Characterization of a new trabectedin-resistant myxoid liposarcoma cell line that shows collateral sensitivity to methylating agents.

Myxoid Liposarcomas (MLS), characterized by the expression of FUS-CHOP fusion gene are clinically very sensitive to the DNA binding antitumor agent, trabectedin. However, resistance eventually occurs, preventing disease eradication. To investigate the mechanisms of resistance, a trabectedin resistant cell line, 402-91/ET, was developed. The resistance to trabectedin was not related to the expression of MDR related proteins, uptake/efflux of trabectedin or GSH levels that were similar in parental and resistant cells. The 402-91/ET cells were hypersensitive to UV light because of a nucleotide excision repair defect: XPG complementation decreased sensitivity to UV rays, but only partially to trabectedin. 402-91/ET cells showed collateral sensitivity to temozolomide due to the lack of O(6) -methylguanine-DNA-methyltransferase (MGMT) activity, related to the hypermethylation of MGMT promoter. In 402-91 cells chromatin immunoprecipitation (ChIP) assays showed that FUS-CHOP was bound to the PTX3 and FN1 gene promoters, as previously described, and trabectedin caused FUS-CHOP detachment from DNA. Here we report that, in contrast, in 402-91/ET cells, FUS-CHOP was not bound to these promoters. Differences in the modulation of transcription of genes involved in different pathways including signal transduction, apoptosis and stress response between the two cell lines were found. Trabectedin activates the transcription of genes involved in the adipogenic-program such as c/EBPα and ß, in 402-91 but not in 402-91/ET cell lines. The collateral sensitivity of 402-91/ET to temozolomide provides the rationale to investigate the potential use of methylating agents in MLS patients resistant to trabectedin.

A genomic view of the microbiome of coral reef demosponges.

Sponges underpin the productivity of coral reefs, yet few of their microbial symbionts have been functionally characterised. Here we present an analysis of ~1200 metagenome-assembled genomes (MAGs) spanning seven sponge species and 25 microbial phyla. Compared to MAGs derived from reef seawater, sponge-associated MAGs were enriched in glycosyl hydrolases targeting components of sponge tissue, coral mucus and macroalgae, revealing a critical role for sponge symbionts in cycling reef organic matter. Further, visualisation of the distribution of these genes amongst symbiont taxa uncovered functional guilds for reef organic matter degradation. Genes for the utilisation of sialic acids and glycosaminoglycans present in sponge tissue were found in specific microbial lineages that also encoded genes for attachment to sponge-derived fibronectins and cadherins, suggesting these lineages can utilise specific structural elements of sponge tissue. Further, genes encoding CRISPR and restriction-modification systems used in defence against mobile genetic elements were enriched in sponge symbionts, along with eukaryote-like gene motifs thought to be involved in maintaining host association. Finally, we provide evidence that many of these sponge-enriched genes are laterally transferred between microbial taxa, suggesting they confer a selective advantage within the sponge niche and therefore play a critical role in host ecology and evolution.

Simulated future conditions of ocean warming and acidification disrupt the microbiome of the calcifying foraminifera Marginopora vertebralis across life stages.

Foraminifera host diverse microbial communities that can shift in response to changing environmental conditions. To characterize climate change impacts on the foraminifera microbiome across life stages, we exposed adult Marginopora vertebralis (Large Benthic Foraminifera) to pCO2 and temperature scenarios representing present-day, 2050 and 2100 levels and raised juveniles under present-day and 2050 conditions. While treatment condition had no significant effect on the seawater microbial communities, exposure to future scenarios significantly altered both adult and juvenile microbiomes. In adults, divergence between present-day and 2050 or 2100 conditions was primarily driven by a reduced relative abundance of Oxyphotobacteria under elevated temperature and pCO2 . In juveniles, the microbial shift predominantly resulted from changes in the proportion of Proteobacteria. Indicator species analysis identified numerous treatment-specific indicator taxa, most of which were indicative of present-day conditions. Oxyphotobacteria, previously reported as putative symbionts of foraminifera, were indicative of present-day and 2050 conditions in adults, but of present-day conditions only in juveniles. Overall, we show that the sensitivity of the M. vertebralis microbiome to climate change scenarios extends to both life stages and primarily correlates with declines in Oxyphotobacteria and shifts in Proteobacteria under elevated temperature and pCO2 .

Field-assisted paper spray mass spectrometry for therapeutic drug monitoring: 1. the case of imatinib in plasma.

The field-assisted paper spray (FAPS) - mass spectrometric method has been employed to quantify the imatinib (IMT) plasma levels in treated patients. The quantitative measurements have been performed on the collisionally generated fragment at m/z 394 of the protonated molecules of IMT and deuterated IMT (d3 -IMT), used as internal standard. The FAPS-tandem mass spectrometry (MS/MS) method exhibits some limitations, because of the high number of operative parameters that need to be carefully controlled. For this aim, papers of different geometry, thickness, and porosity were tested. To obtain a more focalized and intense electrical field, a stainless steel needle was mounted axially and placed at 4 kV voltage. The variability observed in the measurements was ascribed either to the inter-individual variability (e.g. the concomitant presence of other compounds such as proteins, lipids, drugs and/or salts in the plasma of different patients) or to the uncontrollable variables in the instrumental set-up (e.g. sample deposition, changes in paper spray conditions). Furthermore, the manual sample deposition and solvent dripping strongly affects the measure reproducibility. Despite this, it is interesting to observe that, once applied in blind on 24 real plasma samples, FAPS-MS/MS led to results analogous to those obtained by the well-consolidated liquid chromatography-MS/MS, even if the mean coefficient of variation % (CV%) values of 20.4% and 2.6% were observed for the two methods, respectively. In conclusion, despite CV values are relatively high, it is worth noting that the FAPS-MS/MS method is much more straightforward, rapid and economical than the liquid chromatography-MS/MS one, and it appears therefore very promising for applications where a high precision is not always a required task, as e.g. in some cases of therapeutic drug monitoring. Copyright © 2017 John Wiley & Sons, Ltd.

The effects of metal ions on the structure and stability of the DNA gyrase B protein.

The effects of mono- and divalent metal ions on the DNA gyrase B subunit, on its 43 kDa and 47 kDa domains, and on two mutants in the Toprim domain (D498A and D500C) were investigated by means of circular dichroism and protein melting experiments. Both types of metal ion, with the notable exception of Mn2+, did not affect the conformational properties of the enzyme subunit at room temperature, but were able to produce selective and differential effects on protein stability. In particular, monovalent (K+) ions increased the stability of the gyrase B structure, whereas destabilising effects were most prominent using Mn2+ as the metal ion. Ca2+ and Mg2+ produced comparable changes in the gyrase B melting profile. Additionally, we found that monovalent (K+) ions were more effective in the 43 kDa N-terminal domain where ATP binding occurs, whereas divalent ions caused large modifications in the conformational stability of the 47 kDa C-terminal domain. Our results on gyrase B mutants indicate that D498 interacts with Mn2+, whereas it has little effect on the binding of the other ions tested. A D500C mutation, in contrast, effectively impairs Mg2+ affinity, suggesting effective contacts between this ion and D500 in the wild-type enzyme. Hence, the sites of metal ion complexation within the Toprim domain are modulated by the nature of the ion species. These results suggest a double role played by metal ions in the catalytic steps involving DNA gyrase B. One has to do with direct involvement of cations complexed to the Toprim domain in the DNA cutting-rejoining process, the other, until now overlooked, is connected to the dramatic changes in protein flexibility produced by ion binding, which reduces the energy required for the huge conformational changes essential for the catalytic cycle to occur.

Modulation of gene transcription by natural products--a viable anticancer strategy.

Drug design based on the structure of specific enzymes playing a role in carcinogenesis, e.g. tyrosine kinases, has been successful at identifying novel effective anticancer drugs. In contrast, no success has been achieved in drug design attempts, in which transcription factors or DNA-transcription factor complexes involved in the pathogenesis of human neoplasms were targeted. This failure is likely to be due to the fact that the mechanism of transcription regulation is probably too complex and still too inadequately understood to be a suitable target for drug design. It seems plausible that the high selectivity of some human tumors to some DNA-interactive anticancer drugs, e.g. cisplatin, is related to an effect on the transcription of genes that are crucial for those tumors. In this article we propose that some natural products have evolutionarily evolved to exert highly specialized functions, including modulation of the transcriptional regulation of specific genes. We discuss in detail the marine natural product Yondelis (Trabectedin, ET-743) that is effective against some soft tissue sarcoma, possibly because it interferes with the aberrant transcription mechanism in these tumors. In addition we highlight the existing evidence that many different natural products are effective inhibitors of NF-kB, a transcription factor that plays a crucial role in inflammation and cancer, indicating that some of these compounds might possess antitumor properties. We propose that large-scale characterization of natural products acting as potential modulators of gene transcription is a realistic and attractive approach to discover compounds therapeutically effective against neoplastic diseases characterized by specific aberrations of transcriptional regulation.

Pharmacokinetics and metabolism in mice of IDN 5390 (13-(N-Boc-3-i-butylisoserinoyl)-C-7,8-seco-10-deacetylbaccatin III), a new oral c-seco-taxane derivative with antiangiogenic property effective on paclitaxel-resistant tumors.

IDN 5390 (13-(N-Boc-3-i-butylisoserinoyl)-C-7,8-seco-10-deacetylbaccatin III) is a new taxane, derived from 7,8-C-seco-10-deacetylbaccatin, selected for its ability to inhibit angiogenesis, mainly by acting on endothelial cell motility, and for its selective activity on class III beta-tubulin. In vivo, IDN 5390 shows activity against paclitaxel-sensitive and -resistant tumors when administered on a prolonged, continuous dosage schedule. We studied the pharmacokinetics and bioavailabilty of the drug in mice after single and repeated oral treatment. IDN 5390 was rapidly absorbed after oral administration, with good bioavailability (43%). After intravenous injection, it was extensively distributed in tissue, mainly the liver, kidney, and heart, with low but persistent levels in brain. The kinetics appear dose-dependent with a clearance of 2.6, 1.4, and 0.9 l/kg at, respectively, 60, 90, and 120 mg/kg, and a half-life 24, 36, and 54 min. After prolonged daily oral doses given for 2 weeks, we found that there was a decrease in drug availability; i.e., the area under the concentration-time curve value after p.o. daily administration on day 14 was 2-fold lower than that on day 1. Metabolism plays a major role in elimination of the drug, and at least 12 metabolites were identified in feces and urine. The percentage excreted as metabolites after an oral dose (42%) was higher than that after the i.v. dose (33%), suggesting a first-pass effect. Four metabolites were found in plasma at detectable levels; one of them, with restored taxane scaffold, is a species 3 times more potent than IDN 5390, possibly contributing to the observed anti-tumor activity.