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Rabies virus (RABV), which is transmitted via a bite wound caused by a rabid animal, infects peripheral nerves and then spreads to the central nervous system (CNS) before causing severe neurological symptoms and death in the infected individual. Despite the importance of this ability of the virus to spread from a peripheral site to the CNS (neuroinvasiveness) in the pathogenesis of rabies, little is known about the mechanism underlying the neuroinvasiveness of RABV. In this study, to obtain insights into the mechanism, we conducted comparative analysis of two fixed RABV strains, Nishigahara and the derivative strain Ni-CE, which cause lethal and asymptomatic infections, respectively, in mice after intramuscular inoculation. Examination of a series of chimeric viruses harboring the respective genes from Nishigahara in the genetic background of Ni-CE revealed that the Nishigahara phosphoprotein (P) gene plays a major role in the neuroinvasiveness by mediating infection of peripheral nerves. The results obtained from both in vivo and in vitro experiments strongly suggested that the Nishigahara P gene, but not the Ni-CE P gene, is important for stable viral replication in muscle cells. Further investigation based on the previous finding that RABV phosphoprotein counteracts the host interferon (IFN) system demonstrated that the Nishigahara P gene, but not the Ni-CE P gene, functions to suppress expression of the beta interferon (IFN-β) gene ( Ifn -β) and IFN-stimulated genes in muscle cells. In conclusion, we provide the first data strongly suggesting that RABV phosphoprotein assists viral replication in muscle cells by counteracting the host IFN system and, consequently, enhances infection of peripheral nerves.

Using 11 consensus primer pairs designed from S-linked F-box genes of apple and Japanese pear, 10 new F-box genes (MdFBX21 to 30) were isolated from the apple cultivar 'Spartan' (S(9)S(10)). MdFBX21 to 23 and MdFBX24 to 30 were completely linked to the S(9) -RNase and S(10-)RNase, respectively, and showed pollen-specific expression and S-haplotype-specific polymorphisms. Therefore, these 10 F-box genes are good candidates for the pollen determinant of self-incompatibility in apple. Phylogenetic analysis and comparison of deduced amino acid sequences of MdFBX21 to 30 with those of 25 S-linked F-box genes previously isolated from apple showed that a deduced amino acid identity of greater than 88.0 % can be used as the tentative criterion to classify F-box genes into one type. Using this criterion, 31 of 35 F-box genes of apple were classified into 11 types (SFBB1-11). All types included F-box genes derived from S(3-) and S(9-)haplotypes, and seven types included F-box genes derived from S(3-), S(9-), and S(10-)haplotypes. Moreover, comparison of nucleotide sequences of S-RNases and multiple F-box genes among S(3-), S(9-), and S(10-)haplotypes suggested that F-box genes within each type showed high nucleotide identity regardless of the identity of the S-RNase. The large number of F-box genes as candidates for the pollen determinant and the high degree of conservation within each type are consistent with the collaborative non-self-recognition model reported for Petunia. These findings support that the collaborative non-self-recognition system also exists in apple.

SummaryMost cultivars of European pear (Pyrus communis L.) exhibit S-RNase-based self-incompatibility. Their S-genotypes have mainly been assigned by analysing PCR-amplified S-RNase alleles from the genomic DNA of each cultivar. In this study, we identified eight European pear cultivars that had only one S-RNase allele amplified by conventional genomic PCR using a consensus primer pair. Rapid amplification of cDNA ends (RACE) on stylar RNAs of the cultivars was used to obtain the full-length sequence of the putative S 25-RNase. Long-PCR successfully amplified the S25-RNase allele, including a long 3,131 bp intron, the longest intron reported so far among S-RNase alleles in fruit tree species in the sub-tribe Pyrinae. Conventional PCR using a consensus primer and a primer designed from the intron sequence amplified a 385 bp fragment of the S25-RNase allele from the genomic DNA of all eight cultivars. An S-RNase-based, cleaved amplified polymorphic sequence (CAPS) marker system and S25-RNase allele-specific...

SummarySeveral cultivars of European pear (Pyrus communis L.) are triploid (2n = 51), produce little viable pollen, and exhibit S-RNase-based cross-incompatibility. In orchards where triploids are grown, two diploids (one to pollinate the triploids, and the other to pollinate the other diploid) must be inter-planted. In this study, seven European pear cultivars were confirmed to be triploid by flow cytometry analysis. An S-RNase-based, cleaved amplified polymorphic sequence (CAPS) marker system and S-RNase allele-specific PCR were used to genotype these triploids. A comparison of S-genotypes between the triploids and diploids of European pear identified those diploid(s) that could not be used to pollinate the triploids. Pollination tests confirmed the cross-incompatibility between a triploid and a diploid. The S-genotypes of the seven triploids will be useful for pollination management in orchards, and for breeding new cultivars using the triploids as seed parents. In addition, rapid amplification of cDNA...

Genome editing is a powerful technique for genome modification in molecular research and crop breeding and has the great advantage of imparting novel desired traits to genetic resources. However, the genome editing of fruit tree plantlets remains to be established. In this study, we describe induction of a targeted gene mutation in the endogenous apple phytoene desaturase (PDS) gene using the CRISPR/Cas9 system. Four guide RNAs (gRNAs) were designed and stably transformed with Cas9 separately in apple. Clear and partial albino phenotypes were observed in 31.8% of regenerated plantlets for one gRNA and bi-allelic mutations in apple PDS were confirmed by DNA sequencing. In addition, an 18-bp gRNA also induced a targeted mutation. These CRIPSR/Cas9 induced-mutations in the apple genome suggest activation of the NHEJ pathway, but with some involvement also of the HR pathway. Our results demonstrate that genome editing can be practically applied to modify the apple genome.

Ljungan virus (LV) has been isolated/detected from rodents in a limited area including European countries and the USA. In this study, we isolated an LV strain from faecal samples of wild birds that had been collected in Japan, and determined the nearly complete sequence of the genome. Sequence analyses showed that the isolate possesses an LV-like genomic organization: 5UTR-VP0-VP3-VP1-2A1-2A2-2B-2C-3A-3B-3C-3D-3UTR. Phylogenetic and similarity analyses based on the VP1 region indicated that the strain constitutes a novel genotype within LV. In addition, we identified species origin of the faeces as gull species by using the DNA barcoding technique. These data suggested that the novel LV strain infected a gull species, in which the virus had not been identified. Taken together, this study has provided the first evidence of the presence of a novel LV in Japan, highlighting the possibility of LV infection in birds.

The current live rabies vaccine SAG2 is attenuated by only one mutation (Arg-to-Glu) at position 333 in the glycoprotein (G333). This fact generates a potential risk of the emergence of a pathogenic revertant by a back mutation at this position during viral propagation in the body. To circumvent this risk, it is desirable to generate a live vaccine strain highly and stably attenuated by multiple mutations. However, the information on attenuating mutations other than that at G333 is very limited. We previously reported that amino acids at positions 273 and 394 in the nucleoprotein (N273/394) (Leu and His, respectively) of fixed rabies virus Ni-CE are responsible for the attenuated phenotype by enhancing interferon (IFN)/chemokine gene expressions in infected neural cells. In this study, we found that amino acid substitutions at N273/394 (Phe-to-Leu and Tyr-to-His, respectively) attenuated the pathogenicity of the oral live vaccine ERA, which has a virulent-type Arg at G333. Then we generated ERA-N273/394-G333 attenuated by the combination of the above attenuating mutations at G333 and N273/394, and checked its safety. Similar to the ERA-G333, which is attenuated by only the mutation at G333, ERA-N273/394-G333 did not cause any symptoms in adult mice after intracerebral inoculation, indicating a low level of residual pathogenicity of ERA-N273/394-G333. Further examination revealed that infection with ERA-N273/394-G333 induces IFN-β and CXCL10 mRNA expressions more strongly than ERA-G333 infection in a neuroblastoma cell line. Importantly, we found that the ERA-N273/394-G333 stain has a lower risk for emergence of a pathogenic revertant than does the ERA-G333. These results indicate that ERA-N273/394-G333 has a potential to be a promising candidate for a live rabies vaccine strain with a high level of safety.

To obtain complete genome sequences of turkey rotavirus A strains Ty-1 and Ty-3, we sequenced the gene segments that had not been decoded previously. The genotype constellations of the respective strains were determined to be G17-P[38]-I4-R4-C4-M4-A16-N4-T4-E4-H4 and G7-P[35]-I4-R4-C4-M4-A16-N4-T4-E11-H14. Notably, their VP4 and NSP5 genes were classified into novel genotypes.

In situ observation of the formation process of mesophase of isotactic polypropylene (iPP) is reported in structural point of view. Combining a rapid temperature jump and a high-flux synchrotron radiation X-ray scattering techniques, very rapid transformation from the molten amorphous state to the mesophase has been observed. The transformation proceeded very quickly in a narrow temperature range accompanied by instantaneous fluctuations in micrometer scale, suggesting the mesophase formation proceeds similarly to spinodal decomposition.

SummaryTo elucidate the influence of temperature on water flow from soil to stem in a dwarfing rootstock, sap flow was measured under different temperature conditions in 1-year-old trees of apple rootstocks of different vigour. Sap flow was largely determined by solar radiation, and increased linearly as solar radiation increased when the temperatures above and below ground were held constant. The adjusted means of sap flow in the trees tested were not significantly influenced by the total leaf area, but were significantly influenced by the root mass. The adjusted means of sap flow at 30oC were two- to three-times greater than those at 20oC in all rootstocks tested, except one super-dwarfing rootstock, during the period of shoot extension. As the degree of rootstock vigour increased, the adjusted means of sap flow increased. The difference in sap flow between an invigorating rootstock and a dwarfing one increased under high temperature conditions. Because the degree of dwarfing was clearly expressed as th...

SummaryWe have determined the S-RNase genotypes of 33 new apple cultivars and lineages produced in Japan, 44 unknown cultivars and two lineages, and 22 triploid progenies. We have speculated on the putative parentage of new cultivars and lineages based on their S-RNase genotypes and also identified mistaken parents. In the case of the triploid progenies, the breeding of new cultivars using a triploid paternal parent may pose problems due to its low pollen viability. Nevertheless, diploid and triploid progenies were obtained using a triploid maternal parent. We have compiled a database of 516 apple S-RNase genotypes, including those previously investigated, which included a survey system for cultivar combinations, showing those that were fully-incompatible, semi-compatible, or fully-compatible, together with information on the PCR-RFLP method used for the identification of S-RNase genotypes and S-RNase allele designation (available at http://www.agr.nagoya-u.ac.jp/~0hort/apple/).

Differentiation of cultivars with simple sequence repeat (SSR) markers is a very useful technique for the true-to-type characterization of cultivars and clarification of parent-offspring relationships. We developed an SSR marker set for cultivar identification comprising 15 markers that were screened from 46 previously published SSRs. This marker set could be used for apple varieties including Malus × domestica and/or other Malus species. These SSRs successfully characterized 95 apples, including the leading and major founding cultivars used worldwide for modern apple breeding. Therefore, this marker set could be applied to almost all apple cultivars. We also analyzed the parent-offspring relationships of 69 cultivars by considering allele transmissions. This analysis revealed the true parentage of the following seven cultivars: ‘Kizashi’, ‘Chinatsu’, ‘Honey Queen’, ‘Haruka’, ‘Seirin’, ‘Ozenokurenai’, and Morioka #48. This analysis also revealed a parentage discrepancy for ‘Hacnine’. From the parent-offspring analysis, two microsatellite mutation events at alleles inherited from pollen parents were observed.

Article, This paper describes a least complex, high speed decoding method named multi-stage threshold decoding (MTD-DR). Each stage of MTD-DR is formed by the traditional threshold decoder with a special shift register, called difference register (DR). After flipping each information bit, DR helps to shorten the Hamming and the Euclidian distance between a received word and the decoded codeword for hard and soft decoding, respectively. However, the MTD-DR with self-orthogonal convolutional codes (SOCCs), type 1 in this paper, makes an unavoidable error group, which depends on the tap connection patterns in the encoder, and limits the error performance. This paper introduces a class of SOCCs type 2 which can breakdown that error group, as a result, MTD-DR gives better error performance. For a shorter code (code length =4200), hard and soft decoding MTD-DR achieves 4.7dB and 6.5dB coding gain over the additive white Gaussian noise (AWGN) channel at the bit error rate (BER) 10-5, respectively. In addition, hard and soft decoding MTD-DR for a longer code (code length =80000) give 5.3dB and 7.1dB coding gain under the same condition, respectively. The hard and the soft decoding MTD-DR experiences error flooring at high Eb/N0 region. For improving overall error performance of MTD-DR, this paper proposes parity check codes concatenation with soft decoding MTD-DR as well., This article is approved by all authors.

Japanese pear exhibits gametophytic self-incompatibility controlled by a single S-locus with multiple alleles. The S-locus encodes an S-RNase as a stylar product, and 10 S-RNase alleles (S1 to S9, and Sk) have been cloned from major cultivars. To investigate the diversity of S-alleles in Japanese pear, we analyzed the S-genotypes of three local cultivars (‘Senryo’, ‘Kuroki’, and ‘Hogyoku’). Two S-RNase fragments were amplified from each cultivar by genomic PCR with S-RNase-specific primers. A cleaved amplified polymorphic sequence (CAPS) marker system to distinguish S1- to S9-RNases assigned S3- and S2-RNase alleles to ‘Senryo’ and ‘Kuroki’, respectively. Cloning and sequencing of the other S-RNase genes identified Se-RNase of European pear in ‘Senryo’, S12-RNase of Chinese pear in ‘Kuroki’, and S30-RNase of Chinese pear and Sk-RNase in ‘Hogyoku’. Therefore, S-genotypes of ‘Senryo’, ‘Kuroki’, and ‘Hogyoku’ were assigned as S3Se, S2S12, and SkS30, respectively. These results revealed that Japanese pear has some of the same S-alleles as European and Chinese pears as well as S1- to S9- and Sk-alleles.

SummaryMost cultivars of European pear (Pyrus communis L.) exhibit S-RNase-based gametophytic self-incompatibility (SI), but the cultivar, ‘Grand Champion’, is partially self-compatible (SC). We used pollination and molecular genetic approaches to study the cause of the partial SC, and its effects on fruit set and quality. ‘Grand Champion’ was genotyped to SbSe by an S-RNase-based cleaved amplified polymorphic sequence marker system. Crossing ‘Grand Champion’ with pollen from an SI cultivar, ‘California’ (SbSe), showed that partial SC was caused by the disruption of pistil function. Of the Sb- and Se-RNase alleles cloned from ‘Grand Champion’, the Sb-RNase allele had two nonsynonymous nucleotide substitutions compared with the Sb-RNase allele previously cloned from ‘Doyenne du Comice’, but it retained the typical primary structure of S-RNases of the Maloideae. The sequence of the Sb-RNase allele from ‘Grand Champion’ was also obtained from two SI cultivars: ‘Josephine de Malines’ and ‘Urbaniste’. Similar ...

Vegetative propagation enables the multiplication of plants that are genetically identical to the original. Hardwood cuttings have a low cost advantage and are used widely for the propagation of JM series apple rootstocks, which root well from hardwood cuttings. To breed new apple rootstocks with similarly high rooting capabilities, the development of a marker-assisted selection method is desirable. Therefore, it is important to understand the genetic mechanisms behind the rooting capabilities of hardwood cuttings. We identified quantitative trait loci (QTLs) for the rooting capability of ‘JM7’ using a QTL analysis with phenotypic data obtained under two environmental conditions. The QTLs for rooting rate and root quantity co-located. The QTL for rooting rate explained 66 % of the genetic variance and 57 % of the phenotypic variance. Graphical genotyping and database searching revealed candidate genes, including three related to auxin response, one related to ethylene response, and a gene encoding a WRKY transcription factor.

Nine full-length cDNAs of S ribonucleases (S-RNases) were cloned from stylar RNA of European pear cultivars by RT-PCR and 3' and 5' RACE. Comparison of the nucleotide sequences between the nine S-RNases cloned and 13 putative S alleles previously amplified by genomic PCRs revealed that seven corresponded to Sa, Sb, Sd, Se, Sh, Sk and Sl alleles, and the other two were new S alleles (designated as Sq and Sr alleles). Genomic PCR with a set of a8FTQQYQa9 and a8EP-anti-IIWPNVa9 primers was used to amplify nine S alleles; 1,414 bp (Sl), ca. 1.3 kb (Sk and Sq), 998 bp (Se), 440 bp (Sb) and ca. 350 bp (Sa, Sd, Sh and Sr). Among these, S alleles of similar size were discriminated by digestion with BaeI, BglII, BssHII, HindIII, EcoO109I and SphI. The PCR amplification of S allele following digestion with the restriction enzymes provided a PCR-RFLP system for rapid S-genotyping European pear cultivars harboring nine S alleles. The PCR-RFLP system assigned a total of 63 European pear cultivars to 25 genotypes. Among these, 14 genotypes were shared by two or more cultivars, which were cross-incompatible. These results suggested that the genes cloned represented the S-RNases from European pear, and that there were many cross-incompatible combinations among European pear varieties.

A new S9-allele was discovered in 6 Japanese pear cultivars, ‘Shinkou’, ‘Shinsei’, ‘Niitaka’, ‘Amanogawa’, ‘Nangetsu’ and ‘Nansui’. cDNA encoding S9-RNase, a stylar product of S9-allele, was cloned from pistils of ‘Shinkou’ and ‘Shinsei’ by 3' and 5' RACE. The S9-RNase gene had an open reading frame of 684 nucleotides encoding 228 amino acid residues. S9-RNase had a hypervariable (HV) region different from S1- to S8-RNase and shared higher similarity (95.2%) with apple S3-RNase than with 8 Japanese pear S-RNases (from 61.0% to 70.7%). Genomic PCR with primers ‘FTQQYQ’ and ‘anti-(I/T) IWPNV’ provided S1- to S9-amplicon (product), but could not discriminate the S2 from the S9 of ca. 1.3 kb. The S2 and S9 were distinguished by digestion with AflII and BstBI, respectively. The digestion with nine S-allele-specific restriction endonucleases, SfcI, AflII, PpuMI, NdeI,AlwNI, HincII, AccII, NruI and BstBI, distinguished S1 to S9, establishing that this PCR-RFLP system is useful for S-genotype assignments in Japanese pear harboring S1- to S9-allele. ‘Shinkou’, ‘Shinsei’, ‘Nangetsu’ and ‘Nansui’ assigned as S4S9 were determined to be cross incompatible.

A Japanese pear 'Kumoi' was previously determined as S3S4 by pollination tests, but its S-genotype was reconsidered following our PCR-RFLP (S1 to S9) analyses and pollination tests. Based on its compatibility with 'Seigyoku' (S3S4), and PCR-RFLP analysis, 'Kumoi' was classified as S1S3 for the first time. Additional pollination tests were necessary to prove our contention, but 'Kumoi' did not supply sufficient flowers. 'Sekaiichi' was also assigned as S1S3 by PCR-RFLP analysis, and incompatibility with 'Kumoi'. Instead of 'Kumoi', 'Sekaiichi' was pollinated with the pollen from an 55-homozygote and that from an Ssm4-homozygote. The lack of fruit set revealed that 'Sekaiichi' was incompatible with the S3 and Ssm4 pollen, leading us to predict that the 5-genotype of 'Sekaiichi' was S1S3 or 8384. Two S-genotypes with S1S3 and S2S3 segregated in hybrid progenies between 'Doitsu' (S1S2) and 'Sekaiichi', indicating that S1 was present in 'Sekaiichi'. These results of pollination tests with 'Sekaiichi' indicated the S-genotype of 'Kumoi' was S1S3.

Longer- and/or branched-chain polyamines are unique polycations found in thermophiles. N 4 -aminopropylspermine is considered a major polyamine in Thermococcus kodakarensis . To determine whether a quaternary branched penta-amine, N 4 -bis(aminopropyl)spermidine, an isomer of N 4 -aminopropylspermine, was also present, acid-extracted cytoplasmic polyamines were analyzed by high-pressure liquid chromatography, gas chromatography (HPLC), and gas chromatography-mass spectrometry. N 4 -bis(aminopropyl)spermidine was an abundant cytoplasmic polyamine in this species. To identify the enzyme that catalyzes N 4 -bis(aminopropyl)spermidine synthesis, the active fraction was concentrated from the cytoplasm and analyzed by linear ion trap–time of flight mass spectrometry with an electrospray ionization instrument after analysis by the MASCOT database. TK0545, TK0548, TK0967, and TK1691 were identified as candidate enzymes, and the corresponding genes were individually cloned and expressed in Escherichia coli . Recombinant forms were purified, and their N 4 -bis(aminopropyl)spermidine synthesis activity was measured. Of the four candidates, TK1691 (BpsA) was found to synthesize N 4 -bis(aminopropyl)spermidine from spermidine via N 4 -aminopropylspermidine. Compared to the wild type, the bpsA -disrupted strain DBP1 grew at 85°C with a slightly longer lag phase but was unable to grow at 93°C. HPLC analysis showed that both N 4 -aminopropylspermidine and N 4 -bis(aminopropyl)spermidine were absent from the DBP1 strain grown at 85°C, demonstrating that the branched-chain polyamine synthesized by BpsA is important for cell growth at 93°C. Sequence comparison to orthologs from various microorganisms indicated that BpsA differed from other known aminopropyltransferases that produce spermidine and spermine. BpsA orthologs were found only in thermophiles, both in archaea and bacteria, but were absent from mesophiles. These findings indicate that BpsA is a novel aminopropyltransferase essential for the synthesis of branched-chain polyamines, enabling thermophiles to grow in high-temperature environments.

On the basis of computer simulations of the time-dependent Ginzburg - Landau model including thermal noise effects, we evaluate the surface barrier against magnetic flux penetration in type-II superconductors. We discuss several parameter dependencies of the first field for flux penetration, which is estimated from the first peak position of the magnetization curve in the presence of an external field slowly increasing over time. It is shown that the first penetration field is a decreasing function of temperature and of the degree of sample shape deformation. The surface barrier is also found to be suppressed by the surface irregularity on the scale of the magnetic penetration depth.

The ac response of type-II superconductors to an alternating magnetic field is numerically studied on the basis of the time-dependent Ginzburg - Landau equations. We examine the temperature dependence of the ac susceptibility associated with a small ac magnetic field in the absence of a bias dc field. It is shown that with increasing temperature the in-phase component of the fundamental susceptibility exhibits a step-like change from a negative constant value to zero, while the out-of-phase component of the fundamental susceptibility and the third-harmonic component have a peak at a certain temperature near the superconducting transition temperature. These results are in qualitative agreement with those of recent experiments on high- superconductors.

Most fruit trees in the Rosaceae exhibit self-incompatibility, which is controlled by the pistil S gene, encoding a ribonuclease (S-RNase), and the pollen S gene at the S-locus. The pollen S in Prunus is an F-box protein gene (SLF/SFB) located near the S-RNase, but it has not been identified in Pyrus and Malus. In the Japanese pear, various F-box protein genes (PpSFBB(-α-γ)) linked to the S-RNase are proposed as the pollen S candidate. Two bacterial artificial chromosome (BAC) contigs around the S-RNase genes of Japanese pear were constructed, and 649 kb around S(4)-RNase and 378 kb around S(2)-RNase were sequenced. Six and 10 pollen-specific F-box protein genes (designated as PpSFBB(4-u1-u4, 4-d1-d2) and PpSFBB(2-u1-u5,) (2-d1-d5), respectively) were found, but PpSFBB(4-α-γ) and PpSFBB(2-γ) were absent. The PpSFBB(4) genes showed 66.2-93.1% amino acid identity with the PpSFBB(2) genes, which indicated clustering of related polymorphic F-box protein genes between haplotypes near the S-RNase of the Japanese pear. Phylogenetic analysis classified 36 F-box protein genes of Pyrus and Malus into two major groups (I and II), and also generated gene pairs of PpSFBB genes and PpSFBB/Malus F-box protein genes. Group I consisted of gene pairs with 76.3-94.9% identity, while group II consisted of gene pairs with higher identities (92%) than group I. This grouping suggests that less polymorphic PpSFBB genes in group II are non-S pollen genes and that the pollen S candidates are included in the group I PpSFBB genes.

This report describes the development of novel wired chip devices for mu-HPLC analyses. The monolithic capillary column to be wired was prepared using a tri-functional epoxy monomer, tris(2,3-epoxypropyl)isocyanurate with a diamine, 4-[(4-aminocyclohexyl)methyl]cyclohexylamine. The prepared column was evaluated by SEM observation of the sectional structure of column and micro-HPLC. In addition, the reproducibility in the preparation of long capillary columns having nearly 1 m length was extensively examined for applications of novel wired chip devices. The authors demonstrated that the monolithic structure of the prepared long capillary could be finely controlled under the strictly maintained operational conditions and thus the relative standard deviation (RSD) of the column properties such as the number of theoretical plates, retention factor, and permeability could be well controlled to become less than 10%. Furthermore, the wired chip device column showed that its high performance was kept even after chip preparation.

Utilisation du DNA et du RNA comme support de traces de metaux dans l'eau pour la preconcentration par coprecipitation. La methode permet de concentrer les ions metalliques sauf le fer, le calcium et le magnesium qui sont contenus intrinsequement dans les deux acides nucleiques utilises

Japanese pear (Pyrus pyrifolia Nakai) has a gametophytic self-incompatibility (GSI) mechanism controlled by a single S-locus with multiple S-haplotypes, each of which contains separate genes that determine the allelic identity of pistil and pollen. The pistil S gene is the S-ribonuclease (S-RNase) gene, whereas good candidates for the pollen S gene are the F-box protein genes. A self-compatible (SC) cultivar, ‘Osa-Nijisseiki’, which is a bud mutant of ‘Nijisseiki’ (S 2 S 4), has a stylar-part mutant $$ S^{{sm}}_{4} $$ -haplotype, which lacks the S 4-RNase gene but retains the pollen S gene. To delineate the deletion breakpoint in the $$ S^{{sm}}_{4} $$ -haplotype, we constructed a bacterial artificial chromosome (BAC) library from an S 4-homozygote, and assembled a BAC contig of 570 kb around the S 4-RNase. Genomic PCR of DNA from S 4- and $$ S^{{sm}}_{4} $$ -homozygotes and the DNA sequence of the BAC contig allowed the identification of a deletion of 236 kb spanning from 48 kb upstream to 188 kb downstream of S 4-RNase. The $$ S^{{sm}}_{4} $$ -haplotype lacks 34 predicted open reading frames (ORFs) including the S 4-RNase and a pollen-specific F-box protein gene (termed as S 4 F-box0). Genomic PCR with a primer pair designed from the deletion junctions yielded a product specific for the $$ S^{{sm}}_{4} $$ -haplotype. The product could be useful as a maker for early selection of SC cultivars harboring the $$ S^{{sm}}_{4} $$ -haplotype.

Lyssaviruses, including rabies virus (RABV) and Australian bat lyssavirus (ABL) are a globally distributed genus of zoonotic pathogens that cause rabies disease with a case-fatality rate in humans of 100%, resulting in over 61,000 deaths/year. To understand the mechanisms underlying lyssavirus evasion of interferon (IFN)-mediated antiviral immune responses, we have used a multidisciplinary approach combining techniques including viral reverse genetics and in vivo infection, next-generation sequencing/bioinformatics, single live-cell imaging (including novel live-cell microtubule-association assays), super-resolution dSTORM microscopy, immune signalling assays, and protein–protein interaction analysis. This work has determined that the lyssavirus IFN-antagonist P-protein forms a complex array of molecular interactions in host cells, including with STATs 1 and 2, microtubules, nuclear import and export receptors, and intranuclear structures [1] , [2] , [3] , [4] . Recently, we have identified novel interactions with STAT3, which enable inhibition of signalling by several cytokines [3] , and with nucleoli, and have found that P-protein specifically alters microtubule structure and dynamics. Using mutagenesis to modify specific association sequences in P-protein, we have determined that many of these interactions are critical to infection in vivo [1] , [4] . In particular, we found that interactions of lyssavirus P-proteins with STATs are conserved across the lyssavirus genus [2] , and that mutagenic inhibition of these interactions results in strongly reduced growth of virus in the CNS of infected mice to attenuate pathogenicity, such that the mutated virus causes no neurological symptoms or death compared with the invariably lethal wild-type strain [1] . Similarly, our recent studies using mutation of virus to prevent P-protein subcellular trafficking and modulation of microtubule structure indicate that this strongly impairs IFN-antagonist function, resulting in attenuation in vivo. Together, these studies demonstrate critical roles in disease of diverse interactions that underlie viral antagonism of cytokine signalling; this identifies novel targets for the generation of attenuated vaccines and antiviral drugs. Our current studies, which include structural analysis using NMR and crystallography, seek to exploit this knowledge to generate new approaches to combat rabies disease.