Craig Brook; two fishermen bringing in their nets

Archival Collection

Identification of cryptic Anopheles mosquito species by molecular protein profiling.

Vector control is the mainstay of malaria control programmes. Successful vector control profoundly relies on accurate information on the target mosquito populations in order to choose the most appropriate intervention for a given mosquito species and to monitor its impact. An impediment to identify mosquito species is the existence of morphologically identical sibling species that play different roles in the transmission of pathogens and parasites. Currently PCR diagnostics are used to distinguish between sibling species. PCR based methods are, however, expensive, time-consuming and their development requires a priori DNA sequence information. Here, we evaluated an inexpensive molecular proteomics approach for Anopheles species: matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). MALDI-TOF MS is a well developed protein profiling tool for the identification of microorganisms but so far has received little attention as a diagnostic tool in entomology. We measured MS spectra from specimens of 32 laboratory colonies and 2 field populations representing 12 Anopheles species including the A. gambiae species complex. An important step in the study was the advancement and implementation of a bioinformatics approach improving the resolution over previously applied cluster analysis. Borrowing tools for linear discriminant analysis from genomics, MALDI-TOF MS accurately identified taxonomically closely related mosquito species, including the separation between the M and S molecular forms of A. gambiae sensu stricto. The approach also classifies specimens from different laboratory colonies; hence proving also very promising for its use in colony authentication as part of quality assurance in laboratory studies. While being exceptionally accurate and robust, MALDI-TOF MS has several advantages over other typing methods, including simple sample preparation and short processing time. As the method does not require DNA sequence information, data can also be reviewed at any later stage for diagnostic or functional patterns without the need for re-designing and re-processing biological material.

Triple-peptide receptor targeting in vitro allows detection of all tested gut and bronchial NETs.

UNLABELLED A high proportion of gut and bronchial neuroendocrine tumors (NETs) overexpresses somatostatin receptors, especially the sst2 subtype. It has also recently been observed that incretin receptors, namely glucagonlike peptide 1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) receptors, can be overexpressed in gut and bronchial NETs. However, because not all tumors can express these receptors in sufficient amounts, in vivo imaging with a single radioligand may not always be successful. We therefore evaluated with in vitro methods whether a cocktail of radioligands targeting these 3 receptors would improve tumor labeling. METHODS In vitro receptor autoradiography was performed on 55 NETs, comparing in each successive section of tumor the binding with a single radioligand, either (125)I-Tyr(3)-octreotide, (125)I-GLP-1(7-36)amide, or (125)I-GIP(1-30), with the binding using a cocktail of all 3 radioligands, given concomitantly under identical experimental conditions. RESULTS Using the cocktail of radioligands, all tumors without exception showed moderate to very high binding, with a receptor density corresponding to 1,000-10,000 dpm/mg of tissue; conversely, single-ligand binding, although identifying most tumors as receptor-positive, failed to detect receptors or measured only a low density of receptors below 1,000 dpm/mg in a significant number of tumors. In addition, the cocktail of radioligands always provided a homogeneous labeling of the whole tumor, whereas single radioligands occasionally showed heterogeneous labeling. CONCLUSION The study suggests that the use of a cocktail of 3 radioligands binding to somatostatin receptors, GLP-1 receptors, and GIP receptors would allow detecting virtually all NETs and labeling them homogeneously in vivo, representing a significant improvement for imaging and therapy in NETs.

Proteomic analysis of Plasmodium in the mosquito: progress and pitfalls

Here we discuss proteomic analyses of whole cell preparations of the mosquito stages of malaria parasite development (i.e. gametocytes, microgamete, ookinete, oocyst and sporozoite) of Plasmodium berghei. We also include critiques of the proteomes of two cell fractions from the purified ookinete, namely the micronemes and cell surface. Whereas we summarise key biological interpretations of the data, we also try to identify key methodological constraints we have met, only some of which we were able to resolve. Recognising the need to translate the potential of current genome sequencing into functional understanding, we report our efforts to develop more powerful combinations of methods for the in silico prediction of protein function and location. We have applied this analysis to the proteome of the male gamete, a cell whose very simple structural organisation facilitated interpretation of data. Some of the in silico predictions made have now been supported by ongoing protein tagging and genetic knockout studies. We hope this discussion may assist future studies.