bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of Chrysanthemum stunt viroid and Potato spindle tuber viroid by return‐polyacrylamide gel electrophoresis*
Authors: Roenhorst J. W. , Butôt R. P. T. , van Der Heijden K. A. , Hooftman M. , van Zaayen A.
Published: April 2008

Return‐polyacrylamide gel electrophoresis (R‐PAGE) is one of the methods used for the detection of viroids, in particular Chrysanthemum stunt viroid (CSVd) and Potato spindle tuber viroid (PSTVd). The method is based on temperature‐related conformational changes of the circular single‐stranded RNA molecule. This results in a characteristic electrophoretic behaviour, providing a clear separation between the viroid and nucleic acids of the plant. Since the description of R‐PAGE, several modifications of the procedure have been published. In addition, several molecular techniques have been applied for the detection of viroids. However, R‐PAGE is still particularly suitable for phytosanitary purposes because detection is not influenced by variations in the nucleotide sequence. This means that any Pospiviroid will be detected, provided the amount is above the detection threshold. In addition, relatively large amounts of leaf material can be handled in one test. This paper presents a recently developed R‐PAGE procedure, based on the use of a horizontal electrophoresis system and paper wicks as buffer reservoirs. In comparison with the existing procedures, the new procedure is easy to perform and the use of chemicals is decreased. Results of the new procedure are shown and discussed in relation to those obtained by the previous procedure.
Herbaceous test plants for the detection of quarantine viruses of potato*
Authors: Verhoeven J. Th. J. , Roenhorst J. W.
Published: April 2008

To prevent the introduction and spread of non‐indigenous potato viruses in the European Union, the import of potato breeding material is regulated by a Directive prescribing post‐entry testing under quarantine. This testing includes serological screening and mechanical inoculation to herbaceous test plants. A list of appropriate test‐plant species was published by EPPO in 1984, but some other promising test‐plant species have been described since. This paper reports the reactions of nine plant species to six non‐European potato viruses, including all the seed‐borne viruses. Different isolates of all these viruses were detected on Nicotiana hesperis‐67A as well as on N. occidentalis PI. This means that all relevant viruses are detected if one of these indicators is used to test plants grown from true potato seed. The inoculation experiments will be continued with potato viruses that already occur in Europe. The results so far provide a good basis for improving the use of test plants in post‐entry quarantine testing.
Immunomonitoring air‐borne fungal plant pathogens: Mycosphaerella brassicicola*
Authors: Wakeham A. J. , Kennedy R. , Byrne K. G. , Keane G. , Dewey F. M.
Published: April 2008

Detection and enumeration of air‐borne spores of fungal plant pathogens has generally been achieved by microscopic examination of surfaces on which spores have been impacted and conventional agar‐plating techniques. However, this requires considerable amounts of time and expertise. Such methods are inaccurate for obligate pathogens and unrealistic where there is no selective medium or where there are spore types of similar morphology (such as those produced by ascosporic fungi). In field disease‐transmission studies, the use of a Burkard 7‐day volumetric spore trap combined with an immunofluorescence test has, however, enabled the detection and quantification of field‐trapped ascosporic inoculum of Mycosphaerella brassicicola (the ringspot pathogen of brassicas). This test has also been found useful in the validation of more rapid user‐friendly immunoassay‐based trapping procedures. A microtitre immunospore trapping device, which uses a suction system to trap air particulates directly by impaction into microtitre wells, has been used successfully for the rapid detection and quantification of ascosporic inoculum of M. brassicicola. The system shows potential for field detection of air‐borne ascosporic inoculum of the ringspot pathogen.
Detection of soil‐borne pathogens in glasshouse‐grown ornamentals*
Authors: Wubben J. P. , Numansen A. , Kerssies A.
Published: April 2008

The Research Station for Floriculture and Glasshouse Vegetables focuses its activities on the entire glasshouse horticulture chain. Research involves many different ornamental crops, each with its own specific problems with respect to pest control. Correct diagnosis of diseases is very important in order to give good advice for control strategies. With the introduction of integrated and supervised control strategies, pathogen monitoring has become even more important. In general, diagnosis is carried out by conventional methods using selective media to isolate and identify the pathogen. These methods can be time‐consuming and result in the identification of a single pathogen at a time. Diagnostic methods based on isozyme patterns have also been used for the detection of fungal pathogens in infected plant material. Specific isozyme methods have been developed for the diagnosis of Fusarium oxysporum f. sp. cyclaminis in cyclamen, F. oxysporum f. sp. gladioli in gladiolus, Gnomonia radicicola in rose, and Phytophthora nicotianae, Phytophthora sp. and Cylindrocladium spathiphylli in spathiphyllum. Current and future work is focussing on identification of plant pathogens in contained environment‐culturing systems using molecular techniques.
Diagnosis and detection of host‐specific forms of Fusarium oxysporum*
Authors: Baayen R. P.
Published: April 2008

Diagnosis and detection of host‐specific forms of Fusarium oxysporum are traditionally based on the combination of diagnostic symptoms on the host with the presence of the fungus in the affected tissues. The classical approach is becoming increasingly problematic because more than one forma specialis may occur on a given host, along with non‐pathogenic strains which are common soil and rhizosphere inhabitants. Neither formae speciales nor pathogenic races within formae speciales can be distinguished morphologically. Although united by joint pathogenicity to a given host, strains belonging to the same forma specialis need not be phylogenetically related. Development of diagnostics for host‐specific groups in F. oxysporum requires monophyletic target groups. Recent studies on gene‐genealogy and AFLP‐based phylogenies show that the majority of formae speciales in F. oxysporum are polyphyletic (unnatural) and do not offer any prospects for the development of molecular diagnostics. In contrast, highly specific PCR primers have been developed for formae speciales (or races) that consist of a single clonal lineage, and for monophyletic groups of lineages within a forma specialis. Among others, specific PCR primers have thus been developed for F. oxysporum f. sp. basilici, specific races in F. oxysporum ff. spp. dianthi and gladioli, and for the EPPO A2 (EU II/A1) quarantine fungus F. oxysporum f. sp. albedinis which can reliably replace conventional isolation and pathogenicity testing procedures.
Application de la variabilité génétique de l'ADNr chez Monilinia laxa, Monilinia fructigena et Monilinia fructicolaà l'identification des espèces par PCR*
Authors: Ioos R. , Frey P.
Published: April 2008

Monilinia laxa, Monilinia fructigena et Monilinia fructicola sont des agents de pourriture de fruit et de chancre sur rameau des arbres fruitiers. La région des ITS (internal transcribed spacer) située entre les gènes codant pour les sous‐unités ribosomiques 18S et 28S de quatre isolats de M. fructigena et quatre isolats de M. fructigena collectés en France a été amplifiée par PCR et séquencée. L'alignement multiple de ces séquences d'ITS et leur comparaison avec les séquences d'ITS de Monilinia spp. publiées a révélé une très faible variabilité intraspécifique. Par contre, un faible polymorphisme interspécifique a été localisé au niveau de deux régions, respectivement dans l'ITS1 et l'ITS2. Ces deux régions ont été utilisées pour définir des couples d'oligonucléotides espèce‐spécifiques. Ces couples d'amorces ont permis d'amplifier par PCR un fragment de 356 pb pour chacune des trois espèces. La spécificité des trois couples d'amorces a été vérifiée avec succès sur une collection de 17 isolats de M. laxa, 18 isolats de M. fructigena et 6 isolats de M. fructicola isolés de différents hôtes. En utilisant des conditions stringentes lors de la PCR, aucune réaction croisée n'a été observée avec les isolats testés. La spécificité du test a été d'autre part vérifiée avec l'ADN total extrait de différentes espèces de champignons, soit phylogénétiquement proche du genre Monilinia soit fréquemment isolées de fruits malades. L'utilisation de cette technique permet d'identifier avec fiabilité un isolaat indéterminé en une seule amplification, en le testant simultanément avec chacun des trois couples d'amorces. De plus, la détection et l'identification des espèces de Monilinia peuvent être réalisés directement à partir de fruits présentant des symptômes. Cette méthode simple et rapide pourrait être particulièrement utile pour détecter M. fructicola qui est un organisme de quarantaine pour l'Europe.
Comparative testing of nested PCR‐based methods with bait‐plant tests for detecting Phytophthora fragariae var. fragariae in infected strawberry roots from fruit crops in the UK*
Authors: Hughes K. J. D. , Inman A. J. , Cooke D. E. L.
Published: April 2008

This study compared nested polymerase chain reaction (PCR) methods with the traditional Duncan bait test for the detection of Phytophthora fragariae var. fragariae in 86 root samples representing 30 strawberry fruit crops in England and Wales. DNA was extracted directly from the sampled roots using a cetyltrimethyl ammonium bromide‐based (CTAB) extraction method. Dilutions of the extracted DNA were then amplified using a nested PCR protocol. The PCR results correlated strongly with those of the bait tests, with the PCR methodology appearing to be the more reliable detection method on this type of field material. The PCR test also had the advantage of being a more rapid, less labour and time‐intensive test, compared with the 5‐6 week bait test. Comparisons were also made with a second set of nested PCR primers on DNA extracted from infected roots from the same field material; both primer sets performed equally well.
Differentiation of Tilletia taxa by rep‐PCR genomic fingerprinting*
Authors: McDonald J. G. , Wong E. , White G. P.
Published: April 2008

The potential of repetitive‐sequence‐based polymcrase chain reaction (rep‐PCR) fingerprinting of fungal genomic DNA as a rapid and simple alternative to random amplified polymorphic DNA (RAPD) analysis in the study of phylogenetic relationships, and also as a diagnostic method, was investigated with species of Tilletia. DNA primers (BOX, ERIC and REP) corresponding to conserved repetitive element motifs, originally described in prokaryotes, were used to generate genomic fingerprints of T. indica, T. walkeri, T. controversa, T. laevis, T. tritici, T. goloskokovii, T. barclayana and members of the T. fusca complex. Computer‐assisted analysis of the database of combined fingerprints clearly distinguished each taxon and indicated phylogenetic relationships consistent with previously reported RAPD analyses. There were three main cluster groupings where isolates showed 35–40% similarity. Group 1 included T. indica and T. walkeri, group 2 included members of the T. fusca complex, as well as T. controversa, T. laevis, T. tritici and T. goloskokovii, and group 3 included only T. barclayana. If, as is likely, the conserved repetitive element motifs on which this technique is based are widespread or universal in fungal species, rep‐PCR shows strong potential, not only as a simple generic taxonomic tool, but also as a diagnostic method.
Citrus leprosis virus: properties, diagnosis, agro‐ecology and phytosanitary importance
Authors: Lovisolo O.
Published: April 2008

Citrus leprosis disease, caused by citrus leprosis virus (CiLV), had severe effects on sweet oranges in Florida (US) until the 1920s, after which it became rare. In South America, it appeared in the 1930s, first in Argentina and then in Brazil, where it is now widespread and very dangerous. It has also been reported in Paraguay, Peru, Uruguay and Venezuela. CiLV is transmitted by three species of Brevipalpus, mainly Brevipalpus phoenicis. The virus mainly attacks sweet orange, but also citrange, citron, Cleopatra mandarin, grapefruit, lemon, mandarin, sour orange and tangor. CiLV is a non‐enveloped rhabdovirus characterized by bacilliform particles measuring 120–130 × 50–55 nm, present in mesophyll and vascular parenchyma within cisternae of the endoplasm reticulum. Viroplasm structures are present in the infected cells. CiLV has been partially purified and its dsRNA as been investigated. It is mechanical transmissible to 13 test plant species belonging to the genera Atriplex, Beta and Chenopodium (Chenopodiaceae), Gomphrena (Amaranthaceae), and Tetragonia (Tetragoniaceae). Using some of these herbaceous test plants, grown at a suitable temperature, it is possible to diagnose CiLV in 3–4 days. CiLV is covered by lists and requirements in phytosanitary regulations, but the information given is often misleading. For phytosanitary purposes, it is important to consider the following main points: (a) both CiLV and its vectors need to be considered; (b) sweet orange fruits can be infected even more than propagation material; and (c) CiLV does not infect susceptible citrus systemically, or any of its known hosts.
Le dépérissement de l'olivier issu de boutures herbacées en Tunisie
Authors: Boulila M.
Published: April 2008

Quatre espèces fongiques ont été isolées à partir de jeunes oliviers (en phase de multiplication et en plein champ) dépéris et issus de boutures herbacées. Il s'agit de: Macrophomina phaseolina, Nectria haematococca, Armillaria mellea, Corticium rolfsii. Des enquêtes de terrain et des analyses de laboratoire ont permis de les repérer et de les identifier.
Identification of wood‐decay fungi infecting various forest trees
Authors: Krimi Z. , Mehdid S.
Published: April 2008

Fomes fomentarius, Coriolus versicolor and Inonotus hispidus were the commonest wood‐decay fungi on declining trees in the Tlemcen area of Algeria, associated principally with Populus spp., Quercus suber and Fraxinus spp. respectively.
Registration of plant protection products in EPPO countries: current status and possible approaches to harmonization
Authors: Rüegg J. , Siegfried W. , Raisigl U. , Viret O. , Steffek R. , Reisenzein H. , Persen U.
Published: April 2008

A survey concerning registration of plant protection products was carried out in 41 EPPO member countries. Twenty six countries responded and results are summarized in three groups. Various models are discussed which seek to adapt product dosage to the crowns of fruit‐tree crops. The tree row volume model (TRV) is favoured by the authors since it can adequately cope with the wide variability of orchards encountered across Europe. It is suggested that experimenters who carry out registration field trials should measure the height and width of the tree crowns and the distance between the tree rows to facilitate comparison of registration trials including residue data.
Thrips palmi: pathways and possibilities for spread
Authors: Vierbergen G.
Published: April 2008

An analysis was made of 406 detections of Thrips palmi in imported consignments at points of entry in Europe and in the USA. This thrips pest commonly occurred in products for final consumption, but was rarely found in plants for planting. Most detections related to ornamentals (79% in Europe and 85% in USA), and especially orchid cut flowers imported from Thailand. Aubergine (Solanum melongena) and balsam pear (Momordica charantia) were the vegetables most often found to be infested. From experience in The Netherlands, introductions of T. palmi and subsequent outbreaks are assumed to result from the import of infested plants for planting, and from exchange of plant material between growers. There is no indication that products for final consumption have a role in the entry of T. palmi.
Amplification de séquences (RT‐PCR) et analyse du polymorphisme des fragments de restriction (RFLP) de quelques isolats du Prunus necrotic ringspot ilarvirus
Authors: Boulila M. , Marrakchi M.
Published: April 2008

L'approche moléculaire basée sur l'usage de l'amplification génique (RT‐PCR) et l'analyse du polymorphisme des fragments de restriction (RFLP) a été utilisée pour la détection et le typage de trois isolats (Peerless, Texas et Khoukhi) du Prunus necrotic ringspot ilarvirus (PNRSV) sévissant dans les vergers et les parcs à bois d'amandier en Tunisie. Une comparaison a été effectuée avec deux autres isolats provenant, en l'occurrence, de France (isolat de cerisier) et d'Italie (isolat d'abricotier). La RT‐PCR a permis de mettre en évidence la présence de ce virus dans le matériel végétal testé en amplifiant une portion génomique de 282 pb localisée sur l'ARN‐3 de ce virus. L'analyse RFLP a permis de distinguer deux groupes d'isolats (Khoukhi et Cerisier, d'une part et Texas et Abricotier, d'autre part). L'isolat Peerless appartient au deux groupes. Un travail antérieur de caractérisation sérologique basée sur l'emploi d'anticorps monoclonaux a permis de conclure à ce que l'analyse RFLP est moins sensible que la sérologie quant à la distinction entre divers isolats de PNRSV. Un travail ultérieur, utilisant le séquençage de produits de PCR, fournirait des informations encore plus précises et détaillées.
Pest risk assessment for the countries of the European Union (as PRA area) on Monilinia fructicola
Authors: van Leeuwen G. C. M. , Baayen R. P. , Jeger M. J.
Published: April 2008

A pest risk assessment was performed according to the EPPO Standard PM 5/3 to determine the probability of introduction of Monilinia fructicola into the countries of the European Union (EU) and its potential economic impact. Data on the biology of the pathogen were combined with trade pathways and information on the use of commodities in order to quantify risk. On the basis of the EPPO Standard, we concluded that there is a serious risk that M. fructicola could become established in the EU, with significant economic losses as a result. This justifies the phytosanitary measures currently in place in the EU.
A comparison of observed and simulated site response in the Rhône valley
Authors: Roten D. , Fäh D. , Olsen K. B. , Giardini D.
Published: April 2008

Site effects in the city of Sion in the Rhône valley are analysed from weak motion signals recorded on a dense temporary array. We simulate the recorded events with a 3-D finite difference method for frequencies up to 4 Hz using a recently developed velocity model of the Sion basin.

Site-to-reference Fourier spectral ratios are computed from 16 local and regional events. All sites exhibit amplification factors of up to 12 between 0.5 and 0.6 Hz, which can be reproduced by the numerical simulations. By rotating the weak motion to directions parallel and perpendicular to the valley axis, we show that this low-frequency amplification is caused by the SH00 and SV0 fundamental modes of 2-D resonance.

Additional peaks of amplification can be observed at higher frequencies, with amplification factors of up to 20 at some sites.

Application of the high-resolution frequency-wavenumber and the multiple signal characterization method to the vertical component of recorded and simulated signals show that edge-generated surface waves arriving from almost all directions dominate the wavefield at 1.25 and 2.50 Hz.

Peak ground velocities computed from the simulated ground motion show interference patterns that depend strongly on the incidence direction, and the computed amplification of peak ground velocities are generally in agreement with the observations.

We conclude that the complex 3-D geometry of the basin needs to be considered to evaluate site effects up to at least 2.5 Hz.
Global‐scale seismic interferometry: theory and numerical examples
Authors: Ruigrok Elmer , Draganov Deyan , Wapenaar Kees
Published: April 2008

Progress in the imaging of the mantle and core is partially limited by the sparse distribution of natural sources; the earthquake hypocenters are mainly along the active lithospheric plate boundaries. This problem can be approached with seismic interferometry. In recent years, there has been considerable progress in the development of seismic interferometric techniques. The term seismic interferometry refers to the principle of generating new seismic responses by cross‐correlating seismic observations at different receiver locations. The application of interferometric techniques on a global scale could create sources at locations where no earthquakes occur. In this way, yet unknown responses would become available for the application of travel‐time tomography and surface‐wave dispersion studies. The retrieval of a dense‐enough sampling of source gathers would largely benefit the application of reflection imaging.

We derive new elastodynamic representation integrals for global‐scale seismic interferometry. The relations are different from other seismic interferometry relations for transient sources, in the sense that they are suited for a rotating closed system like the Earth. We use a correlation of an observed response with a response to which free‐surface multiple elimination has been applied to account for the closed system. Despite the fact that the rotation of the Earth breaks source‐receiver reciprocity, the seismic interferometry relations are shown to be valid. The Coriolis force is included without the need to evaluate an extra term.

We synthesize global‐scale earthquake responses and use them to illustrate the acoustic versions of the new interferometric relations. When the sampling of real source locations is dense enough, then both the responses with and without free‐surface multiples are retrieved. When we do not take into account the responses from the sources in the direct neighborhood of the seismic interferometry‐constructed source location, the response with free‐surface multiples can still be retrieved. Even when only responses from sources at a certain range of epicentral distances are available, some events in the Green's function between two receiver locations can still be retrieved. The retrieved responses are not perfect, but the artefacts can largely be ascribed to numerical errors. The reconstruction of internal events – the response as if there was a source and a receiver on (major) contrasts within the model – could possibly be of use for imaging.

With modelling it is possible to discover in which region of the correlation panel stationary phases occur that contribute to the retrieval of events. This knowledge opens up a new way of filtering out undesired events and of discovering whether specific events could be retrieved with a given source‐receiver configuration.
Consistency of the spatial autocorrelation method with seismic interferometry and its consequence
Authors: Yokoi Toshiaki , Margaryan Sos
Published: April 2008

We have cross‐checked the conventional theory of the spatial autocorrelation method and the consequence of seismic interferometry: the retrieval of the elastodynamic Green's function. Their mutual consistency is almost complete. The basic formulas of the conventional spatial autocorrelation theory can be derived by an alternative approach based on the retrieval of the elastodynamic Green's function. The only discrepancy is found with the average of the complex coherence function over azimuth in a wavefield dependent on azimuth. It is hypothesized, in discussion, that this discrepancy is due to the way of representing the wavefield in the background theory of seismic interferometry that can produce only wavefields moderately dependent on azimuth and that the mentioned consequence of seismic interferometry can also only make sense in a wavefield moderately dependent on azimuth.

Our field experiment with a wavefield dependent on azimuth showed that the consequence of seismic interferometry in the logical framework of the conventional spatial autocorrelation theory is appropriate under such degrees of approximation as the measure proposed in this study, i.e., the deviation of the total dispersion curves is between about 10 and 16 per cent at the maximum from those averaged over azimuth.

The acceptance of the retrieval of Green's function gives a proper physical meaning to the complex coherence function: the real part of the elastodynamic Green's function normalized by its zero‐offset version. This makes it possible to take a deterministic approach rather than the statistical one on which the conventional spatial autocorrelation method is based and gives fruitful new aspects and perspectives. For example, the formula for the multi‐mode case is given and the possibility of exploration of two or three dimensional velocity structures is suggested.
Endothelial nitric oxide synthase is a critical factor in experimental liver fibrosis
Authors: Leung Tung‐Ming , Tipoe George L. , Liong Emily C. , Lau Thomas Y.H. , Fung Man‐Lung , Nanji Amin A.
Published: April 2008

Reduced expression of endothelial nitric oxide synthase (eNOS) in chronic liver disease can reduce hepatic perfusion and accelerate fibrosis. The relationship between eNOS expression and liver fibrogenesis remains unclear. We investigated whether l‐arginine attenuated chronic liver fibrosis through eNOS expression. Chronic liver injury was induced by administration of carbon tetrachloride (CCl4) to mice for 8 weeks. 5‐Methylisothiourea hemisulphate (SMT), an iNOS inhibitor, or l‐arginine, a NOS substrate were injected subcutaneously. CCl4‐induced hepatotoxicity, oxidative stress and accumulation of collagen were detected in the liver. The expression levels of inducible NOS (iNOS) and nuclear factor kappa‐B (NF‐κB) activity in the liver after CCl4 treatment were increased but eNOS expression and activator protein‐1 (AP‐1) activity were decreased. Both SMT and l‐arginine effectively reduced CCl4 induced oxidative stress and collagen formation, but l‐arginine showed a significantly greater suppression of collagen formation, iNOS expression and NF‐κB activity. l‐Arginine also restored the level of eNOS and AP‐1 activity. l‐Arginine was more effective than SMT in suppressing liver fibrosis. l‐Arginine might improve NO production which facilitates hepatic blood flow and thus retards liver fibrogenesis. Our results showed that the reduced eNOS expression in CCl4‐treated mice was reversed by l‐arginine. Furthermore, l‐arginine also reversed the reduced AP‐1 activity, an eNOS promoter.
Freeze‐substitution: the addition of water to polar solvents enhances the retention of structure and acts at temperatures around –60°C
Authors: BUSER C. , WALTHER P.
Published: April 2008

High‐pressure freezing followed by freeze substitution and plastic embedding is becoming a more widely used method for TEM sample preparation. Here, we have investigated the influence of solvents, fixative concentrations and water content in the substitution medium on the sample quality of high‐pressure frozen, freeze‐substituted and plastic embedded mammalian cell culture monolayers. We found that the visibility of structural details was optimal with acetone and that extraction increased with both increasing and decreasing solvent polarity. Interestingly, the addition of water to polar solvents increased the sample quality, while being destructive when added to apolar solvents. The positive effect of water addition is saturable in acetone and ethanol at 5%(v/v), but even addition of up to 20% water has no negative effect on the sample structure. Therefore, a medium based on acetone containing fixatives and 5% water is most optimal for the substitution of mammalian cell cultures. In addition, our results suggest that the presence of water is critical for the retention of structure at temperatures around –60°C.
SHORT COMMUNICATION: Microcarriers for high‐pressure freezing and cryosectioning of adherent cells
Authors: HAGEN C. , GRÜNEWALD K.
Published: April 2008

A method is described employing microcarrier spheres of cross‐linked dextran for obtaining ultra‐ and semithin vitreous sections from high‐pressure frozen anchorage‐dependent (mammalian) cells. Avoiding trypsination or scraping cells off from the culture surface, the presented approach allows for cryoimmobilization, cryosectioning and cryoelectron microscopy/tomography of frozen‐hydrated cells in an unperturbed manner which is important to preserve the native state of, for instance, the cytoskeleton. Furthermore, our studies on the ‘life cycle’ of Herpes simplex virus in Vero cells demonstrate that cell monolayers on microcarrier beads are well suited for fluorescence microscopic characterization of the sample prior to high‐pressure freezing.
The use of CT scan based planning for oral rehabilitation by means of implants and its transfer to the surgical field: a critical review on accuracy
Authors: VERCRUYSSEN M. , JACOBS R. , VAN ASSCHE N. , Van STEENBERGHE D.
Published: April 2008

Summary  The purpose of the present paper is to review the literature on the use of CT scan based planning for oral rehabilitation and its transfer to the surgical field by means of a surgical guide. The first part will deal with surgical guides based on tomographies or CT scan data often using dedicated software, but manually fabricated. In the second part, CT‐derived drill guides are discussed, which are fabricated by means of CAD/CAM technology or other computer‐controlled technology. The deviations between the position of the implants at the planning stage and after the surgery are of utmost importance, especially when flapless procedures are applied. The maximal deviations are often not stated in the literature. This should be taken into account when a system is applied clinically.
The effects of a maximal jaw clench on an inhibitory jaw reflex
Authors: MAILLOU P. , CADDEN S. W.
Published: April 2008

Summary  The purpose of this study was to determine whether mimicking symptoms of temporomandibular disorders by experimentally activating deep nociceptors in the oro‐facial region, can modulate an inhibitory jaw reflex. In human subjects, electromyograms were recorded from one (eight subjects) or both (16 subjects) active masseter muscles and electrical stimuli were applied to the upper lip. This procedure was performed before and after a 30‐s conditioning period in which the subjects maximally clenched the jaw. In all subjects, the electrical stimuli produced an inhibition of masseter activity. Following conditioning, there was a small but not statistically significant decrease in the mean size of this inhibition (anova: P = 0·066 and P = 0·077, for responses recorded ipsi‐ and contralaterally to the stimulus). There was no relationship between changes in the reflex and the levels of pain induced by the conditioning procedure and recorded on 100 mm visual analogue scales (range = 0–64 mm, median = 11 mm) (Spearman’s correlation test: P = 0·412). These findings suggest that inhibitory jaw reflexes are little if at all affected by this conditioning procedure. The hypothesis that temporomandibular disorders both cause and are sustained by a decrease in protective jaw reflexes is not supported by these findings.
High population differentiation and unusual haplotype structure in a shade‐intolerant pioneer tree species, Zanthoxylum ailanthoides (Rutaceae) revealed by analysis of DNA polymorphism at four nuclear loci
Authors: KAMIYA K. , MORITSUKA E. , YOSHIDA T. , YAHARA T. , TACHIDA H.
Published: April 2008

Differences in demographic history, life‐history traits, and breeding systems affect nucleotide variation patterns. It is expected that shade‐intolerant pioneer tree species have different patterns of genetic polymorphism and population structure than climax species. We studied patterns of nucleotide polymorphism at four putative starch pathway loci (agpSA, agpSB, agpL, and GBSSI) in Zanthoxylum ailanthoides, a shade‐intolerant pioneer tree species that occupies forest gaps in warm‐temperate forests of East Asia. Genetic diversity was lower within each population than among populations, and differentiation among populations was high across the loci (FST = 0.32–0.64), as expected from the insect‐pollinated breeding system and the metapopulation structure of this pioneer species. Numbers of haplotypes were smaller than those expected from the observed numbers of segregating sites. Single haplotypes accounted for more than 47% of all the sampled genes at the respective loci. These variation patterns were incompatible with neutral predictions for populations of a finite island model. Complex population dynamics, such as bottleneck and/or admixture, in the history of this pioneer tree species might have resulted in the observed patterns of genetic variation and population structure, which are different from those of climax wind‐pollinated tree species, such as conifers. In contrast to the other loci investigated in this study, agpL showed nearly no variation in Z. ailanthoides (one singleton only), but there was some extent of variation in a closely related species, Zanthoxylum schinifolium. This suggests possibly a recent selective sweep at or near the locus in Z. ailanthoides.
Temporal changes in kin structure through a population cycle in a territorial bird, the red grouse Lagopus lagopus scoticus
Authors: PIERTNEY STUART B. , LAMBIN XAVIER , MACCOLL ANDREW D. C. , LOCK KERRY , BACON PHILIP J. , DALLAS JOHN F. , LECKIE FIONA , MOUGEOT FRANCOIS , RACEY PAUL A. , REDPATH STEVE , MOSS ROBERT
Published: April 2008

Populations of red grouse (Lagopus lagopus scoticus) undergo regular multiannual cycles in abundance. The ‘kinship hypothesis’ posits that such cycles are caused by changes in kin structure among territorial males producing delayed density‐dependent changes in aggressiveness, which in turn influence recruitment and regulate density. The kinship hypothesis makes several specific predictions about the levels of kinship, aggressiveness and recruitment through a population cycle: (i) kin structure will build up during the increase phase of a cycle, but break down prior to peak density; (ii) kin structure influences aggressiveness, such that there will be a negative relationship between kinship and aggressiveness over the years; (iii) as aggressiveness regulates recruitment and density, there will be a negative relationship between aggressiveness in one year and both recruitment and density in the next; (iv) as kin structure influences recruitment via an affect on aggressiveness, there will be a positive relationship between kinship in one year and recruitment the next. Here we test these predictions through the course of an 8‐year cycle in a natural population of red grouse in northeast Scotland, using microsatellite DNA markers to resolve changing patterns of kin structure, and supra‐orbital comb height of grouse as an index of aggressiveness. Both kin structure and aggressiveness were dynamic through the course of the cycle, and changing patterns were entirely consistent with the expectations of the kinship hypothesis. Results are discussed in relation to potential drivers of population regulation and implications of dynamic kin structure for population genetics.
A Csr‐type regulatory system, including small non‐coding RNAs, regulates the global virulence regulator RovA of Yersinia pseudotuberculosis through RovM
Authors: Heroven Ann Kathrin , Böhme Katja , Rohde Manfred , Dersch Petra
Published: April 2008

The MarR‐type regulator RovA controls expression of virulence genes of Yersinia pseudotuberculosis in response to environmental signals. Using a genetic strategy to discover components that influence rovA expression, we identified new regulatory factors with homology to components of the carbon storage regulator system (Csr). We showed that overexpression of a CsrB‐ or a CsrC‐type RNA activates rovA, whereas a CsrA‐like protein represses RovA synthesis. We further demonstrate that influence of the Csr system on rovA is indirect and occurs through control of the LysR regulator RovM, which inhibits rovA transcription. The CsrA protein had also a major influence on the motility of Yersinia, which was independent of RovM. The CsrB and CsrC RNAs are differentially expressed in Yersinia. CsrC is highly induced in complex but not in minimal media, indicating that medium‐dependent rovM expression is mediated through CsrC. CsrB synthesis is generally very low. However, overexpression of the response regulator UvrY was found to activate CsrB production, which in turn represses CsrC synthesis independent of the growth medium. In summary, the post‐transcriptional Csr‐type components were shown to be key regulators in the co‐ordinated environmental control of physiological processes and virulence factors, which are crucial for the initiation of Yersinia infections.
Systematic functional analysis of calcium‐signalling proteins in the genome of the rice‐blast fungus,Magnaporthe oryzae, using a high‐throughput RNA‐silencing system
Authors: Nguyen Quoc Bao , Kadotani Naoki , Kasahara Seiji , Tosa Yukio , Mayama Shigeyuki , Nakayashiki Hitoshi
Published: April 2008

We developed an RNA‐silencing vector, pSilent‐Dual1 (pSD1), with a convergent dual promoter system that provides a high‐throughput platform for functional genomics research in filamentous fungi. In the pSD1 system, the target gene was designed to be transcribed as a chimeric RNA with enhanced green fluorescent protein (eGFP) RNA. This enabled us to efficiently screen the resulting transformants using GFP fluorescence as an indicator of gene silencing. A model study with the eGFP gene showed that pSD1‐based vectors induced gene silencing via the RNAi pathway with slightly lower efficiency than did hairpin eGFP RNA‐expressing vectors. To demonstrate the applicability of the pSD1 system for elucidating gene function in the rice‐blast fungusMagnaporthe oryzae, 37 calcium signalling‐related genes that include almost all known calcium‐signalling proteins in the genome were targeted for gene silencing by the vector. Phenotypic analyses of the silenced transformants showed that at least 26, 35 and 15 of the 37 genes examined were involved in hyphal growth, sporulation and pathogenicity, respectively, inM. oryzae.These included several novel findings such as thatPmc1‐,Spf1‐ andNeo1‐like Ca2+pumps, calreticulin and calpactin heavy chain were essential for fungal pathogenicity.
An amphiphilic region in the cytoplasmic domain of KdpD is recognized by the signal recognition particle and targeted to the Escherichia coli membrane
Authors: Maier Katja S. , Hubich Stefanie , Liebhart Helga , Krauss Susanne , Kuhn Andreas , Facey Sandra J.
Published: April 2008

The sensor protein KdpD of Escherichia coli is composed of a large N‐terminal hydrophilic region (aa 1–400), four transmembrane regions (aa 401–498) and a large hydrophilic region (aa 499–894) at the C‐terminus. KdpD requires the signal recognition particle (SRP) for its targeting to the membrane. Deletions within KdpD show that the first 50 residues are required for SRP‐driven membrane insertion. A fusion protein of the green fluorescent protein (GFP) with KdpD is found localized at the membrane only when SRP is present. The membrane targeting of GFP was not observed when the first 50 KdpD residues were deleted. A truncated mutant of KdpD containing only the first 25 amino acids fused to GFP lost its ability to specifically interact with SRP, whereas a specific interaction between SRP and the first 48 amino acids of KdpD fused to GFP was confirmed by pull‐down experiments. Conclusively, a small amphiphilic region of 27 residues within the amino‐terminal domain of KdpD (aa 22–48) is recognized by SRP and targets the protein to the membrane. This shows that membrane proteins with a large N‐terminal region in the cytoplasm can be membrane‐targeted early on to allow co‐translational membrane insertion of their distant transmembrane regions.
Plasmodium falciparum antigenic variation. Mapping mosaic var gene sequences onto a network of shared, highly polymorphic sequence blocks
Authors: Bull Peter C. , Buckee Caroline O. , Kyes Sue , Kortok Moses M. , Thathy Vandana , Guyah Bernard , Stoute José A. , Newbold Chris I. , Marsh Kevin
Published: April 2008

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a potentially important family of immune targets, encoded by an extremely diverse gene family called var. Understanding of the genetic organization of var genes is hampered by sequence mosaicism that results from a long history of non‐homologous recombination. Here we have used software designed to analyse social networks to visualize the relationships between large collections of short var sequences tags sampled from clinical parasite isolates. In this approach, two sequences are connected if they share one or more highly polymorphic sequence blocks. The results show that the majority of analysed sequences including several var‐like sequences from the chimpanzee parasite Plasmodium reichenowi can be either directly or indirectly linked together in a single unbroken network. However, the network is highly structured and contains putative subgroups of recombining sequences. The major subgroup contains the previously described group A var genes, previously proposed to be genetically distinct. Another subgroup contains sequences found to be associated with rosetting, a parasite virulence phenotype. The mosaic structure of the sequences and their division into subgroups may reflect the conflicting problems of maximizing antigenic diversity and minimizing epitope sharing between variants while maintaining their host cell binding functions.
Ancestral roles of eukaryotic frataxin: mitochondrial frataxin function and heterologous expression of hydrogenosomal Trichomonas homologues in trypanosomes
Authors: Long Shaojun , Jirků Milan , Mach Jan , Ginger Michael L. , Sutak Robert , Richardson Des , Tachezy Jan , Lukeš Julius
Published: April 2008

Frataxin is a small conserved mitochondrial protein; in humans, mutations affecting frataxin expression or function result in Friedreich's ataxia. Much of the current understanding of frataxin function comes from informative studies with yeast models, but considerable debates remain with regard to the primary functions of this ubiquitous protein. We exploit the tractable reverse genetics of Trypanosoma brucei in order to specifically consider the importance of frataxin in an early branching lineage. Using inducible RNAi, we show that frataxin is essential in T. brucei and that its loss results in reduced activity of the marker Fe–S cluster‐containing enzyme aconitase in both the mitochondrion and cytosol. Activities of mitochondrial succinate dehydrogenase and fumarase also decreased, but the concentration of reactive oxygen species increased. Trypanosomes lacking frataxin also exhibited a low mitochondrial membrane potential and reduced oxygen consumption. Crucially, however, iron did not accumulate in frataxin‐depleted mitochondria, and as T. brucei frataxin does not form large complexes, it suggests that it plays no role in iron storage. Interestingly, RNAi phenotypes were ameliorated by expression of frataxin homologues from hydrogenosomes of another divergent protist Trichomonas vaginalis. Collectively, the data suggest trypanosome frataxin functions primarily only in Fe–S cluster biogenesis and protection from reactive oxygen species.
Amino acid‐dependent growth of Campylobacter jejuni: key roles for aspartase (AspA) under microaerobic and oxygen‐limited conditions and identification of AspB (Cj0762), essential for growth on glutamate
Authors: Guccione Edward , Del Rocio Leon‐Kempis Maria , Pearson Bruce M. , Hitchin Edward , Mulholland Francis , Van Diemen Pauline M. , Stevens Mark P. , Kelly David J.
Published: April 2008

Amino acids are key carbon and energy sources for the asaccharolytic food‐borne human pathogen Campylobacter jejuni. During microaerobic growth in amino acid rich complex media, aspartate, glutamate, proline and serine are the only amino acids significantly utilized by strain NCTC 11168. The catabolism of aspartate and glutamate was investigated. An aspartase (aspA) mutant (unable to utilize any amino acid except serine) and a Cj0762c (aspB) mutant lacking aspartate:glutamate aminotransferase (unable to utilize glutamate), were severely growth impaired in complex media, and an aspA sdaA mutant (also lacking serine dehydratase) failed to grow in complex media unless supplemented with pyruvate and fumarate. Aspartase was shown by activity and proteomic analyses to be upregulated by oxygen limitation, and aspartate enhanced oxygen‐limited growth of C. jejuni in an aspA‐dependent manner. Stoichiometric aspartate uptake and succinate excretion involving the redundant DcuA and DcuB transporters indicated that in addition to a catabolic role, AspA can provide fumarate for respiration. Significantly, an aspA mutant of C. jejuni 81‐176 was impaired in its ability to persist in the intestines of outbred chickens relative to the parent strain. Together, our data highlight the dual function of aspartase in C. jejuni and suggest a role during growth in the avian gut.
Relationship of cytochrome P450 pharmacogenetics to the effects of yohimbine on gastrointestinal transit and catecholamines in healthy subjects
Authors: Bharucha A. E. , Skaar T. , Andrews C. N. , Camilleri M. , Philips S. , Seide B. , Burton D. , Baxter K. , Zinsmeister A. R.
Published: April 2008

Abstract  Alpha‐2 adrenergic receptors tonically inhibit colonic motility and the α2‐adrenergic antagonist yohimbine, given intravenously, increased colonic tone in humans. However, the effect of yohimbine on colonic transit in humans is unknown. In this study, 30 healthy volunteers were randomized to yohimbine 16.2 mg p.o. t.i.d. or identical placebo for 7 days. We evaluated gastric emptying, small intestinal, and colonic transit by scinitigraphy, bowel habits, haemodynamics and plasma catecholamines. As cytochrome P450 enzymes metabolize yohimbine, P450 genotypes (CYP2D6 and CYP3A4) were determined in 25 of 30 subjects who consented to genetic studies. The relationship between drug metabolizer status predicted by CYP2D6 and CYP3A4 and effects of yohmibine were assessed. Compared to placebo, yohimbine increased (P ≤ 0.02) diastolic blood pressure, plasma noradrenaline concentrations and maximum tolerated volume during the satiation test [yohimbine (1241 ± 88, mean ± SEM) vs placebo (1015 ± 87), P = 0.054]. However, yohimbine did not affect gastrointestinal transit. Based on CYP2D6 and CYP3A4 alleles, seven and 18 subjects were, respectively, extensive (EM) and poor (PM) metabolizers of yohimbine. Compared to EM, PM of yohimbine had a greater increase in plasma noradrenaline (P = 0.1 for PM vs EM), lower maximum tolerated volumes (1120 ± 95 vs 1484 + 131 mL, P = 0.02), and faster colonic transit (i.e. GC24 was 3.0 ± 0.4 vs 2.1 ± 0.5, P = 0.1). These data suggest that CYP2D6 and CYP3A4 genotypes which determine the metabolism of yohimbine may influence its sympathetic and gastrointestinal effects.