Personalised proteome analysis by means of protein microarrays made from individual patient samples

DNA sequencing has advanced to a state that permits studying the genomes of individual patients as nearly a matter of routine. Towards analysing a tissue's protein content in a similar manner, we established a method for the production of microarrays that represent full-length proteins as they are encoded in individual specimens, exhibiting the particular variations, such as mutations or splice variations, present in these samples. From total RNA isolates, each transcript is copied to a specific location on the array by an on-chip polymerase elongation reaction, followed by in situ cell-free transcription and translation. These microarrays permit parallel analyses of variations in protein structure and interaction that are specific to particular samples

Micro RNA expression profiles in peripheral blood cells of rats that were experimentally infected with Trypanosoma congolense and different Trypanosoma brucei subspecies

To identify miRNAs whose expression are differentially regulated during trypanosome infections a microarray targeting more than 600 rat miRNA was used to analyze the miRNA expression profiles between uninfected rats and animals infected by Trypanosoma congolense and Trypanosoma brucei s.l. The potential targets of dysregulated miRNAs as well as their biological pathways and functions were predicted using several bioinformatics software tools. Irrespective of the infecting trypanosome species, eight miRNAs (seven up- and one down-regulated) were dysregulated during infections. Moreover, other miRNAs were differentially regulated in rats infected by specific trypanosome species. Functional analyses of differentially regulated miRNAs indicated their involvement in diverse biological processes. Among these, transcription repressor activity, gene expression control as well as protein transporter activity were predominant. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis of dysregulated miRNAs revealed their involvement in several biological pathways and disease conditions. This suggests possible modulation of such pathways following trypanosome infection; for example, the MAPK signaling pathway which is known to play vital roles in apoptosis, innate immune response and response to viral infections was highly affected. Axon guidance was equally highly impacted and may indicate a cross reactivity between pathogen proteins and guidance molecules representing one pathological mechanism as it has been observed with influenza HA. Furthermore, Ingenuity pathway analyses of dysregulated miRNAs and potential targets indicated strong association with inflammatory responses, cell death and survival as well as infectious diseases. The data generated here provide valuable information to understand the regulatory function of miRNAs during trypanosome infections. They improved our knowledge on host-parasite cross-talks and provide a framework for investigations to understand the development of trypanosomes in their hosts as well as the differences in the clinical and pathological evolutions of the disease

Population genetic structure of Central African Trypanosoma brucei gambiense isolates using microsatellite DNA markers

Genetic variation of microsatellite loci is a widely used method for the analysis of population genetic structure of microorganisms. Seven microsatellite markers were used here to characterize Trypanosoma brucei gambiense isolates from Central Africa sub-region in order to improve knowledge on the population genetic structure of this subspecies. These markers confirmed the low genetic polymorphism within Central African T. b. gambiense isolates from the same focus and strong differentiation between different foci. The presence of many multilocus genotypes of T. b. gambiense and the excess of heterozygotes found in this study play in favour of a clonal reproduction of this parasite. But some data may be indicative of a unique recombination event in one subsample. The high F-ST value indicates low migration rates between T. b. gambiense subpopulations (foci). Very negative F-IS suggests fairly small clonal population sizes of this pathogen in the different human trypanosomosis foci of Central Africa

The tethering assay: a simple method for the characterization of mRNA-fate regulators

In trypanosomatids, post-transcriptional controls are very important in regulation of individual gene expression. These are achieved through combinatorial sets of RNA-binding proteins (RBPs) which recognize RNA regulatory motifs or regions of secondary structure within RNAs. To analyze the potential functional impact of an RBP on their mRNA targets, we have applied a robust technique called tethering assay. In this method, the protein under study is attached to an mRNA reporter through an artificial RNA-protein interaction. Therefore, the functional activity of a protein can be analyzed independently of its intrinsic ability to bind to RNA. By making use of a cell line expressing a chloramphenicol acetyltransferase (CAT) reporter mRNA, we have characterized dozens of novel mRNA-fate regulators in cultured Trypanosoma brucei. After induction of the candidate fusion protein, the effect on the reporter expression is determined by a rapid CAT assay. The protocol is simple and typically takes one working day for analysis of a single protein and controls. In this chapter, we provide a description of materials and methods for the tethering method and should allow the assay to be successfully deployed in any laboratory with minimal user training.Fil: Mugo, Elisha. University of Pretoria; SudáfricaFil: Erben, Esteban Daniel. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; Argentin