RIP death domain structural interactions implicated in TNF-mediated proliferation and survival

Death domain (DD)-containing proteins are involved in both apoptosis and survival/proliferation signaling induced by activated death receptors. Here, a phylogenetic and structural analysis was performed to highlight differences in DD domains and their key regulatory interaction sites. The phylogenetic analysis shows that receptor DDs are more conserved than DDs in adaptors. Adaptor DDs can be subdivided into those that activate or inhibit apoptosis. Modeling of six homotypic DD interactions involved in the TNF signaling pathway implicates that the DD of RIP (Receptor interacting protein kinase 1) is capable of interacting with the DD of TRADD (TNFR1-associated death domain protein) in two different, exclusive ways: one that subsequently recruits CRADD (apoptosis/inflammation) and another that recruits NFkappaB (survival/proliferation).     

Tomato Bushy Stunt Virus Recombination Guided by Introduced MicroRNA Target Sequences

Previously we described Tomato bushy stunt virus (TBSV) vectors, which retained their capsid protein gene and were engineered with magnesium chelatase (ChlH) and phytoene desaturase (PDS) gene sequences from Nicotiana benthamiana. Upon plant infection, these vectors eventually lost the inserted sequences, presumably as a result of recombination. Here, we modified the same vectors to also contain the plant miR171 or miR159 target sequences immediately 3′ of the silencing inserts. We inoculated N. benthamiana plants and sequenced recombinant RNAs recovered from noninoculated upper leaves. We found that while some of the recombinant RNAs retained the microRNA (miRNA) target sites, most retained only the 3′ 10 and 13 nucleotides of the two original plant miRNA target sequences, indicating in planta miRNA-guided RNA-induced silencing complex cleavage of the recombinant TBSV RNAs. In addition, recovered RNAs also contained various fragments of the original sequence (ChlH and PDS) upstream of the miRNA cleavage site, suggesting that the 3′ portion of the miRNA-cleaved TBSV RNAs served as a template for negative-strand RNA synthesis by the TBSV RNA-dependent RNA polymerase (RdRp), followed by template switching by the RdRp and continued RNA synthesis resulting in loss of nonessential nucleotides.Several plant viruses have been developed as tools for various biotechnology applications, including expression platforms for protein production in plants (1, 2, 6) and as gene silencing systems as part of reverse genetics approaches toward understanding host plant gene function (4, 5). For both of these applications, nonviral sequences conferring the desired function are cloned into the virus genome in order to be expressed during replication in plants. One advantage of using viruses engineered with nonviral sequences is flexibility in manipulating these “extra” sequences, which are not essential for viral replication or movement (1). However, recombinant viruses also tend to lose these sequences, causing instability at the insertion site and resulting in loss of function of the recombinant viral vector. The relatively high error rates of viral replicases (7, 14, 24) and the propensity for recombination events (9) contribute to the instability often seen with some viral vector systems.Recombination events in RNA viruses typically result in joining of two noncontiguous RNA segments (16). These could be sequences from two separate RNA molecules or distant regions of the same molecule. Retention by viruses of favorable sequences is selection driven and eliminates sequences that are unnecessary or negatively affect fitness (11, 31), hence making recombination critical to virus evolution (13, 29). Although phylogenetic analyses predict that recombination events have affected evolution for essentially all groups of RNA viruses (3), some viruses appear to be more prone to recombination than others. For example, plant-infecting supergroup II viruses of the family Tombusviridae appear to undergo frequent recombination, as is supported by the many well-characterized defective-interfering (DI) RNAs of Tomato bushy stunt virus (TBSV) (10, 30). The TBSV DI RNAs, derived entirely from the parental viral RNA, are not replication competent alone and depend on the parent virus to replicate them in trans. Recent developments of in vitro systems (19, 21) have further enhanced dissection of recombination mechanisms giving rise to TBSV DI RNAs.Of the proposed mechanisms for viral recombination (12, 20), the copy choice or template-switching mechanism is the most widely reported (8, 16, 18). This occurs when the viral replicase and its attached nascent polynucleotide chain switches viral RNA templates (or jumps locations on the same template) when making cRNA. Some properties for preferred donor/acceptor sites (sequences in the RNA molecule at which viral replicase switches from one template to another) are known for various RNA viruses (3, 20, 27), suggesting that recombination is not entirely random.The previously described TBSV vectors were efficient silencers of host genes but only while the inserted sequences were retained (23). Thus, optimizing viral vectors requires a better understanding of factors responsible for recombination and consequent loss of insert sequences. In order to address possible recombination mechanisms, we used previously characterized sequence-specific microRNA (miRNA)-guided cleavage determinants as parts of our TBSV vectors. We introduced the miRNA target site sequences for miR171 or miR159 3′ to the silencing inserts of our TBSV vectors (23). After plant inoculation, we analyzed recombinant virus sequences, determining specific recombination patterns, and demonstrated miRNA-mediated recombination events in vivo for the recombinant TBSV vectors. We also showed miRNA-guided RNA-induced silencing complex (RISC)-mediated cleavage for recombinant TBSV RNA and evidence supporting the TBSV RNA-dependent RNA polymerase (RdRp) switching templates during cRNA synthesis.     

Detecting species-site dependencies in large multiple sequence alignments

Multiple sequence alignments (MSAs) are one of the most important sources of information in sequence analysis. Many methods have been proposed to detect, extract and visualize their most significant properties. To the same extent that site-specific methods like sequence logos successfully visualize site conservations and sequence-based methods like clustering approaches detect relationships between sequences, both types of methods fail at revealing informational elements of MSAs at the level of sequence–site interactions, i.e. finding clusters of sequences and sites responsible for their clustering, which together account for a high fraction of the overall information of the MSA. To fill this gap, we present here a method that combines the Fisher score-based embedding of sequences from a profile hidden Markov model (pHMM) with correspondence analysis. This method is capable of detecting and visualizing group-specific or conflicting signals in an MSA and allows for a detailed explorative investigation of alignments of any size tractable by pHMMs. Applications of our methods are exemplified on an alignment of the Neisseria surface antigen LP2086, where it is used to detect sites of recombinatory horizontal gene transfer and on the vitamin K epoxide reductase family to distinguish between evolutionary and functional signals.     

Predicting Neuropathy and Reactions in Leprosy at Diagnosis and Before Incident Events—Results from the INFIR Cohort Study

Background

Leprosy is a disease of skin and peripheral nerves. The process of nerve injury occurs gradually through the course of the disease as well as acutely in association with reactions. The INFIR (ILEP Nerve Function Impairment and Reactions) Cohort was established to identify clinically relevant neurological and immunological predictors for nerve injury and reactions.

Methodology/Principal Findings

The study, in two centres in India, recruited 188 new, previously untreated patients with multi-bacillary leprosy who had no recent nerve damage. These patients underwent a series of novel blood tests and nerve function testing including motor and sensory nerve conduction, warm and cold detection thresholds, vibrometry, dynamometry, monofilament sensory testing and voluntary muscle testing at diagnosis and at monthly follow up for the first year and every second month for the second year. During the 2 year follow up a total of 74 incident events were detected. Sub-clinical changes to nerve function at diagnosis and during follow-up predicted these new nerve events. Serological assays at baseline and immediately before an event were not predictive; however, change in TNF alpha before an event was a statistically significant predictor of that event.

Conclusions/Significance

These findings increase our understanding of the processes of nerve damage in leprosy showing that nerve function impairment is more widespread than previously appreciated. Any nerve involvement, including sub-clinical changes, is predictive of further nerve function impairment. These new factors could be used to identify patients at high risk of developing impairment and disability.     

Genomic acquisition of a capsular polysaccharide virulence cluster by non-pathogenic Burkholderia isolates

Background

Burkholderia thailandensis is a non-pathogenic environmental saprophyte closely related to Burkholderia pseudomallei, the causative agent of the often fatal animal and human disease melioidosis. To study B. thailandensis genomic variation, we profiled 50 isolates using a pan-genome microarray comprising genomic elements from 28 Burkholderia strains and species.

Results

Of 39 genomic regions variably present across the B. thailandensis strains, 13 regions corresponded to known genomic islands, while 26 regions were novel. Variant B. thailandensis isolates exhibited isolated acquisition of a capsular polysaccharide biosynthesis gene cluster (B. pseudomallei-like capsular polysaccharide) closely resembling a similar cluster in B. pseudomallei that is essential for virulence in mammals; presence of this cluster was confirmed by whole genome sequencing of a representative variant strain (B. thailandensis E555). Both whole-genome microarray and multi-locus sequence typing analysis revealed that the variant strains formed part of a phylogenetic subgroup distinct from the ancestral B. thailandensis population and were associated with atypical isolation sources when compared to the majority of previously described B. thailandensis strains. In functional assays, B. thailandensis E555 exhibited several B. pseudomallei-like phenotypes, including colony wrinkling, resistance to human complement binding, and intracellular macrophage survival. However, in murine infection assays, B. thailandensis E555 did not exhibit enhanced virulence relative to other B. thailandensis strains, suggesting that additional factors are required to successfully colonize and infect mammals.

Conclusions

The discovery of such novel variant strains demonstrates how unbiased genomic surveys of non-pathogenic isolates can reveal insights into the development and emergence of new pathogenic species.     

Protecs,a comprehensive and powerful storage and analysis system for OMICS data,applied for profiling the anaerobiosis response of Staphylococcus aureus COL

Broad functional genomic studies call for comprehensive and powerful data repositories for storage of genome sequences, experimental information, protein identification data, protein properties and expression values. The better such data repositories can integrate and display complex data in a clear and structured way the more biologically meaningful conclusions or novel hypotheses can be derived from extensive omics data sets. This work presents the web accessible database system Protecs and how it was used to support analysis of 50 samples drawn from four Staphylococcus aureus cultivations under anaerobiosis. Protecs incorporates findings from visualization science, e.g. micro charts and heat maps in the user interface. Its integrated tools for expression data analysis in combination with TIGR Multi Experiment Viewer were used to highlight similar gene expression profiles in single or multiple experiments based on the continuously updated S. aureus master gel. Raw data analysis results are available online at www.protecs.uni‐greifswald.de . Our meta‐study revealed that S. aureus responds in different anaerobiotic experimental setups (growth without oxygen; growth without oxygen but with supplemental pyruvate and uracil; growth without oxygen but with NO; growth without oxygen but with NO and without functional nreABC genes) with a general anaerobiosis response. Among others, this response is characterized by an induction of fermentation enzymes (PflB, Ldh1, SACOL0135, SACOL0660) as well as the response regulator SrrA. Interestingly, especially genes with a high codon adaptation index highly overlap with anaerobically induced genes.     

Proteomic Analysis of Tardigrades: Towards a Better Understanding of Molecular Mechanisms by Anhydrobiotic Organisms

Background

Tardigrades are small, multicellular invertebrates which are able to survive times of unfavourable environmental conditions using their well-known capability to undergo cryptobiosis at any stage of their life cycle. Milnesium tardigradum has become a powerful model system for the analysis of cryptobiosis. While some genetic information is already available for Milnesium tardigradum the proteome is still to be discovered.

Principal Findings

Here we present to the best of our knowledge the first comprehensive study of Milnesium tardigradum on the protein level. To establish a proteome reference map we developed optimized protocols for protein extraction from tardigrades in the active state and for separation of proteins by high resolution two-dimensional gel electrophoresis. Since only limited sequence information of M. tardigradum on the genome and gene expression level is available to date in public databases we initiated in parallel a tardigrade EST sequencing project to allow for protein identification by electrospray ionization tandem mass spectrometry. 271 out of 606 analyzed protein spots could be identified by searching against the publicly available NCBInr database as well as our newly established tardigrade protein database corresponding to 144 unique proteins. Another 150 spots could be identified in the tardigrade clustered EST database corresponding to 36 unique contigs and ESTs. Proteins with annotated function were further categorized in more detail by their molecular function, biological process and cellular component. For the proteins of unknown function more information could be obtained by performing a protein domain annotation analysis. Our results include proteins like protein member of different heat shock protein families and LEA group 3, which might play important roles in surviving extreme conditions.

Conclusions

The proteome reference map of Milnesium tardigradum provides the basis for further studies in order to identify and characterize the biochemical mechanisms of tolerance to extreme desiccation. The optimized proteomics workflow will enable application of sensitive quantification techniques to detect differences in protein expression, which are characteristic of the active and anhydrobiotic states of tardigrades.     

inGeno – an integrated genome and ortholog viewer for improved genome to genome comparisons

Background  

Systematic genome comparisons are an important tool to reveal gene functions, pathogenic features, metabolic pathways and genome evolution in the era of post-genomics. Furthermore, such comparisons provide important clues for vaccines and drug development. Existing genome comparison software often lacks accurate information on orthologs, the function of similar genes identified and genome-wide reports and lists on specific functions. All these features and further analyses are provided here in the context of a modular software tool "inGeno" written in Java with Biojava subroutines.     

An integrated view of gene expression and solute profiles of Arabidopsis tumors: a genome-wide approach

Transformation of plant cells with T-DNA of virulent agrobacteria is one of the most extreme triggers of developmental changes in higher plants. For rapid growth and development of resulting tumors, specific changes in the gene expression profile and metabolic adaptations are required. Increased transport and metabolic fluxes are critical preconditions for growth and tumor development. A functional genomics approach, using the Affymetrix whole genome microarray (approximately 22,800 genes), was applied to measure changes in gene expression. The solute pattern of Arabidopsis thaliana tumors and uninfected plant tissues was compared with the respective gene expression profile. Increased levels of anions, sugars, and amino acids were correlated with changes in the gene expression of specific enzymes and solute transporters. The expression profile of genes pivotal for energy metabolism, such as those involved in photosynthesis, mitochondrial electron transport, and fermentation, suggested that tumors produce C and N compounds heterotrophically and gain energy mainly anaerobically. Thus, understanding of gene-to-metabolite networks in plant tumors promotes the identification of mechanisms that control tumor development.