An experimental system to study responses of Medicago truncatula roots to chitin oligomers of high degree of polymerization and other microbial elicitors.

KEY MESSAGE: A fully acetylated, soluble CO preparation of mean DP of ca. 7 was perceived with high sensitivity by M. truncatula in a newly designed versatile root elicitation assay. The root system of legume plants interacts with a large variety of microorganisms, either pathogenic or symbiotic. Understanding how legumes recognize and respond specifically to pathogen-associated or symbiotic signals requires the development of standardized bioassays using well-defined preparations of the corresponding signals. Here we describe the preparation of chitin oligosaccharide (CO) fractions from commercial chitin and their characterization by a combination of liquid-state and solid-state nuclear magnetic resonance spectroscopy. We show that the CO fraction with highest degree of polymerization (DP) became essentially insoluble after lyophilization. However, a fully soluble, fully acetylated fraction with a mean DP of ca. 7 was recovered and validated by showing its CERK1-dependent activity in Arabidopsis thaliana. In parallel, we developed a versatile root elicitation bioassay in the model legume Medicago truncatula, using a hydroponic culture system and the Phytophthora ß-glucan elicitor as a control elicitor. We then showed that M. truncatula responded with high sensitivity to the CO elicitor, which caused the production of extracellular reactive oxygen species and the transient induction of a variety of defense-associated genes. In addition, the bioassay allowed detection of elicitor activity in culture filtrates of the oomycete Aphanomyces euteiches, opening the way to the analysis of recognition of this important legume root pathogen by M. truncatula.

Non-invasive assessment of motor unit anatomy in jaw-elevator muscles.

The estimation of fibre length in jaw-elevator muscles is important for modelling studies and clinical applications. The objective of this study was to identify, from multi-channel surface EMG recordings, the main innervation zone(s) of the superficial masseter and anterior temporalis muscles, and to estimate the fibre length of these muscles. Surface EMG signals were collected from 13 subjects with a 16-electrode linear array. The innervation zones of the masseter and anterior temporalis were identified and their variability intra- and inter-subject outlined. More than one main innervation zone location was identified in the masseter of all subjects and in the temporalis anterior of 12 subjects. Average estimated fibre lengths, for the right (left) side, were (mean+/-SD) 27.3+/-2.4 mm (27.0+/-1.7 mm) and 25.9+/-2.3 mm (26.6+/-1.6 mm), for the superficial masseter and temporalis anterior muscle, respectively. The range of innervation zone locations was up to approximately 50% of the fibre length, both within and between subjects. Fibre length estimates well matched with published data on cadavers. It was concluded that multi-channel surface EMG provides important and reliable information on the anatomy of single motor units in jaw-elevator muscles.

Surface EMG of jaw elevator muscles: effect of electrode location and inter-electrode distance.

This study addresses methodological issues on surface electromyographic (EMG) signal recording from jaw elevator muscles. The aims were (i) to investigate the sensitivity to electrode displacements of amplitude and spectral surface EMG variables, (ii) to analyse if this sensitivity is affected by the inter-electrode distance of the bipolar recording, and (iii) to investigate the effect of inter-electrode distance on the estimated amplitude and spectral EMG variables. The superficial masseter and anterior temporalis muscles of 13 subjects were investigated by means of a linear electrode array. The percentage difference in EMG variable estimates from signals detected at different locations over the muscle was larger than 100% of the estimated value. Increasing the inter-electrode distance resulted in a significant reduction of the estimation variability because of electrode displacement. A criterion for electrode placement selection is suggested, with which the sensitivity of EMG variables to small electrode displacements was of the order of 2% for spectral and 6% for amplitude variables. Finally, spectral and, in particular, amplitude EMG variables were very sensitive to inter-electrode distance, which thus should be fixed when subjects or muscles are compared in the same or different experimental conditions.

Sixty-four channel wearable acquisition system for long-term surface electromyogram recording with electrode arrays.

The use of mono- and bi-dimensional electromyogram (EMG) electrode arrays for the assessment of the neuromuscular system can provide an insight into muscle physiology not achieved with classical bipolar surface EMG. Among the advantages of multichannel EMG detection, there is a) the possibility of estimating muscle fibre conduction velocity, even during motor tasks, and b) the possibility to increase the number of detection points on a muscle, improving the performance of pattern-based EMG decomposition methods. For these reasons, the development and use of multichannel surface EMG devices and techniques were chosen as the primary goals within the European RTD Project 'Neuromuscular assessment in the elderly worker' (NEW). The specific requirements of Project NEW called for the availability of a user-friendly, small-sized EMG acquisition system for field use, suitable for multichannel EMG recording using electrode arrays from one or more muscles. A market survey established that none of the commercially available EMG acquisition systems featured all the desired specifications, nor could they be easily adapted for specific use. The paper describes the design of an innovative acquisition system for long-term multichannel EMG recording fulfilling these requirements and comprising adhesive electrode arrays for artifact-free EMG acquisition during work activity and a portable, user-friendly, battery-powered acquisition system for multichannel EMG recording and storage on a removable PCMCIA card. The system has been used extensively within Project NEW for laboratory and field tests and can find applications in other fields of basic and applied research, including ergonomics, occupational and sports medicine.

Multichannel surface EMG for the non-invasive assessment of the anal sphincter muscle.

BACKGROUND/AIMS: This work focuses on recording, processing and interpretation of multichannel surface EMG detected from the external anal sphincter muscle. The aim is to describe the information that can be extracted from signals recorded with such a technique. METHODS: The recording of many signals from different locations on a muscle allows the extraction of additional information on muscle physiology and anatomy with respect to that obtained by classic bipolar recordings. Multichannel EMG methods have been recently developed for the assessment of the external anal sphincter. An anal probe was used in this study to record signals at different depths within the anal canal during contractions at different effort levels. The plug is 150 mm in length and 14 mm in diameter, holding a circumferential array of 16 equally spaced silver bar electrodes, located at a distance of 20 mm from the probe tip and aligned with the probe axis. RESULTS: Information about localization of the innervation zone, fiber length, EMG amplitude, muscle fiber conduction velocity and single motor unit analysis can be obtained from the signals recorded with the circumferential array by means of innovative signal processing techniques. CONCLUSIONS: The type of information extracted from multichannel surface EMG signals cannot be obtained with other currently available techniques. The technological innovation described in this work is promising for a further insight into the investigation of pelvic floor pathologies and rehabilitation treatments.

Innervation zones of the external anal sphincter in healthy male and female subjects. Preliminary results.

OBJECTIVES: The objective of this work was to investigate the distribution of the innervation zones of the motor units that make up the external anal sphincter (EAS) in healthy males and females. METHODS: A cylindrical probe carrying a circumferential array of 16 electrodes was used to detect the generation, propagation and extinction of individual motor unit action potentials (MUAPs) at 1, 2, and 3 cm depth from the orifice of the anal canal during maximal voluntary contractions of the EAS. Fifteen healthy males and 37 healthy nulliparous females were investigated. RESULTS: IZs could be detected in all males and in 34 out of 37 females. In the males, the IZs are scattered in the right and left hemisphincter at each of the three levels and their distribution is not affected by depth. In the females, the distribution is also concentrated in the right and left hemisphincter at depth 1 cm but is more uniform at depth 2 cm and more concentrated in the dorsal and ventral regions at depth 3 cm. ANOVA shows a statistically significant dependence of the IZ distribution on depth only in females and not in males. CONCLUSIONS: It is concluded that (a) IZs of the EAS can indeed be detected with a circumferential array placed at different depths along the anal canal; (b) large individual variability is observed, and (c) IZs show similar distribution at the three depth levels in males and different distributions in females.

Surface EMG alterations induced by underwater recording.

BACKGROUND AND PURPOSE: This study aims to verify if amplitude and spectral characteristics of surface EMG signal are modified due to recording in a wet environment. METHODS: Isometric contractions of the biceps brachii muscle of ten subjects were performed in several different set-up combinations, both in dry (D) and in water from hydrotherapy pools (PW), with (PWM) or without moving the pool water and with (T) or without water-resistant adhesive taping. RESULTS: In PW condition the amplitude of the recorded signal is reduced to 5-10% of the corresponding signal recorded in D condition. In PWM the power spectrum is drastically reduced and altered by the water movement that introduces an increase of spectral power in the frequency range 0-20 Hz. The use of T modality allows to record signals with both amplitude and spectral frequencies comparable with those obtained in the D conditions. DISCUSSION AND CONCLUSION: This work demonstrates the need for water resistant taping when EMG signals are recorded in water. Signals recorded without such a protective film are strongly affected in their amplitude and frequency characteristics by the conductivity and the movement of water.

Green fluorescent protein (GFP) as gene expression reporter and vital marker for studying development and microbe-plant interaction in the tobacco pathogen Phythphthora parasitica var. nicotianae.

PEG-mediated transformation of protoplasts in the presence of lipofectin was achieved in Phytophthora parasitica var. nicotianae, an oomycete pathogen of tobacco. Using oomycete promoter and terminator sequences, a plant-adapted green fluorescent protein (GFP) was introduced into the microorganism. The data show for the first time that this eukaryotic gene reporter can be used in an oomycete, both as a quantitative reporter of gene induction and as a vital marker allowing the study of development of Phytophthora in vitro and in the host plant.

Purification, Elicitor Activity, and Cell Wall Localization of a Glycoprotein from Phytophthora parasitica var. nicotianae, a Fungal Pathogen of Tobacco.

ABSTRACT A glycoprotein of 34 kDa (GP 34) was solubilized at acidic pH from the mycelium of Phytophthora parasitica var. nicotianae and was purified by ion exchange and gel permeation chromatography. Whole tobacco plants treated with GP 34 through their roots showed an enhanced lipoxygenase activity as well as hydroxyproline-rich glycoprotein accumulation, indicating that this molecule had elicitor properties. An antiserum raised against the pure glycoprotein allowed localization of GP 34 by immunogold-labeling on the cell surface of the mycelium when the fungus was grown in vitro. In the wall-less zoospores, GP 34 was limited to the flagellum surface. It was then abundantly synthesized at the onset of encystment. During infection of tobacco plants, labeling was very faint at early stages of colonization, particularly in the susceptible host cultivar. It appeared earlier in the resistant host cultivar and was restricted to the living fungus, declining with mycelium cell death.

Isolation of a carbon source-regulated gene from Ustilago maydis.

We have isolated a carbon source-regulated gene from the phytopathogenic fungus Ustilago maydis by use of a promoter-probe vector. This gene, called crg1, is strongly induced by L-arabinose and efficiently repressed by D-glucose and D-xylose. The predicted 36.5-kDa mature crg1 gene product lacks similarity to known proteins but is likely to be secreted. Sequences required for regulated expression of a reporter gene are contained within a 3.6-kb fragment upstream of the crg1 gene. The promoter of crg1 fulfils requirements for an efficient controllable gene expression system in U. maydis

Green fluorescent protein (GFP) as a new vital marker in the phytopathogenic fungus Ustilago maydis.

Pathogenic development of Ustilago maydis, the causative agent of corn smut disease, is a multistep process. Compatible yeast-like cells fuse and this generates the infectious dikaryon which grows filamentously. Having entered the plant the dikaryon induces tumors in its host in which massive proliferation of fungal material, karyogamy and spore formation occur. In order to follow fungal development from the initial steps to the final stage we have expressed the green fluorescent protein (GFP) from Aequorea victoria as a vital marker in U. maydis and demonstrate that GFP-tagged strains can be used to study host-pathogen interactions in vivo.