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Abstract: In an article published in Small, researchers successfully applied a new qualitative and quantitative method for the detection of a DNA sequence characteristic of Leishmania infantum kinetoplast, a frequent parasite in veterinary that affects humans too. The work was led from the Catalan Institute of Nanoscience and Nanotechnology (ICN2), a research Center placed in the Campus of the Autonomous University of Barcelona (UAB) in Bellaterra, and the UAB Spin Off company Vetgenomics. This work was coordinated at ICN2 by ICREA Prof Arben Merkoçi, Leader of the ICN2 Nanobioelectronics & Biosensors Group, and Dr. Alfredo de la Escosura-Muñiz (first author of the article) with collaboration of Luis Pires, PhD student from the same group. The work was carried out in collaboration with Prof. Armand Sanchez, Dr. Olga Francino, Dr. Laura Altet and Lorena Serrano from Vetgenomics. Their results are part of the bio-applications defined in the ICN2 Severo Ochoa Program "Devices for Social Challenges". The present work has been published within the POC4PETS European Project, coordinated by the Alma Mater Studiorum - Università di Bologna (Italy) and aimed to improving the speed and accuracy of current diagnostics for veterinary pathogens. Overcoming the classical PCR technique The Polymerase Chain Reaction (PCR) is today's standard method to identify the presence of a specific DNA sequence in a sample. The PCR uses enzymes and two primers, strands of short nucleic acid sequences that serve as a starting point for DNA copy. When the detection is positive, this technique produces millions of copies of the problem sequence to facilitate its detection. This DNA amplification involves precise thermal changes (thermocycling) and sophisticate and expensive equipment which are overcome by an alternative approach called isothermal amplification, performed at constant temperature. In this context, the authors of the article present a novel design of isothermal amplification using primers labelled with both gold nanoparticles and magnetic microbeads. The amplified product carries both labels allowing a rapid purification and quantification. The magnetic properties of the first primer facilitate the purification/preconcentration of the amplified DNA through magnetic separation methods. On the other hand, the gold nanoparticles are easily quantified by simple electrocatalytic detection methods. Thus, the use of primers labelled with gold nanoparticles and magnetic microbeads turns isothermal amplification into a faster and easier qualitative and quantitative diagnostic method. Nanoparticles for Leishmania detection and other point-of-care tests This approach was successfully applied for the detection of a DNA sequence characteristic of Leishmania infantum kinetoplast, a parasite responsible of a disease in domestic dogs, wild canids and humans. The electrochemical method exhibited a good reproducibility and sensitivity. Furthermore, amplified DNA from dogs without Leishmania was perfectly discriminated, demonstrating the specificity of both the amplification procedure and the electrochemical detection. In fact, the performance of the proposed approach is better than the obtained with other point-of-care tests for Leishmania detection, offering also a quantitative tool for parasite determination. This method represents a universal methodology that could be applied for any isothermal DNA amplification design. The technology presented in this article is under patenting process (EP14382266.6/P10398EP00 (09/07/14); PCT/EP2015/065742 (09/07/15)). ### About POC4PETS: Veterinary diagnostics is a key tool in the prevention and control of infectious diseases in animals. It is increasingly recognized that there is the urgent need for innovation in the animal infectious diseases testing field, bringing to new market applications in the development of specific, rapid and efficiently validated diagnostic tests. The European FP7 project Point of Care Diagnostics for rapid and cheap pathogen detection of companion animals (POC4PETS) aims to apply the most promising new technologies to improving both the speed and accuracy of the current diagnostics for veterinary pathogens of companion animal species. ICN2's Nanobioelectronics & Biosensors Group, led by ICREA Research Professor Arben Merkoçi, is one of the POC4PETS partners. Their research focuses in the development of novel nanotechnology and nanomaterials based sensors for DNA, protein and cells detection. The project is particularly focused in filling the gaps of diagnostics availability and enhancing technology transfer to increase competitiveness of the Veterinary Diagnostic industry. Vetgenomics is a SME spin-off of the UAB (Universitat Autònoma de Barcelona) founded in 2010 and devoted to molecular diagnostic in companion animals. The research activity is mainly focused in the animal genomics and veterinary genetic diagnostic fields. The main goal is becoming a company focused in innovation and with the flexibility to adapt technology to customer needs, increasing the added value of their products and becoming a partner of choice in R+D+i. For more information, please click If you have a comment, please us. Issuers of news releases, not 7th Wave, Inc. or Nanotechnology Now, are solely responsible for the accuracy of the content.

Dunay I.R.,Albert Ludwigs University of Freiburg | Bouthillier A.,University of Montreal | Moumdjian R.,University of Montreal | Fontana A.,University of Zurich | And 5 more authors.
Annals of Neurology

Objective: Blood-derived myeloid antigen-presenting cells (APCs) account for a significant proportion of the leukocytes found within lesions of multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE). These APCs along with activated microglia are thought to be pivotal in the initiation of the central nervous system (CNS)-targeted immune response in MS and EAE. However, the exact molecules that direct the migration of myeloid cells from the periphery across the blood-brain barrier (BBB) remain largely unknown. Methods: We identified Ninjurin-1 in a proteomic screen of human BBB endothelial cells (ECs). We assessed the expression of Ninjurin-1 by BBB-ECs and immune cells, and we determined the role of Ninjurin-1 in immune cell migration to the CNS in vivo in EAE mice. Results: Ninjurin-1 was found to be weakly expressed in the healthy human and mouse CNS but upregulated on BBB-ECs and on infiltrating APCs during the course of EAE and in active MS lesions. In human peripheral blood, Ninjurin-1 was predominantly expressed by monocytes, whereas it was barely detectable on T and B lymphocytes. Moreover, Ninjurin-1 neutralization specifically abrogated the adhesion and migration of human monocytes across BBB-ECs, without affecting lymphocyte recruitment. Finally, Ninjurin-1 blockade reduced clinical disease activity and histopathological indices of EAE and decreased infiltration of macrophages, dendritic cells, and APCs into the CNS. Interpretation: Our study uncovers an important cell-specific role for Ninjurin-1 in the transmigration of inflammatory APCs across the BBB and further emphasizes the importance of myeloid cell recruitment during the development of neuroinflammatory lesions.ANN NEUROL 2011 Copyright © 2011 American Neurological Association. Source

Santana H.,Center for Genetic Engineering and Biotechnology | Avila C.L.,Instituto Superior Of Investigaciones Biologicas Insibio | Cabrera I.,Bellaterra | Cabrera I.,CIBER ISCIII | And 9 more authors.
Soft Matter

Growth hormone releasing peptide, GHRP-6, a hexapeptide (His-(d-Trp)-Ala-Trp-(d-Phe)-Lys-NH2, MW = 872.44 Da) that belongs to a class of synthetic growth hormone secretagogues, can stimulate growth hormone secretion from somatotrophs in several species including humans. In the present study, we demonstrate that GHRP-6 dispersed in aqueous solution, at pH 7.0, room temperature of 22 °C, is able to form long nanotubes, which is evidenced by combining small angle X-ray scattering (SAXS), transmission electron microscopy and molecular dynamics simulation results. Such nanotubes possess inner and outer cross-sections equal to 6.7(2) nm and 13.4(5) nm, respectively. The mechanism of peptide self-assembly was determined by molecular dynamics simulations revealing that the peptides self-assemble like amphiphilic molecules in aqueous solution in a partially interdigitated structure. In this case, the position of the positively charged amino terminus is located at the peptide-water interface, whereas the neutral NH2-capped carboxy terminus remains buried at the hydrophobic core. In contrast, the long side chain of Lys-6 stretches out of the hydrophobic core positioning its positive charge near the cylinder surface. The peptide configuration in the nanotube wall comes from the interplay between the hydrophobic interactions of the aromatic side chains of GHRP-6 and the electrostatic repulsion of its cationic charges. On increasing the peptide concentration, the long nanotubes self-arrange in solution displaying a bi-dimensional hexagonal-like packing in the SAXS curves, with a center-to-center distance of ∼15 nm. Further, we also show that the nanostructure formed in solution is quite stable and is preserved following transfer to a solid support. © 2014 The Royal Society of Chemistry. Source

Elizondo E.,Bellaterra | Elizondo E.,CIBER ISCIII | Larsen J.,Copenhagen University | Hatzakis N.S.,Copenhagen University | And 8 more authors.
Journal of the American Chemical Society

A confocal fluorescence microscopy-based assay was used for studying the influence of the preparation route on the supramolecular organization of lipids in a vesicular system. In this work, vesicles composed of cholesterol and CTAB (1/1 mol %) or cholesterol and DOPC (2/8 mol %) and incorporating two membrane dyes were prepared by either a compressed fluid (CF)-based method (DELOS-susp) or a conventional film hydration procedure. They were subsequently immobilized and imaged individually using a confocal fluorescence microscope. Two integrated fluorescence intensities, I dye1 and I dye2, were assigned to each tracked vesicle, and their ratio, I dye1/I dye2, was used for quantifying the degree of membrane inhomogeneity between individual vesicles within each sample. A distribution of I dye1/I dye2 values was obtained for all the studied vesicular systems, indicating intrasample heterogeneity. The degree of inhomogeneity (DI) was similar for Chol/DOPC vesicles prepared by both procedures. In contrast, DI was more than double for the hydration method compared to the CF-based method in the case of Chol/CTAB vesicles, which can suffer from lipid demixing during film formation. These findings reveal a more homogeneous vesicle formation path by CFs, which warranted good homogeneity of the vesicular system, independently of the lipid mixture used. © 2011 American Chemical Society. Source

Soler J.,Bellaterra | Saura P.,Bellaterra | Saura P.,Autonomous University of Barcelona | Garcia-Lopez D.,Bellaterra | And 5 more authors.
Journal of Physical Chemistry B

The most common substrate of mammalian lipoxygenases (LOXs) is arachidonic acid (AA). However, 15-LOXs can present dual substrate specificity. These LOXs catalyze the peroxidation of AA, initiated by a H-abstraction step (mainly H13-abstraction) by the Fe(III)-OH- cofactor, and the peroxidation of linoleic acid (LA) after H11-abstraction. In this paper, QM(B3LYP)/MM(CHARMM) calculations of the rate-limiting H11-abstraction process of LA catalyzed by rabbit 15-LOX-1 (15-rLOX-1) have been carried out using a complete model of the solvated 15-rLOX-1:LA complex. A total of 26 QM/MM potential energy profiles as a function of the H-transfer reaction coordinate have been computed along with one QM/MM free energy profile obtained using the Free Energy Perturbation method. The molecular origin of substrate specificity of 15-rLOX-1 for LA in comparison with AA has been analyzed. In many of the QM/MM reactive 15-rLOX-1:LA energy minima, LA adopts more elongated conformations than AA, although having a shorter carbon chain, because LA has one double bond between C1 and C11 whereas AA has three double bonds between C1 and C13. Consequently, C11 of LA can be located in the same region of the active site as C13 of AA, a zone where H11-abstraction from LA as well as H13-abstraction from AA is not hindered by bulky residue side chains. This explains at a molecular level how 15-LOXs might accommodate and recognize for catalysis two substrates that are different in length by two carbons. Our results also explain why (9Z,11E)-13-hydro(pero)xyoctadeca-9,11-dienoic acid is the major product of the peroxidation and why LA is the preferential substrate of 15-rLOX-1. © 2015 American Chemical Society. Source

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