Molecular evolution of the genomic RNA of Apple stem grooving capillovirus.

The complete genome of the German isolate AC of Apple stem grooving virus (ASGV) was sequenced. It encodes two overlapping open reading frames (ORFs), similarly to previously described ASGV isolates. Two regions of high variability were detected between the ASGV isolates, variable region 1 (V1, from amino acids (aa) 532 to 570), and variable region 2 (V2, from aa 1,583 to 1,868). The phylogenetic analysis of the V1 and V2 regions suggested that the ASGV diversity was structured by host plant species rather than geographical origin. The dN/dS ratio between nonsynonymous and synonymous nucleotide substitution rates varied greatly along the ASGV genome. Most of ORF1 showed predominant negative selection except for the two regions V1 and V2. V1 showed an elevated dN and an average dS when compared to the ORF1 background but no significant positive selection was detected. The V2 region of ORF1 showed an elevated dN and a low dS when compared to the ORF1 background with an average dN/dS ≈ 3.0 indicative of positive selection. However, the V2 area includes overlapping ORFs, making the dN/dS estimate biased. Joint estimates of the selection intensity in the different ORFs by a recent method indicated that this region of ORF1 was in fact evolving close to neutrality. This was convergent with previous results showing that introduction of stop codons in this region of ORF1 did not impair plant infection. These data suggest that the elimination of a stop codon caused the overprinting of a novel coding region over the ancestral ORF.

Integration of decentralized clinical data in a data warehouse: a service-oriented design and realization.

OBJECTIVES: In this paper we present a general concept and describe the difficulties for the integration of data from various clinical partners in one data warehouse using the Open European Nephrology Science Center (OpEN.SC) as an example. This includes a requirements analysis of the data integration process and also the design according to these requirements. METHODS: This conceptual approach based on the Rational Unified Process (RUP) and paradigm of Service-Oriented Architecture (SOA). RESULTS: Because we have to enhance the confidence of our partners in the OpEN.SC system and with this the willingness of them to participate, important requirements are controllability, transparency and security for all partners. Reusable and fine-grained components were found to be necessary when working with diverse data sources. With SOA the requested reusability is implemented easily. CONCLUSIONS: A key step in the development of a data integration process within such a health information system like OpEN.SC is to analyze the requirements. And to show that this is not only a theoretical work, we present a design - developed with RUP and SOA - which fulfills these requirements.

Configurational probabilities for monomers, dimers and trimers in fluids.

A new analytical approach is proposed to model aggregation of molecules with isotropic, nearest-neighbor, attractive interactions. By treating the clustering process as a chain reaction, equations with the exact high temperature limit are derived by evaluating the occupation probabilities of nearest neighbors based on the Ono-Kondo approach for a hexagonal lattice to calculate the configurational probabilities of i-mers (i = 1, 2, 3). Equilibrium constants for dimers and trimers are calculated based on the configurational probability data. The proposed model agrees well with Monte Carlo simulations at medium and high temperatures. At low temperatures, the model can be improved by considering the full set of site densities in the first shell of a central trimer. Approximate analytical solutions derived from exact calculations of the grand partition function for monomer adsorption on a 4 x N hexagonal lattice with cylindrical boundary conditions also are presented.

Monte Carlo simulations of phase transitions and adsorption isotherm discontinuities on surface compression.

Low temperature, Grand Canonical Monte Carlo simulations were used to study the adsorption of fluid layers on cubic, hexagonal, and atomically smooth substrates to determine the effects of registry and surface compression on the system. The size of the fluid molecules was fixed to be 20% larger than the substrate molecules in order to observe the transition from an expanded to commensurate and finally to an incommensurate monolayer. For relatively weak fluid-substrate interactions, the cubic system underwent a first-order phase transition. As the strength of the fluid-substrate interactions increased, the molecules became fixed at commensurate locations and the transition from low density to commensurate packing became continuous. The strong fluid-substrate interactions lead to the development of a kink in the adsorption isotherm that showed the increased stability of the commensurate phase. This kink became more pronounced as the system temperature was decreased. The hexagonal system showed less dramatic results due to a decrease in the substrate well depth of the relative to the cubic system. The system did experience a first-order phase transition for a weak fluid-substrate interactions and the transition became much more gradual as the fluid-substrate interaction increased. The molecules became fixed to commensurate substrate locations, but the surface was not corrugated sufficiently to have a stable commensurate phase. The atomically smooth substrate showed the first-order phase transition expected of a low temperature system with no effects of registry.

Complete nucleotide sequence of a putative new cytorhabdovirus infecting lettuce.

The full-length nucleotide sequence of the genomic RNA of a new cytorhabdovirus infecting lettuce was determined. Six open reading frames were found in the antigenomic sequence of the 12,926-nt negative-sense viral RNA genome. The genomic organisation was similar to that of lettuce necrotic yellows virus (LNYV), the type member of the genus Cytorhabdovirus: 3'-N-P-3-M-G-L-5', where N is the capsid protein gene, P the putative phosphoprotein gene, 3 a gene coding for a putative protein of unknown function, M the putative matrix protein gene, G the glycoprotein gene, and L the putative polymerase gene. Amino acid sequence comparison with the corresponding sequences of other rhabdoviruses revealed the closest relationship to LNYV, with identities ranging from 41% for the matrix proteins and 65% for the L polymerase proteins. These results indicate that this virus may be a member of a new cytorhabdovirus species, for which the name Lettuce yellow mottle virus (LYMoV) is proposed.

Cheravirus and Sadwavirus: two unassigned genera of plant positive-sense single-stranded RNA viruses formerly considered atypical members of the genus Nepovirus (family Comoviridae).

The genus Nepovirus (family Comoviridae) was known both for a good level of homogeneity and for the presence of atypical members. In particular, the atypical members of the genus differed by the number of capsid protein (CP) subunits. While typical nepoviruses have a single CP subunit with three structural domains, atypical nepoviruses have either three small CP subunits, probably corresponding to the three individual domains, or a large and a small subunit, probably containing two and one structural domains, respectively. These differences are corroborated by hierarchical clustering based on sequences derived from both genomic RNAs. Therefore, these atypical viruses are now classified in two distinct genera, Cheravirus (three CP subunits; type species Cherry rasp leaf virus) and Sadwavirus (two CP subunits; type species Satsuma dwarf virus).

Generalization of Kelvin's equation for compressible liquids in nanoconfinement.

The Kelvin equation for a compressible liquid in nanoconfinement is written in a form that takes into account not only Laplace's pressure, but also the oscillatory compression pressure. This leads to a simple analytical equation for pressure in nanocapillaries. The corrected equation is used to analyze properties of aqueous systems, including the oscillatory structural forces between attractive surfaces and inert surfaces, repulsive "hydration" forces between hydrophilic surfaces, and attractive "hydrophobic" forces between hydrophobic surfaces. Relative vapor pressure in a nanocapillary also is discussed.

A RT/PCR-partial restriction enzymatic mapping (PREM) method for the molecular characterisation of the large satellite RNAs of Arabis mosaic virus isolates.

The satellite RNA of the grapevine isolate NW of Arabis mosaic virus (ArMV) was cloned and sequenced, and showed 75% identity at the nucleotide level to the satellite RNA of the lilac isolate of ArMV. In order to survey ArMV isolates from various geographical origins and natural hosts for the presence of large satellite RNAs and analyse their degree of variability, a RT/PCR-partial restriction enzymatic mapping (PREM) method was developed. The method is based on the incorporation of 5-methyl-dCTP in the RT/PCR reaction, and the subsequent digestion of the RT/PCR products by methyl-sensitive restriction enzymes. Satellites RNAs were detected by RT/PCR in eight isolates out of 47, six of them originating from grapevine, one from hop and one from lilac. The partial restriction digestion patterns allowed to distinguish six different types of satellites. Cloning and sequencing of the different satellites confirmed these results, the PREM proving able to discriminate sequences with 96% identity. The sizes of the different satellites varied between 1092 and 1139 nucleotides, their encoded proteins between 338 and 360 amino acids. Conserved domains were found in the amino and carboxy-termini between the sequences of the proteins encoded by the satellites of the different isolates of ArMV.

Sequence analysis of grapevine isolates of Raspberry ringspot nepovirus.

The nucleotide sequences of RNAs 1 and 2 of a German isolate of Raspberry ringspot virus (RpRSV) infecting grapevine (RpRSV-Grapevine), as well as partial sequences of another grapevine isolate from Switzerland (RAC815) were determined. The sequences of the protease-polymerase region encoded by RNA1, and the movement protein and coat protein genes encoded by RNA 2, of these isolates were compared with those of other isolates available in databases. The coat proteins of the grapevine isolates formed a sister group to all those from other RpRSV isolates, but whether this resulted from divergence or recombination was uncertain.

Nucleotide sequence of a new isolate of ribgrass mosaic tobamovirus infecting Impatiens New Guinea.

The complete nucleotide sequence of a tobamovirus isolated from Impatiens New Guinea was determined. The genome was 6302 nt long, and its genomic organisation was similar to those of other crucufer tobamoviruses. Sequence comparisons with the corresponding sequences of other crucifer tobamoviruses revealed highest levels of identity with the ribgrass mosaic virus (Shanghai isolate). A small open reading frame putatively encoding a 4.5-kDa protein with a low degree of similarity to the ORF6 of tobacco mosaic virus was found nested in the movement protein gene.

Adsorption behavior of repulsive molecules.

Recently, it has been shown that adsorption of gases on solid surfaces often leads to repulsive forces between adsorbate molecules. In this paper, adsorption of molecules on a one-dimensional lattice is considered for repulsive interactions between adsorbate molecules. Exact adsorption isotherms are calculated and analyzed for finite and infinite chains of active sites (i.e., a one-dimensional lattice). Although the mathematical solution for the one-dimensional lattice is known for attractive and repulsive systems, the effects of intermolecular repulsions on adsorption behavior have not been studied in detail previously. Similarly, though the mathematics for the one-dimensional lattice has been solved for any arbitrary lattice length, the effect of finite size on adsorption isotherms for repulsive adsorbate interactions has never been examined. This paper shows that spatial confinement and strong attraction to active sites can cause compression of an adsorbed phase and that repulsive interactions between adsorbed molecules result in steps in the adsorption isotherms. For higher chemical potentials, the density increases until saturating at the lattice capacity. These steps in the adsorption isotherm have not been observed in previous studies of lattice systems. For small lattices, the adsorption behavior was found to be fundamentally different for even and odd values of lattice length. Lattices with an even number of lattice sites can have two steps in the adsorption isotherm, whereas systems with an odd number of sites only have a single step occurring at a coverage slightly greater than half the lattice capacity.

Monte Carlo simulations on the effect of substrate geometry on adsorption and compression.

Canonical Monte Carlo simulations were used to study the adsorption and compression of fluid layers on model substrates with cubic, (111) fcc, and graphite geometries. The effect of the relative size of the fluid and substrate molecules on adsorption was considered for strong molecule-surface interactions. In the case of monolayer formation, it was found that the surface geometry and the size of the adsorbate molecules had a significant effect on the structure of the adsorbed layer. These structures varied from well-ordered, commensurate layers to liquid-like structures. Lateral compression was observed for certain fluid to substrate molecule sizes. For the interactions studied in this work, it was found that maximum lateral compression occurred on the cubic surface when adsorbate molecules had a diameter approximately 15% larger than the substrate diameter. In the case of multilayer formation, it was found that second and higher adsorbed layers could compress into the adsorbed layers below them. For cubic substrates, the interlayer compression was predicted analytically with reasonable accuracy, with maximum interlayer compression found for fluid diameters approximately 90% the size of substrate molecule diameters.

Complete nucleotide sequence of the RNA 1 of a grapevine isolate of Arabis mosaic virus.

The complete nucleotide sequence of the genomic RNA 1 of the grapevine isolate NW (Neustadt an der Weinstrasse) of Arabis mosaic virus (ArMV) was determined. It is 7334 nucleotides long excluding the poly(A) tail, and contains one long open reading frame encoding a polypeptide of 2284 amino acids. The 5' and 3' non-coding regions were 227 and 252 nucleotides long respectively, and showed stretches of high identity with the corresponding 5' and 3' non-coding regions of ArMV-NW RNA 2. The analysis of the amino acid sequence of the polyprotein encoded by the RNA 1 of the ArMV-NW showed that the conserved amino acid motifs, characteristic for the viral protease co-factor, the NTP-binding protein, the cystein protease, and the RdRp core domains, were all present. Amino acid sequence comparisons between the polyproteins encoded by the RNAs 1 of ArMV-NW and other nepoviruses showed 75% identity with the GFLV-F13, and up to 36% with other nepoviruses.

Complete nucleotide sequence of an isolate of the nepovirus raspberry ringspot virus from grapevine.

The complete nucleotide sequence of the RNAs 1 and 2 of the nepovirus Raspberry ringspot virus cherry isolate (RpRSV-ch) from grapevine was determined. The RNA 1 is 7935 nucleotides (nt) long excluding the poly(A) tail, and contains one long open reading frame (ORF) encoding a polypeptide of 2367 amino acids. This ORF is preceeded by a 136nt 5' non-coding region, and followed by a 695nt 3' non-coding region. Conserved amino acid motifs, characteristic of the viral protease cofactor, the NTP-binding protein, proteinase and polymerase, were found in the sequence of the RNA 1-encoded polyprotein. The RNA 2 is 3915nt long excluding the poly(A) tail, and contains one long ORF encoding a polypeptide of 1106 amino acids. This ORF is preceeded by a 203nt 5' non-coding region, and followed by a 390nt 3' non-coding region. When compared to the corresponding sequences of other nepoviruses, a maximum level of 34% identity was found between the RNA 1-encoded polypetides of RpRSV-ch and other nepoviruses. For the RNA 2-encoded polypeptide, 88% identity was found between RpRSV-ch and RpRSV-S, a Scottish isolate of RpRSV from raspberry, and a maximum 29% identity between RpRSV-ch and other nepoviruses.

Simultaneous RT/PCR detection and differentiation of arabis mosaic and grapevine fanleaf nepoviruses in grapevines with a single pair of primers.

The movement protein genes from several isolates of ArMV and GFLV of different geographical origins were amplified by RT/PCR using degenerate primers, cloned and sequenced. A single pair of degenerate primers was designed from these sequences to allow the simultaneous amplification of parts of the movement protein genes of ArMV and GFLV. Their use in an immunocapture-RT/PCR for the detection of ArMV or GFLV in infected grapevines proved to be ten times more sensitive than the corresponding ArMV or GFLV ELISA tests. A Sph1 restriction site found in the sequences corresponding to the amplified products from the GFLV isolates, but not in the amplified products from the ArMV isolates, allowed the differentiation between ArMV and GFLV in the infected grapevines by a Sph1 restriction digestion of the amplified products.

Complete nucleotide sequences of the RNAs 2 of German isolates of grapevine fanleaf and Arabis mosaic nepoviruses.

The RNAs 2 of an Arabis mosaic virus (ArMV) and a grapevine fanleaf virus (GFLV) isolate, originating from South West of Germany near Neustadt an der Weinstrasse (NW), were sequenced. They are 3820 and 3775 nucleotides long respectively, and both contain one open reading frame encoding a polypeptide of 1110 amino acids. Their 5' non-coding regions contain conserved and repeated sequences, which are able to form stem-loop structures. Nucleotide sequence comparisons between the full-length RNAs 2 revealed homology levels of 84 and 82% between the ArMV-NW and the ArMV-L and -U, respectively, 90% between GFLV-NW and GFLV-F13, and 72% between ArMV-NW and GFLV-NW. Amino acid sequence comparisons showed that the greatest difference was found between the 2A proteins of the different ArMV isolates, the 2A protein of the ArMV-NW showing more similarity to the 2A protein of GFLV-NW than to those of ArMV-L2 or -U2.

Towards a general procedure for sequencing single DNA molecules.

In this paper we report on the latest technical advances towards single molecule sequencing, a useful method currently developed especially for fast and easy de novo sequencing. Different approaches for complete labeling of DNA with fluorescent dyes are described. In addition, the experimental set-up for the sequencing process is shown. We demonstrate the ability to purify the buffer and enzyme solutions. Inorganic buffers were purified down to at least 20 fM of remaining fluorescent impurities. The exonuclease buffer solution could be cleaned down to 0.8 pM whereby its full activity was kept. Finally, we show a selection procedure for beads and present the data of a model experiment, in which immobilized DNA is degraded by an exonuclease within a polymethylmethacrylate (PMMA) microstructure. Furthermore, the mathematical processing of the obtained raw data is described. A first complete experimental cycle is shown, combining all preparatory steps which are necessary for single molecule sequencing in microstructures.