Transcriptomes of <i>Trypanosoma brucei</i> rhodesiense from sleeping sickness patients, rodents and culture:Effects of strain, growth conditions and RNA preparation methods

All of our current knowledge of African trypanosome metabolism is based on results from trypanosomes grown in culture or in rodents. Drugs against sleeping sickness must however treat trypanosomes in humans. We here compare the transcriptomes of Trypanosoma brucei rhodesiense from the blood and cerebrospinal fluid of human patients with those of trypanosomes from culture and rodents. The data were aligned and analysed using new user-friendly applications designed for Kinetoplastid RNA-Seq data. The transcriptomes of trypanosomes from human blood and cerebrospinal fluid did not predict major metabolic differences that might affect drug susceptibility. Usefully, there were relatively few differences between the transcriptomes of trypanosomes from patients and those of similar trypanosomes grown in rats. Transcriptomes of monomorphic laboratory-adapted parasites grown in in vitro culture closely resembled those of the human parasites, but some differences were seen. In poly(A)-selected mRNA transcriptomes, mRNAs encoding some protein kinases and RNA-binding proteins were under-represented relative to mRNA that had not been poly(A) selected; further investigation revealed that the selection tends to result in loss of longer mRNAs

Structural Mass Spectrometry: Rapid Methods for Separation and Analysis of Peptide Natural Products

A significant challenge in natural product discovery is the initial discrimination of discrete secondary metabolites alongside functionally similar primary metabolic cellular components within complex biological samples. A property that has yet to be fully exploited for natural product identification and characterization is the gas-phase collision cross section, or, more generally, the mobility–mass correlation. Peptide natural products possess many of the properties that distinguish natural products, as they are frequently characterized by a high degree of intramolecular bonding and possess extended and compact conformations among other structural modifications. This report describes a rapid structural mass spectrometry technique based on ion mobility–mass spectrometry for the comparison of peptide natural products to their primary metabolic congeners using mobility–mass correlation. This property is empirically determined using ion mobility–mass spectrometry, applied to the analysis of linear versus modified peptides, and used to discriminate peptide natural products in a crude microbial extract. Complementary computational approaches are utilized to understand the structural basis for the separation of primary metabolism derived linear peptides from secondary metabolite cyclic and modified cyclic species. These findings provide a platform for enhancing the identification of secondary metabolic peptides with distinct mobility–mass ratios within complex biological samples

Combined Elemental and Biomolecular Mass Spectrometry Imaging for Probing the Inventory of Tissue at a Micrometer Scale

Several complementary mass spectrometric imaging techniques allow mapping of various analytes within biological tissue sections. Laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS) quantitatively detects elements and isotopes with very high sensitivity and a particularly high dynamical range. Matrix-assisted laser desorption/ionization ion mobility mass spectrometry (MALDI-IM-MS) allows a pixel-by-pixel classification and identification of biomolecules. In order to dispose of the healthy hemisphere as an internal calibrant in addition to routinely used external standards, adjacent brain sections of mice with a unilateral 6-OHDA lesion of the medial forebrain bundle were chosen as exemplary samples. We demonstrate a comprehensive way of data acquisition and analysis by coregistering mass spectrometric data on photomicrographs as common reference space and thus providing trimodal spatial information. Registering subsequent planar element maps yielded continuous 3-dimensional data sets. Furthermore, we introduce a correction of MSI data for variable slice thickness applicable to all MSI techniques. In the present case, we observed increased concentrations of iron, manganese, and copper in the lesioned substantia nigra while monounsaturated lipid levels were decreased in the identical region of interest. Our techniques provide new insights into the intricate spatial relationship of morphology and chemistry within tissue