Biochemical and functional characterization of mutant KRAS epitopes validates this oncoprotein for immunological targeting.

Activating RAS missense mutations are among the most prevalent genomic alterations observed in human cancers and drive oncogenesis in the three most lethal tumor types. Emerging evidence suggests mutant KRAS (mKRAS) may be targeted immunologically, but mKRAS epitopes remain poorly defined. Here we employ a multi-omics approach to characterize HLA class I-restricted mKRAS epitopes. We provide proteomic evidence of mKRAS epitope processing and presentation by high prevalence HLA class I alleles. Select epitopes are immunogenic enabling mKRAS-specific TCRαß isolation. TCR transfer to primary CD8+ T cells confers cytotoxicity against mKRAS tumor cell lines independent of histologic origin, and the kinetics of lytic activity correlates with mKRAS peptide-HLA class I complex abundance. Adoptive transfer of mKRAS-TCR engineered CD8+ T cells leads to tumor eradication in a xenograft model of metastatic lung cancer. This study validates mKRAS peptides as bona fide epitopes facilitating the development of immune therapies targeting this oncoprotein.

Inherent ER stress in pancreatic islet ß cells causes self-recognition by autoreactive T cells in type 1 diabetes.

Type 1 diabetes (T1D) is an autoimmune disease characterized by pancreatic ß cell destruction induced by islet reactive T cells that have escaped central tolerance. Many physiological and environmental triggers associated with T1D result in ß cell endoplasmic reticulum (ER) stress and dysfunction, increasing the potential for abnormal post-translational modification (PTM) of proteins. We hypothesized that ß cell ER stress induced by environmental and physiological conditions generates abnormally-modified proteins for the T1D autoimmune response. To test this hypothesis we exposed the murine CD4(+) diabetogenic BDC2.5 T cell clone to murine islets in which ER stress had been induced chemically (Thapsigargin). The BDC2.5 T cell IFNγ response to these cells was significantly increased compared to non-treated islets. This ß cell ER stress increased activity of the calcium (Ca(2+))-dependent PTM enzyme tissue transglutaminase 2 (Tgase2), which was necessary for full stress-dependent immunogenicity. Indeed, BDC2.5 T cells responded more strongly to their antigen after its modification by Tgase2. Finally, exposure of non-antigenic murine insulinomas to chemical ER stress in vitro or physiological ER stress in vivo caused increased ER stress and Tgase2 activity, culminating in higher BDC2.5 responses. Thus, ß cell ER stress induced by chemical and physiological triggers leads to ß cell immunogenicity through Ca(2+)-dependent PTM. These findings elucidate a mechanism of how ß cell proteins are modified and become immunogenic, and reveal a novel opportunity for preventing ß cell recognition by autoreactive T cells.

Geographic and temporal dynamics of a global radiation and diversification in the killer whale.

Global climate change during the Late Pleistocene periodically encroached and then released habitat during the glacial cycles, causing range expansions and contractions in some species. These dynamics have played a major role in geographic radiations, diversification and speciation. We investigate these dynamics in the most widely distributed of marine mammals, the killer whale (Orcinus orca), using a global data set of over 450 samples. This marine top predator inhabits coastal and pelagic ecosystems ranging from the ice edge to the tropics, often exhibiting ecological, behavioural and morphological variation suggestive of local adaptation accompanied by reproductive isolation. Results suggest a rapid global radiation occurred over the last 350 000 years. Based on habitat models, we estimated there was only a 15% global contraction of core suitable habitat during the last glacial maximum, and the resources appeared to sustain a constant global effective female population size throughout the Late Pleistocene. Reconstruction of the ancestral phylogeography highlighted the high mobility of this species, identifying 22 strongly supported long-range dispersal events including interoceanic and interhemispheric movement. Despite this propensity for geographic dispersal, the increased sampling of this study uncovered very few potential examples of ancestral dispersal among ecotypes. Concordance of nuclear and mitochondrial data further confirms genetic cohesiveness, with little or no current gene flow among sympatric ecotypes. Taken as a whole, our data suggest that the glacial cycles influenced local populations in different ways, with no clear global pattern, but with secondary contact among lineages following long-range dispersal as a potential mechanism driving ecological diversification.

Adsorption of Benzene on Copper, Silver, and Gold Surfaces.

The adsorption of benzene on the Cu(111), Ag(111), Au(111), and Cu(110) surfaces at low coverage is modeled using density-functional theory (DFT) using periodic-slab models of the surfaces as well as using both DFT and complete-active-space self-consistent field theory with second-order Møller-Plesset perturbation corrections (CASPT2) for the interaction of benzene with a Cu13 cluster model for the Cu(110) surface. For the binding to the (111) surfaces, key qualitative features of the results such as weak physisorption, the relative orientation of the adsorbate on the surface, and surface potential changes are in good agreement with experimental findings. Also, the binding to Cu(110) is predicted to be much stronger than that to Cu(111) and much weaker than that seen in previous calculations for Ni(110), as observed. However, a range of physisorptive-like and chemisorptive-like structures are found for benzene on Cu(110) that are roughly consistent with observed spectroscopic data, with these structures differing dramatically in geometry but trivially in energy. For all systems, the bonding is found to be purely dispersive in nature with minimal covalent character. As dispersive energies are reproduced very poorly by DFT, the calculated binding energies are found to dramatically underestimate the observed ones, while CASPT2 calculations indicate that there is no binding at the Hartree-Fock level and demonstrate that the expected intermolecular correlation (dispersive) energy is of the correct order to explain the experimental binding-energy data. DFT calculations performed for benzene on Cu(110) and for benzene on the model cluster indicate that this cluster is actually too reactive and provides a poor chemical model for the system.

Self-aspirating atmospheric pressure chemical ionization source for direct sampling of analytes on surfaces and in liquid solutions.

A self-aspirating heated nebulizer probe is described and demonstrated for use in the direct analysis of analytes on surfaces and in liquid samples by atmospheric pressure chemical ionization (APCI) mass spectrometry. Functionality and performance of the probe as a self-aspirating APCI source is demonstrated using reserpine and progesterone as test compounds. The utility of the probe to sample analytes directly from surfaces was demonstrated first by scanning development lanes of a reversed-phase thin-layer chromatography plate in which a three-component dye mixture, viz., Fat Red 7B, Solvent Green 3, and Solvent Blue 35, was spotted and the components were separated. Development lanes were scanned by the sampling probe operated under computer control (x, y plane) while full-scan mass spectra were recorded using a quadrupole ion trap mass spectrometer. In addition, the ability to sample the surface of pharmaceutical tablets (viz., Extra Strength Tylenol and Evista tablets) and to detect the active ingredients (acetaminophen and raloxifene, respectively) selectively was demonstrated using tandem mass spectrometry (MS/MS). Finally, the capability to sample analyte solutions from the wells of a 384-well microtiter plate and to perform quantitative analyses using MS/MS detection was illustrated with cotinine standards spiked with cotinine-d3 as an internal standard.