Expression of Cytosolic Peroxiredoxins in Plasmodium Berghei Ookinetes is Regulated by Environmental Factors in the Mosquito bloodmeal

The Plasmodium ookinete develops over several hours in the bloodmeal of its mosquito vector where it is exposed to exogenous stresses, including cytotoxic reactive oxygen species (ROS). How the parasite adapts to these challenging conditions is not well understood. We have systematically investigated the expression of three cytosolic antioxidant proteins, thioredoxin-1 (Trx-1), peroxiredoxin-1 (TPx-1), and 1-Cys peroxiredoxin (1-Cys Prx), in developing ookinetes of the rodent parasite Plasmodium berghei under various growth conditions. Transcriptional profiling showed that tpx-1 and 1-cys prx but not trx-1 are more strongly upregulated in ookinetes developing in the mosquito bloodmeal when compared to ookinetes growing under culture conditions. Confocal immunofluorescence imaging revealed comparable expression patterns on the corresponding proteins. 1-Cys Prx in particular exhibited strong expression in mosquito-derived ookinetes but was not detectable in cultured ookinetes. Furthermore, ookinetes growing in culture upregulated tpx-1 and 1-cys prx when challenged with exogenous ROS in a dose-dependent fashion. This suggests that environmental factors in the mosquito bloodmeal induce upregulation of cytosolic antioxidant proteins in Plasmodium ookinetes. We found that in a parasite line lacking TPx-1 (TPx-1KO), expression of 1-Cys Prx occurred significantly earlier in mosquito-derived TPx-1KO ookinetes when compared to wild type (WT) ookinetes. The protein was also readily detectable in cultured TPx-1KO ookinetes, indicating that 1-Cys Prx at least in part compensates for the loss of TPx-1 in vivo. We hypothesize that this dynamic expression of the cytosolic peroxiredoxins reflects the capacity of the developing Plasmodium ookinete to rapidly adapt to the changing conditions in the mosquito bloodmeal. This would significantly increase its chances of survival, maturation and subsequent escape. Our results also emphasize that environmental conditions must be taken into account when investigating Plasmodium-mosquito interactions

Natalizumab treatment shows low cumulative probabilities of confirmed disability worsening to EDSS milestones in the long-term setting.

Abstract Background Though the Expanded Disability Status Scale (EDSS) is commonly used to assess disability level in relapsing-remitting multiple sclerosis (RRMS), the criteria defining disability progression are used for patients with a wide range of baseline levels of disability in relatively short-term trials. As a result, not all EDSS changes carry the same weight in terms of future disability, and treatment benefits such as decreased risk of reaching particular disability milestones may not be reliably captured. The objectives of this analysis are to assess the probability of confirmed disability worsening to specific EDSS milestones (i.e., EDSS scores ≥3.0, ≥4.0, or ≥6.0) at 288 weeks in the Tysabri Observational Program (TOP) and to examine the impact of relapses occurring during natalizumab therapy in TOP patients who had received natalizumab for ≥24 months. Methods TOP is an ongoing, open-label, observational, prospective study of patients with RRMS in clinical practice. Enrolled patients were naive to natalizumab at treatment initiation or had received ≤3 doses at the time of enrollment. Intravenous natalizumab (300 mg) infusions were given every 4 weeks, and the EDSS was assessed at baseline and every 24 weeks during treatment. Results Of the 4161 patients enrolled in TOP with follow-up of at least 24 months, 3253 patients with available baseline EDSS scores had continued natalizumab treatment and 908 had discontinued (5.4% due to a reported lack of efficacy and 16.4% for other reasons) at the 24-month time point. Those who discontinued due to lack of efficacy had higher baseline EDSS scores (median 4.5 vs. 3.5), higher on-treatment relapse rates (0.82 vs. 0.23), and higher cumulative probabilities of EDSS worsening (16% vs. 9%) at 24 months than those completing therapy. Among 24-month completers, after approximately 5.5 years of natalizumab treatment, the cumulative probabilities of confirmed EDSS worsening by 1.0 and 2.0 points were 18.5% and 7.9%, respectively (24-week confirmation), and 13.5% and 5.3%, respectively (48-week confirmation). The risks of 24- and 48-week confirmed EDSS worsening were significantly higher in patients with on-treatment relapses than in those without relapses. An analysis of time to specific EDSS milestones showed that the probabilities of 48-week confirmed transition from EDSS scores of 0.0–2.0 to ≥3.0, 2.0–3.0 to ≥4.0, and 4.0–5.0 to ≥6.0 at week 288 in TOP were 11.1%, 11.8%, and 9.5%, respectively, with lower probabilities observed among patients without on-treatment relapses (8.1%, 8.4%, and 5.7%, respectively). Conclusions In TOP patients with a median (range) baseline EDSS score of 3.5 (0.0–9.5) who completed 24 months of natalizumab treatment, the rate of 48-week confirmed disability worsening events was below 15%; after approximately 5.5 years of natalizumab treatment, 86.5% and 94.7% of patients did not have EDSS score increases of ≥1.0 or ≥2.0 points, respectively. The presence of relapses was associated with higher rates of overall disability worsening. These results were confirmed by assessing transition to EDSS milestones. Lower rates of overall 48-week confirmed EDSS worsening and of transitioning from EDSS score 4.0–5.0 to ≥6.0 in the absence of relapses suggest that relapses remain a significant driver of disability worsening and that on-treatment relapses in natalizumab-treated patients are of prognostic importance

Genetic architecture of subcortical brain structures in 38,851 individuals

Subcortical brain structures are integral to motion, consciousness, emotions and learning. We identified common genetic variation related to the volumes of the nucleus accumbens, amygdala, brainstem, caudate nucleus, globus pallidus, putamen and thalamus, using genome-wide association analyses in almost 40,000 individuals from CHARGE, ENIGMA and UK Biobank. We show that variability in subcortical volumes is heritable, and identify 48 significantly associated loci (40 novel at the time of analysis). Annotation of these loci by utilizing gene expression, methylation and neuropathological data identified 199 genes putatively implicated in neurodevelopment, synaptic signaling, axonal transport, apoptosis, inflammation/infection and susceptibility to neurological disorders. This set of genes is significantly enriched for Drosophila orthologs associated with neurodevelopmental phenotypes, suggesting evolutionarily conserved mechanisms. Our findings uncover novel biology and potential drug targets underlying brain development and disease

Challenge of ookinete cultures with increasing concentrations of the ROS producing agent paraquat (PQ).

<p>RT-qPCR data showing dose-response modulation of target gene expression in ookinete cultures in the presence of increasing concentrations of the superoxide-producing compound Paraquat (PQ, Viologen, Aldrich). Increasing concentrations of PQ were added to 12-hours <i>P. berghei</i> ookinete cultures as indicated. Shown are the ratios of normalized relative transcript quantities (RQ) of treated vs. non-treated samples (non-treated RQ = 1, represented by the dashed line). All data were normalized against the expression of 18 s rRNA A-type <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003136#ppat.1003136-Yano1" target="_blank">[29]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003136#ppat.1003136-Thompson1" target="_blank">[34]</a>. (*) indicates statistical significance (p<0.1). Shown are mean values of 3 independent experiments. Error bars indicate STDEV.</p

Candidate genes of the cytosolic thioredoxin system of <i>P. berghei</i>.

<p>Candidate genes of the cytosolic thioredoxin system of <i>P. berghei</i>.</p

Time dependent transcription profiles of the cytosolic thioredoxin system in culture-derived and mosquito-derived parasites.

<p>RT-qPCR data showing relative target gene expression as fold increase over time. The 3-hour time point was used as a reference. All data was normalized against the expression of 18 s rRNA A-type <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003136#ppat.1003136-Yano1" target="_blank">[29]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003136#ppat.1003136-Thompson1" target="_blank">[34]</a>. The Mann-Whitney U test was conducted on each candidate gene from both mosquito-derived and from culture-derived parasites. Significance was assessed at p<0.1(*) due to the small sample sizes. Shown are mean values of 4 independent blood feeding experiments (n = 55 mosquitoes/time point/experiment) and 3 independent ookinete culture setups, respectively. Error bars show STDEV.</p

Expression of the Trx-1, TPx-1 and 1-Cys Prx in developing ookinetes from culture or from mosquito bloodmeal.

<p>Samples were isolated from ookinete cultures and blood fed mosquitoes at indicated time points and prepared as described in material and methods. Fixed samples were probed with <b>A</b>) anti-PbTrx-1, <b>B</b>) anti-Pf TPx-1 or <b>C</b>) anti-PbTPx-1. Primary antibodies were probed with secondary antibody coupled to AF 488 (Molecular Probes). Samples were counterstained with the nuclear dye TO-PRO-3 (Life technologies). The source of the ookinetes from either ookinete cultures or bloodfed mosquito midguts is indicated. Images are merged and overlaid onto the respective DIC image. White arrows indicate accumulation of Trx-1, TPx-1 or 1-Cys Prx at the apical ends of developing ookinetes. Scale bar = 5 µM. Quantitative <i>Relative Fluorescence Analysis</i>: Shown is the median boxed by the first and third quartiles with minimum and maximum values displayed as whiskers. Mann-Whitney U-Test was performed with samples of culture-derived ookinetes compared to mosquito-derived ookinetes (p = 0.4551 for Trx-1; p = 0.1061 for TPx-1; p = 0.0413 for 1-Cys Prx). Below each graph are representative images from IFA-epifluorescence experiments.</p