The absence of invariant chain in MHC II cancer vaccines enhances the activation of tumor-reactive type 1 CD4<Superscript>+</Superscript> T lymphocytes

Activation of tumor-reactive T lymphocytes is a promising approach for the prevention and treatment of patients with metastatic cancers. Strategies that activate CD8⁺ T cells are particularly promising because of the cytotoxicity and specificity of CD8⁺ T cells for tumor cells. Optimal CD8⁺ T cell activity requires the co-activation of CD4⁺ T cells, which are critical for immune memory and protection against latent metastatic disease. Therefore, we are developing “MHC II” vaccines that activate tumor-reactive CD4⁺ T cells. MHC II vaccines are MHC class I⁺ tumor cells that are transduced with costimulatory molecules and MHC II alleles syngeneic to the prospective recipient. Because the vaccine cells do not express the MHC II-associated invariant chain (Ii), we hypothesized that they will present endogenously synthesized tumor peptides that are not presented by professional Ii⁺ antigen presenting cells (APC) and will therefore overcome tolerance to activate CD4⁺ T cells. We now report that MHC II vaccines prepared from human MCF10 mammary carcinoma cells are more efficient than Ii⁺ APC for priming and boosting Type 1 CD4⁺ T cells. MHC II vaccines consistently induce greater expansion of CD4⁺ T cells which secrete more IFNγ and they activate an overlapping, but distinct repertoire of CD4⁺ T cells as measured by T cell receptor Vβ usage, compared to Ii⁺ APC. Therefore, the absence of Ii facilitates a robust CD4⁺ T cell response that includes the presentation of peptides that are presented by traditional APC, as well as peptides that are uniquely presented by the Ii⁻ vaccine cells.     

Accelerated HER-2 degradation enhanced ovarian tumor recognition by CTL. Implications for tumor immunogenicity

We investigated the ubiquitination and degradation of a tumor antigen, the HER-2/neu (HER-2) protooncogene product which is overexpressed in epithelial cancers. HER-2 degradation was investigated in the ovarian tumor line, SKOV3.A2, that constitutively overexpressed long-life HER-2. We used as agonist geldanamycin (GA), which initiated downmodulation of HER-2 from the cell surface. HER-2 was polyubiquitinated and degraded faster in the presence than in the absence of GA. GA did not decrease HLA-A2 expression. Presentation of the immunodominant cytotoxic T lymphocyte (CTL) epitope, E75 (369–377) from SKOV.A2 was inhibited by proteasome inhibitors, such as LLnL but was enhanced by cysteine protease inhibitors such as E64, indicating that both the proteasome and cysteine proteases are involved in epitope formation but have different effects. Enhanced tumor recognition was not an immediate or early effect of GA treatment, but was evident after 20 h of GA treatment. In contrast, 20 h GA treatment did not increase tumor sensitivity to LAK cell lysis. Twenty hour GA-treated SKOV3.A2 cells expressed an unstable HER-2 protein synthesized in the presence of GA, of faster electrophoretic mobility than control HER-2. This suggested that the newly synthesized HER-2 in the presence of GA was the main source of epitopes recognized by CTL. Twenty hour GA-treated SKOV3.A2 cells were better inducers of CTL activity directed to a number of HER-2 CTL epitopes, in peripheral blood mononuclear cells compared with control untreated SKOV3.A2 cells. Thus, induction of HER-2 protein instability enhanced the sensitivity of tumor for CTL lysis. Increased HER-2 CTL epitopes presentation may have implications for overcoming the poor immuno-genicity of human tumors, and design of epitope precursors for cancer vaccination.     

Opportunities for renewable bioenergy using microorganisms

Global warming can be slowed, and perhaps reversed, only when society replaces fossil fuels with renewable, carbon-neutral alternatives. The best option is bioenergy: the sun's energy is captured in biomass and converted to energy forms useful to modern society. To make a dent in global warming, bioenergy must be generated at a very high rate, since the world today uses approximately 10 TW of fossil-fuel energy. And, it must do so without inflicting serious damage on the environment or disrupting our food supply. While most bioenergy options fail on both counts, several microorganism-based options have the potential to produce large amounts of renewable energy without disruptions. In one approach, microbial communities convert the energy value of various biomass residuals to socially useful energy. Biomass residuals come from agricultural, animal, and a variety of industrial operations, as well as from human wastes. Microorganisms can convert almost all of the energy in these wastes to methane, hydrogen, and electricity. In a second approach, photosynthetic microorganisms convert sunlight into biodiesel. Certain algae (eukaryotes) or cyanobacteria (prokaryotes) have high lipid contents. Under proper conditions, these photosynthetic microorganisms can produce lipids for biodiesel with yields per unit area 100 times or more than possible with any plant system. In addition, the non-lipid biomass can be converted to methane, hydrogen, or electricity. Photosynthetic microorganisms do not require arable land, an advantage because our arable land must be used to produce food. Algae or cyanobacteria may be the best option to produce bioenergy at rates high enough to replace a substantial fraction of our society's use of fossil fuels.     

A novel mode of action for a microbial-derived immunotoxin: the cytolethal distending toxin subunit B exhibits phosphatidylinositol 3,4,5-triphosphate phosphatase activity

The Actinobacillus actinomycetemcomitans cytolethal distending toxin (Cdt) is a potent immunotoxin that induces G(2) arrest in human lymphocytes. We now show that the CdtB subunit exhibits phosphatidylinositol (PI)-3,4,5-triphosphate phosphatase activity. Breakdown product analysis indicates that CdtB hydrolyzes PI-3,4,5-P(3) to PI-3,4-P(2) and therefore functions in a manner similar to phosphatidylinositol 5-phosphatases. Conserved amino acids critical to catalysis in this family of enzymes were mutated in the cdtB gene. The mutant proteins exhibit reduced phosphatase activity along with decreased ability to induce G(2) arrest. Consistent with this activity, Cdt induces time-dependent reduction of PI-3,4,5-P(3) in Jurkat cells. Lymphoid cells with defects in SHIP1 and/or ptase and tensin homolog deleted on chromosome 10 (PTEN) (such as Jurkat, CEM, Molt) and, concomitantly, elevated PI-3,4,5-P(3) levels were more sensitive to the toxin than HUT78 cells which contain functional levels of both enzymes and low levels of PI-3,4,5-P(3). Finally, reduction of Jurkat cell PI-3,4,5-P(3) synthesis using the PI3K inhibitors, wortmannin and LY290004, protects cells from toxin-induced cell cycle arrest. Collectively, these studies show that the CdtB not only exhibits PI-3,4,5-P(3) phosphatase activity, but also that toxicity in lymphocytes is related to this activity.     

Enzymatic microreactors in chemical analysis and kinetic studies

The fields of application of microreactors are becoming wider every year. A considerable number of papers have been published recently reporting successful application of enzymatic microreactors in chemistry and biochemistry. Most are devices with enzymes immobilized on beads or walls of microfluidic channels, whilst some use dissolved enzymes to run a reaction in the microfluidic system. Apart from model systems, mostly with glucose oxidase, horseradish peroxidase and alkaline phosphatase, the principal fields of application of microreactors are tryptic digestion of proteins and polymerase chain reaction in automated analyses of proteomic and genetic material, respectively. Enzymatic microreactors also facilitate characterization of enzyme activity as a function of substrate concentration, and enable fast screening of new biocatalysts and their substrates. They may constitute key parts of lab-on-a-chip and muTAS, assisting the analysis of biomolecules. This review provides systematic coverage of examples of reports on enzymatic microreactors published recently, as well as relevant older papers.     

Phylogenetic and population genetic analyses of Phaeosphaeria nodorum and its close relatives indicate cryptic species and an origin in the Fertile Crescent

The origin of the fungal wheat pathogen Phaeosphaeria nodorum remains unclear despite earlier intensive global population genetic and phylogeographical studies. We sequenced 1683 bp distributed across three loci in 355 globally distributed Phaeosphaeria isolates, including 74 collected in Iran near the center of origin of wheat. We identified nine phylogenetically distinct clades, including two previously unknown species tentatively named P1 and P2 collected in Iran. Coalescent analysis indicates that P1 and P2 are sister species of P. nodorum and the other Phaeosphaeria species identified in our analysis. Two species, P. nodorum and P. avenaria f. sp. tritici 1 (Pat1), comprised ～85% of the sampled isolates, making them the dominant wheat-infecting pathogens within the species complex. We designed a PCR-RFLP assay to distinguish P. nodorum from Pat1. Approximately 4% of P. nodorum and Pat1 isolates showed evidence of hybridization. Measures of private allelic richness at SSR and sequence loci suggest that the center of origin of P. nodorum coincides with its host in the Fertile Crescent. We hypothesize that the origin of this species complex is also in the Fertile Crescent, with four species out of nine found exclusively in the Iranian collections.     

Validation of elk resource selection models with spatially independent data

Knowledge of how landscape features affect wildlife resource use is essential for informed management. Resource selection functions often are used to make and validate predictions about landscape use; however, resource selection functions are rarely validated with data from landscapes independent of those from which the models were built. This problem has severely limited the application of resource selection functions over larger geographic areas for widely distributed species. North American elk (Cervus elaphus) is an example of a widely-distributed species of keen interest to managers and for which validation of resource selection functions over large geographic areas is important. We evaluated the performance of resource selection functions developed for elk on one landscape in northeast Oregon with independent data from a different landscape in the same region. We compared predicted versus observed elk resource use for 9 monthly or seasonal periods across 3 yr. Results showed strong, positive agreement between predicted and observed use for 2 spring and 3 late summer-early fall models (3-yr r = 0.81–0.95). Predicted versus observed use was negatively or weakly positively correlated for 3 summer models and 1 mid-fall model (3-yr r = −0.57–0.14). Predicted and observed use correlated well when forage was limited (spring and late summer or early fall), corresponding to important biological stages for elk (parturition and breeding seasons). For these seasonal periods, model covariates such as rate of motorized traffic and canopy closure often were effective predictors of elk resource selection. The models we validated for spring and late summer-early fall may be used to evaluate management activities in areas with similar landscape characteristics. © 2010 The Wildlife Society.     

Relationship Between Curved DNA Conformations and Slow Gel Migration

Abstract

We propose some specific DNA conformations that explain, in terms of molecular conformations, the anomalous gel electrophoretic behavior of the sequences (VA₄T₄X)₁, and (V₂A₃X₂)₁ where V and X are either G or C. Previously (J. Biomole. Struct. Dyn. 4, 41, 1986) we considered hydrophobic interactions a mong aliphatic hydrocarbon groups in A/T sequences. In the sequences (T)_n · (A)_n, the T's are slightly bent to yield structures with tightly stacked methyl groups along one side of the major groove. By folding together the two pairs of stacked methyls on the opposite sides of the major groove, TTAA might yield a relatively sharp bend. On this basis, we show below that the sequences (VT₄A₄X)₁ might form a very tightly coiled super-helix whereas the sequences (VA₄T₄X)₁ form a broad super-helix of radius ~ 120 A for i = 25. The sequence (V₂A₃T₃X₂)₁ forms a slightly smaller radius super-helix. The time of passage through the gel has been taken to be inversely proportional to the smallesuiimension of the molecule. Specifically we are taking the ratio of the apparent molecular weight to the actual molecular weight to be related to the moment of inertia I₁ about the smallest principal axis of the molecular conformation. We find a good fit to the experimental gel mobility data of Hagerman (2) if we assume this ratio to be proportional to (I₁)^1/5.     

Metabolomics: building on a century of biochemistry to guide human health

Medical diagnosis and treatment efficacy will improve significantly when a more personalized system for health assessment is implemented. This system will require diagnostics that provide sufficiently detailed information about the metabolic status of individuals such that assay results will be able to guide food, drug and lifestyle choices to maintain or improve distinct aspects of health without compromising others. Achieving this goal will use the new science of metabolomics – comprehensive metabolic profiling of individuals linked to the biological understanding of human integrative metabolism. Candidate technologies to accomplish this goal are largely available, yet they have not been brought into practice for this purpose. Metabolomic technologies must be sufficiently rapid, accurate and affordable to be routinely accessible to both healthy and acutely ill individuals. The use of metabolomic data to predict the health trajectories of individuals will require bioinformatic tools and quantitative reference databases. These databases containing metabolite profiles from the population must be built, stored and indexed according to metabolic and health status. Building and annotating these databases with the knowledge to predict how a specific metabolic pattern from an individual can be adjusted with diet, drugs and lifestyle to improve health represents a logical application of the biochemistry knowledge that the life sciences have produced over the past 100 years.