Analysis of the Binding Moiety Mediating the Interaction between Monocarboxylate Transporters and Carbonic Anhydrase II

Proton-coupled monocarboxylate transporters (MCTs) mediate the exchange of high energy metabolites like lactate between different cells and tissues. We have reported previously that carbonic anhydrase II augments transport activity of MCT1 and MCT4 by a noncatalytic mechanism, while leaving transport activity of MCT2 unaltered. In the present study, we combined electrophysiological measurements in Xenopus oocytes and pulldown experiments to analyze the direct interaction between carbonic anhydrase II (CAII) and MCT1, MCT2, and MCT4, respectively. Transport activity of MCT2-WT, which lacks a putative CAII-binding site, is not augmented by CAII. However, introduction of a CAII-binding site into the C terminus of MCT2 resulted in CAII-mediated facilitation of MCT2 transport activity. Interestingly, introduction of three glutamic acid residues alone was not sufficient to establish a direct interaction between MCT2 and CAII, but the cluster had to be arranged in a fashion that allowed access to the binding moiety in CAII. We further demonstrate that functional interaction between MCT4 and CAII requires direct binding of the enzyme to the acidic cluster ⁴³¹EEE in the C terminus of MCT4 in a similar fashion as previously shown for binding of CAII to the cluster ⁴⁸⁹EEE in the C terminus of MCT1. In CAII, binding to MCT1 and MCT4 is mediated by a histidine residue at position 64. Taken together, our results suggest that facilitation of MCT transport activity by CAII requires direct binding between histidine 64 in CAII and a cluster of glutamic acid residues in the C terminus of the transporter that has to be positioned in surroundings that allow access to CAII.     

Assessment of Heavy Metals and Metalloids in Solanum tuberosum and Pisum sativum Irrigated with Urban Wastewater in the Suburbs of Sargodha City,Pakistan

Dietary exposure to heavy metals (viz., Ni, As, Fe, Cr, Mn, Co, Mo, Cu, Zn, Se, Cd, and Pb) has been recognized as a potential hazard to human health. This study investigates the level of contamination at two different sites in Pakistan, one irrigated with canal water (Site-I) and the other with urban wastewater (Site-II). At Site-II, irrigation with wastewater resulted in a significant increase in heavy metals and metalloids in soil and a subsequent build-up in two vegetables selected for study (Solanum tuberosum [potato] and Pisum sativum [pea]). Results showed that mean concentrations of heavy metals and metalloids in soil at Site-I were lower than those of Site-II. Mean concentrations of As and Cd in soil at both sites and for both vegetables were found above maximum permissible levels, while for both vegetables As at both sites and Cd, Mo, and Pb exceeded the suggested maximum levels for vegetables. High levels of some metals in the soils and vegetables could be due to unnecessary use of fertilizers and disposable water for irrigating the soils and the environmental cues prevalent in the areas, such as presence of ions that may bind the metals, often play an important role in uptake.     

Optimization of Pre-transplantation Conditions to Enhance the Efficacy of Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are considered a potential tool for cell based regenerative therapy due to their immunomodulatory property, differentiation potentials, trophic activity as well as large donor pool. Poor engraftment and short term survival of transplanted MSCs are recognized as major limitations which were linked to early cellular ageing, loss of chemokine markers during ex vivo expansion, and hyper-immunogenicity to xeno-contaminated MSCs. These problems can be minimized by ex vivo expansion of MSCs in hypoxic culture condition using well defined or xeno-free media i.e., media supplemented with growth factors, human serum or platelet lysate. In addition to ex vivo expansion in hypoxic culture condition using well defined media, this review article describes the potentials of transient adaptation of expanded MSCs in autologous serum supplemented medium prior to transplantation for long term regenerative benefits. Such transient adaptation in autologous serum supplemented medium may help to increase chemokine receptor expression and tissue specific differentiation of ex vivo expanded MSCs, thus would provide long term regenerative benefits.     

High yield lipase-catalyzed synthesis of Engkabang fat esters for the cosmetic industry

Engkabang fat esters were produced via alcoholysis reaction between Engkabang fat and oleyl alcohol, catalyzed by Lipozyme RM IM. The reaction was carried out in a 500 ml Stirred tank reactor using heptane and hexane as solvents. Response surface methodology (RSM) based on a four-factor-five-level Central composite design (CCD) was applied to evaluate the effects of synthesis parameters, namely temperature, substrate molar ratio (oleyl alcohol: Engkabang fat), enzyme amount and impeller speed. The optimum yields of 96.2% and 91.4% were obtained for heptane and hexane at the optimum temperature of 53.9 °C, impeller speeds of 309.5 and 309.0 rpm, enzyme amounts of 4.82 and 5.65 g and substrate molar ratios of 2.94 and 3.39:1, respectively. The actual yields obtained compared well with the predicted values of 100.0% and 91.5%, respectively. Meanwhile, the properties of the esters show that they are suitable to be used as ingredient for cosmetic applications.     

Mining the plant-herbivore interface with a leafmining Drosophila of Arabidopsis

Experimental infections of Arabidopsis thaliana (Arabidopsis) with genomically characterized plant pathogens such as Pseudomonas syringae have facilitated the dissection of canonical eukaryotic defence pathways and parasite virulence factors. Plants are also attacked by herbivorous insects, and the development of an ecologically relevant genetic model herbivore that feeds on Arabidopsis will enable the parallel dissection of host defence and reciprocal resistance pathways such as those involved in xenobiotic metabolism. An ideal candidate is Scaptomyza flava, a drosophilid fly whose leafmining larvae are true herbivores that can be found in nature feeding on Arabidopsis and other crucifers. Here, we describe the life cycle of S.?flava on Arabidopsis and use multiple approaches to characterize the response of Arabidopsis to S.?flava attack. Oviposition choice tests and growth performance assays on different Arabidopsis ecotypes, defence-related mutants, and hormone and chitin-treated plants revealed significant differences in host preference and variation in larval performance across Arabidopsis accessions. The jasmonate and glucosinolate pathways in Arabidopsis are important in mediating quantitative resistance against S.?flava, and priming with jasmonate or chitin resulted in increased resistance. Expression of xenobiotic detoxification genes was reduced in S.?flava larvae reared on Arabidopsis jasmonate signalling mutants and increased in plants pretreated with chitin. These results and future research directions are discussed in the context of developing a genetic model system to analyse insect-plant interactions.     

Engineering of the E. coli Outer Membrane Protein FhuA to overcome the Hydrophobic Mismatch in Thick Polymeric Membranes

Background

There is an urgent need to develop safe and effective adjuvants for the new generation of subunit vaccines. We developed the tubular immunostimulating complex (TI-complex) as a new nanoparticulate antigen delivery system. The morphology and composition of TI-complexes principally differ from the known vesicular immunostimulating complexes (ISCOMs). However, methodology for the preparation of TI-complexes has suffered a number of shortcomings. The aim of the present work was to obtain an antigen carrier consisting of triterpene glycosides from Cucumaria japonica, cholesterol, and monogalactosyldiacylglycerol from marine macrophytes with reproducible properties and high adjuvant activity.

Results

The cucumarioside A₂-2 - cholesterol - MGalDG ratio of 6:2:4 (by weight) was found to provide the most effective formation of TI-complexes and the minimum hemolytic activity in vitro. Tubules of TI-complexes have an outer diameter of about 16 nm, an inner diameter of 6 nm, and a length of 500 nm. A significant dilution by the buffer gradually destroyed the tubular nanoparticles. The TI-complex was able to increase the immunogenicity of the protein antigens from Yersinia pseudotuberculosis by three to four times.

Conclusions

We propose an optimized methodology for the preparation of homogeneous TI-complexes containing only tubular particles, which would achieve reproducible immunization results. We suggest that the elaborated TI-complexes apply as a universal delivery system for different subunit antigens within anti-infectious vaccines and enhance their economic efficacy and safety.     

The impact of retail-sector delivery of artemether-lumefantrine on malaria treatment of children under five in Kenya: a cluster randomized controlled trial

Background

It has been proposed that artemisinin-based combination therapy (ACT) be subsidised in the private sector in order to improve affordability and access. This study in western Kenya aimed to evaluate the impact of providing subsidized artemether–lumefantrine (AL) through retail providers on the coverage of prompt, effective antimalarial treatment for febrile children aged 3–59 months.

Methods and Findings

We used a cluster-randomized, controlled design with nine control and nine intervention sublocations, equally distributed across three districts in western Kenya. Cross-sectional household surveys were conducted before and after the delivery of the intervention. The intervention comprised provision of subsidized packs of paediatric ACT to retail outlets, training of retail outlet staff, and community awareness activities. The primary outcome was defined as the proportion of children aged 3–59 months reporting fever in the past 2 weeks who started treatment with AL on the same day or following day of fever onset. Data were collected using structured questionnaires and analyzed based on cluster-level summaries, comparing control to intervention arms, while adjusting for other covariates. Data were collected on 2,749 children in the target age group at baseline and 2,662 at follow-up. 29% of children experienced fever within 2 weeks before the interview. At follow-up, the percentage of children receiving AL on the day of fever or the following day had risen by 14.6% points in the control arm (from 5.3% [standard deviation (SD): 3.2%] to 19.9% [SD: 10.0%]) and 40.2% points in the intervention arm (from 4.7% [SD: 3.4%] to 44.9% [SD: 11.7%]). The percentage of children receiving AL was significantly greater in the intervention arm at follow-up, with a difference between the arms of 25.0% points (95% confidence interval [CI]: 14.1%, 35.9%; unadjusted p = 0.0002, adjusted p = 0.0001). No significant differences were observed between arms in the proportion of caregivers who sought treatment for their child''s fever by source, or in the child''s adherence to AL.

Conclusions

Subsidizing ACT in the retail sector can significantly increase ACT coverage for reported fevers in rural areas. Further research is needed on the impact and cost-effectiveness of such subsidy programmes at a national scale.

Trial Registration

Current Controlled Trials ISRCTN59275137 and Kenya Pharmacy and Poisons Board Ethical Committee for Clinical Trials PPB/ECCT/08/07. Please see later in the article for the Editors'' Summary     

FhuA deletion variant Δ1-159 overexpression in inclusion bodies and refolding with Polyethylene-Poly(ethylene glycol) diblock copolymer

Membrane protein isolation is a challenging problem. In fact especially their extraction from the respective membrane is difficult and often goes along with losses in yield. Usually expensive detergents are needed to extract the target protein from the membrane. Therefore finding an efficient overexpression and extraction method and an alternative to detergents is desirable. In this study we describe a new and fast method to express, extract and purify an engineered variant of the FhuA protein (FhuA Δ1-159) that acts as passive diffusion channel, using a diblock copolymer as an alternative to detergents like octyl-POE (n-octylpolyoxyethylene). The N-terminal leader sequence, facilitating the protein's transport to the outer membrane was deleted (FhuA Δ1-159 Δsignal), resulting in protein accumulation in easy to isolate inclusion bodies. Urea was used to solubilise the unfolded protein and dialysis against phosphate-buffer containing the commercially available diblock copolymer PE-PEG[Polyethylene-Poly(ethyleneglycol)] lead to protein refolding. Circular dichroism spectroscopy revealed a high β-sheet percentage within the refolded protein secondary structure indicating the successful reconstitution of FhuA Δ1-159 Δsignal native state. Furthermore the channel functionality of FhuA Δ1-159 Δsignal was verified by measuring the in and out-flux through the protein when inserted into liposome membrane, using the HRP/TMB (HRP=Horse Radish Peroxidase, TMB=3,3',5,5'-tetramethylbenzidine) assay system.     

Temperature and malaria trends in highland East Africa

There has been considerable debate on the existence of trends in climate in the highlands of East Africa and hypotheses about their potential effect on the trends in malaria in the region. We apply a new robust trend test to mean temperature time series data from three editions of the University of East Anglia's Climatic Research Unit database (CRU TS) for several relevant locations. We find significant trends in the data extracted from newer editions of the database but not in the older version for periods ending in 1996. The trends in the newer data are even more significant when post-1996 data are added to the samples. We also test for trends in the data from the Kericho meteorological station prepared by Omumbo et al. We find no significant trend in the 1979-1995 period but a highly significant trend in the full 1979-2009 sample. However, although the malaria cases observed at Kericho, Kenya rose during a period of resurgent epidemics (1994-2002) they have since returned to a low level. A large assembly of parasite rate surveys from the region, stratified by altitude, show that this decrease in malaria prevalence is not limited to Kericho.