Complete genome sequence and integrated protein localization and interaction map for alfalfa dwarf virus, which combines properties of both cytoplasmic and nuclear plant rhabdoviruses

Summary We have determined the full-length 14,491-nucleotide genome sequence of a new plant rhabdovirus, alfalfa dwarf virus (ADV). Seven open reading frames (ORFs) were identified in the antigenomic orientation of the negative-sense, single-stranded viral RNA, in the order 3′-N-P-P3-M-G-P6-L-5′. The ORFs are separated by conserved intergenic regions and the genome coding region is flanked by complementary 3′ leader and 5′ trailer sequences. Phylogenetic analysis of the nucleoprotein amino acid sequence indicated that this alfalfa-infecting rhabdovirus is related to viruses in the genus Cytorhabdovirus. When transiently expressed as GFP fusions in Nicotiana benthamiana leaves, most ADV proteins accumulated in the cell periphery, but unexpectedly P protein was localized exclusively in the nucleus. ADV P protein was shown to have a homotypic, and heterotypic nuclear interactions with N, P3 and M proteins by bimolecular fluorescence complementation. ADV appears unique in that it combines properties of both cytoplasmic and nuclear plant rhabdoviruses.

The effect of 3-5-6 TPA on fruit drop and fruit size in the lychee (Litchi Chinensis) cultivars 'Fay Zee Siu' (feizixiao) , 'Kaimana', 'Kwai Mai Pink', 'Souey Tung' and 'Tai So' (Mauritus)

Fruit drop can cause major yield losses in Australian lychee orchards, the severity varying with cultivar and season. Research in China, South Africa and Israel has demonstrated the potential for synthetic auxins used as foliar sprays to reduce fruit drop in lychee. Trials tested the efficacy of the synthetic auxin 3-5-6 trichloro-2-phridyl-oxyacetic acid (TPA) applied as a foliar spray at 50 ppm on fruit drop and fruit size on the cultivars ‘Fay Zee Siu’, ‘Kaimana’, ‘Kwai Mai Pink’, ‘Souey Tung’ and ‘Tai So’. TPA reduced fruit drop when applied to fruit greater than 12 mm in length but increased fruit drop when fruit were smaller. Fruit size at the time of application had less effect on the response than the level of natural fruit drop. When natural fruit drop was high, TPA significantly reduced it; by up to 18.7 in ‘Fay Zee Siu’, 37.1 in ‘Kaimana’, 39.8 in ‘Kwai Mai Pink’, 15.1 in ‘Souey Tung’ and 7.7 in ‘Tai So’. TPA was less effective when natural fruit drop was low. TPA increased the number of large fruit and frequently increased the number of small fruit at harvest. The small fruit were associated with an increase in the retention of fruit with poorly developed (chicken tongue) seed. Average fruit size was generally larger (up to 12.7 in ‘Souey Tung’ and 22 in ‘Tai So’) with TPA applications.

Evaluation of a multiplex PCR to identify and serotypeActinobacillus pleuropneumoniaeserovars 1, 5, 7, 12 and 15

The aim of this study was to validate a multiplex PCR for the species identification and serotyping of Actinobacillus pleuropneumoniae serovars 1, 5, 7, 12 and 15. All 15 reference strains and 411 field isolates (394 from Australia, 11 from Indonesia, five from Mexico and one from New Zealand) of A. pleuropneumoniae were tested with the multiplex PCR. The specificity of this multiplex PCR was validated on 26 non-A. pleuropneumoniae species. The multiplex PCR gave the expected results with all 15 serovar reference strains and agreed with conventional serotyping for all field isolates from serovars 1 (n = 46), 5 (n = 81), 7 (n = 80), 12 (n = 16) and serovar 15 (n = 117). In addition, a species-specific product was amplified in the multiplex PCR with all 411 A. pleuropneumoniae field isolates. Of 25 nontypeable field isolates only two did not yield a serovar-specific band in the multiplex PCR. This multiplex PCR for serovars 1, 5, 7, 12 and 15 is species specific and capable of serotyping isolates from diverse locations. Significance and Impact of the Study A multiplex PCR that can recognize serovars 1, 5, 7, 12 and 15 of A. pleuropneumoniae was developed and validated. This novel diagnostic tool will enable frontline laboratories to provide key information (the serovar) to guide targeted prevention and control programmes for porcine pleuropneumonia, a serious economic disease of pigs. The previous technology, traditional serotyping, is typically provided by specialized reference laboratories, limiting the capacity to respond to this key disease.

Evaluation of potential candidate genes involved in salinity tolerance in striped catfish (Pangasianodon hypophthalmus) using an RNA-Seq approach

Increasing salinity levels in freshwater and coastal environments caused by sea level rise linked to climate change is now recognized to be a major factor that can impact fish growth negatively, especially for freshwater teleost species. Striped catfish (Pangasianodon hypophthalmus) is an important freshwater teleost that is now widely farmed across the Mekong River Delta in Vietnam. Understanding the basis for tolerance and adaptation to raised environmental salinity conditions can assist the regional culture industry to mitigate predicted impacts of climate change across this region. Attempt of next generation sequencing using the ion proton platform results in more than 174 million raw reads from three tissue libraries (gill, kidney and intestine). Reads were filtered and de novo assembled using a variety of assemblers and then clustered together to generate a combined reference transcriptome. Downstream analysis resulted in a final reference transcriptome that contained 60,585 transcripts with an N50 of 683 bp. This resource was further annotated using a variety of bioinformatics databases, followed by differential gene expression analysis that resulted in 3062 transcripts that were differentially expressed in catfish samples raised under two experimental conditions (0 and 15 ppt). A number of transcripts with a potential role in salinity tolerance were then classified into six different functional gene categories based on their gene ontology assignments. These included; energy metabolism, ion transportation, detoxification, signal transduction, structural organization and detoxification. Finally, we combined the data on functional salinity tolerance genes into a hypothetical schematic model that attempted to describe potential relationships and interactions among target genes to explain the molecular pathways that control adaptive salinity responses in P. hypophthalmus. Our results indicate that P. hypophthalmus exhibit predictable plastic regulatory responses to elevated salinity by means of characteristic gene expression patterns, providing numerous candidate genes for future investigations.

The Structure of the Monosodium Salt of Cytidine(5')diphosphoethanolamine

CI1H19N4OIIP2.Na+.TH2 O, Mr = 594.08, is orthorhombic, space group P21212 l, with a = 6.946 (2), b = 12.503 (4), c = 28.264 (8)/k, U = 2454.6 A, a, D x = 1.61 Mg m -a, Z = 4, ~t(CuKa) = 2.612 mm -1, F(000) = 1244. Final R = 0.101 for 1454 observed reflections. The cytosine base is in the anti conformation with respect to the sugar (ZCN = 62"60) . The ribose exhibits an uncommon C(l')exo-C(2')endo puckering. The pyrophosphate has a characteristic staggered geometry. The conformation about P(2)-O(7') is trans (-103.4°). This makes CDPethanolamine more extended compared to the folded geometry of CDP-choline, which has a gauche conformation (71.3 o). The molecular interactions in the extended crystal structure, however, are similar to those found in CDP-choline, with the CMP-5' portions tightly bound by metal ligation and the phosphorylethanolamine parts only loosely held by water molecules.

1-D hydrogen-bonded organization of hexanuclear {3d-4f-5d} complexes: evidence for slow relaxation of the magnetization for [{LMe2Ni(H2O)Ln(H2O)4.5}2{W(CN)8}2] with Ln = Tb and Dy

Heterometallic {3d-4f-5d} aggregates with formula [{LMe2Ni(H2O)Ln(H2O)4.5}2{W(CN)8}2]·15H2O, (LMe2 stands for N,N-2,2-dimethylpropylenedi(3-methoxysalicylideneiminato) Schiff-base ligand) with Ln = Gd, Tb, Dy, have been obtained by reacting bimetallic [LMe2Ni(H2O)2Ln(NO3)3] and Cs3{W(CN)8} in H2O. The hexanuclear complexes are organized in 1-D arrays by means of hydrogen bonds established between the solvent molecules coordinated to Ln and the CN ligands of an octacyanometallate moiety. The X-ray structure was solved for the Tb derivative. Magnetic behavior indicates ferromagnetic {W–Ni} and {Ni–Ln} interactions (JNiW = 18.5 cm-1, JNiGd = 1.85 cm-1) as well as ferromagnetic intermolecular interactions mediated by the H-bonds. Dynamic magnetic susceptibility studies reveal slow magnetic relaxation processes for the Tb and Dy derivatives, suggesting SMM type behavior for these compounds.

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Purification and kinetic mechanism of 5,10-metliyienetetrahydrofolate reductase from sheep liver

5,10-Methylenetetrahydrofolate reductase (EC 1.1.1.68) was purified from the cytosolic fraction of sheep liver by (NH4)2 SO4 fractionation, acid precipitation, DEAE-Sephacel chromatography and Blue Sepharose affinity chromatography. The homogeneity of the enzyme was established by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, ultracentrifugation and Ouchterlony immunodiffusion test. The enzyme was a dimer of molecular weight 1,66,000 ± 5,000 with a subunit molecular weight of 87,000 ±5,000. The enzyme showed hyperbolic saturation pattern with 5-methyltetrahydrofolate.K 0.5 values for 5-methyltetrahydrofolate menadione and NADPH were determined to be 132 ΜM, 2.45 ΜM and 16 ΜM. The parallel set of lines in the Lineweaver-Burk plot, when either NADPH or menadione was varied at different fixed concentrations of the other substrate; non-competitive inhibition, when NADPH was varied at different fixed concentrations of NADP; competitive inhibition, when menadione was varied at different fixed concentrations of NADP and the absence of inhibition by NADP at saturating concentration of menadione, clearly established that the kinetic mechanism of the reaction catalyzed by this enzyme was ping-pong.

Natural host range, thrips and seed transmission of distinct Tobacco streak virus strains in Queensland, Australia

Diseases caused by Tobacco streak virus (TSV) have resulted in significant crop losses in sunflower and mung bean crops in Australia. Two genetically distinct strains from central Queensland, TSV-parthenium and TSV-crownbeard, have been previously described. They share only 81% total-genome nucleotide sequence identity and have distinct major alternative hosts, Parthenium hysterophorus (parthenium) and Verbesina encelioides (crownbeard). We developed and used strain-specific multiplex Polymerase chain reactions (PCRs) for the three RNA segments of TSV-parthenium and TSV-crownbeard to accurately characterise the strains naturally infecting 41 hosts species. Hosts included species from 11 plant families, including 12 species endemic to Australia. Results from field surveys and inoculation tests indicate that parthenium is a poor host of TSV-crownbeard. By contrast, crownbeard was both a natural host of, and experimentally infected by TSV-parthenium but this infection combination resulted in non-viable seed. These differences appear to be an effective biological barrier that largely restricts these two TSV strains to their respective major alternative hosts. TSV-crownbeard was seed transmitted from naturally infected crownbeard at a rate of between 5% and 50% and was closely associated with the geographical distribution of crownbeard in central Queensland. TSV-parthenium and TSV-crownbeard were also seed transmitted in experimentally infected ageratum (Ageratum houstonianum) at rates of up to 40% and 27%, respectively. The related subgroup 1 ilarvirus, Ageratum latent virus, was also seed transmitted at a rate of 18% in ageratum which is its major alternative host. Thrips species Frankliniella schultzei and Microcephalothrips abdominalis were commonly found in flowers of TSV-affected crops and nearby weed hosts. Both species readily transmitted TSV-parthenium and TSV-crownbeard. The results are discussed in terms of how two genetically and biologically distinct TSV strains have similar life cycle strategies in the same environment.

A note on sampling chironomids for RNA-based studies of natural populations that retains critical morphological vouchers

The rapid uptake of transcriptomic approaches in freshwater ecology has seen a wealth of data produced concerning the ways in which organisms interact with their environment on a molecular level. Typically, such studies focus either at the community level and so don’t require species identifications, or on laboratory strains of known species identity or natural populations of large, easily identifiable taxa. For chironomids, impediments still exist for applying these technologies to natural populations because they are small-bodied and often require time-consuming secondary sorting of stream material and morphological voucher preparation to confirm species diagnosis. These procedures limit the ability to maintain RNA quantity and quality in such organisms because RNA degrades rapidly and gene expression can be altered rapidly in organisms; thereby limiting the inclusion of such taxa in transcriptomic studies. Here, we demonstrate that these limitations can be overcome and outline an optimised protocol for collecting, sorting and preserving chironomid larvae that enables retention of both morphological vouchers and RNA for subsequent transcriptomics purposes. By ensuring that sorting and voucher preparation are completed within <4 hours after collection and that samples are kept cold at all times, we successfully retained both RNA and morphological vouchers from all specimens. Although not prescriptive in specific methodology, we anticipate that this paper will assist in promoting transcriptomic investigations of the sublethal impact on chironomid gene expression of changes to aquatic environments.

Amide-catalysed isomerisation of 5,6-dihydroisoquinolines: a novel synthesis of 1,2-dihydroisoquinolines

Knoevenagel condensation of 2-acylcyclohexanones or 2-ethoxycarbonylcyclohexanone with either cyanoacetamide or malononitrile followed by silver salt alkylation gave the 5,6,7,8-tetrahydroisoquinolines (3a–i). Chromic acid oxidation of the 5,6,7,8-tetrahydroisoquinolines (3a–i) to the corresponding tetralones (4a–i) followed by sodium borohydride reduction and p-toluenesulphonic acid-catalysed dehydration of the resulting alcohols (5a–i) gave the 5,6-dihydroisoquinolines (6a–i). Reaction of 5,6-dihydroisoquinolines (6a–g) with potassium amide in liquid ammonia gave a mixture of the 1,3-dihydroisoquinolines (7a–g) and the isoquinolines (8a–g). The C-1 unsubstituted 1,2-dihydroisoquinoline (7c) was found to be very unstable. In the case of the 5,6-dihydroisoquinolines (6h and 6i), reaction of potassium amide in liquid ammonia resulted in a mixture of 1-aminoisoquinoline (9) and the isoquinolines (8h and 8i). All the above compounds have been characterised by spectral data. A probable pathway for the formation of the 1,2-dihydroisoquinolines (7a–g) and the isoquinolines (8a–i) is suggested.

3,3',5,5'-Tetrabromophenolsulfonphthalein (bromphenol blue), C19H10Br4O5S

M r=670.02, monoclinic, C2/c, a= 31.003(4), b=11.037(2), c=21.183(3)A, fl= 143.7 (1) °, V= 4291.2/k 3, D,n = 2.06, D x = 2.07Mgm -3, Z=8, MoKa, 2=0.7107/k, /~=7.45 mm -1, F(000) = 2560, T= 293 K, R = 0.061 for 1697 observed reflections. The bromphenol blue molecule consists essentially of three planar groupings: the sulfonphthalein ring system and two dibromophenol rings attached to the tetrahedral C atom of the five-membered ring of the sulfonphthalein system. The dibromophenol rings are inclined with resPect to each other at 73 ° whereas they make angles of 85 and 68 ° with respect to the sulfonphthalein system. The molecules aggregate into helical columns with the non-polar regions of the molecules in the interior and the polar regions on the surface. The columns are held together by a network of hydrogen bonds.

Ni(II)chelates of 4,9-dimethyl-5,8-dizadodeca-4,8-diene-2,11-dione-3,10-diphenyl hydrazone and related molecules

Syntheses and structural characterization of Ni(II) chelates of a new series of symmetric and unsymmetric tetradentate linear ligands are described. Preparative routes involve either the direct reaction between a metal complex and arene diazonium diazonium salts or a simple metal incorporation into the independently synthesized ligands. Recent X-ray structure determination of 4,9-dimethyl-5,8-diazadodeca-4,8-diene-2,11-dione-3,10-di(4′-methyl phenyl) hydrazonatonickel(II) complex reveals the geometry around the Ni(II) to be very close to square planar. The expected distortion because of the disposition of bulky aromatic groups on the neighbouring nitrogens is minimized by their projection in the opposite directions from the plane. PMP, IR and electronic spectral data for the complexes are quite in agreement with this structure.

Editorial of "International Journal of Critical Indigenous Studies Volume 5, Number 1, 2012"

The first two articles of this edition of the journal testify to the lengthening reach of the discipline of Critical Indigenous Studies that is, remarkably, still in its nascence. Emiel Martens examines the development of Maori filmmaking since the 1980s and takes the opportunity to explore this Indigenous cinema in the context of developments in the New Zealand film industry generally. Shifting from cultural production to renewable energy, Steven M. Hoffman and Thibault Martin remind us that in the effort to satiate the demands for energy, it is often Indigenous peoples who bear adverse consequences. Using a social capital framework, the authors examine the impact of the development of hydroelectric power upon a displaced Aboriginal community and conclude that displacement has resulted in an erosion of cohesive social bonds that once ensured a sustainable way of life.

Editorial of "International Journal of Critical Indigenous Studies, Volume 5, Number 2, 2012"

This edition is marked by a strong Antipodean focus. The first three articles bring a critical Indigenous perspective to areas previously cosseted by Western understandings. Robyn Moore, using critical discourse analysis, takes Australian Prime Minister Julia Gillard’s 2011 ‘Closing the Gap’ speech to task for naturalising Indigenous Australia’s position on the wrong side of the social and economic ‘gap’. She argues that, far from accepting white culpability, Gillard instead polishes cultural deficit understandings of Indigenous disadvantage by framing the social and economic divide in meritocratic terms. In so doing, Moore further argues, Gillard casts a benevolent light upon white Australia.