Arsenic trioxide and ascorbic acid interfere with the BCL2 family genes in patients with myelodysplastic syndromes: an ex-vivo study

ABSTRACT: BACKGROUND: Arsenic Trioxide (ATO) is effective in about 20% of patients with myelodysplasia (MDS); its mechanisms of action have already been evaluated in vitro, but the in vivo activity is still not fully understood. Since ATO induces apoptosis in in vitro models, we compared the expression of 93 apoptotic genes in patients' bone marrow before and after ATO treatment. For this analysis, we selected 12 patients affected by MDS who received ATO in combination with Ascorbic Acid in the context of the Italian clinical trial NCT00803530, EudracT Number 2005-001321-28. METHODS: Real-time PCR quantitative assays for genes involved in apoptosis were performed using TaqMan(R) Assays in 384-Well Microfluidic Cards "TaqMan(R) Human Apoptosis Array". Quantitative RT-PCR for expression of EVI1 and WT1 genes was also performed. Gene expression values (Ct) were normalized to the median expression of 3 housekeeping genes present in the card (18S, ACTB and GAPDH). RESULTS: ATO treatment induced up-regulation of some pro-apoptotic genes, such as HRK, BAK1, CASPASE-5, BAD, TNFRSF1A, and BCL2L14 and down-regulation of ICEBERG. In the majority of cases with stable disease, apoptotic gene expression profile did not change, whereas in cases with advanced MDS more frequently pro-apoptotic genes were upregulated. Two patients achieved a major response: in the patient with refractory anemia the treatment down-regulated 69% of the pro-apoptotic genes, whereas 91% of the pro-apoptotic genes were up-regulated in the patient affected by refractory anemia with excess of blasts-1. Responsive patients showed a higher induction of BAD than those with stable disease. Finally, WT1 gene expression was down-regulated by the treatment in responsive cases. CONCLUSIONS: These results represent the basis for a possible association of ATO with other biological compounds able to modify the apoptotic pathways, such as inhibitors of the BCL2 family.     

Metal biosorption in lignocellulosic biofuel biorefinery effluent: an initial step towards sustainability of water resources

Biosorption of metals by microorganisms is a promising technology to remove accumulated non-process elements in highly recycled biorefinery process water. Removal of these elements would enable greater water reuse and reduce the environmental impact of effluent discharge. A model lignocellulosic ethanol biorefinery wastewater was created based on pulp mill effluent. This generated a wastewater with an environmentally realistic high loading of dissolved natural organic matter (900?mg/l), a potentially important factor influencing metal biosorption. Analysis of feedstock and pulp mill effluent indicated that Mn and Zn are likely to be problematic in highly recycled lignocellulosic ethanol biorefinery process water. Therefore, the growth of several bacteria and fungi from existing collections, and some isolated from pulp mill effluent were tested in the model wastewater spiked with Mn and Zn (0.2?mM). Wastewater isolates grew the best in the wastewater. Metal uptake varied by species and was much greater for Zn than Mn. A bacterium, Novosphingobium nitrogenifigens Y88(T), removed the most metal per unit biomass, 35 and 17?mg?Mn/g. No other organism tested decreased the Mn concentration. A yeast, Candida tropicalis, produced the most biomass and removed the most total metal (38?% of Zn), while uptake per unit biomass was 24?mg?Zn/g. These results indicate that microorganisms can remove significant amounts of metals in wastewater with high concentrations of dissolved natural organic matter. Metal sorption by autochthonous microorganisms in an anaerobic bioreactor may be able to extend water reuse and therefore lower the water consumption of future biorefineries.     

Protein nitration as footprint of oxidative stress-related nitric oxide signaling pathways in developing Ciona intestinalis

Developmental processes in the ascidian Ciona intestinalis depend on a complex interplay of events including, during metamorphosis, a caspase-dependent apoptosis which is regulated by the nitric oxide (NO)-cGMP signaling pathway. Herein we disclose an alternate NO-mediated signaling pathway during Ciona development which appears to be critically dependent on local redox control. Evidence in support of this conclusion includes: (a) inhibitors of NO synthase (NOS) and scavengers of NO-derived nitrating agents markedly decrease the rate of Ciona metamorphosis; (b) an NO donor or peroxynitrite caused an opposite effect; (c) increased protein nitration is observed at larva stage. Integrated proteomic and immunochemical methodologies identified nitrated tyrosine residues in ERK and snail. Overall, these results point to protein nitration as a hitherto overlooked NO-dependent regulatory mechanism in Ciona which is specifically triggered by elevated ROS production during developmental processes.     

Sequencing intractable DNA to close microbial genomes

Advancement in high throughput DNA sequencing technologies has supported a rapid proliferation of microbial genome sequencing projects, providing the genetic blueprint for in-depth studies. Oftentimes, difficult to sequence regions in microbial genomes are ruled "intractable" resulting in a growing number of genomes with sequence gaps deposited in databases. A procedure was developed to sequence such problematic regions in the "non-contiguous finished" Desulfovibrio desulfuricans ND132 genome (6 intractable gaps) and the Desulfovibrio africanus genome (1 intractable gap). The polynucleotides surrounding each gap formed GC rich secondary structures making the regions refractory to amplification and sequencing. Strand-displacing DNA polymerases used in concert with a novel ramped PCR extension cycle supported amplification and closure of all gap regions in both genomes. The developed procedures support accurate gene annotation, and provide a step-wise method that reduces the effort required for genome finishing.     

Host genetic and environmental effects on mouse intestinal microbiota

The mammalian gut harbors complex and variable microbial communities, across both host phylogenetic space and conspecific individuals. A synergy of host genetic and environmental factors shape these communities and account for their variability, but their individual contributions and the selective pressures involved are still not well understood. We employed barcoded pyrosequencing of V1-2 and V4 regions of bacterial small subunit ribosomal RNA genes to characterize the effects of host genetics and environment on cecum assemblages in 10 genetically distinct, inbred mouse strains. Eight of these strains are the foundation of the Collaborative Cross (CC), a panel of mice derived from a genetically diverse set of inbred founder strains, designed specifically for complex trait analysis. Diversity of gut microbiota was characterized by complementing phylogenetic and distance-based, sequence-clustering approaches. Significant correlations were found between the mouse strains and their gut microbiota, reflected by distinct bacterial communities. Cohabitation and litter had a reduced, although detectable effect, and the microbiota response to these factors varied by strain. We identified bacterial phylotypes that appear to be discriminative and strain-specific to each mouse line used. Cohabitation of different strains of mice revealed an interaction of host genetic and environmental factors in shaping gut bacterial consortia, in which bacterial communities became more similar but retained strain specificity. This study provides a baseline analysis of intestinal bacterial communities in the eight CC progenitor strains and will be linked to integrated host genotype, phenotype and microbiota research on the resulting CC panel.     

Synthesis and proteomic activity evaluation of a new isotope-coded affinity tagging (ICAT) reagent

During recent years, quantitative proteome profiling has taken advantage of incorporating the traditional stable isotope dilution analysis into global scale or discovery-based proteomic experiments that use mass spectrometers as detectors to allow the pairwise study of differently expressed proteins. Quantitative protein analysis by means of the isotope-coded affinity tag (ICAT) method and tandem mass spectrometry (MS) enables the pairwise comparison of protein expression levels in biological samples. Herein, a modified ICAT reagent, named BAA-ICAT (beta-alanine-arm-ICAT) in which the polyether linker is replaced by a more water-soluble polyamide one, was investigated.     

Evaluation of PCR-Generated Chimeras, Mutations, and Heteroduplexes with 16S rRNA Gene-Based Cloning

To evaluate PCR-generated artifacts (i.e., chimeras, mutations, and heteroduplexes) with the 16S ribosomal DNA (rDNA)-based cloning approach, a model community of four species was constructed from alpha, beta, and gamma subdivisions of the division Proteobacteria as well as gram-positive bacterium, all of which could be distinguished by HhaI restriction digestion patterns. The overall PCR artifacts were significantly different among the three Taq DNA polymerases examined: 20% for Z-Taq, with the highest processitivity; 15% for LA-Taq, with the highest fidelity and intermediate processitivity; and 7% for the conventionally used DNA polymerase, AmpliTaq. In contrast to the theoretical prediction, the frequency of chimeras for both Z-Taq (8.7%) and LA-Taq (6.2%) was higher than that for AmpliTaq (2.5%). The frequencies of chimeras and of heteroduplexes for Z-Taq were almost three times higher than those of AmpliTaq. The total PCR artifacts increased as PCR cycles and template concentrations increased and decreased as elongation time increased. Generally the frequency of chimeras was lower than that of mutations but higher than that of heteroduplexes. The total PCR artifacts as well as the frequency of heteroduplexes increased as the species diversity increased. PCR artifacts were significantly reduced by using AmpliTaq and fewer PCR cycles (fewer than 20 cycles), and the heteroduplexes could be effectively removed from PCR products prior to cloning by polyacrylamide gel purification or T7 endonuclease I digestion. Based upon these results, an optimal approach is proposed to minimize PCR artifacts in 16S rDNA-based microbial community studies.     

Molecular Constructions (Superstructures) with Adjustable Properties Based on Double-Stranded Nucleic Acids

We describe formation of a molecular construction that consists of double-stranded molecules of nucleic acids (or synthetic polynucleotides) located at a distance of 30–50 Å in the spatial structure of particles of their cholesteric liquid-crystalline dispersion and crosslinked by polymeric chelate bridges. The resulting superstructure, which possesses peculiar physicochemical properties, can be used as an integral biosensor whose properties depend on temperature, the presence of chemical or biologically active compounds of different nature, etc.