Whole-genome immunoinformatic analysis of F. tularensis: predicted CTL epitopes clustered in hotspots are prone to elicit a T-cell response

The cellular arm of the immune response plays a central role in the defense against intracellular pathogens, such as F. tularensis. To date, whole genome immunoinformatic analyses were limited either to relatively small genomes (e.g. viral) or to preselected subsets of proteins in complex pathogens. Here we present, for the first time, an unbiased bacterial global immunoinformatic screen of the 1740 proteins of F. tularensis subs. holarctica (LVS), aiming at identification of immunogenic peptides eliciting a CTL response. The very large number of predicted MHC class I binders (about 100,000, IC(50) of 1000 nM or less) required the design of a strategy for further down selection of CTL candidates. The approach developed focused on mapping clusters rich in overlapping predicted epitopes, and ranking these "hotspot" regions according to the density of putative binding epitopes. Limited by the experimental load, we selected to screen a library of 1240 putative MHC binders derived from 104 top-ranking highly dense clusters. Peptides were tested for their ability to stimulate IFNγ secretion from splenocytes isolated from LVS vaccinated C57BL/6 mice. The majority of the clusters contained one or more CTL responder peptides and altogether 127 novel epitopes were identified, of which 82 are non-redundant. Accordingly, the level of success in identification of positive CTL responders was 17-25 fold higher than that found for a randomly selected library of 500 predicted MHC binders (IC(50) of 500 nM or less). Most proteins (ca. 2/3) harboring the highly dense hotspots are membrane-associated. The approach for enrichment of true positive CTL epitopes described in this study, which allowed for over 50% increase in the dataset of known T-cell epitopes of F. tularensis, could be applied in immunoinformatic analyses of many other complex pathogen genomes.     

The molecular basis for the broad substrate specificity of human sulfotransferase 1A1

Cytosolic sulfotransferases (SULTs) are mammalian enzymes that detoxify a wide variety of chemicals through the addition of a sulfate group. Despite extensive research, the molecular basis for the broad specificity of SULTs is still not understood. Here, structural, protein engineering and kinetic approaches were employed to obtain deep understanding of the molecular basis for the broad specificity, catalytic activity and substrate inhibition of SULT1A1. We have determined five new structures of SULT1A1 in complex with different acceptors, and utilized a directed evolution approach to generate SULT1A1 mutants with enhanced thermostability and increased catalytic activity. We found that active site plasticity enables binding of different acceptors and identified dramatic structural changes in the SULT1A1 active site leading to the binding of a second acceptor molecule in a conserved yet non-productive manner. Our combined approach highlights the dominant role of SULT1A1 structural flexibility in controlling the specificity and activity of this enzyme.     

Vitamin D sensitizes breast cancer cells to the action of H2O2: mitochondria as a convergence point in the death pathway

Calcitriol, the hormonal form of vitamin D3, sensitizes breast cancer cells to reactive oxygen species (ROS)-dependent cytotoxicity induced by various anticancer modalities. This effect could be due to increased generation of ROS and/ or to increased sensitivity of the target cells to ROS. This work examined the effect of calcitriol on the damage inflicted on breast cancer cells by the direct action of ROS represented by H2O2. Treatment of MCF-7 cells with H2O2 resulted in activation of caspase 7 as well as induction of caspase-independent cell death. Both were enhanced by 48-72 h of pretreatment with calcitriol. This effect was not due to modulation of H2O2 degradation or to a specific effect on *OH-mediated cytotoxicity. The H2O2-induced drop in mitochondrial membrane potential and release of cytochrome c were enhanced by calcitriol. These findings indicate that calcitriol sensitizes breast cancer cells to ROS-induced death by affecting event(s) common to both caspase-dependent and -independent modes of cell death upstream to mitochondrial damage.     

Release of Neurotransmitter Induced by Ca<Superscript>2+</Superscript>-Uncaging: Reexamination of the Ca-Voltage Hypothesis for Release

The primacy of Ca²⁺ in controlling the amount of released neurotransmitter is well established. However, it is not yet clear what controls the time-course (initiation and termination) of release. Various experiments indicated that the time-course is controlled by membrane potential per se. Consequently the phenomenological Ca-Voltage-Hypothesis (CVH) was formulated. The CVH was later embodied in a molecular level mathematical model, whose key predictions were affirmed experimentally. Nonetheless, the single most important basis for the CVH, namely that depolarization per se is needed to induce physiological phasic release, was challenged by two major experimental findings. (i) Release was induced by Ca²⁺ alone by means of Ca²⁺-uncaging. (ii) There was at most a small additional effect when depolarization was applied after release was induced by Ca²⁺-uncaging. Point (i) was dealt with previously, but additional conclusions are drawn here. Here we concentrate on (ii) and show that the experimental results can be fully accounted for by the molecular level CVH model, with essentially the same parameters.Action Editor: G. Bard Ermentrout     

Impact of Admission and Cache Replacement Policies on Response Times of Jobs on Data Grids

Caching techniques have been used widely to improve the performance gaps of storage hierarchies in computing systems. Little is known about the impact of policies on the response times of jobs that access and process very large files in data grids, particularly when data and computations on the data have to be co-located on the same host. In data intensive applications that access large data files over wide area network environment, such as data-grids, the combination of policies for job servicing (or scheduling), caching and cache replacement can significantly impact the performance of grid jobs. We present preliminary results of a simulation study that combines an admission policy with a cache replacement policy when servicing jobs submitted to a storage resource manager.The results show that, in comparison to a first come first serve policy, the response times of jobs are significantly improved, for practical limits of disk cache sizes, when the jobs that are back-logged to access the same files are taken into consideration in scheduling the next file to be retrieved into the disk cache. Not only are the response times of jobs improved, but also the metric measures for caching policies, such as the hit ratio and the average cost per retrieval, are improved irrespective of the cache replacement policy used. Ekow Otoo is research staff scientist with the scientific data management group at Lawrence Berkeley National Laboratory, University of California, Berkeley. He received his B.Sc. degree in Electrical Engineering from the University of Science and Technology, Kumasi, Ghana and a post graduate diploma in Computer Science from the University of Ghana, Legon. In 1977, he received his M.Sc. degree in Computer Science from the University of Newcastle Upon Tyne in Britain and his Ph.D. degree in Computer Science from McGill University, Montreal, Canada in 1983. He joined the faculty of the School of Computer Science, Carleton University, in 1983 and from 1987 to 1999, he was a tenured faculty member of the School of Computer Science, Carleton University, Ottawa, Canada. He has served as research consultant to Bell Northern Research, Ottawa, Canada, and as a research project consultant to the GIS Division, Geomatics Canada, Natural Resources Canada, from 1990 to 1998. Ekow Otoo is a member of the ACM and IEEE. His research interests include database management systems, data structures and algorithms, parallel I/O for high performance computing, parallel and distributed computing. Doron Rotem is currently a senior staff scientist and a member of the Data Management group at the Lawrence Berkeley National Lab. His research interests include Grid Computing, Workflow, Scientific Data Management and Paralled and Distributed Computing and Algorithms. He has published over 80 papers in international journals and conferences in these areas. Prior to that, Dr Rotem co-founded and served as a CTO of a startup company, called CommerceRoute, that made software products in the area of workflow and data integration and before that, he was an Associate Professor in the Department of Computer Science, University of Waterloo, Canada. Dr. Rotem holds a B.Sc degree in Mathematics and Statistics from the Hebrew University, Jerusalem, Israel and a Ph.D. in Computer Science from the University of the Witwatersrand, Johannesburg, South Africa. Arie Shoshani is a senior staff scientist at Lawrence Berkeley National Laboratory. He joined LBNL in 1976. He heads the Scientific Data Management Group. He received his Ph.D. from Princeton University in 1969. From 1969 to 1976, he was a researcher at System Development Corporation, where he worked on the Network Control Program for the ARPAnet, distributed databases, database conversion, and natural language interfaces to data management systems. His current areas of work include data models, query languages, temporal data, statistical and scientific database management, storage management on tertiary storage, and grid storage middleware. Arie is also the director of a Scientific Data Management (SDM) Integrated Software Infrastructure Center (ISIC), one of seven centers selected by the SciDAC program at DOE in 2001. In this capacity, he is coordinating the work of collaborators from 4 DOE laboratories and 4 universities (see: http://sdmcenter.lbl.gov). Dr. Shoshani has published over 65 technical papers in refereed journals and conferences, chaired several workshops, conferences, and panels in database management; and served on numerous program committees for various database conferences. He also served as an associate editor for the ACM Transactions on Database Systems. He was elected a member of the VLDB Endowment Board, served as the Publication Board Chairperson for the VLDB Journal, and as the Vice-President of the VLDB Endowment. His home page is http://www.lbl.gov/arie.