Peptides identified through phage display direct immunogenic antigen to dendritic cells

Dendritic cells (DC) play a critical role in adaptive immunity by presenting Ag, thereby priming naive T cells. Specific DC-binding peptides were identified using a phage display peptide library. DC-peptides were fused to hepatitis C virus nonstructural protein 3 (NS3) while preserving DC targeting selectivity and Ag immunogenicity. The NS3-DC-peptide fusion protein was efficiently presented to CD4+ and CD8+ T cells derived from hepatitis C virus-positive blood cells, inducing their activation and proliferation. This immunogenic fusion protein was significantly more potent than NS3 control fusion protein or NS3 alone. In chimeric NOD-SCID mice transplanted with human cells, DC-targeted NS3 primed naive CD4+ and CD8+ T cells for potent NS3-specific proliferation and cytokine secretion. The capacity of peptides to specifically target immunogenic Ags to DC may establish a novel strategy for vaccine development.     

High Dietary Intake of Sodium Selenite Does Not Affect Gene Mutation Frequency in Rat Colon and Liver

Our previous studies have shown that selenium (Se) is protective against dimethylhydrazine (DMH)-induced preneoplastic colon cancer lesions, and protection against DNA damage has been hypothesized to be one mechanism for the anticancer effect of Se. The present study was designed to determine whether dietary selenite affects somatic mutation frequency in vivo. We used the Big Blue transgenic model to evaluate the in vivo mutation frequency of the cII gene in rats fed either a Se-deficient (0 μg Se/g diet) or Se-supplemented diet (0.2 or 2 μg Se/g diet; n = 3 rats/diet in experiment 1 and n = 5 rats/group in experiment 2) and injected with DMH (25 mg/kg body weight, i.p.). There were no significant differences in body weight between the Se-deficient and Se-supplemented (0.2 or 2 μg Se/g diet) rats, but the activities of liver glutathione peroxidase and thioredoxin reductase and concentration of liver Se were significantly lower (p < 0.0001) in Se-deficient rats compared to rats supplemented with Se. We found no effect of dietary Se on liver 8-hydroxy-2′-deoxyguanosine. Gene mutation frequency was significantly lower in liver (p < 0.001) than that of colon regardless of dietary Se. However, there were no differences in gene mutation frequency in DNA from colon mucosa or liver from rats fed the Se-deficient diet compared to those fed the Se-supplemented (0.2 or 2 μg Se/g diet) diet. Although gene mutations have been implicated in the etiology of cancer, our data suggest that decreasing gene mutation is not likely a key mechanism through which dietary selenite exerts its anticancer action against DMH-induced preneoplastic colon cancer lesions in a Big Blue transgenic rat model. The US Department of Agriculture, Agricultural Research Service, Northern Plains Area, is an equal opportunity/affirmative action employer and all agency services are available without discrimination. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the US Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable. This work was supported by the US Department of Agriculture and National Cancer Institute.     

Crystal structures of nucleosome core particles in complex with minor groove DNA-binding ligands

We determined the crystal structures of three nucleosome core particles in complex with site-specific DNA-binding ligands, the pyrrole-imidazole polyamides. While the structure of the histone octamer and its interaction with the DNA remain unaffected by ligand binding, nucleosomal DNA undergoes significant structural changes at the ligand-binding sites and in adjacent regions to accommodate the ligands. Our findings suggest that twist diffusion occurs over long distances through tightly bound nucleosomal DNA. This may be relevant to the mechanism of ATP-dependent and spontaneous nucleosome translocation, and to the effect of bound factors on nucleosome dynamics.     

Fatal collision? Are wireless headsets a risk in treating patients?

Wireless-enabled headsets that connect to the internet can provide remote transcribing of patient examination notes. Audio and video can be captured and transmitted by wireless signals sent from the computer screen in the frame of the glasses. But using wireless glass-type devices can expose the user to a specific absorption rates (SAR) of 1.11–1.46 W/kg of radiofrequency radiation. That RF intensity is as high as or higher than RF emissions of some cell phones. Prolonged use of cell phones used ipsilaterally at the head has been associated with statistically significant increased risk of glioma and acoustic neuroma. Using wireless glasses for extended periods to teach, to perform surgery, or conduct patient exams will expose the medical professional to similar RF exposures which may impair brain performance, cognition and judgment, concentration and attention and increase the risk for brain tumors. The quality of medical care may be compromised by extended use of wireless-embedded devices in health care settings. Both medical professionals and their patients should know the risks of such devices and have a choice about allowing their use during patient exams. Transmission of sensitive patient data over wireless networks may increase the risk of hacking and security breaches leading to losses of private patient medical and financial data that are strictly protected under HIPPA health information privacy laws.     

A modeled hydrophobic domain on the TCL1 oncoprotein mediates association with AKT at the cytoplasmic membrane

AKT has a critical role in relaying cell survival and proliferation signals initiated by ligand binding to surface receptors in mammalian cells. Induction of AKT serine/threonine kinase activity is augmented by the T-cell leukemia-1 (TCL1) oncoprotein through a physical association requiring the AKT pleckstrin homology domain. Here, we used molecular modeling and identified an exposed hydrophobic patch composed of two discontinuous amino acid stretches near one end of the TCL1 beta-barrel that was required for a TCL1-AKT association. Site-directed mutations of this region did not affect TCL1 secondary structure, yet they disrupted interactions with AKT. This region was found in other members of the TCL1 oncoprotein family, such as TCL1b and MTCP1, and suggested a conserved, novel AKT binding domain. Interestingly, TCL1 and AKT co-localize in multiple cell compartments, but only extracts from the plasma membrane stimulate optimal complex formation in vitro. Identification of an AKT binding domain on TCL1 is an important step in deciphering the complex interactions that regulate AKT kinase activity in lymphocyte development and neoplasia within the immune system.     

Protist community composition during spring in an Arctic flaw lead polynya

The overwintering deployment of an icebreaker during the Canadian Flaw Lead study provided an opportunity to evaluate how protist communities (phytoplankton and other single-celled eukaryotes) respond to changing spring irradiance conditions in flaw lead polynyas, where open water persists between the central pack ice and land fast ice. We combined microscopic analysis of the protist communities (all cell sizes) with clone libraries of 18S rRNA genes and 18S rRNA (from RNA converted to cDNA) of size-fractionated seawater (0.2–3.0 μm) from 10 to 12 m depth in the surface mixed layer. The rRNA gene analysis provided information on the presence of organisms, while the rRNA analysis provided information on the most active members of the community. There was little overlap between the two types of clone libraries, and there were large community shifts over time. Heterotrophic dinoflagellates and ciliates were the most common sequences recovered. The relative proportion of photosynthetic protist sequences increased in March and April, and there was greater representation of Bacillariophyta, Prasinophyta, Haptophyta, and Cryptophyta in the rRNA compared to rRNA gene libraries. Microscopy indicated that large-celled diatoms dominated the community in May, when chlorophyll concentrations were greatest. However, the RNA sequencing showed that heterotrophic and putative parasitic protists were proportionately more active, and the concomitant decrease in nutrients suggested that the spring phytoplankton bloom had begun to decline by this time. These observations provide evidence of substantial changes in protist community structure and function during the spring transition.     

Applying USEPA Risk Assessment Guidance in the 90s

Over the past decade, risk assessment has become increasingly relied upon for helping to make environmental management decisions. This trend has been accompanied by research and refinements in basic risk assessment methodologies to improve our ability to understand and evaluate the human health risks associated with chemical exposures.Despite this progress, significant uncertainties continue to be associated with the risk assessment process. These uncertainties typically derive from gaps in available data regarding chemical toxicity, and from difficulties in reliably estimating the magnitude of chemical exposures. Given these limitations, risk assessment is generally most valuable in evaluating relative risk; for example, when comparing alternatives to achieving a specified goal, setting priorities for protecting human health, or establishing procedures for properly allocating resources. Risk assessment can also be useful for developing regulatory benchmarks such as permit limits for air or water. In many cases, however, the limitations of the risk assessment process make it difficult (if not impossible) to reliably estimate an absolute level of risk, especially for a specific individual in an exposed population. In such cases, risk assessment can be seriously misapplied, and its results misinterpreted.This paper discusses some of the challenges that have been faced by the field of risk assessment during the 1990s. Current trends in risk assessment, and its use by regulatory agencies in making risk management decisions, are also described.     

Generation of reference cell lines,media, and a process platform for CHO cell biomanufacturing

Due to the favorable attributes of Chinese hamster ovary (CHO) cells for therapeutic proteins and antibodies biomanufacturing, companies generate proprietary cells with desirable phenotypes. One key attribute is the ability to stably express multi-gram per liter titers in chemically defined media. Cell, media, and feed diversity has limited community efforts to translate knowledge. Moreover, academic, and nonprofit researchers generally cannot study “industrially relevant” CHO cells due to limited public availability, and the time and knowledge required to generate such cells. To address these issues, a university-industrial consortium (Advanced Mammalian Biomanufacturing Innovation Center, AMBIC) has acquired two CHO “reference cell lines” from different lineages that express monoclonal antibodies. These reference cell lines have relevant production titers, key performance outcomes confirmed by multiple laboratories, and a detailed technology transfer protocol. In commercial media, titers over 2 g/L are reached. Fed-batch cultivation data from shake flask and scaled-down bioreactors is presented. Using productivity as the primary attribute, two academic sites aligned with tight reproducibility at each site. Further, a chemically defined media formulation was developed and evaluated in parallel to the commercial media. The goal of this work is to provide a universal, industrially relevant CHO culture platform to accelerate biomanufacturing innovation.     

The Pseudomonas aeruginosa Type III Translocon Is Required for Biofilm Formation at the Epithelial Barrier

Clinical infections by Pseudomonas aeruginosa, a deadly Gram-negative, opportunistic pathogen of immunocompromised hosts, often involve the formation of antibiotic-resistant biofilms. Although biofilm formation has been extensively studied in vitro on glass or plastic surfaces, much less is known about biofilm formation at the epithelial barrier. We have previously shown that when added to the apical surface of polarized epithelial cells, P. aeruginosa rapidly forms cell-associated aggregates within 60 minutes of infection. By confocal microscopy we now show that cell-associated aggregates exhibit key characteristics of biofilms, including the presence of extracellular matrix and increased resistance to antibiotics compared to planktonic bacteria. Using isogenic mutants in the type III secretion system, we found that the translocon, but not the effectors themselves, were required for cell-associated aggregation on the surface of polarized epithelial cells and at early time points in a murine model of acute pneumonia. In contrast, the translocon was not required for aggregation on abiotic surfaces, suggesting a novel function for the type III secretion system during cell-associated aggregation. Supernatants from epithelial cells infected with wild-type bacteria or from cells treated with the pore-forming toxin streptolysin O could rescue aggregate formation in a type III secretion mutant, indicating that cell-associated aggregation requires one or more host cell factors. Our results suggest a previously unappreciated function for the type III translocon in the formation of P. aeruginosa biofilms at the epithelial barrier and demonstrate that biofilms may form at early time points of infection.     

Quantitative Tumor Segmentation for Evaluation of Extent of Glioblastoma Resection to Facilitate Multisite Clinical Trials

Standard-of-care therapy for glioblastomas, the most common and aggressive primary adult brain neoplasm, is maximal safe resection, followed by radiation and chemotherapy. Because maximizing resection may be beneficial for these patients, improving tumor extent of resection (EOR) with methods such as intraoperative 5-aminolevulinic acid fluorescence-guided surgery (FGS) is currently under evaluation. However, it is difficult to reproducibly judge EOR in these studies due to the lack of reliable tumor segmentation methods, especially for postoperative magnetic resonance imaging (MRI) scans. Therefore, a reliable, easily distributable segmentation method is needed to permit valid comparison, especially across multiple sites. We report a segmentation method that combines versatile region-of-interest blob generation with automated clustering methods. We applied this to glioblastoma cases undergoing FGS and matched controls to illustrate the method's reliability and accuracy. Agreement and interrater variability between segmentations were assessed using the concordance correlation coefficient, and spatial accuracy was determined using the Dice similarity index and mean Euclidean distance. Fuzzy C-means clustering with three classes was the best performing method, generating volumes with high agreement with manual contouring and high interrater agreement preoperatively and postoperatively. The proposed segmentation method allows tumor volume measurements of contrast-enhanced T₁-weighted images in the unbiased, reproducible fashion necessary for quantifying EOR in multicenter trials.