Software tools for simultaneous data visualization and T cell epitopes and disorder prediction in proteins.

We have developed EpDis and MassPred, extendable open source software tools that support bioinformatic research and enable parallel use of different methods for the prediction of T cell epitopes, disorder and disordered binding regions and hydropathy calculation. These tools offer a semi-automated installation of chosen sets of external predictors and an interface allowing for easy application of the prediction methods, which can be applied either to individual proteins or to datasets of a large number of proteins. In addition to access to prediction methods, the tools also provide visualization of the obtained results, calculation of consensus from results of different methods, as well as import of experimental data and their comparison with results obtained with different predictors. The tools also offer a graphical user interface and the possibility to store data and the results obtained using all of the integrated methods in the relational database or flat file for further analysis. The MassPred part enables a massive parallel application of all integrated predictors to the set of proteins. Both tools can be downloaded from http://bioinfo.matf.bg.ac.rs/home/downloads.wafl?cat=Software. Appendix A includes the technical description of the created tools and a list of supported predictors.

Epitope distribution in ordered and disordered protein regions. Part B - Ordered regions and disordered binding sites are targets of T- and B-cell immunity.

Intrinsically disordered proteins exist in highly flexible conformational states linked to different protein functions. In this work, we have presented evidence that HLA class-I- and class-II-binding T-cell epitopes, experimentally verified in several tumor-associated antigens and nuclear systemic autoantigens, are predominantly located in ordered protein regions or at disorder/order borderlines, defined by the majority of analyzed publicly available disorder predictors. We have also observed the overlapping of secondary structural elements and prevalently hydrophobic regions with T-cell epitopes in Epstein Barr Virus (EBV) nuclear antigen 1 (EBNA-1), cancer/testis antigen MAGE-A4, and Sm-B/B', U1 snRNPA (U1A) and U1-70kDa autoantigens. The results are in accordance with the clustering of the predicted HLA class-I and class-II epitopes in protein parts which encompass the consensus of ordered regions, determined by individual disorder predictors. Some HLA class-II epitopes and linear B-cell epitopes were located near the segments predicted to have elevated crystallographic B factor in EBNA-1, Sm-B/B' and U1 snRNP A proteins, suggesting that protein flexibility could influence the structural availability of epitopes. Naturally processed T-cell epitopes and linear B-cell epitopes could also be found within putative disordered binding sites, determined by "dips" in the prevalently disordered parts of prediction profiles of the majority of disorder predictors, and peaks in ANCHOR-prediction profile. Two minor antigenic regions within EBNA-1, mapped to the residues 58-85 and 398-458, encompassing putative disordered binding sites, contain epitopes connected with anti-Ro 60kDa and anti-Sm B/B' autoimmunity in systemic lupus erythematosus. One of these regions overlaps residues 395-450, identified as the binding site of USP7 (HAUSP), which regulates the EBNA-1 replication function. In Sm-B/B', one of the putative disordered binding sites (residues 114-165) encompasses the T-cell epitope 136-153, while another, residues 200-216, flanks two proline-rich B-cell epitopes (residues 190-198 and 216-222), overlapping the preferred CD2BP2-GYF-binding motif (R/K/G)XXPPGX(R/K), characteristic of splicosomal proteins. We have noticed that the same motif (residues 397-403) is mimicked in EBNA-1 and overlaps epitope 398-404, involved in anti-Sm B/B' autoimmunity. The majority of recognized T- and B-cell epitopes in analyzed autoantigens or tumor-associated antigens appertain to the ordered or transient protein structures. The congruence between certain B- and T-cell epitopes and predicted disordered binding sites or protein-binding eukaryotic motifs in the antigens participating in molecular complexes might influence the capture of antigens, their processing and subsequent presentation and immunodominance.

Epitope distribution in ordered and disordered protein regions - part A. T-cell epitope frequency, affinity and hydropathy.

Highly disordered protein regions are prevalently hydrophilic, extremely sensitive to proteolysis in vitro, and are expected to be under-represented as T-cell epitopes. The aim of this research was to find out whether disorder and hydropathy prediction methods could help in understanding epitope processing and presentation. According to the pan-specific T-cell epitope predictors NetMHCpan and NetMHCIIpan and 9 publicly available disorder predictors, frequency of epitopes presented by human leukocyte antigens (HLA) class-I or -II was found to be more than 2.5 times higher in ordered than in disordered protein regions (depending on the disorder predictor). Both HLA class-I and HLA class-II binding epitopes are prevalently hydrophilic in disordered and prevalently hydrophobic in ordered protein regions, whereas epitopes recognized by HLA class-II alleles are more hydrophobic than those recognized by HLA class-I. As regards both classes of HLA molecules, high-affinity binding epitopes display more hydrophobicity than low affinity-binding epitopes (in both ordered and disordered regions). Epitopes belonging to disordered protein regions were not predicted to have poor affinity to HLA class-II molecules, as expected from disorder intrinsic proteolytic instability. The relation of epitope hydrophobicity and order/disorder location was also valid if alleles were grouped according to the HLA class-I and HLA class-II supertypes, except for the class-I supertype A3 in which the main part of recognized epitopes was prevalently hydrophilic. Regarding specific supertypes, the affinity of epitopes belonging to ordered regions varies only slightly (depending on the disorder predictor) compared to the affinity of epitopes in corresponding disordered regions. The distribution of epitopes in ordered and disordered protein regions has revealed that the curves of order-epitope distribution were convex-like while the curves of disorder-epitope distribution were concave-like. The percentage of prevalently hydrophobic epitopes increases with the enhancement of epitope promiscuity level and moving from disordered to ordered regions. These data suggests that reverse vaccinology, oriented towards promiscuous and high-affinity epitopes, is also oriented towards prevalently hydrophobic, ordered regions. The analysis of predicted and experimentally evaluated epitopes of cancer-testis antigen MAGE-A3 has confirmed that the majority of T-cell epitopes, particularly those that are promiscuous or naturally processed, was located in ordered and disorder/order boundary protein regions overlapping hydrophobic regions.

Influence of sunflower oil qualities and antioxidants on oxidative stability on whey-based salad dressings.

The antioxidant effect of ascorbic acid and EDTA (ethylenediaminetetraacetic acid) in food emulsions, based on whey and sunflower oils with enhanced oleic acid, α- and ß- tocopherol content, was not described up to now. Salad dressings based on cold-pressed high-oleic/α-, ß- tocopherol sunflower oil were oxidatively stable after 3 months of storage at 25 °C regarding primary (peroxide value, PV) and secondary (hexanal) lipid oxidation products (PV = 0.34 mmol O2 kg-1, hexanal value = 1.54 mg kg-1). Slight enhancement of PV and hexanal values was recorded in salad dressings prepared with cold-pressed medium-oleic/α-, ß- tocopherol oil, after 3 months of storage at 25 °C, and was inhibited by ascorbic acid or EDTA. Ascorbic acid (0.50 g kg-1) reduced PV by 80% and hexanal value by 32%. EDTA (0.075 g kg-1) reduced PV by 60% and hexanal value by 27%. In salad dressings, containing linoleic/a- tocopherol sunflower oil, the antioxidant effects of ascorbic acid and EDTA were as following: ascorbic acid (0.25-4.00 g kg-1) reduced PV by 83-100% and hexanal value by 82-73%; EDTA (0.075 g kg-1) reduced PV by 75% and hexanal value by 76%, after 12 months of storage at 4 °C.

Synthesis of some steroidal oximes, lactams, thiolactams and their antitumor activities.

The antiproliferative activity of previously synthesized (Z)-cholest-4-en-6-one oxime (1), (E)-cholest-4-en-6-one oxime (2), 7-aza-B-homocholest-4-en-6-one (3) and 6-aza-B-homocholest-4-en-7-one (4) and newly synthesized 6-thioxo-7-aza-B-homocholest-4-ene (5) and 6-aza-7-thioxo-B-homocholest-4-ene (6) was tested for their possible effects against two human tumor cell lines, cervical carcinoma (HeLa) and chronic myelogenous leukemia (K-562). Compounds 1-6, exerted a dose-dependent antiproliferative effect toward cell lines used in experimental design, showing high selectivity in their action for tumor cells in comparison to normal immunocompetent cells (non-stimulated PBMC and PHA-stimulated PBMC). Compounds 2, 3 and 4 exhibited a very high but selective antitumor activity, by inducing apoptosis in sensitive, for that purpose targeted tumor cell line (HeLa cells). Low toxic effect upon both non-stimulated, and PHA stimulated PBMCs from control, healthy volunteers, has been detected for compounds 1, 2, 3 and 4. The possible reasons for profound differences in the effects of this spectrum of steroidal compounds between tumor cell lines and normal stimulated and non-stimulated PBMCs are discussed. The molecular mechanisms for apoptotic events in HeLa cell line are suggested. The guidelines for further research are underlined.

SARS-CoV genome polymorphism: a bioinformatics study.

A dataset of 103 SARS-CoV isolates (101 human patients and 2 palm civets) was investigated on different aspects of genome polymorphism and isolate classification. The number and the distribution of single nucleotide variations (SNVs) and insertions and deletions, with respect to a "profile", were determined and discussed ("profile" being a sequence containing the most represented letter per position). Distribution of substitution categories per codon positions, as well as synonymous and non-synonymous substitutions in coding regions of annotated isolates, was determined, along with amino acid (a.a.) property changes. Similar analysis was performed for the spike (S) protein in all the isolates (55 of them being predicted for the first time). The ratio Ka/Ks confirmed that the S gene was subjected to the Darwinian selection during virus transmission from animals to humans. Isolates from the dataset were classified according to genome polymorphism and genotypes. Genome polymorphism yields to two groups, one with a small number of SNVs and another with a large number of SNVs, with up to four subgroups with respect to insertions and deletions. We identified three basic nine-locus genotypes: TTTT/TTCGG, CGCC/TTCAT, and TGCC/TTCGT, with four subgenotypes. Both classifications proposed are in accordance with the new insights into possible epidemiological spread, both in space and time.