Recycling factors for ribosome disassembly in the apicoplast and mitochondrion of Plasmodium falciparum

The reduced genomes of the apicoplast and mitochondrion of the malaria parasite Plasmodium falciparum are actively translated and antibiotic‐mediated translation inhibition is detrimental to parasite survival. In order to understand recycling of organellar ribosomes, a critical step in protein translation, we identified ribosome recycling factors (RRF) encoded by the parasite nuclear genome. Targeting of PfRRF1 and PfRRF2 to the apicoplast and mitochondrion respectively was established by localization of leader sequence–GFP fusions. Unlike any RRF characterized thus far, PfRRF2 formed dimers with disulphide interaction(s) and additionally localized in the cytoplasm, thus suggesting adjunct functions for the factor. PfRRF1 carries a large 108‐amino‐acid insertion in the functionally critical hinge region between the head and tail domains of the protein, yet complemented Escherichia coli RRF in the LJ14frr^ts mutant and disassembled surrogate E. coli 70S ribosomes in the presence of apicoplast‐targeted EF‐G. Recombinant PfRRF2 bound E. coli ribosomes and could split monosomes in the presence of the relevant mitochondrial EF‐G but failed to complement the LJ14frr^ts mutant. Although proteins comprising subunits of P. falciparum organellar ribosomes are predicted to differ from bacterial and mitoribosomal counterparts, our results indicate that the essential interactions required for recycling are conserved in parasite organelles.     

Water‐miscible ionic liquids as novel effectors for the firefly luciferase reaction

Ionic liquids (IL) are used as a new class of solvents for various reactions. Especially using IL in biocatalysis in an aqueous milieu has attracted considerable attention because enzymes show remarkable differences in their catalytic features in IL‐containing reaction media. Firefly luciferase is widely used in many analytical techniques, because light production of firefly luciferase is one of the most sensitive analytical measures in the ultrasensitive detection of adenosine‐5′‐triphosphate, e.g. for measuring microbial contamination and monitoring gene expression, as well as for monitoring tumor growth and metastasis in whole animals. Firefly luciferase is an unstable enzyme and its inactivation can lead to low sensitivity in the above‐mentioned assays. The present study addresses the comparative influence of six different water‐immiscible IL, the 3‐methylimidazolium derivatives [BMIM]Cl, [HMIM]Cl, [BMIM]Br, [EMIM]Br, [HMIM]Br, and [BMIM]BF₄, on the kinetic properties, structural stability, and function of firefly luciferase from Photinus pyralis using circular dichroism, fluorescence spectroscopy, and a bioluminescence assay. The incubation of luciferase with various IL showed that, with the exception of [BMIM]BF₄, the activity and stability of luciferase was considerably increased in the presence of IL, compared to luciferase in aqueous medium. Moreover, K_m for the substrate adenosine‐5′‐triphosphate in the presence of IL (except for [BMIM]BF₄) decreased while K_m for luciferin remained constant.     

Interaction between sulphur mobilisation proteins SufB and SufC: evidence for an iron-sulphur cluster biogenesis pathway in the apicoplast of Plasmodium falciparum

The plastid of Plasmodium falciparum, the apicoplast, performs metabolic functions essential to the parasite. Various reactions in the plastid require the assembly of [Fe-S] prosthetic groups on participating proteins as well as the reductant activity of ferredoxin that is converted from its apo-form by the assembly of [Fe-S] clusters inside the apicoplast. The [Fe-S] assembly pathway involving sulphur mobilising Suf proteins has been predicted to function in the apicoplast with one component (PfSufB) encoded by the plastid genome itself. We demonstrate the ATPase activity of recombinant P. falciparum nuclear-encoded SufC and its localisation in the apicoplast. Further, an internal region of apicoplast SufB was used to detect PfSufB-PfSufC interaction in vitro; co-elution of SufB from parasite lysate with recombinant PfSufC on an affinity column also indicated an interaction of the two proteins. As a departure from bacterial SufB and similar to reported plant plastid SufB, apicoplast SufB exhibited ATPase activity, suggesting the evolution of specialised functions in the plastid counterparts. Our results provide experimental evidence for an active Suf pathway in the Plasmodium apicoplast.     

Emissions of nitrous oxide from the leaves of grasses

Aims

Nitrous oxide (N₂O) emissions from pastoral agriculture are considered to originate from the soil as a consequence of microbial activity during soil nitrification and denitrification. However, recent studies have identified the plant canopy as a potentially significant source of N₂O emissions to the atmosphere. Understanding the extent and mechanisms of plant emissions may provide new mitigation opportunities as current options only target soil microbial processes.

Methods

We developed an experimental apparatus and protocol to partition N₂O emissions between the leaves of grasses and the soil and measured emissions from ten common grass species found in New Zealand pastures.

Results

The chamber design enabled us to identify measurable changes in N₂O concentration over a period of 1 h and to distinguish a range of emissions from 0.001 to 0.25 mg N₂O-N/m² leaf area/h. There was a 10-fold variation among species; Holcus lanataus, Lolium perenne and Paspalum dilatatum had the highest leaf N₂O emissions and Poa annua the lowest.

Conclusions

Grasses do emit N₂O from their leaves and the rate that this occurs varies among grass species. The emission does not appear to arise from formation of N₂O in plant leaves but more likely reflects transport of N₂O from the soil. Differences in emission rates appear to arise from a plant influence on the rate of formation of N₂O in the soil rather than the rate of transportation through the plant.     

Social mobilisation, drug coverage and compliance and adverse reactions in a Mass Drug Administration (MDA) Programme for the Elimination of Lymphatic Filariasis in Sri Lanka

Background

In Sri Lanka filariasis is endemic in Southern, Western and North Western provinces covering eight districts designated as implementation units in the Programme for the Elimination of Lymphatic Filariasis (PELF). Despite control activities over sixty years including multidose diethylcarbamazine, 6 mg/kg treatment microfilaria rates had persisted at low levels. Following systematic social mobilisation the first MDA with DEC albendazole combination was conducted in 2002.

Methods

We investigated the extent social mobilisation had reached the people, their drug compliance and adverse reactions. Three localities were selected from each district to pick target population samples for pre-tested questionnaire. Three teams each with six people visited one district each day. One team worked from three starting points in one locality. A member applied eight part questionnaire to one family member totalling 150–160 people from one locality. Questions included social mobilisation, drug compliance and adverse reactions.

Results

Information was disseminated by television, radio, banners and leaflets, to a lesser extent by people. Information reached more people in the periphery than in Colombo. 35.2% from Colombo municipality were unaware of the MDA. Drug coverage was 79.6%, home delivery 71.7% and delivery centres 7.9%. 35.6% in Colombo district and 53.4% from Colombo municipality did not receive drugs. Drugs were consumed by 71.4%. 28.6% who did not comply included 20.4% who did not receive them. 91.4% showed no adverse reactions, 7.5% were mild, 1.1% recovered with home remedies.

Conclusion

Drug compliance showed significant positive correlation with awareness of the MDA. Door to door delivery was more successful than delivery from centres. More delivery centres conveniently located would have rectified this disparity. Poor awareness and compliance in Colombo and urban areas could be rectified with separate strategy for urban areas. More time for MDA and trained adequate manpower would ensure coverage to achieve elimination.     

Simultaneous Treatment of Dioxins and Heavy Metals in Tagonoura Harbor Sediment by Two-Step Pyrolysis

This paper describes the simultaneous treatment of PCDD/PCDF and toxic heavy metals contaminated sediment by pyrolysis with emphasis on complete removal and destruction of PCDD/PCDF in Tagonoura harbor sediment during a two-step pyrolysis process. Pyrolysis was conducted at different temperatures and N ₂ gas flow rates. Results showed that almost all PCDD/PCDF (99.998%) can be removed from the sediment by treatment at 800°C for 30 minutes in single-step pyrolysis; however, PCDD/PCDF remained in the gas phase without destruction. Analysis also revealed that some PCDD/PCDF formation occurred during the single-step pyrolysis. However, by the use of two-step pyrolysis, where off-gases from the first furnace are heated at 1000°C, PCDD/PCDF in the final gas effluent was dramatically reduced by a thermal degradation mechanism. Stabilization of toxic heavy metals was also observed. Specifically, their leaching activity drastically decreased with increasing pyrolysis temperature. Significant metal volatilization did not occur. Though the low temperature pyrolysis was effective in removing PCDD/PCDF, the leachability of toxic heavy metals such as Cr and Ni was higher that of 800°C; however, lower than untreated. Two-step pyrolysis at 800°C for 30 minutes in the single-step and 1000°C in the two-step pyrolyzer would be effective for the treatment of sediments contaminated with PCDD/PCDF, toxic heavy metals and organic matters simultaneously.     

Effects of different processing methods of flaxseed on ruminal degradability and in vitro post-ruminal nutrient disappearance

The aim of the study was to determine the effects of different heat-processing methods of flaxseed on the in situ effective dry matter degradability (EDMD) and the in situ effective crude protein degradability (ECPD). The treatments included roasting, steep roasting, rolled roasting, rolled steep roasting, microwave irradiation and extrusion. Three rumen-fistulated sheep were used for in situ incubations. Furthermore, the effects of heat-processing methods on post-ruminal in vitro nutrient disappearance and total tract disappearance were measured by a three-step in vitro technique. The seeds were roasted and extruded at 140°C to 145°C. One lot of roasted seeds was gradually cooled for about 1 h (roasting) and another lot was held in temperature isolated barrels for 45 min (steep roasting). Moreover, roasted and steep roasted flaxseed was rolled in a roller mill. The lowest and highest EDMD was observed for unheated and extruded flaxseed, respectively (p < 0.05). The highest ECPD was observed for extruded flaxseed (p < 0.05). Roasting and microwave irradiation reduced ECPD of flaxseed (p < 0.05). In vitro post-ruminal disappearance of crude nutrients including fibre fractions was highest for rolled-roasted and rolled steep-roasted flaxseed (p < 0.05). The lowest and highest total tract disappearance rates of crude nutrients and fibre fractions were estimated for unheated and extruded flaxseed, respectively (p < 0.05). The post-ruminal disappearance of crude nutrients was also increased by roasting, in which rolling enhanced this effect. In conclusion, all investigated heat treatments had significant effects on in situ and in vitro degradability of nutrients. As well, rolling of roasted flaxseed enhanced the respective effects. Therefore, different methods of heat processing can be used to modify the feed value of flaxseed for specific purposes.     

Bacteria on leaves: a previously unrecognised source of N2O in grazed pastures

Nitrous oxide (N₂O) emissions from grazed pastures are a product of microbial transformations of nitrogen and the prevailing view is that these only occur in the soil. Here we show this is not the case. We have found ammonia-oxidising bacteria (AOB) are present on plant leaves where they produce N₂O just as in soil. AOB (Nitrosospira sp. predominantly) on the pasture grass Lolium perenne converted 0.02–0.42% (mean 0.12%) of the oxidised ammonia to N₂O. As we have found AOB to be ubiquitous on grasses sampled from urine patches, we propose a ‘plant'' source of N₂O may be a feature of grazed grassland.In terms of climate forcing, nitrous oxide (N₂O) is the third most important greenhouse gas (Blunden and Arndt, 2013). Agriculture is the largest source of anthropogenic N₂O (Reay et al., 2012) with about 20% of agricultural emissions coming from grassland grazed by animals (Oenema et al., 2005).Grazed grassland is a major source of N₂O because grazers harvest nitrogen (N) from plants across a wide area but recycle it back onto the pasture, largely as urine, in patches of very high N concentration. The N in urine patches is often in excess of what can be used by plants resulting in losses through leaching as nitrate, as N₂O and through volatilisation as ammonia (NH₃) creating a high NH₃ environment in the soil and plant canopy; an important point that we will return to later. The established wisdom is that N₂O is generated exclusively by soil-based microbes such as ammonia-oxidising bacteria (AOB). This soil biology is represented in models designed to simulate N₂O emissions and the soil is a target for mitigation strategies such as the use of nitrification inhibitors.We have previously shown that pasture plants can emit N₂O largely through acting as a conduit for emissions generated in the soil, which are themselves controlled to some degree by the plant (Bowatte et al., 2014). In this case the origin of the emission is still the soil microbes. However, AOB have been found on the leaves of plants, for example, Norway spruce (Papen et al., 2002; Teuber et al., 2007) and weeds in rice paddies (Bowatte et al., 2006), prompting us to ask whether AOB might be present on the leaves of pasture species and contribute to N₂O emissions as they do in soil.We looked for AOB on plants in situations where NH₃ concentrations were likely to be high, choosing plants from urine patches in grazed pastures and plants from pastures surrounding a urea fertiliser manufacturing plant. DNA was extracted from the leaves (including both the surface and apoplast) and the presence of AOB tested using PCR. AOB were present in all the species we examined—the grasses Lolium perenne, Dactylis glomerata, Anthoxanthum odoratum, Poa pratensis, Bromus wildenowii and legumes Trifolium repens and T. subterraneum.To measure whether leaf AOB produce N₂O, we used intact plants of ryegrass (L. perenne) lifted as cores from a paddock that had been recently grazed by adult sheep. The cores were installed in a chamber system designed to allow sampling of above- and belowground environments separately (Bowatte et al., 2014). N₂O emissions were measured from untreated (control) plants and from plants where NH₃ was added to the aboveground chamber and leaves were either untreated or sterilised by wiping twice with paper towels soaked in 1% hypoclorite (Sturz et al., 1997) and then with sterile water. We tested for the presence and abundance of AOB on the leaves by extracting DNA and using PCR and real-time PCR targeting the ammonia monoxygenase A (amoA) gene, which is characteristic of AOB. AOB identity was established using cloning and DNA sequencing. Further details of these experiments can be found in the Supplementary Information.The addition of NH₃ to untreated plants significantly stimulated N₂O emissions (P<0.001) compared with the controls; by contrast, the plants with sterilised leaves produced significantly less N₂O than controls (P<0.001) even with NH₃ added (Figure 1) providing strong evidence for emissions being associated with bacteria on the leaves. Control plants did emit N₂O suggesting there was either sufficient NH₃ available for bacterially generated emissions and/or other plant-based mechanisms were involved (Bowatte et al., 2014).Open in a separate windowFigure 1Effect of an elevated NH₃ atmosphere and surface sterilisation of leaves on leaf N₂O emissions measured over 1-h periods on three occasions during the day. Values are means (s.e.m.), where n=7.The major AOB species identified was Nitrosospira strain III7 that has been previously shown to produce N₂O (Jiang and Bakken, 1999). We measured 10⁹ AOB cells per m² ryegrass leaf, assuming a specific leaf area of 250 cm² g⁻¹ leaf.The rate of production of N₂O (0.1–0.17 mg N₂O-N per m² leaf area per hour) can be translated to a field situation using the leaf area index (LAI)—1 m² leaf per m² ground would be an LAI of 1. LAI in a pasture can vary from <1 to >6 depending on the management (for example, Orr et al., 1988). At LAI of 1, the AOB leaf emission rate would equate to a N₂O emission rate of about 0.1–0.3 mg N₂O-N per m² ground per hour. By comparison, the emission rates measured after dairy cattle urine (650 kg N ha⁻¹) was applied to freely and poorly drained soil were 0.024–1.55 and 0.048–3.33 mg N₂O-N per m² ground per hour, respectively (Li and Kelliher, 2005).The fraction of the NH₃ that was converted to N₂O by the leaf AOB was 0.02–0.42% (mean 0.12%). The mean value is close to that measured for Nitrosospira strains including strain III7 isolated from acidic, loamy and sandy soils where values ranged from 0.07 to 0.10% (Jiang and Bakken, 1999). This is good evidence that the AOB on leaves have the capacity to produce N₂O at the same rate as AOB in soils. We do not suggest that leaf AOB will produce as much N₂O as soil microbes; however, because leaf AOB have access to a source of substrate—volatilised NH₃—that is unavailable to soil microbes and may constitute 26% (Laubach et al., 2013) to 40% (Carran et al., 1982) of the N deposited in the urine, N₂O emissions from these aboveground AOB are additional to soil emissions. Further research is required to identify the situations in which leaf AOB contribute to total emissions and to quantify this contribution.     

IMPARO: inferring microbial interactions through parameter optimisation

Type 2 diabetes mellitus (T2DM) is a worldwide disease that have an impact on individuals of all ages causing micro and macro vascular impairments due to hyperglycemic internal environment. For ultimate treatment to cure T2DM, association of diabetes with immune components provides a strong basis for immunotherapies and vaccines developments that could stimulate the immune cells to minimize the insulin resistance and initiate gluconeogenesis through an insulin independent route. Immunoinformatics based approach was used to design a polyvalent vaccine for T2DM that involved data accession, antigenicity analysis, T-cell epitopes prediction, conservation and proteasomal evaluation, functional annotation, interactomic and in silico binding affinity analysis. We found the binding affinity of antigenic peptides with major histocompatibility complex (MHC) Class-I molecules for immune activation to control T2DM. We found 13-epitopes of 9 amino acid residues for multiple alleles of MHC class-I bears significant binding affinity. The downstream signaling resulted by T-cell activation is directly regulated by the molecular weight, amino acid properties and affinity of these epitopes. Each epitope has important percentile rank with significant ANN IC50 values. These high score potential epitopes were linked using AAY, EAAAK linkers and HBHA adjuvant to generate T-cell polyvalent vaccine with a molecular weight of 35.6 kDa containing 322 amino acids residues. In silico analysis of polyvalent construct showed the significant binding affinity (− 15.34 Kcal/mol) with MHC Class-I. This interaction would help to understand our hypothesis, potential activation of T-cells and stimulatory factor of cytokines and GLUT1 receptors. Our system-level immunoinformatics approach is suitable for designing potential polyvalent therapeutic vaccine candidates for T2DM by reducing hyperglycemia and enhancing metabolic activities through the immune system.