The roles of WRN and BLM RecQ helicases in the Alternative Lengthening of Telomeres

Approximately 10% of all cancers, but a higher proportion of sarcomas, use the recombination-based alternative lengthening of telomeres (ALT) to maintain telomeres. Two RecQ helicase genes, BLM and WRN, play important roles in homologous recombination repair and they have been implicated in telomeric recombination activity, but their precise roles in ALT are unclear. Using analysis of sequence variation present in human telomeres, we found that a WRN– ALT+ cell line lacks the class of complex telomere mutations attributed to inter-telomeric recombination in other ALT+ cell lines. This suggests that WRN facilitates inter-telomeric recombination when there are sequence differences between the donor and recipient molecules or that sister-telomere interactions are suppressed in the presence of WRN and this promotes inter-telomeric recombination. Depleting BLM in the WRN– ALT+ cell line increased the mutation frequency at telomeres and at the MS32 minisatellite, which is a marker of ALT. The absence of complex telomere mutations persisted in BLM-depleted clones, and there was a clear increase in sequence homogenization across the telomere and MS32 repeat arrays. These data indicate that BLM suppresses unequal sister chromatid interactions that result in excessive homogenization at MS32 and at telomeres in ALT+ cells.     

Lipids of Pinus halepensis pollen

The total lipids of Pinus halepensis pollen were separated into individual classes of neutral and polar lipids and the components of each class were identified and determined quantitatively. Free fatty acids, waxes and triacylglycerols were found as the main constituents of neutral lipids and phosphatidylcholine and phosphatidylethanolamine of polar lipids. Glycerylether derivatives were detected in neutral and polar lipid fractions. Free and esterified volatile fatty acids were also found in pollen and its neutral lipid fraction.     

Breast cancer diagnostics based on extracellular DNA and RNA circulating in blood

Extracellular DNA and RNA were extracted from blood plasma and cell surface-bound fractions of healthy women and patients with fibroadenoma and breast cancer. Frequency of methylation of RASSF1A, Cyclin D2, and RARβ2 genes was detected in the extracellular DNA using methylation-specific PCR. Methylation of at least one of these genes was found in plasma of 13% patients with nonmalignant breast fibroadenoma and in 60% of breast cancer patients. Employment cell-surface bound DNA as the substrate for PCR increased the detection frequency of gene methylation up to 87% in patients with fibroadenoma and 95% in breast cancer patients. In clinically healthy women the methylation markers have not been found in extracellular DNA. GAPDH, RASSF8, Ki-67 mRNAs, and 18S rRNA copies were quantified using RT-qPCR of extracellular RNA circulating in blood of patients with breast tumors and healthy controls. The major part of blood extracellular RNA is associated with cell surface. ROC analysis has shown that differences in concentrations 18S RNA, RASSF8, and Ki-67 mRNAs in blood plasma are highly sensitive and specific in discrimination of benign and malignant breast tumors. Thus, analysis of methylated forms of tumor suppressor genes in blood extracellular and quantification of specific extracellular RNA circulating in blood plasma may detect mammary gland tumors and discriminate malignant and benign neoplasms.     

Investigation of rifampicin resistance mechanisms in Brucella abortus using MS-driven comparative proteomics

Mutations in the rpoB gene have already been shown to contribute to rifampicin resistance in many bacterial strains including Brucella species. Resistance against this antibiotic easily occurs and resistant strains have already been detected in human samples. We here present the first research project that combines proteomic, genomic, and microbiological analysis to investigate rifampicin resistance in an in vitro developed rifampicin resistant strain of Brucella abortus 2308. In silico analysis of the rpoB gene was performed and several antibiotics used in the therapy of Brucellosis were used for cross resistance testing. The proteomic profiles were examined and compared using MS-driven comparative proteomics. The resistant strain contained an already described mutation in the rpoB gene, V154F. A correlation between rifampicin resistance and reduced susceptibility on trimethoprim/sulfamethoxazole was detected by E-test and supported by the proteomics results. Using 12?836 MS/MS spectra we identified 6753 peptides corresponding to 456 proteins. The resistant strain presented 39 differentially regulated proteins most of which are involved in various metabolic pathways. Results from our research suggest that rifampicin resistance in Brucella mostly involves mutations in the rpoB gene, excitation of several metabolic processes, and perhaps the use of the already existing secretion mechanisms at a more efficient level.     

PATTERNS OF OSSIFICATION IN SOUTHERN VERSUS NORTHERN PLACENTAL MAMMALS

Consensus on placental mammal phylogeny is fairly recent compared to that for vertebrates as a whole. A stable phylogenetic hypothesis enables investigation into the possibility that placental clades differ from one another in terms of their development. Here, we focus on the sequence of skeletal ossification as a possible source of developmental distinctiveness in “northern” (Laurasiatheria and Euarchontoglires) versus “southern” (Afrotheria and Xenarthra) placental clades. We contribute data on cranial and postcranial ossification events during growth in Afrotheria, including elephants, hyraxes, golden moles, tenrecs, sengis, and aardvarks. We use three different techniques to quantify sequence heterochrony: continuous method, sequence‐ANOVA (analysis of variance) and event‐paring/Parsimov. We show that afrotherians significantly differ from other placentals by an early ossification of the orbitosphenoid and caudal vertebrae. Our analysis also suggests that both southern placental groups show a greater degree of developmental variability; however, they rarely seem to vary in the same direction, especially regarding the shifts that differ statistically. The latter observation is inconsistent with the Atlantogenata hypothesis in which afrotherians are considered as the sister clade of xenarthrans. Interestingly, ancestral nodes for Laurasiatheria and Euarchontoglires show very similar trends and our results suggest that developmental homogeneity in some ossification sequences may be restricted to northern placental mammals (Boreoeutheria).     

Synthesis and biological activity of water-soluble maleimide derivatives of the anticancer drug carboplatin designed as albumin-binding prodrugs

Four platinum (II) complexes (13-16) were synthesized by reacting either [Pt trans-DACH](NO(3))(2) with a 6-maleimidocaproic acid, a 15-maleimido-4,7,10,13-tetroxapentadecanoic acid, and a 6-maleimido-4-oxacaproic ester derivative of cyclobutane-1,1-dicarboxylic acid (CDBA) or [Pt(NH(3))(2)](NO(3))(2) with a 6-maleimido-4-oxacaproic ester derivative of CBDA. Both complexes containing the 6-maleimido-4-oxacaproic ester (15, 16) showed good water solubility (>/=8 mg/mL) and CE experiments revealed rapid binding to human serum albumin and the formation of biadducts with dGMP and dAMP. In the MaTu xenograft model in nude mice, both complexes showed an improved antitumor effect at their maximum tolerated dose (2 x 50 mg/kg carboplatin equivalents) compared to therapy with carboplatin at equimolar dose or at its optimal dose (2 x 75 mg/kg).