Proteomic identification of small, copper-responsive proteins in germinating embryos of Oryza sativa

Background and Aims

Although copper (Cu) is an essential micronutrient for plants and algae, excess Cu is toxic to most plants and can cause a wide range of deleterious effects. To investigate the response of rice (Oryza sativa) to Cu stress, a proteomic approach was used to analyse Cu stress-induced changes in the expression of low molecular-weight proteins in germinating rice seed embryos.

Methods

Rice seeds were germinated in the presence or absence of 200 µm Cu for 6 d, and embryos, including newly formed shoots and radicles, were isolated. After proteins were extracted from the germinating embryos and separated by two-dimensional PAGE, 16 proteins in the 6- to 25-kDa range were identified using MALDI-TOF mass spectrometry.

Key Results and Conclusions

Thirteen of the proteins identified, including metallothionein-like protein, membrane-associated protein-like protein, putative wall-associated protein kinase, pathogenesis-related proteins and the putative small GTP-binding protein Rab2, were up-regulated by Cu stress. Three proteins, a putative small cytochrome P450 (CYP90D2), a putative thioredoxin and a putative GTPase, were down-regulated by Cu stress. As far as is known, this study provides the first proteomic evidence that metallothionein and CYP90D2 are Cu-responsive proteins in plants. These findings may lead to a better understanding of plant molecular responses to toxic metal exposure.Key words: Copper, metallothionein, rice, Oryza sativa, proteomics, seed germination     

Safety and Immunogenicity of an AMA1 Malaria Vaccine in Malian Children: Results of a Phase 1 Randomized Controlled Trial

Background

The objective was to evaluate the safety and immunogenicity of the AMA1-based malaria vaccine FMP2.1/AS02_A in children exposed to seasonal falciparum malaria.

Methodology/Principal Findings

A Phase 1 double blind randomized controlled dose escalation trial was conducted in Bandiagara, Mali, West Africa, a rural town with intense seasonal transmission of Plasmodium falciparum malaria. The malaria vaccine FMP2.1/AS02_A is a recombinant protein (FMP2.1) based on apical membrane antigen 1 (AMA1) from the 3D7 clone of P. falciparum, formulated in the Adjuvant System AS02_A. The comparator vaccine was a cell-culture rabies virus vaccine (RabAvert®). One hundred healthy Malian children aged 1–6 years were recruited into 3 cohorts and randomized to receive either 10 µg FMP2.1 in 0.1 mL AS02_A, or 25 µg FMP2.1 in 0.25 mL AS02_A, or 50 µg FMP2.1 50 µg in 0.5 mL AS02_A, or rabies vaccine. Three doses of vaccine were given at 0, 1 and 2 months, and children were followed for 1 year. Solicited symptoms were assessed for 7 days and unsolicited symptoms for 30 days after each vaccination. Serious adverse events were assessed throughout the study. Transient local pain and swelling were common and more frequent in all malaria vaccine dosage groups than in the comparator group, but were acceptable to parents of participants. Levels of anti-AMA1 antibodies measured by ELISA increased significantly (at least 100-fold compared to baseline) in all 3 malaria vaccine groups, and remained high during the year of follow up.

Conclusion/Significance

The FMP2.1/AS02_A vaccine had a good safety profile, was well-tolerated, and induced high and sustained antibody levels in malaria-exposed children. This malaria vaccine is being evaluated in a Phase 2 efficacy trial in children at this site.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00358332","term_id":"NCT00358332"}}NCT00358332 [{"type":"clinical-trial","attrs":{"text":"NCT00358332","term_id":"NCT00358332"}}NCT00358332]     

Genetic diversity of selected genes that are potentially economically important in feral sheep of New Zealand

Background

Feral sheep are considered to be a source of genetic variation that has been lost from their domestic counterparts through selection.

Methods

This study investigates variation in the genes KRTAP1-1, KRT33, ADRB3 and DQA2 in Merino-like feral sheep populations from New Zealand and its offshore islands. These genes have previously been shown to influence wool, lamb survival and animal health.

Results

All the genes were polymorphic, but no new allele was identified in the feral populations. In some of these populations, allele frequencies differed from those observed in commercial Merino sheep and other breeds found in New Zealand. Heterozygosity levels were comparable to those observed in other studies on feral sheep. Our results suggest that some of the feral populations may have been either inbred or outbred over the duration of their apparent isolation.

Conclusion

The variation described here allows us to draw some conclusions about the likely genetic origin of the populations and selective pressures that may have acted upon them, but they do not appear to be a source of new genetic material, at least for these four genes.     

A Retrospective Study on Genetic Heterogeneity within Treponema Strains: Subpopulations Are Genetically Distinct in a Limited Number of Positions

Background

Pathogenic uncultivable treponemes comprise human and animal pathogens including agents of syphilis, yaws, bejel, pinta, and venereal spirochetosis in rabbits and hares. A set of 10 treponemal genome sequences including those of 4 Treponema pallidum ssp. pallidum (TPA) strains (Nichols, DAL-1, Mexico A, SS14), 4 T. p. ssp. pertenue (TPE) strains (CDC-2, Gauthier, Samoa D, Fribourg-Blanc), 1 T. p. ssp. endemicum (TEN) strain (Bosnia A) and one strain (Cuniculi A) of Treponema paraluisleporidarum ecovar Cuniculus (TPLC) were examined with respect to the presence of nucleotide intrastrain heterogeneous sites.

Methodology/Principal Findings

The number of identified intrastrain heterogeneous sites in individual genomes ranged between 0 and 7. Altogether, 23 intrastrain heterogeneous sites (in 17 genes) were found in 5 out of 10 investigated treponemal genomes including TPA strains Nichols (n = 5), DAL-1 (n = 4), and SS14 (n = 7), TPE strain Samoa D (n = 1), and TEN strain Bosnia A (n = 5). Although only one heterogeneous site was identified among 4 tested TPE strains, 16 such sites were identified among 4 TPA strains. Heterogeneous sites were mostly strain-specific and were identified in four tpr genes (tprC, GI, I, K), in genes involved in bacterial motility and chemotaxis (fliI, cheC-fliY), in genes involved in cell structure (murC), translation (prfA), general and DNA metabolism (putative SAM dependent methyltransferase, topA), and in seven hypothetical genes.

Conclusions/Significance

Heterogeneous sites likely represent both the selection of adaptive changes during infection of the host as well as an ongoing diversifying evolutionary process.     

Gene Set Enrichment Analysis (GSEA) of Toxoplasma gondii expression datasets links cell cycle progression and the bradyzoite developmental program

Background

Large amounts of microarray expression data have been generated for the Apicomplexan parasite Toxoplasma gondii in an effort to identify genes critical for virulence or developmental transitions. However, researchers’ ability to analyze this data is limited by the large number of unannotated genes, including many that appear to be conserved hypothetical proteins restricted to Apicomplexa. Further, differential expression of individual genes is not always informative and often relies on investigators to draw big-picture inferences without the benefit of context. We hypothesized that customization of gene set enrichment analysis (GSEA) to T. gondii would enable us to rigorously test whether groups of genes serving a common biological function are co-regulated during the developmental transition to the latent bradyzoite form.

Results

Using publicly available T. gondii expression microarray data, we created Toxoplasma gene sets related to bradyzoite differentiation, oocyst sporulation, and the cell cycle. We supplemented these with lists of genes derived from community annotation efforts that identified contents of the parasite-specific organelles, rhoptries, micronemes, dense granules, and the apicoplast. Finally, we created gene sets based on metabolic pathways annotated in the KEGG database and Gene Ontology terms associated with gene annotations available at http://www.toxodb.org. These gene sets were used to perform GSEA analysis using two sets of published T. gondii expression data that characterized T. gondii stress response and differentiation to the latent bradyzoite form.

Conclusions

GSEA provides evidence that cell cycle regulation and bradyzoite differentiation are coupled. Δgcn5A mutants unable to induce bradyzoite-associated genes in response to alkaline stress have different patterns of cell cycle and bradyzoite gene expression from stressed wild-type parasites. Extracellular tachyzoites resemble a transitional state that differs in gene expression from both replicating intracellular tachyzoites and in vitro bradyzoites by expressing genes that are enriched in bradyzoites as well as genes that are associated with the G1 phase of the cell cycle. The gene sets we have created are readily modified to reflect ongoing research and will aid researchers’ ability to use a knowledge-based approach to data analysis facilitating the development of new insights into the intricate biology of Toxoplasma gondii.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-515) contains supplementary material, which is available to authorized users.     

Safety and Immunogenicity of a Recombinant Plasmodium falciparum AMA1 Malaria Vaccine Adjuvanted with Alhydrogel?, Montanide ISA 720 or AS02

Background

Plasmodium falciparum Apical Membrane Antigen 1 (PfAMA1) is a candidate vaccine antigen expressed by merozoites and sporozoites. It plays a key role in red blood cell and hepatocyte invasion that can be blocked by antibodies.

Methodology/Principal Findings

We assessed the safety and immunogenicity of recombinant PfAMA1 in a dose-escalating, phase Ia trial. PfAMA1 FVO strain, produced in Pichia pastoris, was reconstituted at 10 µg and 50 µg doses with three different adjuvants, Alhydrogel™, Montanide ISA720 and AS02 Adjuvant System. Six randomised groups of healthy male volunteers, 8–10 volunteers each, were scheduled to receive three immunisations at 4-week intervals. Safety and immunogenicity data were collected over one year. Transient pain was the predominant injection site reaction (80–100%). Induration occurred in the Montanide 50 µg group, resulting in a sterile abscess in two volunteers. Systemic adverse events occurred mainly in the AS02 groups lasting for 1–2 days. Erythema was observed in 22% of Montanide and 59% of AS02 group volunteers. After the second dose, six volunteers in the AS02 group and one in the Montanide group who reported grade 3 erythema (>50 mm) were withdrawn as they met the stopping criteria. All adverse events resolved. There were no vaccine-related serious adverse events. Humoral responses were highest in the AS02 groups. Antibodies showed activity in an in vitro growth inhibition assay up to 80%. Upon stimulation with the vaccine, peripheral mononuclear cells from all groups proliferated and secreted IFNγ and IL-5 cytokines.

Conclusions/Significance

All formulations showed distinct reactogenicity profiles. All formulations with PfAMA1 were immunogenic and induced functional antibodies.

Trial Registration

Clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00730782","term_id":"NCT00730782"}}NCT00730782     

Agrobacterium-mediated transformation of tomato elicits unexpected flower phenotypes with similar gene expression profiles

Background

Genetic transformation mediated by Agrobacterium tumefaciens is known to cause unexpected phenotypes. Mutations of a specific set of homeotic genes can result in alterred floral structure.

Methodology/Principal Findings

Previously we identified two genes (LeTGA1 and SOLly GLB1) induced by nutrient availability in tomato. To further elucidate their function, we sought to knock out the genes using antisense RNAi. When antisense constructs for the two different tomato genes were each transformed into Micro-Tina tomato plants, one primary transformant with similar mutant flower phenotypes was identified from transformation of each construct. Microarray analysis shows that a similar set of genes were up- or downregulated in both mutants. Sequencing of insertion sites indicates that each is inserted into a repetitive region which could impact expression of affected genes but direct alteration of floral homeotic gene sequences was not detected.

Conclusion

This is the first report that dominant flower mutations could be caused by genetic transformation designed to knock out two nutrient stress related genes.     

Genetic susceptibility for chronic bronchitis in chronic obstructive pulmonary disease

Background

Chronic bronchitis (CB) is one of the classic phenotypes of COPD. The aims of our study were to investigate genetic variants associated with COPD subjects with CB relative to smokers with normal spirometry, and to assess for genetic differences between subjects with CB and without CB within the COPD population.

Methods

We analyzed data from current and former smokers from three cohorts: the COPDGene Study; GenKOLS (Bergen, Norway); and the Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints (ECLIPSE). CB was defined as having a cough productive of phlegm on most days for at least 3 consecutive months per year for at least 2 consecutive years. CB COPD cases were defined as having both CB and at least moderate COPD based on spirometry. Our primary analysis used smokers with normal spirometry as controls; secondary analysis was performed using COPD subjects without CB as controls. Genotyping was performed on Illumina platforms; results were summarized using fixed-effect meta-analysis.

Results

For CB COPD relative to smoking controls, we identified a new genome-wide significant locus on chromosome 11p15.5 (rs34391416, OR = 1.93, P = 4.99 × 10^-8) as well as significant associations of known COPD SNPs within FAM13A. In addition, a GWAS of CB relative to those without CB within COPD subjects showed suggestive evidence for association on 1q23.3 (rs114931935, OR = 1.88, P = 4.99 × 10^-7).

Conclusions

We found genome-wide significant associations with CB COPD on 4q22.1 (FAM13A) and 11p15.5 (EFCAB4A, CHID1 and AP2A2), and a locus associated with CB within COPD subjects on 1q23.3 (RPL31P11 and ATF6). This study provides further evidence that genetic variants may contribute to phenotypic heterogeneity of COPD.

Trial registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00608764","term_id":"NCT00608764"}}NCT00608764, {"type":"clinical-trial","attrs":{"text":"NCT00292552","term_id":"NCT00292552"}}NCT00292552

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0113-2) contains supplementary material, which is available to authorized users.     

An evidence-based approach to identify aging-related genes in Caenorhabditis elegans

Background

Extensive studies have been carried out on Caenorhabditis elegans as a model organism to elucidate mechanisms of aging and the effects of perturbing known aging-related genes on lifespan and behavior. This research has generated large amounts of experimental data that is increasingly difficult to integrate and analyze with existing databases and domain knowledge. To address this challenge, we demonstrate a scalable and effective approach for automatic evidence gathering and evaluation that leverages existing experimental data and literature-curated facts to identify genes involved in aging and lifespan regulation in C. elegans.

Results

We developed a semantic knowledge base for aging by integrating data about C. elegans genes from WormBase with data about 2005 human and model organism genes from GenAge and 149 genes from GenDR, and with the Bio2RDF network of linked data for the life sciences. Using HyQue (a Semantic Web tool for hypothesis-based querying and evaluation) to interrogate this knowledge base, we examined 48,231 C. elegans genes for their role in modulating lifespan and aging. HyQue identified 24 novel but well-supported candidate aging-related genes for further experimental validation.

Conclusions

We use semantic technologies to discover candidate aging genes whose effects on lifespan are not yet well understood. Our customized HyQue system, the aging research knowledge base it operates over, and HyQue evaluations of all C. elegans genes are freely available at http://hyque.semanticscience.org.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-015-0469-4) contains supplementary material, which is available to authorized users.     

Impact of RTS,S/AS02A and RTS,S/AS01B on Genotypes of P. falciparum in Adults Participating in a Malaria Vaccine Clinical Trial

Objective

RTS,S, a candidate vaccine for malaria, is a recombinant protein expressed in yeast containing part of the circumsporozoite protein (CSP) sequence of 3D7 strain of Plasmodium falciparum linked to the hepatitis B surface antigen in a hybrid protein. The RTS,S antigen is formulated with GSK Biologicals'' proprietary Adjuvant Systems AS02_A or AS01_B. A recent trial of the RTS,S/AS02_A and RTS,S/AS01_B vaccines evaluated safety, immunogenicity and impact on the development of parasitemia of the two formulations. Parasite isolates from this study were used to determine the molecular impact of RTS,S/AS02_A and RTS,S/AS01_B on the multiplicity of infection (MOI) and the csp allelic characteristics of subsequent parasitemias.

Design

The distribution of csp sequences and the MOI of the infecting strains were examined at baseline and in break-through infections from vaccinated individuals and from those receiving a non-malarial vaccine.

Setting

The study was conducted in Kombewa District, western Kenya.

Participants

Semi-immune adults from the three study arms provided isolates at baseline and during break-through infections.

Outcome

Parasite isolates used for determining MOI and divergence of csp T cell–epitopes were 191 at baseline and 87 from break-through infections.

Results

Grouping recipients of RTS,S/AS01_A and RTS,S/AS02_B together, vaccine recipients identified as parasite-positive by microscopy contained significantly fewer parasite genotypes than recipients of the rabies vaccine comparator (median in pooled RTS,S groups: 3 versus 4 in controls, P = 0.0313). When analyzed separately, parasitaemic individuals in the RTS,S/AS01_B group, but not the RTS,S/AS02_A group, were found to have significantly fewer genotypes than the comparator group. Two individual amino acids found in the vaccine construct (Q339 in Th2R and D371 in Th3R) were observed to differ in incidence between vaccine and comparator groups but in different directions; parasites harboring Q339 were less common among pooled RTS,S/AS vaccine recipients than among recipients of rabies vaccine, whereas parasites with D371 were more common among the RTS,S/AS groups.

Conclusions

It is concluded that both RTS,S/AS vaccines reduce multiplicity of infection. Our results do not support the hypothesis that RTS,S/AS vaccines elicit preferential effects against pfcsp alleles with sequence similarity to the 3D7 pfcsp sequence employed in the vaccine construct.     

HGTector: an automated method facilitating genome-wide discovery of putative horizontal gene transfers

Background

First pass methods based on BLAST match are commonly used as an initial step to separate the different phylogenetic histories of genes in microbial genomes, and target putative horizontal gene transfer (HGT) events. This will continue to be necessary given the rapid growth of genomic data and the technical difficulties in conducting large-scale explicit phylogenetic analyses. However, these methods often produce misleading results due to their inability to resolve indirect phylogenetic links and their vulnerability to stochastic events.

Results

A new computational method of rapid, exhaustive and genome-wide detection of HGT was developed, featuring the systematic analysis of BLAST hit distribution patterns in the context of a priori defined hierarchical evolutionary categories. Genes that fall beyond a series of statistically determined thresholds are identified as not adhering to the typical vertical history of the organisms in question, but instead having a putative horizontal origin. Tests on simulated genomic data suggest that this approach effectively targets atypically distributed genes that are highly likely to be HGT-derived, and exhibits robust performance compared to conventional BLAST-based approaches. This method was further tested on real genomic datasets, including Rickettsia genomes, and was compared to previous studies. Results show consistency with currently employed categories of HGT prediction methods. In-depth analysis of both simulated and real genomic data suggests that the method is notably insensitive to stochastic events such as gene loss, rate variation and database error, which are common challenges to the current methodology. An automated pipeline was created to implement this approach and was made publicly available at: https://github.com/DittmarLab/HGTector. The program is versatile, easily deployed, has a low requirement for computational resources.

Conclusions

HGTector is an effective tool for initial or standalone large-scale discovery of candidate HGT-derived genes.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-717) contains supplementary material, which is available to authorized users.