Enhanced C/EBP binding to G·T mismatches facilitates fixation of CpG mutations in cancer and adult stem cells.

Somatic mutations in regulatory sites of human stem cells affect cell identity or cause malignant transformation. By mining the human genome for co-occurrence of mutations and transcription factor binding sites, we show that C/EBP binding sites are strongly enriched with [C > T]G mutations in cancer and adult stem cells, which is of special interest because C/EBPs regulate cell fate and differentiation. In vitro protein-DNA binding assay and structural modeling of the CEBPB-DNA complex show that the G·T mismatch in the core CG dinucleotide strongly enhances affinity of the binding site. We conclude that enhanced binding of C/EBPs shields CpG·TpG mismatches from DNA repair, leading to selective accumulation of [C > T]G mutations and consequent deterioration of the binding sites. This mechanism of targeted mutagenesis highlights the effect of a mutational process on certain regulatory sites and reveals the molecular basis of putative regulatory alterations in stem cells.

Recombinant domains III of Tick-Borne Encephalitis Virus envelope protein in combination with dextran and CpGs induce immune response and partial protectiveness against TBE virus infection in mice.

BACKGROUND: E protein of tick-borne encephalitis virus (TBEV) and other flaviviruses is located on the surface of the viral particle. Domain III of this protein seems to be a promising component of subunit vaccines for prophylaxis of TBE and kits for diagnostics of TBEV. METHODS: Three variants of recombinant TBEV E protein domain III of European, Siberian and Far Eastern subtypes fused with dextran-binding domain of Leuconostoc citreum KM20 were expressed in E. coli and purified. The native structure of domain III was confirmed by ELISA antibody kit and sera of patients with tick-borne encephalitis. Immunogenic and protective properties of the preparation comprising these recombinant proteins immobilized on a dextran carrier with CpG oligonucleotides as an adjuvant were investigated on the mice model. RESULTS: All 3 variants of recombinant proteins immobilized on dextran demonstrate specific interaction with antibodies from the sera of TBE patients. Thus, constructed recombinant proteins seem to be promising for TBE diagnostics. The formulation comprising the 3 variants of recombinant antigens immobilized on dextran and CpG oligonucleotides, induces the production of neutralizing antibodies against TBEV of different subtypes and demonstrates partial protectivity against TBEV infection. CONCLUSIONS: Studied proteins interact with the sera of TBE patients, and, in combination with dextran and CPGs, demonstrate immunogenicity and limited protectivity on mice compared with reference "Tick-E-Vac" vaccine.

Probe-Level Universal Search (PLUS) algorithm for gender differentiation in affymetrix datasets.

Affymetrix microarrays measure gene expression based on the intensity of hybridization of a panel of oligonucleotide probes (probe set) with mRNA. The signals from all probes within a probe set are converted into a single measure that represents the expression value of a gene. This step diminishes the number of independently measured parameters and eliminates from consideration individual "good-working" probes. We propose a new feature selection algorithm (Probe Level Universal Search or PLUS algorithm) for probe-level analysis of gene expression datasets. The algorithm evaluates the intensities of perfect-match Affymetrix probes individually and selects probes that allow one to distinguish two given classes of samples. The algorithm was used to differentiate the samples according to their gender ("gender differentiation"). The universal gender differentiating set of 3' Gene Affymetrix microarray probes was selected; the set consists of 38 probes from XIST gene of X-chromosome and 17 probes from five Y-chromosome genes: RPS4Y1, EIF1A, DDX3Y, JARID1D and USP9Y. The selection procedure based on the probes selected by PLUS algorithm differentiates the sex chromosome karyotype of the sample, reveals samples with incorrect gender labels and samples from patients with hereditary syndromes or cancer-associated chromosome abnormalities.