Proteomic analysis of microvesicles from plasma of healthy donors reveals high individual variability

Healthy blood plasma is required for several therapeutic procedures. To maximize successful therapeutic outcomes it is critical to control the quality of blood plasma. Clearly initiatives to improve the safety of blood transfusions will have a high economical and social impact. A detailed knowledge of the composition of healthy blood plasma is essential to facilitate such improvements. Apart from free proteins, lipids and metabolites, blood plasma also contains cell-derived microvesicles, including exosomes and microparticles from several different cellular origins. In this study, we have purified microvesicles smaller than 220nm from plasma of healthy donors and performed proteomic, ultra-structural, biochemical and functional analyses. We have detected 161 microvesicle-associated proteins, including many associated with the complement and coagulation signal-transduction cascades. Several proteases and protease inhibitors associated with acute phase responses were present, indicating that these microvesicles may be involved in these processes. There was a remarkably high variability in the protein content of plasma from different donors. In addition, we report that this variability could be relevant for their interaction with cellular systems. This work provides valuable information on plasma microvesicles and a foundation to understand microvesicle biology and clinical implications.     

Hydrology Affects Environmental and Spatial Structuring of Microalgal Metacommunities in Tropical Pacific Coast Wetlands

The alternating climate between wet and dry periods has important effects on the hydrology and therefore on niche-based processes of water bodies in tropical areas. Additionally, assemblages of microorganism can show spatial patterns, in the form of a distance decay relationship due to their size or life form. We aimed to test spatial and environmental effects, modulated by a seasonal flooding climatic pattern, on the distribution of microalgae in 30 wetlands of a tropical dry forest region: the Pacific coast of Costa Rica and Nicaragua. Three surveys were conducted corresponding to the beginning, the highest peak, and the end of the hydrological year during the wet season, and species abundance and composition of planktonic and benthic microalgae was determined. Variation partitioning analysis (as explained by spatial distance or environmental factors) was applied to each seasonal dataset by means of partial redundancy analysis. Our results show that microalgal assemblages were structured by spatial and environmental factors depending on the hydrological period of the year. At the onset of hydroperiod and during flooding, neutral effects dominated community dynamics, but niche-based local effects resulted in more structured algal communities at the final periods of desiccating water bodies. Results suggest that climate-mediated effects on hydrology can influence the relative role of spatial and environmental factors on metacommunities of microalgae. Such variability needs to be accounted in order to describe accurately community dynamics in tropical coastal wetlands.     

The Mitochondrial Intermembrane Space Oxireductase Mia40 Funnels the Oxidative Folding Pathway of the Cytochrome c Oxidase Assembly Protein Cox19

Mia40-catalyzed disulfide formation drives the import of many proteins into the mitochondria. Here we characterize the oxidative folding of Cox19, a twin CX₉C Mia40 substrate. Cox19 oxidation is extremely slow, explaining the persistence of import-competent reduced species in the cytosol. Mia40 accelerates Cox19 folding through the specific recognition of the third Cys in the second helical CX₉C motif and the subsequent oxidation of the inner disulfide bond. This renders a native-like intermediate that oxidizes in a slow uncatalyzed reaction into native Cox19. The same intermediate dominates the pathway in the absence of Mia40, and chemical induction of an α-helical structure by trifluoroethanol suffices to accelerate productive folding and mimic the Mia40 folding template mechanism. The Mia40 role is to funnel a rough folding landscape, skipping the accumulation of kinetic traps, providing a rationale for the promiscuity of Mia40.     

Elucidating Emergence and Transmission of Multidrug-Resistant Tuberculosis in Treatment Experienced Patients by Whole Genome Sequencing

Background

Understanding the emergence and spread of multidrug-resistant tuberculosis (MDR-TB) is crucial for its control. MDR-TB in previously treated patients is generally attributed to the selection of drug resistant mutants during inadequate therapy rather than transmission of a resistant strain. Traditional genotyping methods are not sufficient to distinguish strains in populations with a high burden of tuberculosis and it has previously been difficult to assess the degree of transmission in these settings. We have used whole genome analysis to investigate M. tuberculosis strains isolated from treatment experienced patients with MDR-TB in Uganda over a period of four years.

Methods and Findings

We used high throughput genome sequencing technology to investigate small polymorphisms and large deletions in 51 Mycobacterium tuberculosis samples from 41 treatment-experienced TB patients attending a TB referral and treatment clinic in Kampala. This was a convenience sample representing 69% of MDR-TB cases identified over the four year period. Low polymorphism was observed in longitudinal samples from individual patients (2-15 SNPs). Clusters of samples with less than 50 SNPs variation were examined. Three clusters comprising a total of 8 patients were found with almost identical genetic profiles, including mutations predictive for resistance to rifampicin and isoniazid, suggesting transmission of MDR-TB. Two patients with previous drug susceptible disease were found to have acquired MDR strains, one of which shared its genotype with an isolate from another patient in the cohort.

Conclusions

Whole genome sequence analysis identified MDR-TB strains that were shared by more than one patient. The transmission of multidrug-resistant disease in this cohort of retreatment patients emphasises the importance of early detection and need for infection control. Consideration should be given to rapid testing for drug resistance in patients undergoing treatment to monitor the emergence of resistance and permit early intervention to avoid onward transmission.     

Drug2ways: Reasoning over causal paths in biological networks for drug discovery

Elucidating the causal mechanisms responsible for disease can reveal potential therapeutic targets for pharmacological intervention and, accordingly, guide drug repositioning and discovery. In essence, the topology of a network can reveal the impact a drug candidate may have on a given biological state, leading the way for enhanced disease characterization and the design of advanced therapies. Network-based approaches, in particular, are highly suited for these purposes as they hold the capacity to identify the molecular mechanisms underlying disease. Here, we present drug2ways, a novel methodology that leverages multimodal causal networks for predicting drug candidates. Drug2ways implements an efficient algorithm which reasons over causal paths in large-scale biological networks to propose drug candidates for a given disease. We validate our approach using clinical trial information and demonstrate how drug2ways can be used for multiple applications to identify: i) single-target drug candidates, ii) candidates with polypharmacological properties that can optimize multiple targets, and iii) candidates for combination therapy. Finally, we make drug2ways available to the scientific community as a Python package that enables conducting these applications on multiple standard network formats.     

Impact of Daily Thermocycles on Hatching Rhythms,Larval Performance and Sex Differentiation of Zebrafish

In the wild, water temperature cycles daily: it warms up after sunrise, and cools rapidly after sunset. Surprisingly, the impact of such daily thermocycles during the early development of fish remains neglected. We investigated the influence of constant vs daily thermocycles in zebrafish, from embryo development to sexual differentiation, by applying four temperature regimens: two constant (24°C and 28°C) and two daily thermocycles: 28:24°C, TC (thermophase coinciding with daytime, and cryophase coinciding with night-time) and 24:28°C, CT (opposite to TC) in a 12:12 h light:dark cycle (LD). Embryo development was temperature-dependent but enhanced at 28°C and TC. Hatching rhythms were diurnal (around 4 h after lights on), but temperature- and cycle-sensitive, since hatching occurred sooner at 28°C (48 hours post fertilization; hpf) while it was delayed at 24°C (96 hpf). Under TC, hatching occurred at 72 hpf, while under CT hatching displayed two peaks (at 70 hpf and 94 hpf). In constant light (LL) or darkness (DD), hatching rhythms persisted with tau close to 24 h, suggesting a clock-controlled “gating” mechanism. Under 28°C or TC, larvae showed the best performance (high growth and survival, and low malformations). The sex ratio was strongly influenced by temperature, as the proportion of females was higher in CT and TC (79 and 83% respectively), contrasting with 28°C and 24°C, which led to more males (83 and 76%). Ovarian aromatase (cyp19a) expression in females was highest in TC and CT (6.5 and 4.6 fold higher than at 28°C, respectively); while anti-müllerian hormone (amh) expression in males increased in testis at 24°C (3.6 fold higher compared to TC) and particularly at 28°C (14.3 fold increase). Taken together, these findings highlight the key role of environmental cycles during early development, which shaped the daily rhythms in fish embryo and larvae, and ultimately influenced sex differentiation.