In situ gastrointestinal protection against anthrax edema toxin by single-chain antibody fragment producing lactobacilli

<p/> <p>Background</p> <p>Anthrax is caused by the bacterium <it>Bacillus anthracis </it>and is regarded as one of the most prominent bioterrorism threats. Anthrax toxicity is induced by the tripartite toxin complex, composed of the receptor-binding anthrax protective antigen and the two enzymatic subunits, lethal factor and edema factor. Recombinant lactobacilli have previously been used to deliver antibody fragments directed against surface epitopes of a variety of pathogens, including <it>Streptococcus mutans, Porphyromonas gingivalis</it>, and rotavirus. Here, we addressed whether or not anthrax toxins could be targeted and neutralised in the gastrointestinal tract by lactobacilli producing recombinant antibody fragments as a model system for toxin neutralisation in the gastrointestinal lumen.</p> <p>Results</p> <p>The neutralising anti-PA scFv, 1H, was expressed in <it>L. paracasei </it>as a secreted protein, a cell wall-anchored protein or both secreted and wall-anchored protein. Cell wall display on lactobacilli and PA binding of the anchored constructs was confirmed by flow cytometry analysis. Binding of secreted or attached scFv produced by lactobacilli to PA were verified by ELISA. Both construct were able to protect macrophages in an <it>in vitro </it>cytotoxicity assay. Finally, lactobacilli producing the cell wall attached scFv were able to neutralise the activity of anthrax edema toxin in the GI tract of mice, <it>in vivo</it>.</p> <p>Conclusion</p> <p>We have developed lactobacilli expressing a neutralising scFv fragment against the PA antigen of the anthrax toxin, which can provide protection against anthrax toxins both <it>in vitro </it>and <it>in vivo</it>. Utilising engineered lactobacilli therapeutically for neutralising toxins in the gastrointestinal tract can potential be expanded to provide protection against a range of additional gastrointestinal pathogens. The ability of lactobacilli to colonise the gastrointestinal tract may allow the system to be used both prophylactically and therapeutically.</p

A Lotus japonicus cytoplasmic kinase connects Nod factor perception by the NFR5 LysM receptor to nodulation

The establishment of nitrogen-fixing root nodules in legume-rhizobia symbiosis requires an intricate communication between the host plant and its symbiont. We are, however, limited in our understanding of the symbiosis signaling process. In particular, how membrane-localized receptors of legumes activate signal transduction following perception of rhizobial signaling molecules has mostly remained elusive. To address this, we performed a coimmunoprecipitation-based proteomics screen to identify proteins associated with Nod factor receptor 5 (NFR5) in Lotus japonicus. Out of 51 NFR5-associated proteins, we focused on a receptor-like cytoplasmic kinase (RLCK), which we named NFR5-interacting cytoplasmic kinase 4 (NiCK4). NiCK4 associates with heterologously expressed NFR5 in Nicotiana benthamiana, and directly binds and phosphorylates the cytoplasmic domains of NFR5 and NFR1 in vitro. At the cellular level, Nick4 is coexpressed with Nfr5 in root hairs and nodule cells, and the NiCK4 protein relocates to the nucleus in an NFR5/NFR1-dependent manner upon Nod factor treatment. Phenotyping of retrotransposon insertion mutants revealed that NiCK4 promotes nodule organogenesis. Together, these results suggest that the identified RLCK, NiCK4, acts as a component of the Nod factor signaling pathway downstream of NFR5

Nano Random Forests to mine protein complexes and their relationships in quantitative proteomics data

Ever-increasing numbers of quantitative proteomics data sets constitute an underexploited resource for investigating protein function. Multiprotein complexes often follow consistent trends in these experiments, which could provide insights about their biology. Yet, as more experiments are considered, a complex’s signature may become conditional and less identifiable. Previously we successfully distinguished the general proteomic signature of genuine chromosomal proteins from hitchhikers using the Random Forests (RF) machine learning algorithm. Here we test whether small protein complexes can define distinguishable signatures of their own, despite the assumption that machine learning needs large training sets. We show, with simulated and real proteomics data, that RF can detect small protein complexes and relationships between them. We identify several complexes in quantitative proteomics results of wild-type and knockout mitotic chromosomes. Other proteins covary strongly with these complexes, suggesting novel functional links for later study. Integrating the RF analysis for several complexes reveals known interdependences among kinetochore subunits and a novel dependence between the inner kinetochore and condensin. Ribosomal proteins, although identified, remained independent of kinetochore subcomplexes. Together these results show that this complex-oriented RF (NanoRF) approach can integrate proteomics data to uncover subtle protein relationships. Our NanoRF pipeline is available online

Validation of the Spanish Version of the ICECAP-O for Nursing Home Residents with Dementia

Background Measurement of health-related quality of life (HRQoL) is important for a chronic disease, such as dementia, which impairs the quality of life of affected patients in addition to their length of life. This is important in the context of economic evaluations when interventions do not (only) affect HRQoL and these other factors also affect overall quality of life. Objective To validate the Spanish translation of the ICECAP-O's capability to measure Health-related quality of life in elderly with dementia who live in nursing homes. Method Cross-sectional study. For 217 residents living in 8 Spanish nursing homes, questionnaires were completed by nursing professionals serving as proxy respondents. We analyzed the internal consistency and other psychometric properties. We investigated the convergent validity of the ICECAP-O with other HRQoL instruments, the EQ-5D extended with a cognitive dimension (EQ-5D+C), the Alzheimer's Disease Related Quality of Life (ADRQL) measures, and the Barthel Index measure of activities of daily living (ADL). Results The ICECAP-O presents satisfactory internal consistency (alpha 0.820). The factorial analysis indicated a structure of five principal dimensions that explain 66.57% of the total variance. Convergent validity between the ICECAP-O, EQ-5D+C, ADRQL, and Barthel Index scores was moderate to good (with correlations of 0.62, 0.61, and 0.68, respectively), but differed between dimensions of the instruments. Discriminant validity was confirmed by finding differences in ICECAP-O scores between subgroups based on ADL scores (0.70 low, 0.59 medium, and 0.39 high level care), dementia severity (0.72 mild, 0.63 medium, and 0.50 severe), and ages (0.59 below 75 years and 0.84 above 75 years). Conclusions This study presented the first use of a Spanish version of the ICECAP-O. The results indicate that the ICECAP-O appears to be a reliable Health-related quality of life measurement instrument showing good convergent and discriminant validity for people with dementia

Insights into hominid evolution from the gorilla genome sequence.

Gorillas are humans' closest living relatives after chimpanzees, and are of comparable importance for the study of human origins and evolution. Here we present the assembly and analysis of a genome sequence for the western lowland gorilla, and compare the whole genomes of all extant great ape genera. We propose a synthesis of genetic and fossil evidence consistent with placing the human-chimpanzee and human-chimpanzee-gorilla speciation events at approximately 6 and 10 million years ago. In 30% of the genome, gorilla is closer to human or chimpanzee than the latter are to each other; this is rarer around coding genes, indicating pervasive selection throughout great ape evolution, and has functional consequences in gene expression. A comparison of protein coding genes reveals approximately 500 genes showing accelerated evolution on each of the gorilla, human and chimpanzee lineages, and evidence for parallel acceleration, particularly of genes involved in hearing. We also compare the western and eastern gorilla species, estimating an average sequence divergence time 1.75 million years ago, but with evidence for more recent genetic exchange and a population bottleneck in the eastern species. The use of the genome sequence in these and future analyses will promote a deeper understanding of great ape biology and evolution

52 Genetic Loci Influencing Myocardial Mass.

BACKGROUND: Myocardial mass is a key determinant of cardiac muscle function and hypertrophy. Myocardial depolarization leading to cardiac muscle contraction is reflected by the amplitude and duration of the QRS complex on the electrocardiogram (ECG). Abnormal QRS amplitude or duration reflect changes in myocardial mass and conduction, and are associated with increased risk of heart failure and death. OBJECTIVES: This meta-analysis sought to gain insights into the genetic determinants of myocardial mass. METHODS: We carried out a genome-wide association meta-analysis of 4 QRS traits in up to 73,518 individuals of European ancestry, followed by extensive biological and functional assessment. RESULTS: We identified 52 genomic loci, of which 32 are novel, that are reliably associated with 1 or more QRS phenotypes at p < 1 × 10(-8). These loci are enriched in regions of open chromatin, histone modifications, and transcription factor binding, suggesting that they represent regions of the genome that are actively transcribed in the human heart. Pathway analyses provided evidence that these loci play a role in cardiac hypertrophy. We further highlighted 67 candidate genes at the identified loci that are preferentially expressed in cardiac tissue and associated with cardiac abnormalities in Drosophila melanogaster and Mus musculus. We validated the regulatory function of a novel variant in the SCN5A/SCN10A locus in vitro and in vivo. CONCLUSIONS: Taken together, our findings provide new insights into genes and biological pathways controlling myocardial mass and may help identify novel therapeutic targets