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Biofilm development is recognized as a major virulence factor underlying most chronic bacterial infections. When a biofilm community is established, planktonic cells growing in the surroundings of a tissue switch to a sessile lifestyle and start producing a biofilm matrix. The initial steps of in vivo biofilm development are poorly characterized and difficult to assess experimentally. A great amount of in vitro evidence has shown that accumulation of high levels of cyclic dinucleotides (c-di-NMPs) is the most prevalent hallmark governing the initiation of biofilm development by bacteria. As mentioned above, recent studies also link detection of c-di-NMPs by host cells with the activation of a type I interferon immune response against bacterial infections. We discuss here c-di-NMP signaling and the host immune response in the context of the initial steps of in vivo biofilm development. © 2013 Elsevier Ltd., J. Valle was supported by Spanish Ministry of Science and Innovation ‘Ramón y Cajal’ contract. We thank Professor Angel L. Corbí and Dr Estanislao Nistal for excellent comments on the manuscript. Work in the Laboratory of Microbial Biofilms is funded by the Spanish Ministry of Economy and Competitiveness grants BIO2011-30503-C02-02, AGL2011-23954, and BFU2011-23222, as well as by ERA-NET Pathogenomics (PIM2010EPA-00606) and grants from the Departamento de Innovación (IIQ14066.RI1 and IIM13329.RI1) and Departamento de Salud (Resolución 1312/2010), Gobierno de Navarra.

Cyclic di-GMP (c-di-GMP) is a secondary messenger that controls a variety of cellular processes, including the switch between a biofilm and a planktonic bacterial lifestyle. This nucleotide binds to cellular effectors in order to exert its regulatory functions. In Salmonella, two proteins, BcsA and YcgR, both of them containing a c-di-GMP binding PilZ domain, are the only known c-di- GMP receptors. BcsA, upon c-di-GMP binding, synthesizes cellulose, the main exopolysaccharide of the biofilm matrix. YcgR is dedicated to c-di-GMP-dependent inhibition of motility through its interaction with flagellar motor proteins. However, previous evidences indicate that in the absence of YcgR, there is still an additional element that mediates motility impairment under high c-di-GMP levels. Here we have uncovered that cellulose per se is the factor that further promotes inhibition of bacterial motility once high c-di-GMP contents drive the activation of a sessile lifestyle. Inactivation of different genes of the bcsABZC operon, mutation of the conserved residues in the RxxxR motif of the BcsA PilZ domain, or degradation of the cellulose produced by BcsA rescued the motility defect of δycgR strains in which high c-di-GMP levels were reached through the overexpression of diguanylate cyclases. High c-di-GMP levels provoked cellulose accumulation around cells that impeded flagellar rotation, probably by means of steric hindrance, without affecting flagellum gene expression, exportation, or assembly. Our results highlight the relevance of cellulose in Salmonella lifestyle switching as an architectural element that is both essential for biofilm development and required, in collaboration with YcgR, for complete motility inhibition. © 2013, American Society for Microbiology., J.V. was supported by a Ramón y Cajal contract from the Ministerio de Economía y Competitividad, Spain. A JAE predoctoral research contract for V.Z. from the Consejo Superior de Investigaciones Científicas (CSIC, Spain) is gratefully acknowledged. This research was supported by a grant from the Departamento de Salud (Resolución 1312/2010), Gobierno de Navarra, Spain.

The study of the anatomy and physiology of the motor unit has important implications in the diagnosis and follow-up of neuromuscular pathologies. Muscle action potentials allow the use of electrophysiological techniques based on electromyography (EMG) to make inferences about muscle structure, state and behaviour. Scanning EMG is one such technique that can record the temporal and spatial distribution of electrical activity of a single motor unit, allowing for deep insight into the structure and function of motor units. In this chapter, we describe the scanning EMG technique in detail, both from a technical and clinical point of view. A brief review of the motor unit anatomy and physiology is provided in Section 2. The technique, the apparatus setup, the recording procedure and the signal processing required are described in Section 3. Key results of studies using scanning EMG are reviewed in Section 4, including findings related to motor unit organisation in normal muscle and how changes due to pathology are reflected using this electrophysiological technique. Finally, Section 5 provides some hints regarding the use of scanning EMG in research. This work was supported by the Regional Health Ministry of the Government of Navarre under the project 1312/2010.