HT-SuperSAGE of the gut tissue of a Vip3Aa-resistant Heliothis virescens (Lepidoptera: Noctuidae) strain provides insights into the basis of resistance.

Multitoxin Bt-crops expressing insecticidal toxins with different modes of action, for example, Cry and Vip, are expected to improve resistance management in target pests. While Cry1A resistance has been relatively well characterized in some insect species, this is not the case for Vip3A, for which no mechanism of resistance has yet been identified. Here we applied HT-SuperSAGE to analyze the transcriptome of the gut tissue of tobacco budworm Heliothis virescens (F.) laboratory-selected for Vip3Aa resistance. From a total of 1 324 252 sequence reads, 5 895 126-bp tags were obtained representing 17 751 nonsingleton unique transcripts (UniTags) from genetically similar Vip3Aa-resistant (Vip-Sel) and susceptible control (Vip-Unsel) strains. Differential expression was significant (≥2.5 fold or ≤0.4; P < 0.05) for 1989 sequences (11.2% of total UniTags), where 420 represented overexpressed (OE) and 1569 underexpressed (UE) genes in Vip-Sel. BLASTN searches mapped 419 UniTags to H. virescens sequence contigs, of which, 416 (106 OE and 310 UE) were unambiguously annotated to proteins in NCBI nonredundant protein databases. Gene Ontology distributed 345 of annotated UniTags in 14 functional categories with metabolism (including serine-type hydrolases) and translation/ribosome biogenesis being the most prevalent. A UniTag homologous to a particular member of the REsponse to PAThogen (REPAT) family was found among most overexpressed, while UniTags related to the putative Vip3Aa-binding ribosomal protein S2 (RpS2) were underexpressed. qRT-PCR of a subset of UniTags validated the HT-SuperSAGE data. This study is the first providing lepidopteran gut transcriptome associated with Vip3Aa resistance and a foundation for future attempts to elucidate the resistance mechanism.

High-throughput SuperSAGE for gene expression analysis of Nicotiana tabacum-Rhizoctonia solani interaction.

OBJECTIVE: The ubiquitous soil pathogen Rhizoctonia solani causes serious diseases in different plant species. Despite the importance of this disease, little is known regarding the molecular basis of susceptibility. SuperSAGE technology and next-generation sequencing were used to generate transcript libraries during the compatible Nicotiana tabacum-R. solani interaction. Also, we used the post-transcriptional silencing to evaluate the function of a group of important genes. RESULTS: A total of 8960 and 8221 unique Tag sequences identified as differentially up- and down-regulated were obtained. Based on gene ontology classification, several annotated UniTags corresponded to defense response, metabolism and signal transduction. Analysis of the N. tabacum transcriptome during infection identified regulatory genes implicated in a number of hormone pathways. Silencing of an mRNA induced by salicylic acid reduced the susceptibility of N. tabacum to R. solani. We provide evidence that the salicylic acid pathway was involved in disease development. This is important for further development of disease management strategies caused by this pathogen.

'Candidatus Liberibacter asiaticus', Causal Agent of Citrus Huanglongbing, Is Reduced by Treatment with Brassinosteroids.

Huanglongbing (HLB) constitutes the most destructive disease of citrus worldwide, yet no established efficient management measures exist for it. Brassinosteroids, a family of plant steroidal compounds, are essential for plant growth, development and stress tolerance. As a possible control strategy for HLB, epibrassinolide was applied to as a foliar spray to citrus plants infected with the causal agent of HLB, 'Candidatus Liberibacter asiaticus'. The bacterial titers were reduced after treatment with epibrassinolide under both greenhouse and field conditions but were stronger in the greenhouse. Known defense genes were induced in leaves by epibrassinolide. With the SuperSAGE technology combined with next generation sequencing, induction of genes known to be associated with defense response to bacteria and hormone transduction pathways were identified. The results demonstrate that epibrassinolide may provide a useful tool for the management of HLB.

Field Trial and Molecular Characterization of RNAi-Transgenic Tomato Plants That Exhibit Resistance to Tomato Yellow Leaf Curl Geminivirus.

RNA interference (RNAi) is a widely used approach to generate virus-resistant transgenic crops. However, issues of agricultural importance like the long-term durability of RNAi-mediated resistance under field conditions and the potential side effects provoked in the plant by the stable RNAi expression remain poorly investigated. Here, we performed field trials and molecular characterization studies of two homozygous transgenic tomato lines, with different selection markers, expressing an intron-hairpin RNA cognate to the Tomato yellow leaf curl virus (TYLCV) C1 gene. The tested F6 and F4 progenies of the respective kanamycin- and basta-resistant plants exhibited unchanged field resistance to TYLCV and stably expressed the transgene-derived short interfering RNA (siRNAs) to represent 6 to 8% of the total plant small RNAs. This value outnumbered the average percentage of viral siRNAs in the nontransformed plants exposed to TYLCV-infested whiteflies. As a result of the RNAi transgene expression, a common set of up- and downregulated genes was revealed in the transcriptome profile of the plants selected from either of the two transgenic events. A previously unidentified geminivirus causing no symptoms of viral disease was detected in some of the transgenic plants. The novel virus acquired V1 and V2 genes from TYLCV and C1, C2, C3, and C4 genes from a distantly related geminivirus and, thereby, it could evade the repressive sequence-specific action of transgene-derived siRNAs. Our findings shed light on the mechanisms of siRNA-directed antiviral silencing in transgenic plants and highlight the applicability limitations of this technology as it may alter the transcriptional pattern of nontarget genes.

First-line eradication rates comparing two shortened non-bismuth quadruple regimens against Helicobacter pylori: an open-label, randomized, multicentre clinical trial.

OBJECTIVES: Helicobacter pylori eradication remains a challenge. Non-bismuth-based quadruple regimens (NBQR) have shown high eradication rates (ER) elsewhere that need to be locally confirmed. The objective of this study was to compare the first-line ER of a hybrid therapy (20 mg of omeprazole twice daily and 1 g of amoxicillin twice daily for 10 days, adding 500 mg of clarithromycin twice daily and 500 mg of metronidazole every 8 h for the last 5 days; OA-OACM) with that of a 10 day concomitant regimen consisting of taking all four drugs twice daily every day (including 500 mg of metronidazole every 12 h; OACM). A 10 day arm with standard triple therapy (OAC; 20 mg of omeprazole/12 h, 1 g of amoxicillin/12 h and 500 mg of clarithromycin/12 h) was included. PATIENTS AND METHODS: Three hundred consecutive patients were randomized (1: 2: 2) into one of the three following regimens: (i) OAC (60); (ii) OA-OACM (120); and (iii) OACM (120). Eradication was generally confirmed by a [(13)C]urea breath test at least 4 weeks after the end of treatment. Adverse events and compliance were assessed. EudraCT: 2011-006258-99. RESULTS: ITT cure rates were: OAC, 70.0% (42/60) (95% CI: 58.3-81.7); OA-OACM, 90.8% (109/120) (95% CI: 85.6-96.0); and OACM, 90.0% (107/119) (95% CI: 84.6-95.4). PP rates were: OAC, 72.4% (42/58) (95% CI: 60.8-84.1); OA-OACM, 93.9% (108/115) (95% CI: 89.5-98.3); and OACM, 90.3% (102/113) (95% CI: 84.8-95.8). Both NBQR significantly improved ER compared with OAC (P < 0.01), but no differences were seen between them. Mean compliance was elevated [98.0% (SD = 9.8)] with no differences between groups. There were more adverse events in the quadruple arms (OACM, 65.8%; OA-OACM, 68.6%; OAC, 46.6%; P < 0.05), but no significant differences between groups in terms of severity were seen. CONCLUSIONS: Hybrid and concomitant regimens show good ER against H. pylori infection with an acceptable safety profile. They clearly displace OAC as first-line regimen in our area.

A methodology based on molecular interactions and pathways to find candidate genes associated to diseases: its application to schizophrenia and Alzheimer's disease.

Experimental techniques for the identification of genes associated with diseases are expensive and have certain limitations. In this scenario, computational methods are useful tools to identify lists of promising genes for further experimental verification. This paper describes a flexible methodology for the in silico prediction of genes associated with diseases combining the use of available tools for gene enrichment analysis, gene network generation and gene prioritization. A set of reference genes, with a known association to a disease, is used as bait to extract candidate genes from molecular interaction networks and enriched pathways. In a second step, prioritization methods are applied to evaluate the similarities between previously selected candidates and the set of reference genes. The top genes obtained by these programs are grouped into a single list sorted by the number of methods that have selected each gene. As a proof of concept, top genes reported a few years ago in SzGene and AlzGene databases were used as references to predict genes associated to schizophrenia and Alzheimer's disease, respectively. In both cases, we were able to predict a statistically significant amount of genes belonging to the updated lists.

BisoGenet: a new tool for gene network building, visualization and analysis.

BACKGROUND: The increasing availability and diversity of omics data in the post-genomic era offers new perspectives in most areas of biomedical research. Graph-based biological networks models capture the topology of the functional relationships between molecular entities such as gene, protein and small compounds and provide a suitable framework for integrating and analyzing omics-data. The development of software tools capable of integrating data from different sources and to provide flexible methods to reconstruct, represent and analyze topological networks is an active field of research in bioinformatics. RESULTS: BisoGenet is a multi-tier application for visualization and analysis of biomolecular relationships. The system consists of three tiers. In the data tier, an in-house database stores genomics information, protein-protein interactions, protein-DNA interactions, gene ontology and metabolic pathways. In the middle tier, a global network is created at server startup, representing the whole data on bioentities and their relationships retrieved from the database. The client tier is a Cytoscape plugin, which manages user input, communication with the Web Service, visualization and analysis of the resulting network. CONCLUSION: BisoGenet is able to build and visualize biological networks in a fast and user-friendly manner. A feature of Bisogenet is the possibility to include coding relations to distinguish between genes and their products. This feature could be instrumental to achieve a finer grain representation of the bioentities and their relationships. The client application includes network analysis tools and interactive network expansion capabilities. In addition, an option is provided to allow other networks to be converted to BisoGenet. This feature facilitates the integration of our software with other tools available in the Cytoscape platform. BisoGenet is available at http://bio.cigb.edu.cu/bisogenet-cytoscape/.

Progressive combinatorial algorithm for multiple structural alignments: application to distantly related proteins.

MALECON is a progressive combinatorial procedure for multiple alignments of protein structures. It searches a library of pairwise alignments for all three-protein alignments in which a specified number of residues is consistently aligned. These alignments are progressively expanded to include additional proteins and more spatially equivalent residues, subject to certain criteria. This action involves superimposing the aligned proteins by their hitherto equivalent residues and searching for additional Calpha atoms that lie close in space. The performance of MALECON is illustrated and compared with several extant multiple structure alignment methods by using as test the globin homologous superfamily, the OB and the Jellyrolls folds. MALECON gives better definitions of the common structural features in the structurally more diverse proteins of the OB and Jellyrolls folds, but it yields comparable results for the more similar globins. When no consistent multiple alignments can be derived for all members of a protein group, our procedure is still capable of automatically generating consistent alignments and common core definitions for subgroups of the members. This finding is illustrated for proteins of the OB fold and SH3 domains, believed to share common structural features, and should be very instrumental in homology modeling and investigations of protein evolution.

Advanced pairwise structure alignments of proteins and analysis of conformational changes.

MOTIVATION: Comparing the 3D structures of two proteins or analyzing the structural changes undergone by a protein upon ligand binding or when it crystallizes under different conditions, can be both tricky and tedious, especially when the two proteins are distantly related, or when the structural changes are complex. Readily accessible tools for performing these tasks automatically and reliably should therefore be welcome. RESULTS: We describe a web interface to several automatic procedures for performing pairwise structure superposition in a flexible manner, for detailed analyses of conformational changes and for displaying the results in a pictorial fashion. AVAILABILITY: This interface can be accessed at the Brussels and Cuba Web sites, respectively: http://www.ucmb.ulb.ac.be/SCMBB/Tools.htmland http://bio.cigb.edu.cu.