Degradation of pesticides by the ligninolytic enzyme Laccase : optimisation of in vitro conditions, immobilisation and screening for natural mediators

Pesticides are widely used in many industries but the majority reaches non-target organisms or locations through point or diffuse sources. Understanding conditions for their degradation is therefore important. The degradation of glyphosate, its metabolite AMPA and isoproturon using the ligninolytic enzyme laccase was studied. Optimisation of in vitro conditions were tested with findings indicating that factors such as altering pH and the concentrations of both manganese and redox mediators can impact degradation giving insight into optimal conditions. A method of encapsulation was used showing it is possible to immobilise laccase suggesting a possibility of its suitability as a co-formulation agent in pesticide applications. The immobilised laccase was applied in a laboratory scale experiment to investigate degradation of glyphosate and AMPA in soil and sand. The findings showed an apparent ability of the encapsulated laccase to be liberated and have an effect on glyphosate degradation, although much work still remains in this area. In the final part of this project ligninolytic substrates were screened for natural and easily extractable mediators. Extracts were used to check enzymatic activity and degradation potential. Candidates that showed promising results included extracts from hemp and wheat

Clinical relevance of DNA microarray analyses using archival formalin-fixed paraffin-embedded breast cancer specimens

Abstract Background The ability of gene profiling to predict treatment response and prognosis in breast cancers has been demonstrated in many studies using DNA microarray analyses on RNA from fresh frozen tumor specimens. In certain clinical and research situations, performing such analyses on archival formalin fixed paraffin-embedded (FFPE) surgical specimens would be advantageous as large libraries of such specimens with long-term follow-up data are widely available. However, FFPE tissue processing can cause fragmentation and chemical modifications of the RNA. A number of recent technical advances have been reported to overcome these issues. Our current study evaluates whether or not the technology is ready for clinical applications. Methods A modified RNA extraction method and a recent DNA microarray technique, cDNA-mediated annealing, selection, extension and ligation (DASL, Illumina Inc) were evaluated. The gene profiles generated from FFPE specimens were compared to those obtained from paired fresh fine needle aspiration biopsies (FNAB) of 25 breast cancers of different clinical subtypes (based on ER and Her2/neu status). Selected RNA levels were validated using RT-qPCR, and two public databases were used to demonstrate the prognostic significance of the gene profiles generated from FFPE specimens. Results Compared to FNAB, RNA isolated from FFPE samples was relatively more degraded, nonetheless, over 80% of the RNA samples were deemed suitable for subsequent DASL assay. Despite a higher noise level, a set of genes from FFPE specimens correlated very well with the gene profiles obtained from FNAB, and could differentiate breast cancer subtypes. Expression levels of these genes were validated using RT-qPCR. Finally, for the first time we correlated gene expression profiles from FFPE samples to survival using two independent microarray databases. Specifically, over-expression of ANLN and KIF2C, and under-expression of MAPT strongly correlated with poor outcomes in breast cancer patients. Conclusion We demonstrated that FFPE specimens retained important prognostic information that could be identified using a recent gene profiling technology. Our study supports the use of FFPE specimens for the development and refinement of prognostic gene signatures for breast cancer. Clinical applications of such prognostic gene profiles await future large-scale validation studies