SIRAC: Supervised Identification of Regions of Aberration in aCGH datasets

Background  

Array comparative genome hybridization (aCGH) provides information about genomic aberrations. Alterations in the DNA copy number may cause the cell to malfunction, leading to cancer. Therefore, the identification of DNA amplifications or deletions across tumors may reveal key genes involved in cancer and improve our understanding of the underlying biological processes associated with the disease.     

Feasibility of an in situ measurement device for bubble size and distribution

The feasibility of in situ measurement device for bubble size and distribution was explored. A novel in situ probe measurement system, the EnviroCam™, was developed. Where possible, this probe incorporated strengths, and minimized weaknesses of historical and currently available real-time measurement methods for bubbles. The system was based on a digital, high-speed, high resolution, modular camera system, attached to a stainless steel shroud, compatible with standard Ingold ports on fermenters. Still frames and/or video were produced, capturing bubbles passing through the notch of the shroud. An LED light source was integral with the shroud. Bubbles were analyzed using customized commercially available image analysis software and standard statistical methods. Using this system, bubble sizes were measured as a function of various operating parameters (e.g., agitation rate, aeration rate) and as a function of media properties (e.g., viscosity, antifoam, cottonseed flour, and microbial/animal cell broths) to demonstrate system performance and its limitations. For selected conditions, mean bubble size changes qualitatively compared favorably with published relationships. Current instrument measurement capabilities were limited primarily to clear solutions that did not contain large numbers of overlapping bubbles.     

Survival of Indicator and Pathogenic Bacteria in Bovine Feces on Pasture

The survival of enteric bacteria was measured in bovine feces on pasture. In each season, 11 cow pats were prepared from a mixture of fresh dairy cattle feces and sampled for up to 150 days. Four pats were analyzed for Escherichia coli, fecal streptococci, and enterococci, and four inoculated pats were analyzed for Campylobacter jejuni and Salmonella enterica. Two pats were placed on drainage collectors, and another pat was fitted with a temperature probe. In the first 1 to 3 weeks, there were increases (up to 1.5 orders of magnitude) in the counts of enterococci (in four seasons), E. coli (three seasons), fecal streptococci (three seasons), and S. enterica (two seasons), but there was no increase in the counts of C. jejuni. Thereafter, the counts decreased, giving an average ranking of the times necessary for 90% inactivation of C. jejuni (6.2 days from deposition) < fecal streptococci (35 days) < S. enterica (38 days) < E. coli (48 days) < enterococci (56 days). The pat temperature probably influenced bacterial growth, but the pattern of increases and decreases was primarily determined by desiccation; growth occurred when the water content was greater than 80%, but at a water content of 70 to 75% counts decreased. E. coli and enterococcus regrowth appeared to result from pat rehydration. Of 20 monthly leaching losses of E. coli, 16 were <10% of the total counts in the pat, and 12 were <1%. Drainage losses of C. jejuni (generally <1%) were detected for only 1 to 2 months. Although enterococci exhibited the best survival rate, higher final counts suggested that E. coli is the more practical indicator of bovine fecal pollution.     

Two routes for production and purification of Fab fragments in biopharmaceutical discovery research: Papain digestion of mAb and transient expression in mammalian cells

Fab (fragment that having the antigen binding site) of a monoclonal antibody (mAb) is widely required in biopharmaceutical research and development. At Centocor, two routes of Fab production and purification were used to enable a variety of research and development efforts, particularly, crystallographic studies of antibody–antigen interactions. One route utilizes papain digestion of an intact monoclonal antibody for Fab fragment production. After digestion, separation of the Fab fragment from the Fc (fragment that crystallizes) and residual intact antibody was achieved using protein A affinity chromatography. In another route, His-tagged Fab fragments were obtained by transient expression of an appropriate construct in mammalian cells, and typical yields are 1–20 mg of Fab fragment per liter of cell culture. The His-tagged Fab fragments were first captured using immobilized metal affinity chromatography (IMAC). To provide high quality protein sample for crystallization, Fabs from either proteolytic digestion or from direct expression were further purified using size-exclusion chromatography (SEC) and/or ion-exchange chromatography (IEC). The purified Fab fragments were characterized by mass spectrometry, SDS–PAGE, dynamic light scattering, and circular dichroism. Crystallization experiments demonstrated that the Fab fragments are of high quality to produce diffraction quality crystals suitable for X-ray crystallographic analysis.     

Linking fish population dynamics to habitat conditions: insights from the application of a process-oriented approach to several Great Lakes species

One of the major challenges facing fishery scientists and managers today is determining how fish populations are influenced by habitat conditions. Many approaches have been explored to address this challenge, all of which involve modeling at one level or another. In this paper, we explore a process-oriented model approach whereby the critical population processes of birth and death rates are explicitly linked to habitat conditions. Application of this approach to five species of Great Lakes fishes including: walleye (Sander vitreus), lake trout (Salvelinus namaycush), smallmouth bass (Micropterus dolomieu), yellow perch (Perca flavescens), and rainbow trout (Onchorynchus mykiss), yielded a number of insights into the modeling process. One of the foremost insights is that processes determining movement and transport of fish are critical components of such models since these processes largely determine the habitats fish occupy. Because of the importance of fish location, an individual-based model appears to be a nearly inescapable modeling requirement. There is, however, a paucity of field-based data directly relating birth, death, and movement rates to habitat conditions experienced by individual fish. There is also a paucity of habitat information at a fine temporal and spatial scale for many important habitat variables. Finally, the general occurrence of strong ontogenetic changes in the response of different life stages to habitat conditions emphasizes the need for a modeling approach that considers all life stages in an integrated fashion.     

A comprehensive sensitivity analysis of microarray breast cancer classification under feature variability

Background  

Large discrepancies in signature composition and outcome concordance have been observed between different microarray breast cancer expression profiling studies. This is often ascribed to differences in array platform as well as biological variability. We conjecture that other reasons for the observed discrepancies are the measurement error associated with each feature and the choice of preprocessing method. Microarray data are known to be subject to technical variation and the confidence intervals around individual point estimates of expression levels can be wide. Furthermore, the estimated expression values also vary depending on the selected preprocessing scheme. In microarray breast cancer classification studies, however, these two forms of feature variability are almost always ignored and hence their exact role is unclear.     

HIV-1 RNA Dysregulates the Natural TLR Response to Subclinical Endotoxemia in Kenyan Female Sex-Workers

Background

Subclinical endotoxemia has been reported in HIV-1 infected persons and may drive systemic immune activation and pathogenesis. Proinflammatory responsiveness to endotoxin (LPS) is mediated by Toll-like receptor 4 (TLR4). We therefore examined the association between plasma LPS levels, HIV RNA, and TLR4 expression and cytokine responses in the blood of HIV infected and uninfected participants in a cohort of female sex-workers in Kenya.

Methodology/Principal Findings

Ex vivo plasma and peripheral blood mononuclear cells (PBMC) were assessed for LPS and TLR mRNA, respectively. The effects of HIV single stranded RNA, a TLR8 ligand, on TLR4 and LPS signaling were further assessed in short term PBMC culture. Both HIV uninfected and infected subjects frequently had low detectable LPS levels in their plasmas. Significantly increased LPS levels were associated with chronic HIV-1 infection, both treated and untreated, but not with other acute or semi-chronic conditions reported. In HIV-uninfected subjects, TLR4 mRNA expression levels correlated inversely with plasma LPS levels, suggesting chronic endotoxin ‘tolerance’ in vivo. A similar effect of reduced TLR4 mRNA was seen in short term PBMC culture after stimulation with LPS. Interestingly, the apparent in vivo tolerance effect was diminished in subjects with HIV infection. Additionally, pre-stimulation of PBMC with LPS lead to proinflammatory (TNF-α) tolerance to subsequent LPS stimulation; however, pre-treatment of PBMC with HIV single-stranded RNA40, could enhance TLR4-mediated LPS responsiveness in vitro.

Conclusions/Significance

Thus, dysregulation of endotoxin tolerance by HIV-1 RNA may exacerbate HIV chronic immune activation and pathogenesis.