Novel AroA from Pseudomonas putida Confers Tobacco Plant with High Tolerance to Glyphosate

Glyphosate is a non-selective broad-spectrum herbicide that inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS, also designated as AroA), a key enzyme in the aromatic amino acid biosynthesis pathway in microorganisms and plants. Previously, we reported that a novel AroA (PpAroA1) from Pseudomonas putida had high tolerance to glyphosate, with little homology to class I or class II glyphosate-tolerant AroA. In this study, the coding sequence of PpAroA1 was optimized for tobacco. For maturation of the enzyme in chloroplast, a chloroplast transit peptide coding sequence was fused in frame with the optimized aroA gene (PparoA1optimized) at the 5′ end. The PparoA1optimized gene was introduced into the tobacco (Nicotiana tabacum L. cv. W38) genome via Agrobacterium-mediated transformation. The transformed explants were first screened in shoot induction medium containing kanamycin. Then glyphosate tolerance was assayed in putative transgenic plants and its T1 progeny. Our results show that the PpAroA1 from Pseudomonas putida can efficiently confer tobacco plants with high glyphosate tolerance. Transgenic tobacco overexpressing the PparoA1optimized gene exhibit high tolerance to glyphosate, which suggest that the novel PpAroA1 is a new and good candidate applied in transgenic crops with glyphosate tolerance in future

Public Health and Air Pollution in Asia (PAPA): A Multicity Study of Short-Term Effects of Air Pollution on Mortality

Background and Objectives: Although the deleterious effects of air pollution from fossil fuel combustion have been demonstrated in many Western nations, fewer studies have been conducted in Asia. The Public Health and Air Pollution in Asia (PAPA) project assessed the effects of short-term exposure to air pollution on daily mortality in Bangkok, Thailand, and in three cities in China: Hong Kong, Shanghai, and Wuhan. Methods: Poisson regression models incorporating natural spline smoothing functions were used to adjust for seasonality and other time-varying covariates that might confound the association between air pollution and mortality. Effect estimates were determined for each city and then for the cities combined using a random effects method. Results: In individual cities, associations were detected between most of the pollutants [nitrogen dioxide, sulfur dioxide, particulate matter ≤ 10 μm in aerodynamic diameter (PM 10), and ozone] and most health outcomes under study (i.e., all natural-cause, cardiovascular, and respiratory mortality). The city-combined effects of the four pollutants tended to be equal or greater than those identified in studies conducted in Western industrial nations. In addition, residents of Asian cities are likely to have higher exposures to air pollution than those in Western industrial nations because they spend more time outdoors and less time in air conditioning. Conclusions: Although the social and environmental conditions may be quite different, it is reasonable apply estimates derived from previous health effect of air pollution studies in the West to Asia.published_or_final_versio

The context of HIV risk behaviours among HIV-positive injection drug users in Viet Nam: Moving toward effective harm reduction

<p>Abstract</p> <p>Background</p> <p>Injection drug users represent the largest proportion of all HIV reported cases in Viet Nam. This study aimed to explore the perceptions of risk and risk behaviours among HIV-positive injection drug users, and their experiences related to safe injection and safe sex practices.</p> <p>Methods</p> <p>This study used multiple qualitative methods in data collection including in-depth interviews, focus group discussions and participant observation with HIV-positive injection drug users.</p> <p>Results</p> <p>The informants described a change in the sharing practices among injection drug users towards more precautions and what was considered 'low risk sharing', like sharing among seroconcordant partners and borrowing rather than lending. However risky practices like re-use of injection equipment and 'syringe pulling' i.e. the use of left-over drugs in particular, were frequently described and observed. Needle and syringe distribution programmes were in place but carrying needles and syringes and particularly drugs could result in being arrested and fined. Fear of rejection and of loss of intimacy made disclosure difficult and was perceived as a major obstacle for condom use among recently diagnosed HIV infected individuals.</p> <p>Conclusion</p> <p>HIV-positive injection drug users continue to practice HIV risk behaviours. The anti-drug law and the police crack-down policy appeared as critical factors hampering ongoing prevention efforts with needle and syringe distribution programmes in Viet Nam. Drastic policy measures are needed to reduce the very high HIV prevalence among injection drug users.</p

Structural and Functional Insights into the Malaria Parasite Moving Junction Complex

Members of the phylum Apicomplexa, which include the malaria parasite Plasmodium, share many features in their invasion mechanism in spite of their diverse host cell specificities and life cycle characteristics. The formation of a moving junction (MJ) between the membranes of the invading apicomplexan parasite and the host cell is common to these intracellular pathogens. The MJ contains two key parasite components: the surface protein Apical Membrane Antigen 1 (AMA1) and its receptor, the Rhoptry Neck Protein (RON) complex, which is targeted to the host cell membrane during invasion. In particular, RON2, a transmembrane component of the RON complex, interacts directly with AMA1. Here, we report the crystal structure of AMA1 from Plasmodium falciparum in complex with a peptide derived from the extracellular region of PfRON2, highlighting clear specificities of the P. falciparum RON2-AMA1 interaction. The receptor-binding site of PfAMA1 comprises the hydrophobic groove and a region that becomes exposed by displacement of the flexible Domain II loop. Mutations of key contact residues of PfRON2 and PfAMA1 abrogate binding between the recombinant proteins. Although PfRON2 contacts some polymorphic residues, binding studies with PfAMA1 from different strains show that these have little effect on affinity. Moreover, we demonstrate that the PfRON2 peptide inhibits erythrocyte invasion by P. falciparum merozoites and that this strong inhibitory potency is not affected by AMA1 polymorphisms. In parallel, we have determined the crystal structure of PfAMA1 in complex with the invasion-inhibitory peptide R1 derived by phage display, revealing an unexpected structural mimicry of the PfRON2 peptide. These results identify the key residues governing the interactions between AMA1 and RON2 in P. falciparum and suggest novel approaches to antimalarial therapeutics

Characterization of Within-Host Plasmodium falciparum Diversity Using Next-Generation Sequence Data

Our understanding of the composition of multi-clonal malarial infections and the epidemiological factors which shape their diversity remain poorly understood. Traditionally within-host diversity has been defined in terms of the multiplicity of infection (MOI) derived by PCR-based genotyping. Massively parallel, single molecule sequencing technologies now enable individual read counts to be derived on genome-wide datasets facilitating the development of new statistical approaches to describe within-host diversity. In this class of measures the FWS metric characterizes within-host diversity and its relationship to population level diversity. Utilizing P. falciparum field isolates from patients in West Africa we here explore the relationship between the traditional MOI and FWS approaches. FWS statistics were derived from read count data at 86,158 SNPs in 64 samples sequenced on the Illumina GA platform. MOI estimates were derived by PCR at the msp-1 and -2 loci. Significant correlations were observed between the two measures, particularly with the msp-1 locus (P = 5.92×10−5). The FWS metric should be more robust than the PCR-based approach owing to reduced sensitivity to potential locus-specific artifacts. Furthermore the FWS metric captures information on a range of parameters which influence out-crossing risk including the number of clones (MOI), their relative proportions and genetic divergence. This approach should provide novel insights into the factors which correlate with, and shape within-host diversity

A Soluble Acetylcholinesterase Provides Chemical Defense against Xenobiotics in the Pinewood Nematode

The pinewood nematode genome encodes at least three distinct acetylcholinesterases (AChEs). To understand physiological roles of the three pinewood nematode AChEs (BxACE-1, BxACE-2, and BxACE-3), BxACE-3 in particular, their tissue distribution and inhibition profiles were investigated. Immunohistochemistry revealed that BxACE-1 and BxACE-2 were distributed in neuronal tissues. In contrast, BxACE-3 was detected from some specific tissues and extracted without the aid of detergent, suggesting its soluble nature unlike BxACE-1 and BxACE-2. When present together, BxAChE3 significantly reduced the inhibition of BxACE-1 and BxACE-2 by cholinesterase inhibitors. Knockdown of BxACE-3 by RNA interference significantly increased the toxicity of three nematicidal compounds, supporting the protective role of BxACE-3 against chemicals. In summary, BxACE-3 appears to have a non-neuronal function of chemical defense whereas both BxACE-1 and BxACE-2 have classical neuronal function of synaptic transmission

Bioactivity of miltefosine against aquatic stages of Schistosoma mansoni, Schistosoma haematobium and their snail hosts, supported by scanning electron microscopy

<p>Abstract</p> <p>Background</p> <p>Miltefosine, which is the first oral drug licensed for the treatment of leishmaniasis, was recently reported to be a promising lead compound for the synthesis of novel antischistosomal derivatives with potent activity <it>in vivo </it>against different developmental stages of <it>Schistosoma mansoni</it>. In this paper an <it>in vitro </it>study was carried out to investigate whether it has a biocidal activity against the aquatic stages of <it>Schistosoma mansoni </it>and its snail intermediate host, <it>Biomphalaria alexandrina </it>, thus being also a molluscicide. Additionally, to see whether miltefosine can have a broad spectrum antischistosomal activity, a similar <it>in vitro </it>study was carried out on the adult stage of <it>Schistosoma haematobium</it>, the second major human species, its larval stages and snail intermediate host, <it>Bulinus truncutes</it>. This was checked by scanning electron microscopy.</p> <p>Results</p> <p>Miltefosine proved to have <it>in vitro </it>ovicidal, schistolarvicidal and lethal activity on adult worms of both <it>Schistosoma </it>species and has considerable molluscicidal activity on their snail hosts. Scanning electron microscopy revealed several morphological changes on the different stages of the parasite and on the soft body of the snail, which further strengthens the current evidence of miltefosine's activity. This is the first report of mollusicidal activity of miltefosine and its <it>in vitro </it>schistosomicidal activity against <it>S.haematobium</it>.</p> <p>Conclusions</p> <p>This study highlights miltefosine not only as a potential promising lead compound for the synthesis of novel broad spectrum schistosomicidal derivatives, but also for molluscicidals.</p

Mechanisms for the Evolution of a Derived Function in the Ancestral Glucocorticoid Receptor

Understanding the genetic, structural, and biophysical mechanisms that caused protein functions to evolve is a central goal of molecular evolutionary studies. Ancestral sequence reconstruction (ASR) offers an experimental approach to these questions. Here we use ASR to shed light on the earliest functions and evolution of the glucocorticoid receptor (GR), a steroid-activated transcription factor that plays a key role in the regulation of vertebrate physiology. Prior work showed that GR and its paralog, the mineralocorticoid receptor (MR), duplicated from a common ancestor roughly 450 million years ago; the ancestral functions were largely conserved in the MR lineage, but the functions of GRs—reduced sensitivity to all hormones and increased selectivity for glucocorticoids—are derived. Although the mechanisms for the evolution of glucocorticoid specificity have been identified, how reduced sensitivity evolved has not yet been studied. Here we report on the reconstruction of the deepest ancestor in the GR lineage (AncGR1) and demonstrate that GR's reduced sensitivity evolved before the acquisition of restricted hormone specificity, shortly after the GR–MR split. Using site-directed mutagenesis, X-ray crystallography, and computational analyses of protein stability to recapitulate and determine the effects of historical mutations, we show that AncGR1's reduced ligand sensitivity evolved primarily due to three key substitutions. Two large-effect mutations weakened hydrogen bonds and van der Waals interactions within the ancestral protein, reducing its stability. The degenerative effect of these two mutations is extremely strong, but a third permissive substitution, which has no apparent effect on function in the ancestral background and is likely to have occurred first, buffered the effects of the destabilizing mutations. Taken together, our results highlight the potentially creative role of substitutions that partially degrade protein structure and function and reinforce the importance of permissive mutations in protein evolution

Massively Parallel Sequencing and Analysis of the Necator americanus Transcriptome

The blood-feeding hookworm Necator americanus infects hundreds of millions of people. To elucidate fundamental molecular biological aspects of this hookworm, the transcriptome of adult Necator americanus was studied using next-generation sequencing and in silico analyses. Contigs (n = 19,997) were assembled from the sequence data; 6,771 of them had known orthologues in the free-living nematode Caenorhabditis elegans, and most encoded proteins with WD40 repeats (10.6%), proteinase inhibitors (7.8%) or calcium-binding EF-hand proteins (6.7%). Bioinformatic analyses inferred that C. elegans homologues are involved mainly in biological pathways linked to ribosome biogenesis (70%), oxidative phosphorylation (63%) and/or proteases (60%). Comparative analyses of the transcriptomes of N. americanus and the canine hookworm, Ancylostoma caninum, revealed qualitative and quantitative differences. Essential molecules were predicted using a combination of orthology mapping and functional data available for C. elegans. Further analyses allowed the prioritization of 18 predicted drug targets which did not have human homologues. These candidate targets were inferred to be linked to mitochondrial metabolism or amino acid synthesis. This investigation provides detailed insights into the transcriptome of the adult stage of N. americanus