Successful heterologous expression of a novel chitinase identified by sequence analyses of the metagenome from a chitin-enriched soil sample.

Chitin and its derivative chitosan are abundant natural polysaccharides with many potential industrial applications. Metagenomic analysis of chitin-enriched soil samples using the Roche Genome Sequencer FLX platform led to the identification of several novel genes for chitin and chitosan modifying enzymes (CCMEs) which may be used to produce novel chitosans. The sequencing approach yielded 2,281,090 reads with an average length of 378 bp amounting to a total sequence information of approximately 851 Mb. Assembly of the obtained sequences comprised 699,710 reads representing 30.68% of all reads. A total of 6625 contigs larger than 500 bp containing 16,289 predicted genes are included in the assembly. Taxonomic profiling of the indigenous microbial community by applying the software CARMA revealed that 96.1% of the reads were of bacterial origin including 17% assigned to the family Xanthomonadaceae. Several putative genes encoding CCMEs were identified by comparison against the GenBank database, inclusive a full-length chitinase gene which was codon optimized for Escherichia coli and heterologously synthesized as a Strep-tagged protein in E. coli Rosetta 2 using the pET vector system. Approximately 5mg of the novel active chitinase was purified as demonstrated by dot assay analysis using glycol chitin as a substrate. Next generation metagenomic sequencing, thus, emerges as a new and powerful tool for the identification of potentially novel biocatalysts of biotechnological value.

Affinity protein-based FRET tools for cellular tracking of chitosan nanoparticles and determination of the polymer degree of acetylation.

Chitosan (CS) is a family of linear polysaccharides with diverse applications in medicine, agriculture, and industry. Its bioactive properties are determined by parameters such as the degree of acetylation (DA), but current techniques to measure the DA are laborious and require large amounts of substrate and sophisticated equipment. It is also challenging to monitor the fate of chitosan-based nanoparticles (CS-NPs) in vitro because current tools cannot measure their enzymatic or chemical degradation. We have developed a method based on the Förster resonance energy transfer (FRET) that occurs between two independent fluorescent proteins fused to a CS-binding domain, who interact with CS polymers or CS-NPs. We used this approach to calibrate a simple and rapid analytical method that can determine the DA of CS substrates. We showed unequivocally that FRET occurs on the surface of CS-NPs and that the FRET signal is quenched by enzymatic degradation of the CS substrate. Finally, we provide in vitro proof-of-concept that these approaches can be used to label CS-NPs and colocalize them following their interactions with mammalian cells.

Analysis of thrombopoietin receptor (c-mpl) mRNA expression in de novo acute myeloid leukemia.

Expression of the thrombopoietin receptor, c-mpl, has been recently suggested to represent an adverse prognostic factor in myelodysplasia and acute myeloid leukemia (AML). To further evaluate this putative correlation, we assessed the c-mpl mRNA expression in blast samples of 53 AML patients. Overall, c-mpl mRNA expression was observed in 27 (51%) patients. No significant difference between c-mpl(+) and c-mpl(-) patients was found with respect to established prognostic factors such as age (50 vs. 53 years) or karyotype, whereas a significant correlation was observed between c-mpl and CD34 expression (P = 0.026). Among 40 patients who completed standard-/high-dose cytarabine-containing induction/consolidation treatment and were evaluable for treatment response, a higher complete remission (CR) rate was achieved in c-mpl- than in c-mpl(+) patients (95 vs. 68%; P = 0.026). Upon multivariate analysis, this relationship was independent from CD34 expression. CR duration was not significantly longer in c-mpl(-) than in c-mpl(+) patients (median: 14 vs. 10 months, P = 0.262). In conclusion, our data strongly support the previously suggested notion that c-mpl expression is of prognostic relevance for CR induction in de novo AML patients, and suggest determination of c-mpl expression within larger prospective studies in the attempt to develop risk-adapted AML treatment strategies.