Performance of Microscopy for the Diagnosis of Malaria and Human African Trypanosomiasis by Diagnostic Laboratories in the Democratic Republic of the Congo: Results of a Nation-Wide External Quality Assessment

The present External Quality Assessment (EQA) assessed microscopy of blood parasites among diagnostic laboratories in the Democratic Republic of the Congo. The EQA addressed 445 participants in 10/11 provinces (October 2013–April 2014). Participants were sent a panel of five slides and asked to return a routinely stained slide which was assessed for quality of preparation and staining. Response rate was 89.9% (400/445). For slide 1 (no parasites), 30.6% participants reported malaria, mostly Plasmodium falciparum. Only 11.0% participants reported slide 2 (Plasmodium malariae) correctly, 71.0% reported “malaria” or “Plasmodium falciparum” (considered acceptable). Slide 3 contained Plasmodium falciparum (109/μl) and Trypanosoma brucei brucei trypomastigotes: they were each reported by 32.5% and 16.5% participants respectively, 6.0% reported both. Slide 4 (Trypanosoma) was recognised by 44.9% participants. Slide 5 (Plasmodium ovale) was correctly reported by 6.2% participants, another 68.8% replied “malaria” or “Plasmodium falciparum” (considered acceptable). Only 13.6% of routine slides returned were correctly prepared and stained. The proportion of correct/acceptable scores for at least 4/5 slides was higher among EQA-experienced participants compared to first time participants (40.9% versus 22.4%, p = 0.001) and higher among those being trained < 2 years ago compared to those who were not (42.9% versus 26.3%, p = 0.01). Among diagnostic laboratories in Democratic Republic of the Congo, performance of blood parasite microscopy including non-falciparum species and Trypanosoma was poor. Recent training and previous EQA participation were associated with a better performance.     

A Bcl-2-related gene is activated in avian cells transformed by the Rous sarcoma virus.

The oncoprotein p60v-src encoded by the Rous sarcoma virus (RSV) genome is the prototype of non-receptor tyrosine kinases. More than 50 targets of p60v-src have been described to date. However, the precise mechanisms of RSV transformation remain to be elucidated. Here, we present the study of a new v-src-activated gene, NR-13, which encodes a protein identified as a new member of the Bcl-2 family. This protein is localized in the membrane with a pattern already observed with Bcl-2. In quail embryos, this gene is mainly expressed in neural and muscular tissues. Its expression is dramatically down-regulated after embryonic day 7 (E7) in the optic tectum. To evaluate a possible role for NR-13 in the control of apoptotic processes in this particular brain area, in situ hybridization and DNA ladder fractionation studies were performed to correlate NR-13 expression with typical situations of apoptosis during brain development. Our results support the idea that RSV could activate anti-apoptotic functions of the host cell resulting in an increase of their lifespan, which could be particularly relevant to tumour formation.     

The Extracellular and Cytoplasmic Domains of Syndecan Cooperate Postsynaptically to Promote Synapse Growth at the Drosophila Neuromuscular Junction

The heparan sulfate proteoglycan (HSPG) Syndecan (Sdc) is a crucial regulator of synapse development and growth in both vertebrates and invertebrates. In Drosophila, Sdc binds via its extracellular heparan sulfate (HS) sidechains to the receptor protein tyrosine phosphatase LAR to promote the morphological growth of the neuromuscular junction (NMJ). To date, however, little else is known about the molecular mechanisms by which Sdc functions to promote synapse growth. Here we show that all detectable Sdc found at the NMJ is provided by the muscle, strongly suggesting a post-synaptic role for Sdc. We also show that both the cytoplasmic and extracellular domains of Sdc are required to promote synapse growth or to rescue Sdc loss of function. We report the results of a yeast two-hybrid screen using the cytoplasmic domains of Sdc as bait, and identify several novel candidate binding partners for the cytoplasmic domains of Sdc. Together, these studies provide new insight into the mechanism of Sdc function at the NMJ, and provide enticing future directions for further exploring how Sdc promotes synapse growth.     

Dinoflagellate chromosome behaviour during stages of replication.

In most dinoflagellate species, chromosomes are characterized by an almost continuous condensation of the nucleofilaments throughout the cell cycle and the absence of longitudinal differentiation as Q, G, or C banding. Their supercoiled architecture is maintained by divalent cations and structural RNAs. Their chromatin is devoid of histones and nucleosomes and their DNA composition is distinctive: in several species, more than 60% of thymines are replaced by a rare base, hydroxymethyluracil. We report here an immunofluorescence (conventional and confocal laser scanning microscopy, CLSM) and immunogold transmission electron microscopy (TEM) analysis of some stages of the early replication process in Prorocentrum micans dinoflagellate cells, after long pulse incorporation (3, 6 or 9 days) with 50 micrograms/ml bromodeoxyuridine (BrdU) in the presence of 5-fluoro-2'-deoxyuridine (FUdR) and BrdU antibody technique (BAT) detection. The large DNA content (45 pg per nucleus) of P. micans cells is compacted on 100 chromosomes, 10 microns in length. In early S-phase, DNA replication sites are revealed as fluorescent domains organized in clusters, which appear in the periphery of the nucleus unlike other eukaryotes. In late S-phase, the number of labelled clusters increased; helically distributed, they did not appear synchronously in the whole chromosome. Under TEM, spherical domains of equivalent diameter appeared located all along the chromosomes after 6 days BrdU pulse. Replication occurs, but in our experimental conditions, segregation of daughter chromosomes was never observed. The blockade of the cell cycle after BrdU incorporation intervening just before the segregation of daughter chromosomes is discussed.     

Protease B from Saccharomyces cerevisiae. Purification and characterization.

Protease B has been isolated from Saccharomyces cerevisiae and purified in six steps as follows: autolysis of the yeast cells, ammonium sulfate fractionation, activation of the proteolytic enzymes, chromatography on DEAE-cellulose, chromatography on CM-cellulose and finally, a second chromatography on DEAE-cellulose. The preparation was shown to be homogeneous on polyacrylamide gels in the absence as well as in the presence of sodium dodecylsulfate. Furthermore, the molecular weight (43,000 daltons) and the isoelectric point (5.45) were in good agreement with earlier published values. The amino acid composition is reported. The absence of disulfide bonds in protease B has to be outlined. The amino acid residues of the protein have been found to be folded nearly quantitatively (at least 80%) in a beta-conformation as deduced from a circular dichroism study. Finally, the tryptophan residues (5 mol/mol protein) are largely buried in the hydrophobic core of the enzyme.     

Reproducible and Consistent Quantification of the Saccharomyces cerevisiae Proteome by SWATH-mass spectrometry

Targeted mass spectrometry by selected reaction monitoring (S/MRM) has proven to be a suitable technique for the consistent and reproducible quantification of proteins across multiple biological samples and a wide dynamic range. This performance profile is an important prerequisite for systems biology and biomedical research. However, the method is limited to the measurements of a few hundred peptides per LC-MS analysis. Recently, we introduced SWATH-MS, a combination of data independent acquisition and targeted data analysis that vastly extends the number of peptides/proteins quantified per sample, while maintaining the favorable performance profile of S/MRM. Here we applied the SWATH-MS technique to quantify changes over time in a large fraction of the proteome expressed in Saccharomyces cerevisiae in response to osmotic stress.We sampled cell cultures in biological triplicates at six time points following the application of osmotic stress and acquired single injection data independent acquisition data sets on a high-resolution 5600 tripleTOF instrument operated in SWATH mode. Proteins were quantified by the targeted extraction and integration of transition signal groups from the SWATH-MS datasets for peptides that are proteotypic for specific yeast proteins. We consistently identified and quantified more than 15,000 peptides and 2500 proteins across the 18 samples. We demonstrate high reproducibility between technical and biological replicates across all time points and protein abundances. In addition, we show that the abundance of hundreds of proteins was significantly regulated upon osmotic shock, and pathway enrichment analysis revealed that the proteins reacting to osmotic shock are mainly involved in the carbohydrate and amino acid metabolism. Overall, this study demonstrates the ability of SWATH-MS to efficiently generate reproducible, consistent, and quantitatively accurate measurements of a large fraction of a proteome across multiple samples.In systems biology and biomedical studies targeted mass spectrometry via selected reaction monitoring (SRM)¹ (also known as multiple reaction monitoring, MRM) has emerged as a powerful technique for the consistent and reproducible quantification of proteins across numerous complex samples (1–6). Optimal sets of precursor/fragment ion pairs, called transitions, uniquely represent a specific peptide. They constitute a definitive mass spectrometric assay for the detection of targeted peptides, and thus the proteins from which they derive, in the complex matrix of trypsinized biological samples (1, 7). Protein quantification is then performed by relating the intensity of the acquired transition signals to suitable reference signals. Most quantification strategies commonly used in proteomics are compatible with this method (8). Recently, the high-throughput development of S/MRM assays has been achieved via the generation of MS/MS spectral libraries from the measurements of thousands of synthetic peptides representing proteotypic peptides (9). Moreover, many experimental and bioinformatics workflows have been developed for assay generation, assay optimization, data evaluation, and the dissemination of optimized S/MRM assays (10–16). In combination, these developments have supported the creation of mass-spectrometric maps of entire proteomes of selected species including Streptococcus pyogenes, Mycobacterium tuberculosis, and Saccharomyces cerevisae (5, 17–19) and the robust use of these resources to quantify specific protein sets across multiple biological samples.Currently, targeted proteomics by S/MRM can be multiplexed to a maximum set of ∼100 proteins that can be measured in a single LC-S/MRM run at optimal quantitative accuracy, limit of detection and dynamic range. The quantification of higher numbers of proteins per run compromises some of the performance parameters of the method because of well understood tradeoffs (8). Attempts have been made to further increase the degree of multiplexing of S/MRM, either by automated adjustment of the scheduled detection windows (20) or by acquiring, in a data-dependent manner, the complete set of precursor-fragment ion pairs of a given assay (21). Alternatively, parallel reaction monitoring (PRM) approach operated on quadrupole-orbitrap mass spectrometer has shown detection and quantification performances similar or better than those obtained in SRM, because of the increased selectivity of the mass analyzer (22–24). These approaches are promising, but their application relies on prior knowledge of the precursor ions that need to be targeted during the data acquisition, and they still are subject of the above-mentioned tradeoffs.Recently, we developed a novel MS strategy that combines data independent acquisition (DIA) of trypsinized protein samples with S/MRM-like, in silico targeted analysis of the acquired complete fragment ion maps (25). We termed the method SWATH-MS, and applied the sequential isolation window acquisition principle (26) to repeatedly cycle, in a single injection, through 32 consecutive 25-Da precursor isolation windows (swaths). The process acquires fragment ion spectra of all precursors in a space defined by the 400–1200 m/z precursor range and a user-specified retention time window. We used the prior information in MS/MS spectral libraries to extract groups of signals that uniquely identify a specific peptide, and to demonstrate that peptides could be identified and quantified over a dynamic range of four orders of magnitude, even when the precursors were not detectable in a survey MS scan. For the 45 proteins involved in the central carbon metabolism of yeast, we demonstrated that the accuracy of quantification was equivalent to that of S/MRM (25). However, because of the lack of adequate software tools at that time, the extensive high-throughput targeted data analysis of the SWATH-MS maps could not be fully demonstrated in that first study.Here we demonstrate the multiplexing capabilities of SWATH-MS for the detection and quantification of significantly larger fractions of a proteome as compared with S/MRM, without compromising reproducibility, consistency, and quantitative accuracy. We describe the large scale deployment of fragment ion spectral libraries and the use of S/MRM-like analysis tools specifically adapted to SWATH-MS data for the detection and quantification of temporal changes of the S. cerevisae proteome in response to osmotic stress.