Universal Primers Used for Species Identification of Foodstuff of Animal Origin: Effects of Oligonucleotide Tails on PCR Amplification and Sequencing Performance

M13 universal non-homologous oligonucleotide tails incorporated into universal primers have been shown to improve amplification and sequencing performance. However, a few protocols use these tails in the field of food inspection. In this study, two types of M13 tails (by Steffens and Messing) were selected to assess their benefits using universal cytochrome oxidase subunit I (COI) and 16S ribosomal RNA gene (16SrRNA) primers in standard procedures. The primer characteristics were tested in silico. Then, using 20 DNA samples of edible species (birds, fishes, and mammals), their performance during PCR amplification (band recovery and intensity) and sequencing (sequence recovery, length, and Phred score) was assessed and compared. While 16SrRNA tailed and non-tailed primers performed similarly, differences were found for COI primers. Messing’s tails negatively affected the reaction outputs, while Steffens’ tails significantly improved the band intensity and the length of the final contigs based on the individual bidirectional read sequence. This different performance could be related to a destabilization effect of certain tails on primers with unfavorable mismatches on the annealing region. Even though our results cannot be generalized because the tail performances are strictly dependent on laboratory conditions, they show that appropriate tails can improve the overall throughput of the analysis, supporting food traceabilit

Detecting single viruses and nanoparticles using whispering gallery microlasers

Detection and characterization of individual nano-scale particles, virions, and pathogens are of paramount importance to human health, homeland security, diagnostic and environmental monitoring[1]. There is a strong demand for high-resolution, portable, and cost-effective systems to make label-free detection and measurement of individual nanoparticles, molecules, and viruses [2-6]. Here, we report an easily accessible, real-time and label-free detection method with single nanoparticle resolution that surpasses detection limit of existing micro- and nano-photonic devices. This is achieved by using an ultra-narrow linewidth whispering gallery microlaser, whose lasing line undergoes frequency splitting upon the binding of individual nano-objects. We demonstrate detection of polystyrene and gold nanoparticles as small as 15 nm and 10 nm in radius, respectively, and Influenza A virions by monitoring changes in self-heterodyning beat note of the split lasing modes. Experiments are performed in both air and aqueous environment. The built-in self-heterodyne interferometric method achieved in a microlaser provides a self-reference scheme with extraordinary sensitivity [7,8], and paves the way for detection and spectroscopy of nano-scale objects using micro- and nano-lasers.Comment: Main Text: 14 pages, 5 figures, 27 references. Supplement: 26 pages, 12 figures, 26 reference

On-chip Single Nanoparticle Detection and Sizing by Mode Splitting in an Ultra-high-Q Microresonator

The ability to detect and size individual nanoparticles with high resolution is crucial to understanding behaviours of single particles and effectively using their strong size-dependent properties to develop innovative products. We report real-time, in-situ detection and sizing of single nanoparticles, down to 30 nm in radius, using mode-splitting in a monolithic ultra-high-Q whispering-gallery-mode (WGM) microtoroid resonator. Particle binding splits a WGM into two spectrally shifted resonance modes, forming a self-referenced detection scheme. This technique provides superior noise suppression and enables extracting accurate size information in a single-shot measurement. Our method requires neither labelling of the particles nor apriori information on their presence in the medium, providing an effective platform to study nanoparticles at single particle resolution.Comment: 23 pages, 8 figure

Lung glutathione adaptive responses to cigarette smoke exposure

<p>Abstract</p> <p>Background</p> <p>Smoking tobacco is a leading cause of chronic obstructive pulmonary disease (COPD), but although the majority of COPD cases can be directly related to smoking, only a quarter of smokers actually develop the disease. A potential reason for the disparity between smoking and COPD may involve an individual's ability to mount a protective adaptive response to cigarette smoke (CS). Glutathione (GSH) is highly concentrated in the lung epithelial lining fluid (ELF) and protects against many inhaled oxidants. The changes in GSH that occur with CS are not well investigated; therefore the GSH adaptive response that occurs with a commonly utilized CS exposure was examined in mice.</p> <p>Methods</p> <p>Mice were exposed to CS for 5 h after which they were rested in filtered air for up to 16 h. GSH levels were measured in the ELF, bronchoalveolar lavage cells, plasma, and tissues. GSH synthesis was assessed by measuring γ-glutamylcysteine ligase (GCL) activity in lung and liver tissue.</p> <p>Results</p> <p>GSH levels in the ELF, plasma, and liver were decreased by as much as 50% during the 5 h CS exposure period whereas the lung GSH levels were unchanged. Next, the time course of rebound in GSH levels after the CS exposure was examined. CS exposure initially decreased ELF GSH levels by 50% but within 2 h GSH levels rebound to about 3 times basal levels and peaked at 16 h with a 6-fold increase and over repeat exposures were maintained at a 3-fold elevation for up to 2 months. Similar changes were observed in tissue GCL activity which is the rate limiting step in GSH synthesis. Furthermore, elevation in ELF GSH levels was not arbitrary since the CS induced GSH adaptive response after a 3d exposure period prevented GSH levels from dropping below basal levels.</p> <p>Conclusions</p> <p>CS exposures evoke a powerful GSH adaptive response in the lung and systemically. These data suggests there may be a sensor that sets the ELF GSH adaptive response to prevent GSH levels from dipping below basal levels. Factors that disrupt GSH adaptive responses may contribute to the pathophysiology of COPD.</p

Whispering gallery microresonators for second harmonic light generation from a low number of small molecules

Unmarked sensitive detection of molecules is needed in environmental pollution monitoring, disease diagnosis, security screening systems and in many other situations in which a substance must be identified. When molecules are attached or adsorbed onto an interface, detecting their presence is possible using second harmonic light generation, because at interfaces the inversion symmetry is broken. However, such light generation usually requires either dense matter or a large number of molecules combined with high-power laser sources. Here we show that using high-Q spherical microresonators and low average power, between 50 and 100 small non-fluorescent molecules deposited on the outer surface of the microresonator can generate a detectable change in the second harmonic light. This generation requires phase matching in the whispering gallery modes, which we achieved using a new procedure to periodically pattern, with nanometric precision, a molecular surface monolayer

Altered adipocyte differentiation and unbalanced autophagy in type 2 Familial Partial Lipodystrophy: an in vitro and in vivo study of adipose tissue browning

Type-2 Familial Partial Lipodystrophy is caused by LMNA mutations. Patients gradually lose subcutaneous fat from the limbs, while they accumulate adipose tissue in the face and neck. Several studies have demonstrated that autophagy is involved in the regulation of adipocyte differentiation and the maintenance of the balance between white and brown adipose tissue. We identified deregulation of autophagy in laminopathic preadipocytes before induction of differentiation. Moreover, in differentiating white adipocyte precursors, we observed impairment of large lipid droplet formation, altered regulation of adipose tissue genes, and expression of the brown adipose tissue marker UCP1. Conversely, in lipodystrophic brown adipocyte precursors induced to differentiate, we noticed activation of autophagy, formation of enlarged lipid droplets typical of white adipocytes, and dysregulation of brown adipose tissue genes. In agreement with these in vitro results indicating conversion of FPLD2 brown preadipocytes toward the white lineage, adipose tissue from FPLD2 patient neck, an area of brown adipogenesis, showed a white phenotype reminiscent of its brown origin. Moreover, in vivo morpho-functional evaluation of fat depots in the neck area of three FPLD2 patients by PET/CT analysis with cold stimulation showed the absence of brown adipose tissue activity. These findings highlight a new pathogenetic mechanism leading to improper fat distribution in lamin A-linked lipodystrophies and show that both impaired white adipocyte turnover and failure of adipose tissue browning contribute to disease.We thank FPLD2 patients for donating biological samples. We thank the Italian Network for Laminopathies and the European Consortium of Lipodystrophies (ECLip) for support and helpful discussion. We thank Aurelio Valmori for the technical support. The studies were supported by Rizzoli Orthopedic Institute “5 per mille” 2014 project to MC, AIProSaB project 2016 and Fondazione Del Monte di Bologna e Ravenna grant 2015–2016 “New pharmacological approaches in bone laminopathies based on the use of antibodies neutralizing TGF beta 2” to GL. GL is also supported by PRIN MIUR project 2015FBNB5Y.S

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)