An Epithelial Serine Protease, AgESP, Is Required for Plasmodium Invasion in the Mosquito Anopheles gambiae

Background: Plasmodium parasites need to cross the midgut and salivary gland epithelia to complete their life cycle in the mosquito. However, our understanding of the molecular mechanism and the mosquito genes that participate in this process is still very limited. Methodology/Principal Findings: We identified an Anopheles gambiae epithelial serine protease (AgESP) that is constitutively expressed in the submicrovillar region of mosquito midgut epithelial cells and in the basal side of the salivary glands that is critical for Plasmodium parasites to cross these two epithelial barriers. AgESP silencing greatly reduces Plasmodium berghei and Plasmodium falciparum midgut invasion and prevents the transcriptional activation of gelsolin, a key regulator of actin remodeling and a reported Plasmodium agonist. AgESP expression is highly induced in midgut cells invaded by Plasmodium, suggesting that this protease also participates in the apoptotic response to invasion. In salivary gland epithelial cells, AgESP is localized on the basal side–the surface with which sporozoites interact. AgESP expression in the salivary gland is also induced in response to P. berghei and P. falciparum sporozoite invasion, and AgESP silencing significantly reduces the number of sporozoites that invade this organ. Conclusion: Our findings indicate that AgESP is required for Plasmodium parasites to effectively traverse the midgut and salivary gland epithelial barriers. Plasmodium parasites need to modify the actin cytoskeleton of mosquito epithelial cells t

Building ProteomeTools based on a complete synthetic human proteome.

We describe ProteomeTools, a project building molecular and digital tools from the human proteome to facilitate biomedical research. Here we report the generation and multimodal liquid chromatography-tandem mass spectrometry analysis of \u3e330,000 synthetic tryptic peptides representing essentially all canonical human gene products, and we exemplify the utility of these data in several applications. The resource (available at http://www.proteometools.org) will be extended to \u3e1 million peptides, and all data will be shared with the community via ProteomicsDB and ProteomeXchange

Submicroscopic Gametocytes and the Transmission of Antifolate-Resistant Plasmodium falciparum in Western Kenya

BACKGROUND: Single nucleotide polymorphisms (SNPs) in the dhfr and dhps genes are associated with sulphadoxine-pyrimethamine (SP) treatment failure and gametocyte carriage. This may result in enhanced transmission of mutant malaria parasites, as previously shown for chloroquine resistant parasites. In the present study, we determine the association between parasite mutations, submicroscopic P. falciparum gametocytemia and malaria transmission to mosquitoes. METHODOLOGY/PRINCIPAL FINDINGS: Samples from children treated with SP alone or in combination with artesunate (AS) or amodiaquine were genotyped for SNPs in the dhfr and dhps genes. Gametocytemia was determined by microscopy and Pfs25 RNA-based quantitative nucleic acid sequence-based amplification (Pfs25 QT-NASBA). Transmission was determined by membrane-feeding assays. We observed no wild type infections, 66.5% (127/191) of the infections expressed mutations at all three dhfr codons prior to treatment. The presence of all three mutations was not related to higher Pfs25 QT-NASBA gametocyte prevalence or density during follow-up, compared to double mutant infections. The proportion of infected mosquitoes or oocyst burden was also not related to the number of mutations. Addition of AS to SP reduced gametocytemia and malaria transmission during follow-up. CONCLUSIONS/SIGNIFICANCE: In our study population where all infections had at least a double mutation in the dhfr gene, additional mutations were not related to increased submicroscopic gametocytemia or enhanced malaria transmission. The absence of wild-type infections is likely to have reduced our power to detect differences. Our data further support the use of ACT to reduce the transmission of drug-resistant malaria parasites

Characterization of a R plasmid-associated, trimethoprim-resistant dihydrofolate reductase and determination of the nucleotide sequence of the reductase gene.

The trimethoprim-resistant dihydrofolate reductase associated with the R plasmid R388 was isolated from strains that over-produce the enzyme. It was purified to apparent homogeneity by affinity chromatography and two consecutive gel filtration steps under native and denaturing conditions. The purified enzyme is composed of four identical subunits with molecular weights of 8300. A 1100 bp long DNA segment which confers resistance to trimethoprim was sequenced. The structural gene was identified on the plasmid DNA by comparing the amino acid composition of the deduced proteins with that of the purified enzyme. The gene is 234 bp long and codes for 78 amino acids. No homology can be found between the deduced amino acid sequence of the R388 dihydrofolate reductase and those of other prokaryotic or eukaryotic dihydrofolate reductases. However, it differs in only 17 positions from the enzyme associated with the trimethoprim-resistance plasmid R67

Inactivation of human immunodeficiency virus type 1 in blood samples stored as high-salt lysates.

Blood samples to be tested for the presence of parasite DNA by using specific DNA probes are routinely stored in our laboratory as high-salt lysates (HSL). To safeguard against the risk of accidental infection with etiological agents such as the human immunodeficiency virus type 1 (HIV-1) while manipulating large numbers of blood samples in preparation for DNA probing, we determined the residual infectivity of HIV-1 after exposure to HSL components. Both high-titer virus stocks or provirus-carrying cells, suspended either in tissue culture medium or freshly drawn blood, were completely inactivated upon contact with the HSL components. This was verified by the absence of any detectable HIV-1-specific antigen in the supernatants of long-term cultures and the absence of virus-specific DNA fragments after amplification by polymerase chain reaction with DNA from such cultures as target DNA. These results support the conclusion that the virus is in fact completely inactivated by contact with the HSL components, rendering blood specimens stored as HSL noninfectious in regard to HIV-1

Diagnosis of rheumatoid arthritis: multivariate analysis of biomarkers.

Item does not contain fulltextOBJECTIVE: To test if a combination of biomarkers can increase the classification power of autoantibodies to cyclic citrullinated peptides (anti-CCP) in the diagnosis of rheumatoid arthritis (RA) depending on the diagnostic situation. METHODS: Biomarkers were subject to three inclusion/exclusion criteria (discrimination between RA patients and healthy blood donors, ability to identify anti-CCP-negative RA patients, specificity in a panel with major non-rheumatological diseases) before univariate ranking and multivariate analysis was carried out using a modelling panel (n = 906). To enable the evaluation of the classification power in different diagnostic settings the disease controls (n = 542) were weighted according to the admission rates in rheumatology clinics modelling a clinic panel or according to the relative prevalences of musculoskeletal disorders in the general population seen by general practitioners modelling a GP panel. RESULT: Out of 131 biomarkers considered originally, we evaluated 32 biomarkers in this study, of which only seven passed the three inclusion/exclusion criteria and were combined by multivariate analysis using four different mathematical models. In the modelled clinic panel, anti-CCP was the lead marker with a sensitivity of 75.8% and a specificity of 94.0%. Due to the lack in specificity of the markers other than anti-CCP in this diagnostic setting, any gain in sensitivity by any marker combination is off-set by a corresponding loss in specificity. In the modelled GP panel, the best marker combination of anti-CCP and interleukin (IL)-6 resulted in a sensitivity gain of 7.6% (85.9% vs. 78.3%) at a minor loss in specificity of 1.6% (90.3% vs. 91.9%) compared with anti-CCP as the best single marker. CONCLUSION: Depending on the composition of the sample panel, anti-CCP alone or anti-CCP in combination with IL-6 has the highest classification power for the diagnosis of established RA