Elucidating fungal Rigidoporus species FMD21 lignin-modifying enzyme genes and 2,3,7,8-tetrachlorodibenzo-p-dioxin degradation by laccase isozymes.

White-rot fungus Rigidoporus sp. FMD21 is a lignin-modifying enzyme producing fungus that can degrade dioxin. Extracellular enzymes from FMD21 include laccase and manganese peroxidase which are promising enzymes for myco-remediation because of their wide substrate specificity and mild catalysis conditions. The FMD21 genome was sequenced using Ion Torrent technology and consists of 38.98 Mbps with a GC content of 47.4 %. Gene prediction using Augustus with Basidiomycota reference setting resulted in 8245 genes. Functional gene annotations were carried out by using several programs and databases. We focused on laccase and ligninolytic peroxidase genes, which are most likely involved in the degradation of aromatic pollutants. The genome of FMD21 contains 12 predicted laccase genes (10 out of 12 predicted as full length) and 13 putative ligninolytic peroxidases which were annotated as MnP or versatile peroxidases. Four predicted laccases showed a higher than 65 % binding chance to 2,3,7,8-TCDD with the highest at 72 % in in silico docking analysis. Heterologous expressed laccases showed activity towards three tested substrates included ABTS, guaiacol and 2,6-DMP. ABTS displayed two-stage oxidation which differed from natural FMD21 laccases. 2,3,7,8-TCDD was degraded by 50 % after two weeks of enzymatic treatment by three out of five laccase isozymes which were natural laccases secreted by FMD21. In this study, we provide direct evidence for the 2,3,7,8-TCDD biodegradation capability of fungal laccases.

Characterization of 2,3,7,8-tetrachlorodibenzo-p-dioxin biodegradation by extracellular lignin-modifying enzymes from ligninolytic fungus.

Ligninolytic fungi secrete extracellular lignin-modifying enzymes (LME) that degrade plant polymers for fungal nutrition but that are, because of their broad substrate specificity, also applicable for the degradation of many hazardous pollutants. Laccase is one of the most well characterized LME and is involved in the removal and degradation of recalcitrant aromatic compounds with or without the assistance of laccase-mediators. The Ligninolytic fungus Rigidoporus sp. FMD21 can degrade 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) with a half-life of 6.2 days. Using Rigidoporus sp. FMD21 crude extracellular enzyme extract (ExE) that mainly consisted of laccase, 77.4% of 2,3,7,8-TCDD was degraded within 36 days. The degradation rate did not depend on the 2,3,7,8-TCDD concentration in the tested range between 0.005 and 0.5 pgTEQ/µL. 2,3,7,8-TCDD was analysed by DR-CALUX® bioassay and the degradation was confirmed by GC-HRMS. In this study, we found evidence for cleavage of the diaryl ether bond in the 2,3,7,8-TCDD molecule and here we propose a new degradation mechanism in which 3,4-dichlorophenol is the main metabolite of 2,3,7,8-TCDD degradation by FMD21's ExE. Six laccase-mediators were tested. Three of them 1-hydroxybenzotriazole (HBT), syringaldehyde (Syr) and violuric acid (Vio) showed an equipotent added effect on 2,3,7,8-TCDD degradation by ExE, however only in case of Vio a level of significance was reached. The others showed no effect or negatively impacted degradation. In conclusion, we have shown that Rigidoporus sp. FMD21 produces extracellular enzymes, mainly laccases that apparently are able to degrade the highly recalcitrant and most toxic 2,3,7,8-congener of TCDD via diaryl bond cleavage into 3,4-dichlorophenol.

Activation of Evi1 inhibits cell cycle progression and differentiation of hematopoietic progenitor cells.

The transcription factor Evi1 has an outstanding role in the formation and transformation of hematopoietic cells. Its activation by chromosomal rearrangement induces a myelodysplastic syndrome with progression to acute myeloid leukemia of poor prognosis. Similarly, retroviral insertion-mediated upregulation confers a competitive advantage to transplanted hematopoietic cells, triggering clonal dominance or even leukemia. To study the molecular and functional response of primary murine hematopoietic progenitor cells to the activation of Evi1, we established an inducible lentiviral expression system. EVI1 had a biphasic effect with initial growth inhibition and retarded myeloid differentiation linked to enhanced survival of myeloblasts in long-term cultures. Gene expression microarray analysis revealed that within 24 h EVI1 upregulated 'stemness' genes characteristic for long-term hematopoietic stem cells (Aldh1a1, Abca1, Cdkn1b, Cdkn1c, Epcam, among others) but downregulated genes involved in DNA replication (Cyclins and their kinases, among others) and DNA repair (including Brca1, Brca2, Rad51). Cell cycle analysis demonstrated EVI1's anti-proliferative effect to be strictly dose-dependent with accumulation of cells in G0/G1, but preservation of a small fraction of long-term proliferating cells. Although confined to cultured cells, our study contributes to new hypotheses addressing the mechanisms and molecular targets involved in preleukemic clonal dominance or leukemic transformation by Evi1.

Meta-analysis of clodronate and breast cancer survival.

Clinical trials have reported conflicting results on whether oral clodronate therapy improves survival in breast cancer patients. This study was undertaken to evaluate further the effect of oral clodronate therapy on overall survival, bone metastasis-free survival and nonskeletal metastasis-free survival among breast cancer patients. An extensive literature search was undertaken for the period 1966 to July 2006 to identify clinical trials examining survival in breast cancer patients who received 2 or 3 years of oral clodronate therapy at 1600 mg day(-1) compared with those without therapy. Meta-analyses were carried out separately for patients diagnosed with advanced breast cancer and early breast cancer. Our meta-analysis found no evidence of any statistically significant difference in overall survival, bone metastasis-free survival or nonskeletal metastasis-free survival in advanced breast cancer patients receiving clodronate therapy or early breast cancer patients receiving adjuvant clodronate treatment compared with those who did not receive any active treatment.