O-glycosylation in Toxoplasma gondii: identification and analysis of a family of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases

The initiation of mucin-type O-glycosylation is catalysed by a family of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (EC 2.4.1.41). These enzymes are responsible for the transfer of N-acetylgalactosamine from the nucleotide sugar donor, UDP-GalNAc, to the hydroxyl group on specific serine or threonine residues in acceptor proteins. By screening a Toxoplasma gondii cDNA library, three distinct isoforms of the ppGalNAc-T gene family were cloned. Two additional isoforms were identified and partially cloned following analysis of the T. gondii genome sequence database. All of the cloned and identified ppGalNAc-T's are type II membrane proteins that share up to 50% amino acid sequence identity within the conserved catalytic domain. They each contain an N-terminal cytoplasmic domain, a hydrophobic transmembrane domain, and a lumenal domain; the latter consists of stem, catalytic, and lectin-like domains. Moreover, each of this ppGalNAc-T's contains important sequence motifs that are typical for this class of glycosyltransferases. These include a glycosyltransferase 1 motif containing the DXH sequence, a Gal/GalNAc-T motif, and the CLD and QXW sequence motifs located in alpha-, beta-, and gamma-repeats present within the lectin-like domain. The coding regions of T. gondii ppGalNAc-T1, -T2, and -T3 reside in multiple exons ranging in number from 6 to 10. Our results demonstrate that mucin-type O-glycosylation in T. gondii is catalysed by a multimember gene family, which is evolutionarily conserved from single-celled eukaryotes through nematodes and insects up to mammals. Taken together, this information creates the basis for future studies of the function of the ppGalNAc-T gene family in the pathobiology of this apicomplexan parasite.     

Molecular phylogenetic analysis of the avian malarial parasite Plasmodium (Novyella) juxtanucleare

Plasmodium (Novyella) juxtanucleare is a widely distributed parasite that primarily infects chickens (Gallus gallus domesticus). All species of Novyella are characterized by very small schizonts, which in the case of P. juxtanucleare are always found juxtaposed to the erythrocyte nucleus, hence its name. Nearly complete small-subunit ribosomal RNA sequences have been obtained from 2 isolates of this species, and comparisons with other Plasmodium species have been made. Phylogenetic analysis reveals that this parasite is closely related to other avian-infecting Plasmodium species and that molecular relationships among the avian-infecting plasmodia do not correspond to their morphology-based subgeneric classifications.     

Many gene and domain families have convergent fates following independent whole-genome duplication events in Arabidopsis, Oryza, Saccharomyces and Tetraodon

Genome duplication is potentially a good source of new genes, but such genes take time to evolve. We have found a group of "duplication-resistant" genes, which have undergone convergent restoration to singleton status following several independent genome duplications. Restoration of duplication-resistant genes to singleton status could be important to long-term survival of a polyploid lineage. Angiosperms show more frequent polyploidization and a higher degree of duplicate gene preservation than other paleopolyploids, making them well-suited to further study of duplication-resistant genes.     

Fragment-based discovery of hepatitis C virus NS5b RNA polymerase inhibitors

Non-nucleoside inhibitors of HCV NS5b RNA polymerase were discovered by a fragment-based lead discovery approach, beginning with crystallographic fragment screening. The NS5b binding affinity and biochemical activity of fragment hits and inhibitors was determined by surface plasmon resonance (Biacore) and an enzyme inhibition assay, respectively. Crystallographic fragment screening hits with 1–10 mM binding affinity (K_D) were iteratively optimized to give leads with 200 nM biochemical activity and low μM cellular activity in a Replicon assay.     

Pyrrolo[1,2-b]pyridazin-2-ones as potent inhibitors of HCV NS5B polymerase

Pyrrolo[1,2-b]pyridazin-2-one analogs were discovered as a novel class of inhibitors of genotype 1 HCV NS5B polymerase. Structure-based design led to the discovery of compound 3 k, which displayed potent inhibitory activities in biochemical and replicon assays (IC(50) (1b)<10nM; EC(50) (1b)=12 nM) as well as good stability towards human liver microsomes (HLM t(1/2)>60 min).     

piggyBacis an effective tool for functional analysis of thePlasmodium falciparumgenome

Background  

Much of thePlasmodium falciparumgenome encodes hypothetical proteins with limited homology to other organisms. A lack of robust tools for genetic manipulation of the parasite limits functional analysis of these hypothetical proteins and other aspects of thePlasmodiumgenome. Transposon mutagenesis has been used widely to identify gene functions in many organisms and would be extremely valuable for functional analysis of thePlasmodiumgenome.     

Real-Time Loop-Mediated Isothermal Amplification (RealAmp) for the Species-Specific Identification of Plasmodium vivax

Plasmodium vivax infections remain a major source of malaria-related morbidity and mortality. Early and accurate diagnosis is an integral component of effective malaria control programs. Conventional molecular diagnostic methods provide accurate results but are often resource-intensive, expensive, have a long turnaround time and are beyond the capacity of most malaria-endemic countries. Our laboratory has recently developed a new platform called RealAmp, which combines loop-mediated isothermal amplification (LAMP) with a portable tube scanner real-time isothermal instrument for the rapid detection of malaria parasites. Here we describe new primers for the detection of P. vivax using the RealAmp method. Three pairs of amplification primers required for this method were derived from a conserved DNA sequence unique to the P. vivax genome. The amplification was carried out at 64°C using SYBR Green or SYTO-9 intercalating dyes for 90 minutes with the tube scanner set to collect fluorescence signals at 1-minute intervals. Clinical samples of P. vivax and other human-infecting malaria parasite species were used to determine the sensitivity and specificity of the primers by comparing with an 18S ribosomal RNA-based nested PCR as the gold standard. The new set of primers consistently detected laboratory-maintained isolates of P. vivax from different parts of the world. The primers detected P. vivax in the clinical samples with 94.59% sensitivity (95% CI: 87.48–98.26%) and 100% specificity (95% CI: 90.40–100%) compared to the gold standard nested-PCR method. The new primers also proved to be more sensitive than the published species-specific primers specifically developed for the LAMP method in detecting P. vivax.     

PlasmoDB: the Plasmodium genome resource. A database integrating experimental and computational data

PlasmoDB (http://PlasmoDB.org) is the official database of the Plasmodium falciparum genome sequencing consortium. This resource incorporates the recently completed P. falciparum genome sequence and annotation, as well as draft sequence and annotation emerging from other Plasmodium sequencing projects. PlasmoDB currently houses information from five parasite species and provides tools for intra- and inter-species comparisons. Sequence information is integrated with other genomic-scale data emerging from the Plasmodium research community, including gene expression analysis from EST, SAGE and microarray projects and proteomics studies. The relational schema used to build PlasmoDB, GUS (Genomics Unified Schema) employs a highly structured format to accommodate the diverse data types generated by sequence and expression projects. A variety of tools allow researchers to formulate complex, biologically-based, queries of the database. A stand-alone version of the database is also available on CD-ROM (P. falciparum GenePlot), facilitating access to the data in situations where internet access is difficult (e.g. by malaria researchers working in the field). The goal of PlasmoDB is to facilitate utilization of the vast quantities of genomic-scale data produced by the global malaria research community. The software used to develop PlasmoDB has been used to create a second Apicomplexan parasite genome database, ToxoDB (http://ToxoDB.org).     

Towards the development of a sustainable soya bean‐based feedstock for aquaculture

Soya bean (Glycine max (L.) Merr.) is sought after for both its oil and protein components. Genetic approaches to add value to either component are ongoing efforts in soya bean breeding and molecular biology programmes. The former is the primary vegetable oil consumed in the world. Hence, its primary usage is in direct human consumption. As a means to increase its utility in feed applications, thereby expanding the market of soya bean coproducts, we investigated the simultaneous displacement of marine ingredients in aquafeeds with soya bean‐based protein and a high Omega‐3 fatty acid soya bean oil, enriched with alpha‐linolenic and stearidonic acids, in both steelhead trout (Oncorhynchus mykiss) and Kampachi (Seriola rivoliana). Communicated herein are aquafeed formulations with major reduction in marine ingredients that translates to more total Omega‐3 fatty acids in harvested flesh. Building off of these findings, subsequent efforts were directed towards a genetic strategy that would translate to a prototype design of an optimal identity‐preserved soya bean‐based feedstock for aquaculture, whereby a multigene stack approach for the targeted synthesis of two value‐added output traits, eicosapentaenoic acid and the ketocarotenoid, astaxanthin, were introduced into the crop. To this end, the systematic introduction of seven transgenic cassettes into soya bean, and the molecular and phenotypic evaluation of the derived novel events are described.