Effect of pH and temperature on protein kinase release by Leishmania donovani

During their life cycle Leishmania are exposed to environments that differ markedly in pH and temperature. The effect of these factors on protein kinase release into the surrounding environment by Leishmania donovani promastigotes was examined. Promastigotes release protein kinase activity both constitutively and following induction by incubation with an exogenous substrate, phosvitin. The substrate specificity of the constitutive and induced activities was similar, unlike that previously described for Leishmania major promastigotes. The Leishmania donovani enzymes phosphorylate phosvitin, but not casein, mixed histones or protamine sulphate, and both activities are shed over a wide pH range from 6 to 9. Transfer of promastigotes from pH 7.4/30 degrees C to pH 5.0-5.5/37 degrees C, conditions that mimic those encountered by parasites following transmission from sandflies to a mammalian host and uptake by macrophages, inhibited release of the constitutive activity. Identical conditions had only a minor effect on induced protein kinase release. Both types of protein kinase activities released at pH 7.4 were still active when assayed at pH 5.0. Characterisation of the constitutive and induced promastigote protein kinases showed that casein kinase 1- and casein kinase 2-like activities are released by Leishmania donovani. Constitutive enzyme release decreased over time, however, the addition of phosvitin to these "casein kinase-depleted" promastigotes induced elevated casein kinase 1 and casein kinase 2 shedding. These results suggest that shed protein kinase might play a role in parasite survival and adaptation to host environments.     

Chemical Composition,Antibacterial, Antibiofilm and Synergistic Properties of Essential Oils from Eucalyptus globulus Labill. and Seven Mediterranean Aromatic Plants

Essential oils (EOs) from Eucalyptus globulus Labill . ssp. globulus and from Mediterranean autochthonous aromatic plants – Thymus mastichina L., Mentha pulegium L., Rosmarinus officinalis L., Calamintha nepeta (L.) Savi ssp. nepeta, Cistus ladanifer L., Foeniculum vulgare L., Dittrichia viscosa (L.) Greuter ssp. viscosa – were extracted by hydrodistillation and characterized by GC‐FID and NMR spectroscopy. EOs were evaluated for antimicrobial properties against several bacterial strains, using diverse methods, namely, the agar disc‐diffusion method, the microdilution method, the crystal violet assay and the Live/Dead staining for assessment of biofilm formation. Potential synergy was assessed by a checkerboard method. EOs of R. officinalis and C. ladanifer showed a predominance in monoterpene hydrocarbons (> 60%); EOs of C. nepeta, M. pulegium, T. mastichina, E. globulus and F. vulgare were rich in oxygenated monoterpenes (62 – 96%) whereas EO of D. viscosa was mainly composed of oxygenated sesquiterpenes (54%). All EOs showed antimicrobial activity; M. pulegium and E. globulus generally had the strongest antimicrobial activity. EO of C. nepeta was the most promising in hampering the biofilm formation. The combinations D. viscosa/C. nepeta and E. globulus/T. mastichina were synergistic against Staphylococcus aureus. These results support the notion that EOs from the aromatic plants herein reported should be further explored as potential pharmaceuticals and/or food preservatives.     

Tissue culture and plant propagation of Gomphrena officinalis — a Brazilian medicinal plant

Leaf and stem segments of Gomphrena officinalis originated from aseptically grown seedlings were used to initiate cultures. Callus production was obtained on gelled Murashige & Skoog medium supplemented with 6-benzylaminopurine alone (1.0, 5.0 or 10.0 mgl^-1) or combined with -naphthalene acetic acid (0.1, 0.5 and 1.0 mgl^-1) after 10 to 15 days of culture, and can be transferred to fresh medium every 30 days. The combinations of 5.0 or 10.0 mgl^-1 of 6-benzylaminopurine with 0.1 mgl^-1 of -naphthalene acetic acid were found to be the best for shoot regeneration. Adventitious shoot formation occurred after 50 to 60 days of culture in leaf and internode stem explants. Nodal segments developed actively growing lateral buds after 30 days of culture. Gelled Murashige & Skoog medium containing 10 mgl^-1 of indole-3-butyric acid was considered optimal for the rooting of shoots. Rooted plants transferred to potting soil could be successfully established.Abbreviations BA 6-benzylaminopurine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige & Skoog - NAA -naphthalene acetic acid     

Biodeterioration of Mayan buildings at uxmal and tulum,Mexico

Uxmal and Tulum are two important Mayan sites in the Yucatan peninsula. The buildings are mainly composed of limestone and grey/black discoloration is seen on exposed walls and copious greenish biofilms on inner walls. The principal microorganisms detected on interior walls at both Uxmal and Tulum were cyanobacteria; heterotrophic bacteria and filamentous fungi were also present. A dark‐pigmented mitosporic fungus and Bacillus cereus, both isolated from Uxmal, were shown to be acidogenic in laboratory cultures. Cyanobacteria belonging to rock‐degrading genera Synechocystis and Gloeocapsa were identified at both sites. Surface analysis previously showed that calcium ions were present in the biofilms on buildings at Uxmal and Tulum, suggesting the deposition of biosolubilized stone. Apart from their potential to degrade the substrate, the coccoid cyanobacteria supply organic nutrients for bacteria and fungi, which can produce organic acids, further increasing stone degradation.     

Microhabitat Variation Explains Local‐scale Distribution of Terrestrial Amazonian Lizards in Rondônia,Western Brazil

We investigate the role of ecology and phylogeny in the association between lizard abundance and microhabitat variables in an Amazon rain forest site. Using pitfall trap arrays, we collected data from 349 individuals belonging to 23 lizard species. After accounting for spatial autocorrelation and using a canonical correspondence analysis (CCA), we found that lizard captures were significantly associated with microhabitat variables, which accounted for 48 percent of the observed variation. Furthermore, a canonical phylogenetic ordination (CPO) indicated that microhabitat variables are more important in determining the distribution of lizard species than phylogenetic relationships among species. Termite nests, canopy openness, and tree circumference were strongly associated with the number of captures of certain lizard species. Our results confirm autecology studies of individual lizard species for which data are available. We suggest that maintaining heterogeneous forested microhabitats should be a central goal for sustaining a high lizard biodiversity in Amazon rain forests.     

Indicator selection in life cycle assessment to enable decision making: issues and solutions

Purpose

With an ever increasing list of indicators available, life cycle assessment (LCA) practitioners face the challenge of effectively communicating results to decision makers. Simplification of LCA is often limited to an arbitrary selection of indicators, use of single scores by using weighted values or single attribute indicators. These solutions are less attractive to decision makers, since value judgments are introduced or multi-indicator information is lost. Normalization could be a means to narrow the list of indicators by ranking indicators vs. a reference system. This paper shows three different normalization approaches that produce very different ranking of indicators. It is explained how normalization helps maintain a multi-indicator approach while keeping the most relevant indicators, allowing effective decision making.

Methods

The approaches are illustrated on a hand dishwashing case study, using ReCiPe as the impact assessment method and taking the European population (year 2000) as the reference situation. Indicators are ranked using midpoint normalization factors, and compared to the ranking from endpoint normalization broken down by midpoint contribution.

Results and discussion

Endpoint normalization shows Resources as the most relevant area of protection for this case, closely followed by Human Health and Ecosystem. Broken down by their key driving midpoints, fossil depletion, climate change and, to a lesser extent, particulate matter formation and metal depletion, are most relevant. Midpoint normalization, however, indicates Freshwater Eutrophication, Natural Land Transformation and Toxicity indicators (marine and freshwater ecotoxicity and human toxicity) are most relevant.

Conclusions

A three-step approach based on endpoint normalization is recommended to present only the most relevant indicators, allowing more effective decision making instead of communicating all LCA indicators. The selection process breaks out the normalized endpoint results into the most contributing midpoints (relevant indicators) and reports results with midpoint level units. Bias due to lack of data completeness is less of an issue in the endpoint normalization process (compared to midpoint normalization), while midpoint results are less subject to uncertainty (compared to endpoint results). Focusing on the relevant indicators and key contributing unit processes has proven to be effective for non-LCA expert decision makers to understand, use, and communicate complex LCA results.     

The N-terminal region of the Oenococcus oeni bacteriophage fOg44 lysin behaves as a bona fide signal peptide in Escherichia coli and as a cis-inhibitory element, preventing lytic activity on oenococcal cells

The function of the N-terminal region of the Oenococcus oeni phage fOg44 lysin (Lys44) as an export signal was investigated. We observed that when induced in Escherichia coli, Lys44 was cleaved between residues 27 and 28 in a SecA-dependent manner. Lys44 processing could be blocked by a specific signal peptidase inhibitor and was severely reduced by modification of the cleavage site. The lethal effect of Lys44 expression observed in E. coli was ascribed to the presence of its N-terminal 27-residue sequence, as its deletion resulted in the production of a nontoxic, albeit active, product. We have further established that lytic activity in oenococcal cells was dependent on Lys44 processing. An active protein with the molecular mass expected for the cleaved enzyme was detected in extracts from O. oeni-infected cells. The temporal pattern of its appearance suggests that synthesis and export of Lys44 in the infected host progress along with phage maturation. Overall, these results provide, for the first time, experimental evidence for the presence of a signal peptide in a bacteriophage lysin. Database searches and alignment of protein sequences support the prediction that other known O. oeni and Lactococcus lactis phages also encode secretory lysins. The evolutionary significance of a putative phage lysis mechanism relying on secretory lytic enzymes is tentatively discussed, on the basis of host cell wall structure and autolytic capacity.     

Structural and Functional Analysis of a Platelet-Activating Lysophosphatidylcholine of Trypanosoma cruzi

Background

Trypanosoma cruzi is the causative agent of the life-threatening Chagas disease, in which increased platelet aggregation related to myocarditis is observed. Platelet-activating factor (PAF) is a potent intercellular lipid mediator and second messenger that exerts its activity through a PAF-specific receptor (PAFR). Previous data from our group suggested that T. cruzi synthesizes a phospholipid with PAF-like activity. The structure of T. cruzi PAF-like molecule, however, remains elusive.

Methodology/Principal findings

Here, we have purified and structurally characterized the putative T. cruzi PAF-like molecule by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). Our ESI-MS/MS data demonstrated that the T. cruzi PAF-like molecule is actually a lysophosphatidylcholine (LPC), namely sn-1 C18:1(delta 9)-LPC. Similar to PAF, the platelet-aggregating activity of C18:1-LPC was abrogated by the PAFR antagonist, WEB 2086. Other major LPC species, i.e., C16:0-, C18:0-, and C18:2-LPC, were also characterized in all T. cruzi stages. These LPC species, however, failed to induce platelet aggregation. Quantification of T. cruzi LPC species by ESI-MS revealed that intracellular amastigote and trypomastigote forms have much higher levels of C18:1-LPC than epimastigote and metacyclic trypomastigote forms. C18:1-LPC was also found to be secreted by the parasite in extracellular vesicles (EV) and an EV-free fraction. A three-dimensional model of PAFR was constructed and a molecular docking study was performed to predict the interactions between the PAFR model and PAF, and each LPC species. Molecular docking data suggested that, contrary to other LPC species analyzed, C18:1-LPC is predicted to interact with the PAFR model in a fashion similar to PAF.

Conclusions/Significance

Taken together, our data indicate that T. cruzi synthesizes a bioactive C18:1-LPC, which aggregates platelets via PAFR. We propose that C18:1-LPC might be an important lipid mediator in the progression of Chagas disease and its biosynthesis could eventually be exploited as a potential target for new therapeutic interventions.     

Disproportionate Contribution of Riparian Inputs to Organic Carbon Pools in Freshwater Systems

A lack of appropriate proxies has traditionally hampered our ability to distinguish riverine organic carbon (OC) sources at the landscape scale. However, the dissection of C₄ grasslands by C₃-enriched riparian vegetation, and the distinct carbon stable isotope signature (δ¹³C) of these two photosynthetic pathways, provides a unique setting to assess the relative contribution of riparian and more distant sources to riverine C pools. Here, we compared δ¹³C signatures of bulk sub-basin vegetation (δ¹³C_VEG) with those of riverine OC pools for a wide range of sites within two contrasting river basins in Madagascar. Although C₃-derived carbon dominated in the eastern Rianala catchment, consistent with the dominant vegetation, we found that in the C₄-dominated Betsiboka basin, riverine OC is disproportionately sourced from the C₃-enriched riparian fringe, irrespective of climatic season, even though δ¹³C_VEG estimates suggest as much as 96% of vegetation cover in some Betsiboka sub-basins may be accounted for by C₄ biomass. For example, δ¹³C values for river bed OC were on average 6.9 ± 2.7‰ depleted in ¹³C compared to paired estimates of δ¹³C_VEG. The disconnection of the wider C₄-dominated basin is considered the primary driver of the under-representation of C₄-derived C within riverine OC pools in the Betsiboka basin, although combustion of grassland biomass by fire is likely a subsidiary constraint on the quantity of terrestrial organic matter available for export to these streams and rivers. Our findings carry implications for the use of sedimentary δ¹³C signatures as proxies for past forest-grassland distribution and climate, as the C₄ component may be considerably underestimated due to its disconnection from riverine OC pools.