Subcellular heterogeneity of ryanodine receptor properties in ventricular myocytes with low T-tubule density

Rationale: In ventricular myocytes of large mammals, not all ryanodine receptor (RyR) clusters are associated with T-tubules (TTs); this fraction increases with cellular remodeling after myocardial infarction (MI). Objective: To characterize RyR functional properties in relation to TT proximity, at baseline and after MI. Methods: Myocytes were isolated from left ventricle of healthy pigs (CTRL) or from the area adjacent to a myocardial infarction (MI). Ca2+ transients were measured under whole-cell voltage clamp during confocal linescan imaging (fluo-3) and segmented according to proximity of TTs (sites of early Ca2+ release, F>F50 within 20 ms) or their absence (delayed areas). Spontaneous Ca2+ release events during diastole, Ca2+ sparks, reflecting RyR activity and properties, were subsequently assigned to either category. Results: In CTRL, spark frequency was higher in proximity of TTs, but spark duration was significantly shorter. Block of Na+/Ca2+ exchanger (NCX) prolonged spark duration selectively near TTs, while block of Ca2+ influx via Ca2+ channels did not affect sparks properties. In MI, total spark mass was increased in line with higher SR Ca2+ content. Extremely long sparks (>47.6 ms) occurred more frequently. The fraction of near-TT sparks was reduced; frequency increased mainly in delayed sites. Increased duration was seen in near-TT sparks only; Ca2+ removal by NCX at the membrane was significantly lower in MI. Conclusion: TT proximity modulates RyR cluster properties resulting in intracellular heterogeneity of diastolic spark activity. Remodeling in the area adjacent to MI differentially affects these RyR subpopulations. Reduction of the number of sparks near TTs and reduced local NCX removal limit cellular Ca2+ loss and raise SR Ca2+ content, but may promote Ca2+ waves

Species identification by experts and non-experts: comparing images from field guides

Accurate species identification is fundamental when recording ecological data. However, the ability to correctly identify organisms visually is rarely questioned. We investigated how experts and non-experts compared in the identification of bumblebees, a group of insects of considerable conservation concern. Experts and non-experts were asked whether two concurrent bumblebee images depicted the same or two different species. Overall accuracy was below 60% and comparable for experts and non-experts. However, experts were more consistent in their answers when the same images were repeated, and more cautious in committing to a definitive answer. Our findings demonstrate the difficulty of correctly identifying bumblebees using images from field guides. Such error rates need to be accounted for when interpreting species data, whether or not they have been collected by experts. We suggest that investigation of how experts and non-experts make observations should be incorporated into study design, and could be used to improve training in species identification

Effectiveness of acupuncture, special dressings and simple, low-adherence dressings for healing venous leg ulcers in primary healthcare: study protocol for a cluster-randomized open-labeled trial

<p>Abstract</p> <p>Background</p> <p>Venous leg ulcers constitute a chronic recurring complaint that affects 1.0–1.3% of the adult population at some time in life, and which corresponds to approximately 75% of all chronic ulcers of the leg. Multilayer compression bandaging is, at present, the only treatment that has been proved to be effective in treating this type of ulcer. There is no consensus, however, about the dressings that may be applied, beneath the compression, to promote the healing of this type of ulcer, as there does not seem to be any added benefit from using special dressings rather than simple, low-adherence ones. As well as analgesia, acupuncture provokes peripheral vasodilation, in skin and muscles – which has been demonstrated both experimentally and in clinical practice – probably due to the axon reflex, among other mechanisms. The aim of the present study is to measure the effectiveness and cost of compression treatment for venous leg ulcers combined with special dressings, in comparison with low-adherence ones and acupuncture.</p> <p>Methods/design</p> <p>Cluster-randomized open-labeled trial, at 15 primary healthcare clinics in the Sevilla-Sur Healthcare District, with a control group treated with compression bandaging and low-adherence dressings; the experiment will consist, on the one hand, of the compression treatment applied in combination with special dressings (Treatment 1), and on the other, the compression treatment applied in association with low-adherence dressings, together with acupuncture (Treatment 2).</p> <p>Discussion</p> <p>The results will be measured and recorded in terms of the median time elapsed until complete healing of the ulcer, and the rate of complete healing at 3 months after beginning the treatment. An economic analysis will also be made.</p> <p>This study, carried out in the context of real clinical practice, will provide information for decision-taking concerning the effectiveness of special dressings. Moreover, for the first time a high-quality study will evaluate the effectiveness of acupuncture in the process of healing venous leg ulcers.</p> <p>Trial registration</p> <p>Current Controlled Trials ISRCTN26438275.</p

Immunocytochemical characterisation of cultures of human bladder mucosal cells

<p>Abstract</p> <p>Background</p> <p>The functional role of the bladder urothelium has been the focus of much recent research. The bladder mucosa contains two significant cell types: urothelial cells that line the bladder lumen and suburothelial interstitial cells or myofibroblasts. The aims of this study were to culture these cell populations from human bladder biopsies and to perform immunocytochemical characterisation.</p> <p>Methods</p> <p>Primary cell cultures were established from human bladder biopsies (n = 10). Individual populations of urothelial and myofibroblast-like cells were isolated using magnetic activated cell separation (MACS). Cells were slow growing, needing 3 to 5 weeks to attain confluence.</p> <p>Results</p> <p>Cytokeratin 20 positive cells (umbrella cells) were isolated at primary culture and also from patients' bladder washings but these did not proliferate. In primary culture, proliferating cells demonstrated positive immunocytochemical staining to cytokeratin markers (AE1/AE3 and A0575) as well fibroblasts (5B5) and smooth muscle (αSMA) markers. An unexpected finding was that populations of presumptive urothelial and myofibroblast-like cells, isolated using the MACS beads, stained for similar markers. In contrast, staining for cytokeratins and fibroblast or smooth muscle markers was not co-localised in full thickness bladder sections.</p> <p>Conclusions</p> <p>Our results suggest that, in culture, bladder mucosal cells may undergo differentiation into a myoepithelial cell phenotype indicating that urothelial cells have the capacity to respond to environmental changes. This may be important pathologically but also suggests that studies of the physiological function of these cells in culture may not give a reliable indicator of human physiology.</p

Nanorings and rods interconnected by self-assembly mimicking an artificial network of neurons

[EN] Molecular electronics based on structures ordered as neural networks emerges as the next evolutionary milestone in the construction of nanodevices with unprecedented applications. However, the straightforward formation of geometrically defined and interconnected nanostructures is crucial for the production of electronic circuitry nanoequivalents. Here we report on the molecularly fine-tuned self-assembly of tetrakis-Schiff base compounds into nanosized rings interconnected by unusually large nanorods providing a set of connections that mimic a biological network of neurons. The networks are produced through self-assembly resulting from the molecular conformation and noncovalent intermolecular interactions. These features can be easily generated on flat surfaces and in a polymeric matrix by casting from solution under ambient conditions. The structures can be used to guide the position of electron-transporting agents such as carbon nanotubes on a surface or in a polymer matrix to create electrically conducting networks that can find direct use in constructing nanoelectronic circuits.The research leading to these results has received funding from ICIQ, ICREA, the Spanish Ministerio de Economia y Competitividad (MINECO) through project CTQ2011-27385 and the European Community Seventh Framework Program (FP7-PEOPLE-ITN-2008, CONTACT consortium) under grant agreement number 238363. We acknowledge E. C. Escudero-Adan, M. Martinez-Belmonte and E. Martin from the X-ray department of ICIQ for crystallographic analysis, and M. Moncusi, N. Argany, R. Marimon, M. Stefanova and L. Vojkuvka from the Servei de Recursos Cientifics i Tecnics from Universitat Rovira i Virgili (Tarragona, Spain).Escarcega-Bobadilla, MV.; Zelada-Guillen, GA.; Pyrlin, SV.; Wegrzyn, M.; Ramos, MMD.; Giménez Torres, E.; Stewart, A.... (2013). Nanorings and rods interconnected by self-assembly mimicking an artificial network of neurons. Nature Communications. 4:2648-2648. https://doi.org/10.1038/ncomms3648S264826484Champness, N. R. Making the right connections. Nat. Chem. 4, 149–150 (2012).Hopfield, J. J. & Tank, D. W. Computing with neural circuits: A model. Science 233, 625–633 (1986).Andres, P. R. et al. Self-assembly of a two-dimensional superlattice of molecularly linked metal clusters. Science 273, 1690–1693 (1996).Eichen, Y., Braun, E., Sivan, U. & Ben-Yoseph, G. Self-assembly of nanoelectronic components and circuits using biological templates. Acta Polym. 49, 663–670 (1998).Kawakami, T. et al. Possibilities of molecule-based spintronics of DNA wires, sheets, and related materials. Int. J. Quantum Chem. 105, 655–671 (2005).Kashtan, N., Itzkovitz, S., Milo, R. & Alon, U. Topological generalizations of network motifs. Phys. Rev. E 70, 031909 (2004).Grill, L. et al. Nano-architectures by covalent assembly of molecular building blocks. Nat. Nanotech. 2, 687–691 (2007).Lafferentz, L. et al. Controlling on-surface polymerization by hierarchical and substrate-directed growth. Nat. Chem. 4, 215–220 (2012).Alivisatos, A. P. et al. From molecules to materials: current trends and future directions. Adv. Mater. 10, 1297–1336 (1998).Pauling, L. The principles determining the structure of complex ionic crystals. J. Am. Chem. Soc. 51, 1010–1026 (1929).Damasceno, P. F., Engel, M. & Glotzer, S. C. Predictive self-assembly of polyhedra into complex structures. Science 337, 453–457 (2012).De Graaf, J. & Manna, L. A roadmap for the assembly of polyhedral particles. Science 337, 417–418 (2012).Percec, V. et al. Controlling polymer shape through the self-assembly of dendritic side-groups. Nature 391, 161–164 (1998).Stupp, S. I. et al. Supramolecular materials: self-organized nanostructures. Science 276, 384–389 (1997).Mann, S. The chemistry of form. Angew. Chem. Int. Ed. 39, 3392–3406 (2000).Sakakibara, K., Hill, J. P. & Ariga, K. Thin-film-based nanoarchitectures for soft matter: controlled assemblies into two-dimensional worlds. Small 7, 1288–1308 (2011).Huang, Z. et al. Pulsating tubules from noncovalent macrocycles. Science 337, 1521–1526 (2012).Ackermann, D., Jester, S.-S. & Famulok, M. Design strategy for DNA rotaxanes with a mechanically reinforced PX100 axle. Angew. Chem. Int. Ed. 27, 6771–6775 (2012).Marx, J. L. Microtubules: versatile organelles. Science 181, 1236–1237 (1973).Heus, H. A. & Pardi, A. Structural features that give rise to the unusual stability of RNA hairpins containing GNRA loops. Science 253, 191–194 (1991).Braun, E., Eichen, Y., Sivan, U. & Ben-Yoseph, G. DNA-templated assembly and electrode attachment of a conducting silver wire. Nature 391, 775–778 (1998).Zhang, S. Fabrication of novel biomaterials through molecular self-assembly. Nat. Biotechnol. 21, 1171–1178 (2003).Cai, X. et al. Integrated compact optical vortex beam emitters. Science 338, 363–365 (2012).Clark, A. W. & Cooper, J. M. Nanogap ring antennae as plasmonically coupled SERRS substrates. Small 7, 119–125 (2011).Armani, A. M., Kulkarni, R. P., Fraser, S. E., Flagan, R. C. & Vahala, K. J. Label-free, single-molecule detection with optical microcavities. Science 317, 783–787 (2007).Frischmann, P. D., Guieu, S., Tabeshi, R. & MacLachlan, M. J. Columnar organization of head-to-tail self-assembled Pt4 rings. J. Am. Chem. Soc. 132, 7668–7675 (2010).Frischmann, P. D. et al. Capsule formation, carboxylate exchange, and DFT exploration of cadmium cluster metallocavitands: highly dynamic supramolecules. J. Am. Chem. Soc. 132, 3893–3908 (2010).Akine, S., Hotate, S. & Nabeshima, T. A molecular leverage for helicity control and helix Inversion. J. Am. Chem. Soc. 133, 13868–13871 (2011).Salassa, G. et al. Extremely strong self-assembly of a bimetallic salen complex visualized at the single-molecule level. J. Am. Chem. Soc. 134, 7186–7192 (2012).Escárcega-Bobadilla, M. V., Salassa, G., Martínez Belmonte, M., Escudero-Adán, E. C. & Kleij, A. W. Versatile switching in substrate topicity: supramolecular chirality induction in di- and trinuclear host complexes. Chem. Eur. J. 18, 6805–6810 (2012).Frischmann, P. D., Jiang, J., Hui, J. K.-H., Grzybowski, J. J. & MacLachlan, M. J. Reversible—irreversible approach to Schiff base macrocycles. Access to isomeric macrocycles with multiple salphen pockets. Org. Lett. 10, 1255–1258 (2008).Glaser, T. Rational design of single-molecule magnets: a supramolecular approach. Chem. Commun. 47, 116–130 (2011).Lee, E. C. et al. Understanding of assembly phenomena by aromatic−aromatic interactions: benzene dimer and the substituted systems. J. Phys. Chem. A 111, 3446–3457 (2007).Grybowski, B. A., Wilmer, C. E., Kim, J., Browne, K. P. & Bishop, K. J. M. Self-assembly: from crystals to cells. Soft Matter. 5, 1110–1128 (2009).Martínez Belmonte, M. et al. Self-assembly of Zn(salphen) complexes: steric regulation, stability studies and crystallographic analysis revealing an unexpected dimeric 3,3′-t-Bu-substituted Zn(salphen) complex. Dalton Trans. 39, 4541–4550 (2010).Salassa, G., Castilla, A. M. & Kleij, A. W. Cooperative self-assembly of a macrocyclic Schiff base complex. Dalton Trans. 40, 5236–5243 (2011).Hormoz, S. & Brenner, M. P. Design principles for self-assembly with short-range interactions. Proc. Natl Acad. Sci. 108, 5193–5198 (2011).Biemans, H. A. M. et al. Hexakis porphyrinato benzenes. A new class of porphyrin arrays. J. Am. Chem. Soc. 120, 11054–11060 (1998).Lensen, M. C. et al. Aided self-assembly of porphyrin nanoaggregates into ring-shaped architectures. Chem. Eur. J. 10, 831–839 (2004).Martin, A., Buguin, A. & Brochard-Wyart, F. Dewetting nucleation centers at soft interfaces. Langmuir. 17, 6553–6559 (2001).Schenning, A. P. H. J., Benneker, F. B. G., Geurts, H. P. M., Liu, X. Y. & Nolte, R. J. M. Porphyrin wheels. J. Am. Chem. Soc. 118, 8549–8552 (1996).Deegan, R. D. et al. Capillary flow as the cause of ring strains from dried liquid drops. Nature 389, 827–829 (1997).Scriven, L. E. & Sternling, C. V. The Marangoni effects. Nature 187, 186–188 (1960).Cai, Y. & Newby, B. Z. Marangoni flow-induced self-assembly of hexagonal and stripe-like nanoparticle patterns. J. Am. Chem. Soc. 130, 6076–6077 (2008).Whitesides, G. M. & Grzybowski, B. Self-assembly at all scales. Science 295, 2418–2421 (2002).Mann, S. Self-assembly and transformation of hybrid nano-objects and nanostructures under equilibrium and non-equilibrium conditions. Nat. Mater. 8, 781–792 (2009).Gröschnel, A. H. et al. Precise hierarchical self-assembly of multicompartment micelles. Nat. Commun. 3, 710 (2012).Adam, M., Dogic, Z., Keller, S. L. & Fraden, S. Entropically driven microphase transitions in mixtures of colloidal rods and spheres. Nature 393, 349–352 (1998).Ohara, P. C., Heath, J. R. & Gelbart, W. M. Self-assembly of submicrometer rings of particles from solutions of nanoparticles. Angew. Chem. Int. Ed. 36, 1077–1080 (1997).Xu, J., Xia, J. & Lin, Z. Evaporation-induced self-assembly of nanoparticles from a sphere-on-flat geometry. Angew. Chem. Int. Ed. 46, 1860–1863 (2007).Yosef, G. & Rabani, E. Self-assembly of nanoparticles into rings: A lattice-gas model. J. Phys. Chem. B 110, 20965–20972 (2006).Khanal, B. P. & Zubarev, E. R. Rings of nanorods. Angew. Chem. Int. Ed. 46, 2195–2198 (2007).Wang, Z. et al. One-step, self-assembly, alignment, and patterning of organic semiconductor nanowires by controlled evaporation of confined microfluids. Angew. Chem. Int. Ed. 50, 2811–2815 (2011).Hong, S. W. et al. Directed self-assembly of gradient concentric carbon nanotube rings. Adv. Func. Mater. 18, 2114–2122 (2008).Palma, M. et al. Controlled formation of carbon nanotube junctions via linker-induced assembly in aqueous solution. J. Am. Chem. Soc. 135, 8440–8443 (2013).Horcas, I. et al. WSXM: A software for scanning probe microscopy and a tool for nanotechnology. Rev. Sci. Instrum. 78, 013705 (2007).Soler, J. M. et al. The SIESTA method for ab initio order-n materials simulation. J. Phys. Cond. Matter 14, 2745–2779 (2002).Haynes, P. D., Mostof, A. A., Skylaris, C. & Payne, M. C. ONETEP: Linear-scaling density-functional theory with plane-waves. J. Phys. Conf. Ser. 26, 143–148 (2006).Valiev, M. et al. NWCHEM: A comprehensive and scalable open-source solution for large scale molecular simulations. Comp. Phys. Commun. 181, 1477–1489 (2010).Plimpton, S. Fast parallel algorithms for short-range molecular dynamics. J. Comp. Phys. 117, 1–19 (1995)

The Canadian Bandaging Trial: Evidence-informed leg ulcer care and the effectiveness of two compression technologies

Background: Objective: To determine the relative effectiveness of evidence-informed practice using two high compression systems: four-layer (4LB) and short-stretch bandaging (SSB) in community care of venous leg ulcers. Design and Setting: Pragmatic, multi-centre, parallel-group, open-label, randomized controlled trial conducted in 10 centres. Cognitively intact adults (≥18 years) referred for community care (home or clinic) with a venous ulceration measuring ≥0.7cm and present for ≥1 week, with an ankle brachial pressure index (ABPI) ≥0.8, without medication-controlled Diabetes Mellitus or a previous failure to improve with either system, were eligible to participate.Methods: Consenting individuals were randomly allocated (computer-generated blocked randomization schedule) to receive either 4LB or SSB following an evidence-informed protocol. Primary endpoint: time-to- healing of the reference ulcer. Secondary outcomes: recurrence rates, health-related quality of life (HRQL), pain, and expenditures.Results: 424 individuals were randomized (4LB n = 215; SSB n = 209) and followed until their reference ulcer was healed (or maximum 30 months). An intent-to-treat analysis was conducted on all participants. Median time to ulcer healing in the 4LB group was 62 days [95% confidence interval (CI) 51 to 73], compared with 77 days (95% CI 63 to 91) in the SSB group. The unadjusted Kaplan-Meier curves revealed the difference in the distribution of cumulative healing times was not significantly different between group (log rank χ2 = 0.001, P = 0.98) nor ulcers recurrence (4LB, 10.1%; SSB, 13.3%; p = 0.345). Multivariable Cox Proportional Hazard Modeling also showed no significant between-bandage differences in healing time after controlling for significant covariates (p = 0.77). At 3-months post-baseline there were no differences in pain (no pain: 4LB, 22.7%; SSB, 26.7%; p = 0.335), or HRQL (SF-12 Mental Component Score: 4LB, 55.1; SSB, 55.8; p = 0.615; SF-12 Physical Component Score: 4LB, 39.0; SSB, 39.6; p = 0.675). The most common adverse events experienced by both groups included infection, skin breakdown and ulcer deterioration.Conclusions: The Canadian Bandaging Trial revealed that in the practice context of trained RNs using an evidence-informed protocol, the choice of bandage system (4LB and SSB) does not materially affect healing times, recurrence rates, HRQL, or pain. From a community practice perspective, this is positive news for patient-centred care allowing individual/family and practitioner choice in selecting compression technologies based on circumstances and context.Trial registration: clinicaltrials.gov Identifier: NCT00202267

Unfolded protein response in cancer: the Physician's perspective

The unfolded protein response (UPR) is a cascade of intracellular stress signaling events in response to an accumulation of unfolded or misfolded proteins in the lumen of the endoplasmic reticulum (ER). Cancer cells are often exposed to hypoxia, nutrient starvation, oxidative stress and other metabolic dysregulation that cause ER stress and activation of the UPR. Depending on the duration and degree of ER stress, the UPR can provide either survival signals by activating adaptive and antiapoptotic pathways, or death signals by inducing cell death programs. Sustained induction or repression of UPR pharmacologically may thus have beneficial and therapeutic effects against cancer. In this review, we discuss the basic mechanisms of UPR and highlight the importance of UPR in cancer biology. We also update the UPR-targeted cancer therapeutics currently in clinical trials