Structural Basis of Drug Resistance by Genetic Variants of HIV Type 1 Clade C Protease from India

Using computer modeling of three-dimensional structures and structural information available on the crystal structures of HIV-1 protease, we investigated the structural effects of mutations, in treatment-naive and treatment-exposed individuals from India and postulated mechanisms of resistance in clade C variants. A large number of models (14) have been generated by computational mutation of the available crystal structures of drug bound proteases. Localized energy minimization was carried out in and around the sites of mutation in order to optimize the geometry of interactions present. Most of the mutations result in structural differences at the flap that favors the semiopen state of the enzyme. Some of the mutations were also found to confer resistance by affecting the geometry of the active site. The E35D mutation affects the flap structure in clade B strains and E35N and E35K mutation, seen in our modeled strains, have a more profound effect. Common polymorphisms at positions 36 and 63 in clade C also affected flap structure. Apart from a few other residues Gln-58, Asn-83, Asn-88, and Gln-92 and their interactions are important for the transition from the closed to the open state. Development of protease inhibitors by structure-based design requires investigation of mechanisms operative for clade C to improve the efficacy of therapy.

Characterisation and early detection of an offtype from micropropagated Lady Finger bananas

An offtype has been identified from micropropagated Lady Finger bananas (Musa spp., AAB group, Pome subgroup) that is characterised by its slow growth and poor bunch size. Bunch weights were approximately 25% those of normal Lady Finger plants and all of the fruit produced was unmarketable. This particular offtype is the most commonly encountered from micropropagated Lady Finger plants and, in 2 instances, blocks of 3000 and 1500 plants were entirely comprised of this single offtype. Detection of offtype plants was possible during establishment and growth of plants in the glasshouse by the presence of chlorotic streaks in the leaves. In more severe cases the streaks coalesced into chlorotic patches that developed thin, necrotic areas that eventually produced holes or splits in the leaves. Symptom expression was not ameliorated by the addition of fertiliser and even though symptoms were similar to severe Ca and B deficiency, both normal and offtype plants had similar levels of these elements in the leaves. The offtype plants were also slow growing in the glasshouse and produced significantly (P<0.05) smaller pseudostems and leaves than normal plants. Offtype plants could be readily detected after 4 weeks deflasking using the presence of chlorotic streaks in the leaves as the main selection criterion. Maximum discrimination was possible between weeks 5–7 and at the 6-leaf stage when all of the offtypes could be detected.

Family type and depression in pregnancy: Factors mediating risk in a community sample

The rate of severe depression among women in single-parent and biological families and in a variety of stepfamilies was examined in a large community sample of 13,088 pregnant women in the United Kingdom. Compared with women in biological families and published population rates, women in single-parent families and step-families reported significantly elevated rates of depression. Family-type differences in several risk factors were examined, including cohabiting (vs. married) status, relationship history, and socioeconomic and psychosocial risks, such as crowding, social support, and stressful life events. Family-type differences in depression were mediated partly by differences in social support, stressful life events, and crowding, but a main effect of family type in predicting depression remained after statistically controlling for these risks.

Electroleaching of Zinc leach residues

A room-temperature cathodic electrolytic process was developed in the laboratory to recover zinc from industrial leach residues. The various parameters affecting the electroleaching process were studied using a statistically designed experiment. To understand the mechanisms behind the electrode processes, cyclic voltammetry and galvanostatic studies were carried out. The role of Einh measurements in monitoring such an electroleaching procedure is also shown. Since significant amounts of iron were also present in the leach liquor, attempts were made to purify it before zinc recovery by electrowinning. Reductive dissolution and creation of anion vacancies were found to be responsible for the dissolution of zinc ferrite present in the leach residue. A flow sheet of the process is given.

Assessing the relationship between patch type and soil mites: A molecular approach

An urgent need exists for indicators of soil health and patch functionality in extensive rangelands that can be measured efficiently and at low cost. Soil mites are candidate indicators, but their identification and handling is so specialised and time-consuming that their inclusion in routine monitoring is unlikely. The aim of this study was to measure the relationship between patch type and mite assemblages using a conventional approach. An additional aim was to determine if a molecular approach traditionally used for soil microbes could be adapted for soil mites to overcome some of the bottlenecks associated with soil fauna diversity assessment. Soil mite species abundance and diversity were measured using conventional ecological methods in soil from patches with perennial grass and litter cover (PGL), and compared to soil from bare patches with annual grasses and/or litter cover (BAL). Soil mite assemblages were also assessed using a molecular method called terminal-restriction fragment length polymorphism (T-RFLP) analysis. The conventional data showed a relationship between patch type and mite assemblage. The Prostigmata and Oribatida were well represented in the PGL sites, particularly the Aphelacaridae (Oribatida). For T-RFLP analysis, the mite community was represented by a series of DNA fragment lengths that reflected mite sequence diversity. The T-RFLP data showed a distinct difference in the mite assemblage between the patch types. Where possible, T-RFLP peaks were matched to mite families using a reference 18S rDNA database, and the Aphelacaridae prevalent in the conventional samples at PGL sites were identified, as were prostigmatids and oribatids. We identified limits to the T-RFLP approach and this included an inability to distinguish some species whose DNA sequences were similar. Despite these limitations, the data still showed a clear difference between sites, and the molecular taxonomic inferences also compared well with the conventional ecological data. The results from this study indicated that the T-RFLP approach was effective in measuring mite assemblages in this system. The power of this technique lies in the fact that species diversity and abundance data can be obtained quickly because of the time taken to process hundreds of samples, from soil DNA extraction to data output on the gene analyser, can be as little as 4 days.

Detection of equine herpesvirus type 1 using a real-time polymerase chain reaction.

Equid herpesvirus 1 (EHV1) is a major disease of equids worldwide causing considerable losses to the horse industry. A variety of techniques, including PCR have been used to diagnose EHV1. Some of these PCRs were used in combination with other techniques such as restriction enzyme analysis (REA) or hybridisation, making them cumbersome for routine diagnostic testing and increasing the chances of cross-contamination. Furthermore, they involve the use of suspected carcinogens such as ethidium bromide and ultraviolet light. In this paper, we describe a real-time PCR, which uses minor groove-binding probe (MGB) technology for the diagnosis of EHV1. This technique does not require post-PCR manipulations thereby reducing the risk of cross-contamination. Most importantly, the technique is specific; it was able to differentiate EHV1 from the closely related member of the Alphaherpesvirinae, equid herpesvirus 4 (EHV4). It was not reactive with common opportunistic pathogens such as Escherichia coli, Klebsiella oxytoca, Pseudomonas aeruginosa and Enterobacter agglomerans often involved in abortion. Similarly, it did not react with equine pathogens such as Streptococcus equi, Streptococcus equisimilis, Streptococcus zooepidemicus, Taylorella equigenitalis and Rhodococcus equi, which also cause abortion. The results obtained with this technique agreed with results from published PCR methods. The assay was sensitive enough to detect EHV1 sequences in paraffin-embedded tissues and clinical samples. When compared to virus isolation, the test was more sensitive. This test will be useful for the routine diagnosis of EHV1 based on its specificity, sensitivity, ease of performance and rapidity.

Completion of the genome sequence of Lettuce necrotic yellows virus, type species of the genus Cytorhabdovirus

We completed the genome sequence of Lettuce necrotic yellows virus (LNYV) by determining the nucleotide sequences of the 4a (putative phosphoprotein), 4b, M (matrix protein), G (glycoprotein) and L (polymerase) genes. The genome consists of 12,807 nucleotides and encodes six genes in the order 3′ leader-N-4a(P)-4b-M-G-L-5′ trailer. Sequences were derived from clones of a cDNA library from LNYV genomic RNA and from fragments amplified using reverse transcription-polymerase chain reaction. The 4a protein has a low isoelectric point characteristic for rhabdovirus phosphoproteins. The 4b protein has significant sequence similarities with the movement proteins of capillo- and trichoviruses and may be involved in cell-to-cell movement. The putative G protein sequence contains a predicted 25 amino acids signal peptide and endopeptidase cleavage site, three predicted glycosylation sites and a putative transmembrane domain. The deduced L protein sequence shows similarities with the L proteins of other plant rhabdoviruses and contains polymerase module motifs characteristic for RNA-dependent RNA polymerases of negative-strand RNA viruses. Phylogenetic analysis of this motif among rhabdoviruses placed LNYV in a group with other sequenced cytorhabdoviruses, most closely related to Strawberry crinkle virus.

Notes on the type, synonyms, and other specimens of the balansioid fungus, Nigrocornus scleroticus

The type, and other specimens of the balansioid fungus, Nigrocornus scleroticus, its synonyms, and similar fungi studied during extensive research on the taxonomy and biology of the fungus, are described. Two hypocrealean fungi found parasitising the ascostromata of N. scleroticus are also discussed.

Characterization of disease resistance gene candidates of the nucleotide binding site (NBS) type from banana and correlation of a transcriptional polymorphism with resistance to Fusarium oxysporum f.sp. cubense race 4

Most plant disease resistance (R) genes encode proteins with a nucleotide binding site and leucine-rich repeat structure (NBS-LRR). In this study, degenerate primers were used to amplify genomic NBS-type sequences from wild banana (Musa acuminata ssp. malaccensis) plants resistant to the fungal pathogen Fusarium oxysporum formae specialis (f. sp.) cubense (FOC) race 4. Five different classes of NBS-type sequences were identified and designated as resistance gene candidates (RGCs). The deduced amino acid sequences of the RGCs revealed the presence of motifs characteristic of the majority of known plant NBS-LRR resistance genes. Structural and phylogenetic analyses grouped the banana RGCs within the non-TIR (homology to Toll/interleukin-1 receptors) subclass of NBS sequences. Southern hybridization showed that each banana RGC is present in low copy number. The expression of the RGCs was assessed by RT-PCR in leaf and root tissues of plants resistant or susceptible to FOC race 4. RGC1, 3 and 5 showed a constitutive expression profile in both resistant and susceptible plants whereas no expression was detected for RGC4. Interestingly, RGC2 expression was found to be associated only to FOC race 4 resistant lines. This finding could assist in the identification of a FOC race 4 resistance gene.

Effect of Structural Modification on the Quantum-Size Effect in II-VI Semiconducting Nanocrystals

The electronic structure of group II-VI semiconductors in the stable wurtzite form is analyzed using state-of-the-art ab initio approaches to extract a simple and chemically transparent tight-binding model. This model can be used to understand the variation in the bandgap with size, for nanoclusters of these compounds. Results complement similar information already available for same systems in the zinc blende structure. A comparison with all available experimental data on quantum size effects in group II-VI semiconductor nanoclusters establishes a remarkable agreement between theory and experiment in both structure types, thereby verifying the predictive ability of our approach. The significant dependence of the quantum size effect on the structure type suggests that the experimental bandgap change at a given size compared to the bulk bandgap, may be used to indicate the structural form of the nanoclusters, particularly in the small size limit, where broadening of diffraction features often make it difficult to unambiguously determine the structure.

An eels study of water, methanol, formaldehyde and formic acid adsorbed on clean and oxygen-covered zinc(0001) surfaces

Water adsorbs molecularly on a clean Zn(0001) surface; on a surface covered with atomic oxygen, however, hydroxyl species is produced due to proton abstraction by the surface oxygen atoms. Methanol, molecularly adsorbed on a clean surface at 80 K, transforms to methoxy species above 110 K. On an atomic oxygen-covered surface, adsorbed methanol gives rise to methoxy species and water, the latter arising from proton abstraction. HCHO adsorbs molecularly at 80 K on both clean as well as oxygen-covered surfaces and polymerizes at higher temperatures. Formic acid does not adsorb on a clean Zn surface, but on an oxygen-covered surface gives rise to formate and hydroxyl species.

HNbWO6 and HTaWO6: Novel oxides related to ReO3 formed by ion exchange of rutile-type LiNbWO6 and LiTaWO6

Both LiNbWO6 and LiTaWO6 undergo ion exchange in hot aqueous H2SO4 yielding the hydrates HMWO6 · H2O (M = Nb or Ta). The reaction is accompanied by a structural transformation from the rutile to the ReO3 structure. The cell constants are a = 3.783(3)Å for HNbWO6 · H2O and a = 3.785(5)Å for HTaWO6 · H2O. The ReO3 structure is retained by the dehydration products HMWO6 and MWO5.5 as well. HMWO6 phases yield H1+xMWO6 hydrogen bronzes on exposure to hydrogen in the presence of platinum catalyst.

Identification of two distinct bovine parainfluenza virus type 3 genotypes

The partial gene sequencing of the matrix (M) protein from seven clinical isolates of bovine parainfluenza virus type 3 (BPIV-3), and the complete sequencing of a representative isolate (Q5592) was completed in this study. Nucleotide sequence analysis was initiated because of the failure of in-house BPIV-3 RT-PCR methods to yield expected products for four of the isolates. Phylogenetic reconstructions based on the nucleotide sequences for the M-protein and the entire genome, using all of the available BPIV-3 nucleotide sequences, demonstrated that there were two distinct BPIV-3 genotypes (BPIV-3a and BPIV-3b). These newly identified genotypes have implications for the development of BPIV-3 molecular detection methods and may also impact on BPIV-3 vaccine formulations.

Bovine mucopolysaccharidosis type IIIB

Mucopolysaccharidosis IIIB, an autosomal recessive lysosomal storage disorder of heparan sulfate caused by mutations in the α-N-acetylglucosaminidase (NAGLU) gene, was recently discovered in cattle. Clinical signs include progressive ataxia, stumbling gait, swaying and difficulty in balance and walking. These clinical signs are usually first observed at approximately 2 years of age and then develop progressively over the lifespan of the animals. Affected bulls were found to be homozygous for the missense mutation E452K (c.1354G>A). The availability of mutational analysis permits screening for the NAGLU mutation to eradicate this mutation from the cattle breeding population.

Models for the field-based toxicity of copper and zinc salts to wheat in 11 Australian soils and comparison to laboratory-based models

Laboratory-based relationships that model the phytotoxicity of metals using soil properties have been developed. This paper presents the first field-based phytotoxicity relationships. Wheat(Triticum aestivum L) was grown at 11 Australian field sites at which soil was spiked with copper (Cu) and zinc (Zn) salts. Toxicity was measured as inhibition of plant growth at 8 weeks and grain yield at harvest. The added Cu and Zn EC10 values for both endpoints ranged from approximately 3 to 4760 mg/kg. There were no relationships between field-based 8-week biomass and grain yield toxicity values for either metal. Cu toxicity was best modelled using pH and organic carbon content while Zn toxicity was best modelled using pH and the cation exchange capacity. The best relationships estimated toxicity within a factor of two of measured values. Laboratory-based phytotoxicity relationships could not accurately predict field-based phytotoxicity responses.