Split-seed: a new tool for maize researchers

Until recently, immature embryos have been a choice tissue for manipulation in culture for regeneration and production of transgenic maize plants. The utility of this explant has been compromised by low output, genotype dependence and time-consuming incubation in tissue culture. We have developed a new explant, the split-seed, which addresses these limitations by formally treating each seed as though it were a “dicot”. By splitting maize seed longitudinally, three different tissues: the scutellum, the coleoptilar-ring and the shoot apical meristems are simultaneously exposed. The cells of these tissues can be made competent to enhance the regeneration, given that the molecular networks resulting from exposure of the split-seed to hormones is likely to be different from whole seed and, in turn, affects the in vitro response. Using this explant, callus induction frequency exceeded 92% and the regeneration frequency was 76%. The mean number of shoots regenerated via callus was 11 shoots per callus clump and 28 shoots per explant at first sub-culture. All of the regenerated plants survived and were 95% fertile. The large numbers of fertile plants produced were regenerated in 6–8 weeks. Finally, the incidence of regenerated plants varies as a function of growth regulator profile.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Jane: a new tool for the cophylogeny reconstruction problem

Background  

This paper describes the theory and implementation of a new software tool, called Jane, for the study of historical associations. This problem arises in parasitology (associations of hosts and parasites), molecular systematics (associations of orderings and genes), and biogeography (associations of regions and orderings). The underlying problem is that of reconciling pairs of trees subject to biologically plausible events and costs associated with these events. Existing software tools for this problem have strengths and limitations, and the new Jane tool described here provides functionality that complements existing tools.     

Panalysis: a new spreadsheet-based tool for pandemic planning

Publicly available influenza modeling tools are of limited use to hospitals and local communities in planning for a severe pandemic. We developed Panalysis, a new tool to estimate the likely healthcare consequences of a pandemic and to aid hospitals in the development of mitigation and response strategies. By way of example, we demonstrate how Panalysis can be used to plan for a 1918-like flu pandemic. We discuss potential future applications of this tool.     

Recombineering: a powerful new tool for mouse functional genomics

Highly efficient phage-based Escherichia coli homologous recombination systems have recently been developed that enable genomic DNA in bacterial artificial chromosomes to be modified and subcloned, without the need for restriction enzymes or DNA ligases. This new form of chromosome engineering, termed recombinogenic engineering or recombineering, is efficient and greatly decreases the time it takes to create transgenic mouse models by traditional means. Recombineering also facilitates many kinds of genomic experiment that have otherwise been difficult to carry out, and should enhance functional genomic studies by providing better mouse models and a more refined genetic analysis of the mouse genome.     

Benchmarking of single-virus genomics: a new tool for uncovering the virosphere

Metagenomics and single-cell genomics have enabled the discovery of relevant uncultured microbes. Recently, single-virus genomics (SVG), although still in an incipient stage, has opened new avenues in viral ecology by allowing the sequencing of one single virus at a time. The investigation of methodological alternatives and optimization of existing procedures for SVG is paramount to deliver high-quality genomic data. We report a sequencing dataset of viral single-amplified genomes (vSAGs) from cultured and uncultured viruses obtained by applying different conditions in each SVG step, from viral preservation and novel whole-genome amplification (WGA) to sequencing platforms and genome assembly. Sequencing data showed that cryopreservation and mild fixation were compatible with WGA, although fresh samples delivered better genome quality data. The novel TruPrime WGA, based on primase-polymerase features, and WGA-X employing a thermostable phi29 polymerase, were proven to be with sufficient sensitivity in SVG. The Oxford Nanopore (ON) sequencing platform did not provide a significant improvement of vSAG assembly compared to Illumina alone. Finally, the SPAdes assembler performed the best. Overall, our results represent a valuable genomic dataset that will help to standardized and advance new tools in viral ecology.     

Quantum dots: a new tool for anti-malarial drug assays

Background

Malaria infects over 300 million people every year and one of the major obstacles for the eradication of the disease is parasite's resistance to current chemotherapy, thus new drugs are urgently needed. Quantum dot (QD) is a fluorescent nanocrystal that has been in the spotlight as a robust tool for visualization of live cell processes in real time. Here, a simple and efficient method using QD to directly label Plasmodium falciparum-infected erythrocytes (iRBCs) was searched in order to use the QD as a probe in an anti-malarial drug-screening assay.

Methods

A range of QDs with different chemical coatings were tested for their ability to specifically bind iRBCs by immunofluorescence assay (IFA). One QD was selected and used to detect parasite growth and drug sensitivity by flow cytometry.

Results

PEGylated-cationic QD (PCQD) was found to specifically label infected erythrocytes preferentially with late stage parasites. The detection of QD-labelled infected erythrocytes by flow cytometry was sensitive enough to monitor chloroquine anti-malarial toxicity with a drug incubation period as short as 24 h (EC₅₀ = 113nM). A comparison of our assay with another widely used anti-malarial drug screening assay, the pLDH assay, showed that PCQD-based assay had 50% improved sensitivity in detecting drug efficacy within a parasite life cycle. An excellent Z-factor of 0.8 shows that the QD assay is suitable for high-throughput screening.

Conclusions

This new assay can offer a rapid and robust platform to screen novel classes of anti-malarial drugs.

     

Mini-TnhlyAs: a new tool for the construction of secreted fusion proteins

A simple and efficient procedure for the construction of secreted fusion proteins inEscherichia coli is described that uses a new minitransposon, termed TnhlyAs, carrying the secretion signal (HlyAs) ofE. coli hemolysin (HlyA). This transposon permits the generation of random gene fusions encoding proteins that carry the HlyAs at their C-termini. For the construction of model gene fusions we usedlacZ, encoding the cytoplasmicβ-galactosidase (β-Gal), andphoA, encoding the periplasmic alkaline phosphatase, as target genes. Our data suggest that allβ-Gal-HlyAs fusion proteins generated are secreted, albeit with varying efficiencies, by the HlyB/HlyD/TolC hemolysin secretion machinery under Sec-proficient conditions. In contrast, the PhoA-HlyAs fusion proteins are efficiently secreted in asecA mutant strain only under SecA-deficient conditions.     

Molecular topology: a useful tool for the search of new antibacterials

Molecular topology has been applied to find new lead antibacterial compounds. Among the selected compounds, hesperidin, neohesperidin and Mordant Brown 24 stand out, with minimum inhibitory concentrations 90, MIC90 < 0.3 mg/mL.     

ElliPro: a new structure-based tool for the prediction of antibody epitopes

Background  

Reliable prediction of antibody, or B-cell, epitopes remains challenging yet highly desirable for the design of vaccines and immunodiagnostics. A correlation between antigenicity, solvent accessibility, and flexibility in proteins was demonstrated. Subsequently, Thornton and colleagues proposed a method for identifying continuous epitopes in the protein regions protruding from the protein's globular surface. The aim of this work was to implement that method as a web-tool and evaluate its performance on discontinuous epitopes known from the structures of antibody-protein complexes.     

Single virus genomics: a new tool for virus discovery

Whole genome amplification and sequencing of single microbial cells has significantly influenced genomics and microbial ecology by facilitating direct recovery of reference genome data. However, viral genomics continues to suffer due to difficulties related to the isolation and characterization of uncultivated viruses. We report here on a new approach called 'Single Virus Genomics', which enabled the isolation and complete genome sequencing of the first single virus particle. A mixed assemblage comprised of two known viruses; E. coli bacteriophages lambda and T4, were sorted using flow cytometric methods and subsequently immobilized in an agarose matrix. Genome amplification was then achieved in situ via multiple displacement amplification (MDA). The complete lambda phage genome was recovered with an average depth of coverage of approximately 437X. The isolation and genome sequencing of uncultivated viruses using Single Virus Genomics approaches will enable researchers to address questions about viral diversity, evolution, adaptation and ecology that were previously unattainable.     

Single-cell MALDI: a new tool for direct peptide profiling

Matrix-assisted laser desorption-ionization (MALDI) mass spectrometry (MS) is a rapid and sensitive analytical approach that is well suited for obtaining molecular weights of peptides and proteins from complex samples. MALDI-MS can profile the peptides and proteins from single-cell and small tissue samples without the need for extensive sample preparation, except for the cell isolation and matrix application. Strategies for peptide identification and characterization of post-translational modifications are presented. Furthermore, several recent enhancements in MALDI-MS technology, including in situ peptide sequencing as well as the direct spatial mapping of peptides in cells and tissues are discussed.     

Immuno-PCR--a new tool for paleomicrobiology: the plague paradigm

Background

The cause of past plague pandemics was controversial but several research teams used PCR techniques and dental pulp as the primary material to reveal that they were caused by Yersinia pestis. However, the degradation of DNA limits the ability to detect ancient infections.

Methods

We used for the first time immuno-PCR to detect Yersinia pestis antigens; it can detect protein concentrations 70 times lower than the standard ELISA. After determining the cut-off value, we tested 34 teeth that were obtained from mass graves of plague, and compared previous PCR results with ELISA and immuno-PCR results.

Results

The immuno-PCR technique was the most sensitive (14 out of 34) followed by the PCR technique (10 out of 34) and ELISA (3 out of 34). The combination of these three methods identified 18 out of 34 (53%) teeth as presumably being from people with the plague.

Conclusion

Immuno-PCR is specific (no false-positive samples were found) and more sensitive than the currently used method to detect antigens of ancient infections in dental pulp. The combination of three methods, ELISA, PCR and immuno-PCR, increased the capacity to identify ancient pathogens in dental pulp.     

Centromere plasmid: a new genetic tool for the study of Plasmodium falciparum

The introduction of transgenes into Plasmodium falciparum, a highly virulent human malaria parasite, has been conducted either by single crossover recombination or by using episomal plasmids. However, these techniques remain insufficient because of the low transfection efficiency and the low frequency of recombination. To improve the genetic manipulation of P. falciparum, we developed the centromere plasmid as a new genetic tool. First, we attempted to clone all of the predicted centromeres from P. falciparum into E. coli cells but failed because of the high A/T contents of these sequences. To overcome this difficulty, we identified the common sequence features of the centromere of Plasmodium spp. and designed a small centromere that retained those features. The centromere plasmid constructed with the small centromere sequence, pFCEN, segregated into daughter parasites with approximately 99% efficiency, resulting in the stable maintenance of this plasmid in P. falciparum even in the absence of drug selection. This result demonstrated that the small centromere sequence harboured in pFCEN could function as an actual centromere in P. falciparum. In addition, transgenic parasites were more rapidly generated when using pFCEN than when using the control plasmid, which did not contain the centromere sequence. Furthermore, in contrast to the control plasmid, pFCEN did not form concatemers and, thus, was maintained as a single copy over multiple cell divisions. These unique properties of the pFCEN plasmid will solve the current technical limitations of the genetic manipulation of P. falciparum, and thus, this plasmid will become a standard genetic tool for the study of this parasite.     

Expressed protein ligation: a new tool for the biosynthesis of cyclic polypeptides

The present paper reviews the use of expressed protein ligation for the biosynthesis of backbone cyclized polypeptides. This general method allows the in vivo and in vitro biosynthesis of cyclic polypeptides using recombinant DNA expression techniques. Biosynthetic access to backbone cyclic peptides opens the possibility to generate cell-based combinatorial libraries that can be screened inside living cells for their ability to attenuate or inhibit cellular processes.