molecule detection and manipulation. We show that these hybrid sensors have perfectly aligned nanopores and field-effect transistor components making it possible to detect molecular events with up to near 100% synchronization. Furthermore, we show that the transport across the nanopore can be voltage-gated to switch on/off translocations in real time. Finally, surface functionalization of the gate electrode can also be used to fine tune transport properties enabling more active control over the translocation velocity and capture rates.
Vaneev AN, Alova A, Erofeev AS, et al., 2018, DETECTING REACTIVE OXYGEN SPECIES IN BIOLOGICAL FLUIDS BY PLATINUM NANOELECTRODE APPLYING AMPEROMETRIC METHOD, BULLETIN OF RUSSIAN STATE MEDICAL UNIVERSITY, Pages: 157-163, ISSN: 2500-1094
Donatien P, Anand U, Yiangou Y, et al., 2018, Granulocyte-macrophage colony-stimulating factor receptor expression in clinical pain disorder tissues and role in neuronal sensitization, PAIN Reports, Vol: 3, Pages: e676-e676, ISSN: 2471-2531
Introduction: Granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR) is highly expressed in peripheral macrophages and microglia, and is involved in arthritis and cancer pain in animal models. However, there is limited information on GM-CSFR expression in human central nervous system (CNS), peripheral nerves, or dorsal root ganglia (DRG), particularly in chronic pain conditions. Objectives: Immunohistochemistry was used to quantify GM-CSFR expression levels in human tissues, and functional sensory effects of GM-CSF were studied in cultured DRG neurons. Results: Granulocyte-macrophage colony-stimulating factor receptor was markedly increased in microglia at lesional sites of multiple sclerosis spinal cords (P = 0.01), which co-localised with macrophage marker CD68 (P = 0.009). In human DRG, GM-CSFR was expressed in a subset of small/medium diameter cells (30%) and few large cells (10%), with no significant change in avulsion-injured DRG. In peripheral nerves, there was a marked decrease in axonal GM-CSFR after chronic painful nerve injury (P = 0.004) and in painful neuromas (P = 0.0043); CD-68-positive macrophages were increased (P = 0.017) but did not appear to express GM-CSFR. Although control synovium showed absent GM-CSFR immunostaining, this was markedly increased in macrophages of painful osteoarthritis knee synovium. Granulocyte-macrophage colony-stimulating factor receptor was expressed in 17 ± 1.7% of small-/medium-sized cultured adult rat DRG neurons, and in 27 ± 3.3% of TRPV1-positive neurons. Granulocyte-macrophage colony-stimulating factor treatment sensitized capsaicin responses in vitro, which were diminished by p38 MAPK or TrkA inhibitors. Conclusion: Our findings support GM-CSFR as a therapeutic target for pain and hypersensitivity in clinical CNS and peripheral inflammatory conditions. Although GM-CSFR was decreased in chronic painful injured peripheral nerves, it could mediate CNS neuroinflammatory effects, which deserves
Gorelkin P, Erofeev A, Alova A, et al., 2018, Nanopipette navigation system as a new tool for nanoscale investigation of living cells, FEBS OPEN BIOMED, Publisher: WILEY, Pages: 480-480, ISSN: 2211-5463
Erofeev A, Gorelkin P, Garanina A, et al., 2018, Smart nanopipette for single cell analysis, Publisher: WILEY, Pages: 480-481, ISSN: 2211-5463
Anand U, Yiangou Y, Akbar A, et al., 2018, Glucagon-like peptide 1 receptor (GLP-1R) expression by nerve fibres in inflammatory bowel disease and functional effects in cultured neurons, PLoS ONE, Vol: 13, ISSN: 1932-6203
IntroductionGlucagon like-peptide 1 receptor (GLP-1R) agonists diminish appetite and may contribute to the weight loss in inflammatory bowel disease (IBD).ObjectivesThe aim of this study was to determine, for the first time, the expression of GLP-1R by colon nerve fibres in patients with IBD, and functional effects of its agonists in cultured rat and human sensory neurons.MethodsGLP-1R and other nerve markers were studied by immunohistochemistry in colon biopsies from patients with IBD (n = 16) and controls (n = 8), human dorsal root ganglia (DRG) tissue, and in GLP-1R transfected HEK293 cells. The morphological effects of incretin hormones oxyntomodulin, exendin-4 and glucagon were studied on neurite extension in cultured DRG neurons, and their functional effects on capsaicin and ATP signalling, using calcium imaging.ResultsSignificantly increased numbers of colonic mucosal nerve fibres were observed in IBD biopsies expressing GLP-1R (p = 0.0013), the pan-neuronal marker PGP9.5 (p = 0.0008), and sensory neuropeptide CGRP (p = 0.0014). An increase of GLP-1R positive nerve fibres in IBD colon was confirmed with a different antibody to GLP-1R (p = 0.016). GLP-1R immunostaining was intensely positive in small and medium-sized neurons in human DRG, and in human and rat DRG cultured neurons. Co-localization of GLP-1R expression with neuronal markers in colon and DRG confirmed the neural expression of GLP-1R, and antibody specificity was confirmed in HEK293 cells transfected with the GLP-1R. Treatment with oxyntomodulin, exendin-4 and GLP-1 increased neurite length in cultured neurons compared with controls, but did not stimulate calcium influx directly, or affect capsaicin responses. However, exendin-4 significantly enhanced ATP responses in human DRG neurons.ConclusionOur results show that increased GLP-1R innervation in IBD bowel could mediate enhanced visceral afferent signalling, and provide a peripheral target for therapeutic intervention. The differential effect of
Erofeev A, Gorelkin P, Garanina A, et al., 2018, Novel method for rapid toxicity screening of magnetic nanoparticles, SCIENTIFIC REPORTS, Vol: 8, ISSN: 2045-2322
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- Citations: 57
Zhou Y, Saito M, Miyamoto T, et al., 2018, Nanoscale imaging of primary cilia with scanning ion conductance microscopy, Analytical Chemistry, Vol: 90, Pages: 2891-2895, ISSN: 0003-2700
Primary cilia are hair-like sensory organelles whose dimensions and location vary with cell type and culture condition. Herein, we employed scanning ion conductance microscopy (SICM) to visualize the topography of primary cilia from different cell types. By combining SICM with fluorescence imaging, we successfully distinguished between surface cilia that project outward from the cell surface and subsurface cilia that are trapped below it. The nanoscale structure of the ciliary pocket, which cannot be easily identified using a confocal fluorescence microscope, was observed in SICM images. Furthermore, we developed a topographic reconstruction method using current-distance profiles to evaluate the relationship between set point and topographic image and found that a low set point is important for detecting the true topography of a primary cilium using hopping mode SICM.
Ren R, Zhang Y, Paulose Nadappuram B, et al., 2017, Nanopore extended field effect transistor for selective single molecule biosensing, Nature Communications, Vol: 8, Pages: 1-9, ISSN: 2041-1723
There has been a significant drive to deliver nanotechnological solutions to biosensing, yet there remains an unmet need in the development of biosensors that are affordable, integrated, fast, capable of multiplexed detection, and offer high selectivity for trace analyte detection in biological fluids. Herein, some of these challenges are addressed by designing a new class of nanoscale sensors dubbed nanopore extended field-effect transistor (nexFET) that combine the advantages of nanopore single-molecule sensing, field-effect transistors, and recognition chemistry. We report on a polypyrrole functionalized nexFET, with controllable gate voltage that can be used to switch on/off, and slow down single-molecule DNA transport through a nanopore. This strategy enables higher molecular throughput, enhanced signal-to-noise, and even heightened selectivity via functionalization with an embedded receptor. This is shown for selective sensing of an anti-insulin antibody in the presence of its IgG isotype.
Gorelkin P, Erofeev A, Komarova A, et al., 2017, Nanopipette biosensors as a new tool for metabolism and signaling investigation, 42nd Congress of the Federation-of-European-Biochemical-Societies (FEBS) on From Molecules to Cells and Back, Publisher: Wiley, Pages: 372-372, ISSN: 0014-2956
Cadinu P, Paulose Nadappuram B, Lee DJ, et al., 2017, Single molecule trapping and sensing using dual nanopores separated by a zeptoliter nanobridge, Nano Letters, Vol: 17, Pages: 6376-6384, ISSN: 1530-6984
There is a growing realization, especially within the diagnostic and therapeutic community, that the amount of information enclosed in a single molecule can not only enable a better understanding of biophysical pathways, but also offer exceptional value for early stage biomarker detection of disease onset. To this end, numerous single molecule strategies have been proposed, and in terms of label-free routes, nanopore sensing has emerged as one of the most promising methods. However, being able to finely control molecular transport in terms of transport rate, resolution, and signal-to-noise ratio (SNR) is essential to take full advantage of the technology benefits. Here we propose a novel solution to these challenges based on a method that allows biomolecules to be individually confined into a zeptoliter nanoscale droplet bridging two adjacent nanopores (nanobridge) with a 20 nm separation. Molecules that undergo confinement in the nanobridge are slowed down by up to 3 orders of magnitude compared to conventional nanopores. This leads to a dramatic improvement in the SNR, resolution, sensitivity, and limit of detection. The strategy implemented is universal and as highlighted in this manuscript can be used for the detection of dsDNA, RNA, ssDNA, and proteins.
Komarova A, Gorelkin P, Erofeev A, et al., 2017, Decrease of external oxygen concentration as an early response to cell wall injury of Chara corallina, 42nd Congress of the Federation-of-European-Biochemical-Societies (FEBS) on From Molecules to Cells and Back, Publisher: WILEY, Pages: 229-229, ISSN: 1742-464X
Usmanov AR, Erofeev AS, Gorelkin PV, et al., 2017, Nanocapillaries: A Universal Tool for Modern Biomedical Applications, NANOTECHNOLOGIES IN RUSSIA, Vol: 12, Pages: 448-460, ISSN: 1995-0780
Komarova A, Gorelkin P, Erofeev A, et al., 2017, Oxygen gulp in microwounded cells of Chara corallina detected by novel O2 nanosensors, 19th IUPAB Congress / 11th EBSA Congress, Publisher: SPRINGER, Pages: S245-S245, ISSN: 0175-7571
Vivekananda U, Novak P, Bello OD, et al., 2017, Kv1.1 channelopathy abolishes presynaptic spike width modulation by subthreshold somatic depolarization, Proceedings of the National Academy of Sciences of the United States of America, Vol: 114, Pages: 2395-2400, ISSN: 0027-8424
Although action potentials propagate along axons in an all-or-none manner, subthreshold membrane potential fluctuations at the soma affect neurotransmitter release from synaptic boutons. An important mechanism underlying analog–digital modulation is depolarization-mediated inactivation of presynaptic Kv1-family potassium channels, leading to action potential broadening and increased calcium influx. Previous studies have relied heavily on recordings from blebs formed after axon transection, which may exaggerate the passive propagation of somatic depolarization. We recorded instead from small boutons supplied by intact axons identified with scanning ion conductance microscopy in primary hippocampal cultures and asked how distinct potassium channels interact in determining the basal spike width and its modulation by subthreshold somatic depolarization. Pharmacological or genetic deletion of Kv1.1 broadened presynaptic spikes without preventing further prolongation by brief depolarizing somatic prepulses. A heterozygous mouse model of episodic ataxia type 1 harboring a dominant Kv1.1 mutation had a similar broadening effect on basal spike shape as deletion of Kv1.1; however, spike modulation by somatic prepulses was abolished. These results argue that the Kv1.1 subunit is not necessary for subthreshold modulation of spike width. However, a disease-associated mutant subunit prevents the interplay of analog and digital transmission, possibly by disrupting the normal stoichiometry of presynaptic potassium channels.
Marquitan M, Clausmeyer J, Actis P, et al., 2016, Intracellular Hydrogen Peroxide Detection with Functionalised Nanoelectrodes, CHEMELECTROCHEM, Vol: 3, Pages: 2125-2129, ISSN: 2196-0216
Sanchez-Alonso JL, Bhargava A, O'Hara T, et al., 2016, Microdomain-specific modulation of L-type calcium channels leads to triggered ventricular arrhythmia in heart failure, Circulation Research, Vol: 119, Pages: 944-955, ISSN: 1524-4571
RATIONALE: Disruption in subcellular targeting of Ca(2+) signaling complexes secondary to changes in cardiac myocyte structure may contribute to the pathophysiology of a variety of cardiac diseases, including heart failure (HF) and certain arrhythmias. OBJECTIVE: To explore microdomain-targeted remodeling of ventricular L-type Ca(2+) channels (LTCCs) in HF. METHODS AND RESULTS: Super-resolution scanning patch-clamp, confocal and fluorescence microscopy were used to explore distribution of single LTCCs in different membrane microdomains of non-failing and failing human and rat ventricular myocytes. Disruption of membrane structure in both species led to re-distribution of functional LTCCs from their canonical location in transversal tubules (T-tubules) to the non-native crest of the sarcolemma, where their open probability (Po) was dramatically increased (0.034±0.011 vs 0.154±0.027, P<0.001). High Po was linked to enhanced calcium-calmodulin kinase II (CaMKII)-mediated phosphorylation in non-native microdomains and resulted in an elevated ICa,L window current which contributed to the development of early afterdepolarizations (EADs). A novel model of LTCC function in HF was developed; following its validation with experimental data, the model was used to ascertain how HF-induced T-tubule loss led to altered LTCC function and EADs. The HF myocyte model was then implemented in a 3D left ventricle model, demonstrating that such EADs can propagate and initiate reentrant arrhythmias. CONCLUSIONS: Microdomain-targeted remodeling of LTCC properties is an important event in pathways that may contribute to ventricular arrhythmogenesis in the settings of HF-associated remodeling. This extends beyond the classical concept of electrical remodelling in HF and adds a new dimension to cardiovascular disease.
Anand P, Yiangou Y, Anand U, et al., 2016, Nociceptin/Orphanin FQ receptor expression in clinical pain disorders and functional effects in cultured neurons, Pain, Vol: 157, Pages: 1960-1969, ISSN: 1872-6623
The Nociceptin/Orphanin FQ peptide receptor (NOP), activated by its endogenous peptide ligand Nociceptin/Orphanin FQ (N/OFQ), exerts several effects including modulation of pain signalling. We have examined, for the first time, the tissue distribution of the NOP receptor in clinical visceral and somatic pain disorders by immunohistochemistry, and assessed functional effects of NOP and [micro] opioid receptor activation in cultured human and rat dorsal root ganglion (DRG) neurons. Quantification of NOP-positive nerve fibres within the bladder sub-urothelium revealed a remarkable several-fold increase in Detrusor Overactivity (p<0.0001) and Painful Bladder Syndrome patient specimens (p=0.0014), compared to controls. In post-mortem control human DRGs, 75-80% of small/medium neurons (<=50 [micro]m diameter) in the lumbar (somatic) and sacral (visceral) DRG were positive for NOP, and fewer large neurons; avulsion-injured cervical human DRG neurons showed similar numbers. NOP-immunoreactivity was significantly decreased in injured peripheral nerves (p=0.0004), and also in painful neuromas (p=0.025). Calcium imaging studies in cultured rat DRG neurons demonstrated dose-dependent inhibition of capsaicin responses in the presence of N/OFQ, with an IC50 of 8.6 pM. In cultured human DRG neurons, 32% inhibition of capsaicin responses was observed in the presence of 1 pM N/OFQ (p<0.001). The maximum inhibition of capsaicin responses was greater with N/OFQ than [mu]-opioid receptor agonist DAMGO. Our findings highlight the potential of NOP agonists, particularly in urinary bladder overactivity and pain syndromes. The regulation of NOP expression in visceral and somatic sensory neurons by target-derived neurotrophic factors deserves further study, and the efficacy of NOP selective agonists in clinical trials.
Anand U, Sinisi M, Fox M, et al., 2016, Mycolactone mediated neurite degeneration and functional effects in cultured human and rat DRG neurons: mechanisms underlying hypoalgesia in Buruli Ulcer, Molecular Pain, Vol: 12, ISSN: 1744-8069
Background: Mycolactone (ML) is a polyketide toxin secreted by the mycobacterium M.ulcerans, responsible for the extensive hypoalgesic skin lesions characteristic of patients withBuruli Ulcer. A recent pre-clinical study proposed that ML may produce analgesia via activationof the angiotensin II type 2 receptor (AT2R). In contrast, AT2R antagonist EMA401 has shownanalgesic efficacy in animal models and clinical trials for neuropathic pain. We thereforeinvestigated the morphological and functional effects of ML in cultured human and rat dorsal rootganglia (DRG) neurons, and the role of AT2R using EMA401. Primary sensory neurons wereprepared from avulsed cervical human DRG, and rat DRG. 24 hours after plating, neurons wereincubated for 24 to 96 hours with synthetic ML A/B, followed by immunostaining with antibodiesto PGP9.5, Gap43, tubulin, or Mitotracker dye staining. Acute functional effects wereexamined by measuring capsaicin responses with calcium imaging in DRG neuronal culturestreated with ML.Results: Morphological effects: ML treated cultures showed dramatically reduced numbers ofsurviving neurons and non-neuronal cells, reduced Gap43 and tubulin expression, degeneratingneurites and reduced cell body diameter, compared with controls. Dose related reduction ofneurite length was observed in ML treated cultures. Mitochondria were distributed throughout thelength of neurites and soma of control neurons, but clustered in the neurites and soma of MLtreated neurons. Functional effects: ML treated human and rat DRG neurons showed doserelated inhibition of capsaicin responses, which were reversed by calcineurin inhibitorcyclosporine and phosphodiesterase inhibitor IBMX, indicating involvement of cAMP/ATPreduction. The morphological and functional effects of ML were not altered by Angiotensin II orAT2R antagonist EMA401.3Conclusion: ML induces toxic effects in DRG neurons, leading to impaired nociceptor function,neurite degeneration and cell death, resembling the cuta
Shevchuk A, Tokar S, Gopal S, et al., 2016, Angular approach Scanning Ion Conductance Microscopy, Biophysical Journal, Vol: 110, Pages: 2252-2265, ISSN: 1542-0086
Scanning ion conductance microscopy (SICM) is a super-resolution live imagingtechnique that uses a glass nanopipette as an imaging probe to produce 3D images of cell surface.SICM can be used to analyze cell morphology at nanoscale, follow membrane dynamics, preciselyposition an imaging nanopipette close to a structure of interest, and use it to obtain ion channelrecordings or locally apply stimuli or drugs. Practical implementations of these SICM advantages,however, are often complicated due to the limitations of currently available SICM systems that“inherited” their design from other scanning probe microscopes in which the scan assembly isplaced right above the specimen. Such arrangement makes the setting of optimal illuminationnecessary for phase contrast or the use of high magnification upright optics difficult. Here wedescribe the designs that allow mounting SICM scanhead on a standard patch-clampmicromanipulator and imaging the sample at an adjustable approach angle. This angle could be asshallow as the approach angle of a patch-clamp pipette between a water immersion objective andthe specimen. Using this angular approach SICM, we obtained topographical images of cells grownon non-transparent nanoneedle arrays, of islets of Langerhans, and of hippocampal neurons under 2upright optical microscope. We also imaged previously inaccessible areas of cells such as the sidesurfaces of the hair cell stereocilia and the intercalated disks of isolated cardiac mocytes, andperformed targeted patch-clamp recordings from the latter. Thus, our new angular approach SICMallows imaging of living cells on non-transparent substrates and a seamless integration with mostpatch-clamp setups on either inverted or upright microscopes, which would facilitate research incell biophysics and physiology.
Velez-Ortega AC, Belov O, Novak P, et al., 2016, Adaptive Hopping Probe Ion Conductance Microscopy of Live Cells at ∼5-10 NM Resolution, 60th Annual Meeting of the Biophysical-Society, Publisher: CELL PRESS, Pages: 517A-517A, ISSN: 0006-3495
Leo-Macias A, Agullo-Pascual E, Sanchez-Alonso JL, et al., 2016, Nanoscale visualization of functional adhesion/excitability nodes at the intercalated disc (vol 6, 10342, 2015), NATURE COMMUNICATIONS, Vol: 7, ISSN: 2041-1723
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Zhang Y, Clausmeyer J, Babakinejad B, et al., 2016, Spearhead Nanometric Field-Effect Transistor Sensors for Single-Cell Analysis., ACS Nano, Vol: 10, Pages: 3214-3221, ISSN: 1936-086X
Nanometric field-effect-transistor (FET) sensors are made on the tip of spear-shaped dual carbon nanoelectrodes derived from carbon deposition inside double-barrel nanopipettes. The easy fabrication route allows deposition of semiconductors or conducting polymers to comprise the transistor channel. A channel from electrodeposited poly pyrrole (PPy) exhibits high sensitivity toward pH changes. This property is exploited by immobilizing hexokinase on PPy nano-FETs to give rise to a selective ATP biosensor. Extracellular pH and ATP gradients are key biochemical constituents in the microenvironment of living cells; we monitor their real-time changes in relation to cancer cells and cardiomyocytes. The highly localized detection is possible because of the high aspect ratio and the spear-like design of the nano-FET probes. The accurately positioned nano-FET sensors can detect concentration gradients in three-dimensional space, identify biochemical properties of a single living cell, and after cell membrane penetration perform intracellular measurements.
Schobesberger S, Jonsson P, Buzuk A, et al., 2016, Nanoscale, Voltage-Driven Application of Bioactive Substances onto Cells with Organized Topography, Biophysical Journal, Vol: 110, Pages: 141-146, ISSN: 1542-0086
With scanning ion conductance microscopy (SICM), a noncontact scanning probe technique, it is possible both toobtain information about the surface topography of live cells and to apply molecules onto specific nanoscale structures. Thetechnique is therefore widely used to apply chemical compounds and to study the properties of molecules on the surfaces ofvarious cell types. The heart muscle cells, i.e., the cardiomyocytes, possess a highly elaborate, unique surface topographyincluding transverse-tubule (T-tubule) openings leading into a cell internal system that exclusively harbors many proteins necessaryfor the cell’s physiological function. Here, we applied isoproterenol into these surface openings by changing the appliedvoltage over the SICM nanopipette. To determine the grade of precision of our application we used finite-element simulationsto investigate how the concentration profile varies over the cell surface. We first obtained topography scans of the cardiomyocytesusing SICM and then determined the electrophoretic mobility of isoproterenol in a high ion solution to be 7 10 9 m2/V s.The simulations showed that the delivery to the T-tubule opening is highly confined to the underlying Z-groove, and especially tothe first T-tubule opening, where the concentration is ~6.5 times higher compared to on a flat surface under the same deliverysettings. Delivery to the crest, instead of the T-tubule opening, resulted in a much lower concentration, emphasizing the importanceof topography in agonist delivery. In conclusion, SICM, unlike other techniques, can reliably deliver precise quantities ofcompounds to the T-tubules of cardiomyocytes
Leo-Macias A, Agullo-Pascual E, Sanchez-Alonso JL, et al., 2016, Nanoscale visualization of functional adhesion/excitability nodes at the intercalated disc, NATURE COMMUNICATIONS, Vol: 7, ISSN: 2041-1723
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Leo-Macias A, Agullo-Pascual E, Sanchez-Alonso JL, et al., 2015, Nanoscale Visualization of Functional Adhesion/Excitability Nodes at the Intercalated Disc., 69th Annual Meeting of the Society-of-General-Physiologists - Macromolecular Local Signaling Complexes, Publisher: ROCKEFELLER UNIV PRESS, Pages: 9A-10A, ISSN: 0022-1295
Seifert J, Rheinlaender J, Novak P, et al., 2015, Comparison of atomic force microscopy and scanning ion conductance microscopy for live cell imaging, 10th European-Biophysical-Societies-Association (EBSA) European Biophysics Congress, Publisher: SPRINGER, Pages: S83-S83, ISSN: 0175-7571
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
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