Discovery of biological networks from diverse functional genomic data

We have developed a general probabilistic system for query-based discovery of pathway-specific networks through integration of diverse genome-wide data. This framework was validated by accurately recovering known networks for 31 biological processes in Saccharomyces cerevisiae and experimentally verifying predictions for the process of chromosomal segregation. Our system, bioPIXIE, a public, comprehensive system for integration, analysis, and visualization of biological network predictions for S. cerevisiae, is freely accessible over the worldwide web.     

Drug resistance testing in PBMCs of HIV infected patients.

HIV infected patients are considered a sort of reservoir having different genetically distinct viral variants (quasispecies), that evolve from the starting virus inoculum. Frequently, during replication, HIV can generate nucleotide differences in the new viral population; such genetic changes may be uninfluential in viral "fitness" (replication capacity) or give the virus some advantages under a selective pressure, due to immune response or drug treatment. The use of potent combination therapy for the treatment of HIV infections has certainly improved the "quality of life" for patients, decreasing the viral load in the plasma (HIV RNA). In our study, we investigated whether detection of drug resistance-related mutations was possible in circulating PBMCs, which represent a sort of genetic archive of viral drug resistances, when the levels of viral RNA were reduced to below 400 or 50 copies/ml, since, generally, plasma samples with more than 1,000 copies/ml of HIV RNA are needed to generate some results. The study was successfully performed sequencing proviral HIV DNA in PBMCs from 32 samples belonging to 25 patients, using a new modified protocol, that showed a good reproduciblity and very interesting data, also in patients with low or without circulating HIV RNA levels.     

Presence of Yersinia specific antibodies in hospitalized patients with arthritis and Reiter syndrome

A number of 50 patients admitted with arthritis and Reiter syndrome were investigated to detect the presence of Yersinia specific antibodies. The specific antibodies were evidenced in 5 patients (10%)--3 with antibodies to Y. enterocolitica and 2 with antibodies to Y. pseudotuberculosis, in titers greater than or equal to 1/160. The patients were followed in dynamics up to 3-4 months. The antibodies to Y. enterocolitica persisted in time longer than those to Y. pseudotuberculosis. For a better knowledge concerning the germs responsible for sero-negative arthritis, serological investigations for Yersinia, in the patients with arthritis, appeared as necessary.     

Blister‐inducing antibodies target multiple epitopes on collagen VII in mice

Epidermolysis bullosa acquisita (EBA) is an autoimmune subepidermal blistering disease of mucous membranes and the skin caused by autoantibodies against collagen VII. In silico and wet laboratory epitope mapping studies revealed numerous distinct epitopes recognized by EBA patients' autoantibodies within the non‐collagenous (NC)1 and NC2 domains of collagen VII. However, the distribution of pathogenic epitopes on collagen VII has not yet been described. In this study, we therefore performed an in vivo functional epitope mapping of pathogenic autoantibodies in experimental EBA. Animals (n = 10/group) immunized against fragments of the NC1 and NC2 domains of collagen VII or injected with antibodies generated against the same fragments developed to different extent experimental EBA. Our results demonstrate that antibodies targeting multiple, distinct epitopes distributed over the entire NC1, but not NC2 domain of collagen VII induce blistering skin disease in vivo. Our present findings have crucial implications for the development of antigen‐specific B‐ and T cell‐targeted therapies in EBA.     

High Resolution Helium Ion Scanning Microscopy of the Rat Kidney

Helium ion scanning microscopy is a novel imaging technology with the potential to provide sub-nanometer resolution images of uncoated biological tissues. So far, however, it has been used mainly in materials science applications. Here, we took advantage of helium ion microscopy to explore the epithelium of the rat kidney with unsurpassed image quality and detail. In addition, we evaluated different tissue preparation methods for their ability to preserve tissue architecture. We found that high contrast, high resolution imaging of the renal tubule surface is possible with a relatively simple processing procedure that consists of transcardial perfusion with aldehyde fixatives, vibratome tissue sectioning, tissue dehydration with graded methanol solutions and careful critical point drying. Coupled with the helium ion system, fine details such as membrane texture and membranous nanoprojections on the glomerular podocytes were visualized, and pores within the filtration slit diaphragm could be seen in much greater detail than in previous scanning EM studies. In the collecting duct, the extensive and striking apical microplicae of the intercalated cells were imaged without the shrunken or distorted appearance that is typical with conventional sample processing and scanning electron microscopy. Membrane depressions visible on principal cells suggest possible endo- or exocytotic events, and central cilia on these cells were imaged with remarkable preservation and clarity. We also demonstrate the use of colloidal gold probes for highlighting specific cell-surface proteins and find that 15 nm gold labels are practical and easily distinguishable, indicating that external labels of various sizes can be used to detect multiple targets in the same tissue. We conclude that this technology represents a technical breakthrough in imaging the topographical ultrastructure of animal tissues. Its use in future studies should allow the study of fine cellular details and provide significant advances in our understanding of cell surface structures and membrane organization.     

A Multifactor Analysis of Fungal and Bacterial Community Structure in the Root Microbiome of Mature Populus deltoides Trees

Bacterial and fungal communities associated with plant roots are central to the host health, survival and growth. However, a robust understanding of the root-microbiome and the factors that drive host associated microbial community structure have remained elusive, especially in mature perennial plants from natural settings. Here, we investigated relationships of bacterial and fungal communities in the rhizosphere and root endosphere of the riparian tree species Populus deltoides, and the influence of soil parameters, environmental properties (host phenotype and aboveground environmental settings), host plant genotype (Simple Sequence Repeat (SSR) markers), season (Spring vs. Fall) and geographic setting (at scales from regional watersheds to local riparian zones) on microbial community structure. Each of the trees sampled displayed unique aspects to its associated community structure with high numbers of Operational Taxonomic Units (OTUs) specific to an individual trees (bacteria >90%, fungi >60%). Over the diverse conditions surveyed only a small number of OTUs were common to all samples within rhizosphere (35 bacterial and 4 fungal) and endosphere (1 bacterial and 1 fungal) microbiomes. As expected, Proteobacteria and Ascomycota were dominant in root communities (>50%) while other higher-level phylogenetic groups (Chytridiomycota, Acidobacteria) displayed greatly reduced abundance in endosphere compared to the rhizosphere. Variance partitioning partially explained differences in microbiome composition between all sampled roots on the basis of seasonal and soil properties (4% to 23%). While most variation remains unattributed, we observed significant differences in the microbiota between watersheds (Tennessee vs. North Carolina) and seasons (Spring vs. Fall). SSR markers clearly delineated two host populations associated with the samples taken in TN vs. NC, but overall host genotypic distances did not have a significant effect on corresponding communities that could be separated from other measured effects.