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The variant antigen Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), which is expressed on the surface of P. falciparum-infected red blood cells, is a critical virulence factor for malaria (1). Each parasite has 60 antigenically distinct var genes that each code for a different PfEMP1 protein. During infection the clonal parasite population expresses only one gene at a time before switching to the expression of a new variant antigen as an immune-evasion mechanism to avoid the host antibody response (2,3). The mechanism by which 59 of the 60 var genes are silenced remains largely unknown (4-7). Here we show that knocking out the P. falciparum variant-silencing SET gene (here termed PfSETvs), which encodes an orthologue of Drosophila melanogaster ASH1 and controls histone H3 lysine 36 trimethylation (H3K36me3) on var genes, results in the transcription of virtually all var genes in the single parasite nuclei and their expression as proteins on the surface of individual infected red blood cells. PfSETvs-dependent H3K36me3 is present along the entire gene body, including the transcription start site, to silence var genes. With low occupancy of PfSETvs at both the transcription start site of var genes and the intronic promoter, expression of var genes coincides with transcription of their corresponding antisense long noncoding RNA. These results uncover a previously unknown role of PfSETvs-dependent H3K36me3 in silencing vargenes in P. falciparum that might provide a general mechanism by which orthologues of PfSETvs repress gene expression in other eukaryotes. PfSETvs knockout parasites expressing all PfEMP1 proteins may also be applied to the development of a malaria vaccine., In addition to histone deacetylases (HDACs) (8,9), histone lysine methyl-transferases (HKMTs) or histone lysine demethylases (HKDMs) may have critical roles in controlling gene expression in P. falciparum (4-7,10,11). There are [...]

We examined the retention ability of a New Zealand dairy farm soil amended with 3 types of biochar produced from a variety of feedstocks for a steroid hormone (oestradiol, E2) and its primary transformation product (estrone, El). Biochars produced from corn cob (CC), pine sawdust (PSD) and green waste (GW) were characterised by scanning electron microscopy, Fourier-transform infrared spectroscopy, X-ray diffraction, and solid-state [sup.13]C nuclear magnetic resonance spectroscopy. Batch sorption studies were performed on soil amended with each biochar (0.5% and 1% by weight) using a complex solvent extraction scheme, and isotherms were fitted to the Freundlich model. All isotherms were highly non-linear, with N values in the range 0.46-0.83 (E2) and 0.664).88 (El) in soil amended with different percentages of biochars. Overall, addition of all 3 biochars was found to increase the soil sorption affinity for the hormones, with E2 sorption being the highest in the soil amended with 1% PSD biochar. There was no marked difference in hormone sorption ability in the other 2 treatments (soil treated with 1% CC biochar and 1% GW biochar). Overall, the effective distribution coefficient ([K.sub.d.sup.cfr]) values for E2 at the lowest equilibrium concentration ([C.sub.w] 0.5 mg/L) ranged from 35 to 311 L/kg in soil amended with the 3 types of biochar. Addition of 0.5% of PSD biochar resulted in ~560% increase in the [K.sub.d.sup.efr] value for E2, while at 1% addition of PSD biochar, uptake of E2 was nearly 1400% higher than the control. For E1, the percentage increase in [K.sub.d.sup.elf] was comparatively smaller than E2; however, it still ranged from 40 to 280%, and 60 to >320% at addition of 0.5% and 1% PSD biochar, respectively, compared with the control soil. Highest treatment temperature and associated greater surface area, low ash content, higher carbon content, and the abundance of polar functional groups (e.g. 43H, C=O) may explain why the soil amended with PSD biochar exhibited high sorptive capacity for the hormones. Additional keywords: effective distribution coefficient, estradiol, estrone, FTIR, NMR spectroscopy, SEM., Introduction Steroid hormones such as 17β-oestradiol (E2) and its primary metabolite estrone (El) are naturally excreted in the faeces and urine of dairy cows and other livestock animals (Hanselman et [...]

Background: Bloodstream malaria parasites require Ca++ for their development, but the sites and mechanisms of Ca++ utilization are not well understood. We hypothesized that there may be differences in Ca++ uptake or utilization by genetically distinct lines of P. falciparum. These differences, if identified, may provide insights into molecular mechanisms. Results: Dose response studies with the Ca++ chelator EGTA (ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid) revealed stable differences in Ca++ requirement for six geographically divergent parasite lines used in previous genetic crosses, with the largest difference seen between the parents of the HB3 x Dd2 cross. Genetic mapping of Ca++ requirement yielded complex inheritance in 34 progeny clones with a single significant locus on chromosome 7 and possible contributions from other loci. Although encoded by a gene in the significant locus and a proposed Ca++ target, PfCRT (P. falciparum chloroquine resistance transporter), the primary determinant of clinical resistance to the antimalarial drug chloroquine, does not appear to contribute to this quantitative trait. Stage-specific application of extracellular EGTA also excluded determinants associated with merozoite egress and erythrocyte reinvasion. Conclusions: We have identified differences in Ca++ utilization amongst P. falciparum lines. These differences are under genetic regulation, segregating as a complex trait in genetic cross progeny. Ca++ uptake and utilization throughout the bloodstream asexual cycle of malaria parasites represents an unexplored target for therapeutic intervention. [ABSTRACT FROM AUTHOR]

In this study, we pyrolysed six waste derived biomass: pine sawdust (PSD), paunch grass (PG), broiler litter (BL), sewage sludge (SS), dewatered pond sludge (DWP), and dissolved air-floatation sludge (DAF) into biochar. Biochars were characterized using scanning electron microscopy, energy dispersive X-ray spectrometry, X-ray diffraction, Fourier transform infrared spectroscopy, inductively-coupled plasma mass spectrometry, 13 C-solid-state nuclear magnetic resonance spectroscopy, and X-ray photoelectron spectroscopy to evaluate their feasibility for potential agronomic and environmental applications. Syngas produced during the pyrolysis process was also analyzed to determine the energy values. Results show that PSD biochar has the utmost potential for carbon sequestration and contaminant remediation due to its high surface area, aromaticity and carbon content. Additionally given its low ash content, PSD biochar could also potentially be used as filler in wood plastic biocomposites. Low levels of heavy metals (Cr, Cu, Zn, As, Cd, Hg, and Pb) in all biochars suggest that biochars are also applicable for land application according to the United States Environmental Protection Agency regulation 40 CFR part 503. The composition of syngas evolved during the pyrolysis of feedstocks showed little difference in the calorific values, ranging from 12–16 MJ/dsm with PSD having the maximum calorific value of 16 MJ/dsm. [ABSTRACT FROM AUTHOR]

We investigated the effects of feedstock type and pyrolysis temperatures on the sorptive potential of a model pastoral soil amended with biochars for sulfamethoxazole (SMO), using laboratory batch sorption studies. The results indicated that high temperature chars exhibited enhanced adsorptive potential, compared to low temperature chars. Pine sawdust (PSD) biochar produced at 700 °C using the steam gasification process exhibited the highest sorptive capacity (2-fold greater than the control treatment) for SMO among the three biochars used. Soils amended with green waste (GW) biochars produced at three different pyrolysis temperatures showed a small increase in SMO sorption with the increases in temperature. The NMR spectra, the elemental molar ratios (H/C, O/C) and polarity index (O + N)/C of the biochars revealed that PSD biochar possessed the highest degree of aromatic condensation compared to CC and GW chars. These results correlated well with the sorption affinity of each biochar, with effective distribution coefficient ( K d eff ) being highest for PSD and lowest for GW biochars. X-ray photoelectron spectroscopy results for the biochars showed a relatively large difference in oxygen containing surface functional groups amongst the GW biochars. However, they exhibited nearly identical sorption affinity to SMO, indicating negligible role of oxygen containing surface functional groups on SMO sorption. These observations provide important information on the use of biochars as engineered sorbents for environmental applications, such as reducing the bioavailability of antibiotics and/or predicting the fate of sulfonamides in biochar-amended soils. [ABSTRACT FROM AUTHOR]

We investigated the sorption potential and transport behaviour of three sulfonamides, namely, sulfamethoxazole (SMO), sulfachloropyridazine (SCP) and sulfamethazine (SM), and a conservative bromide tracer (Br-) in two undisturbed soil columns collected from the dairy farming regions in the North Island of New Zealand. Based on the low log Koc values obtained from the sorption study, all three sulfonamides are likely to have high mobility, making them a potential threat to surface and ground water. Soil column studies also showed that the mobility of the sulfonamides varied among soils and antibiotic type. Sulfonamides exhibited a mobility pattern similar to that of conservative Br- tracer. Considerable retardation was observed for the Hamilton soil, and the delayed peak arrival time (or maxima) was due to the role of sorption-related retention processes under saturated flow conditions. Residual antibiotic concentrations for SMO and SCP were detected in all soil sections including at 18 cm depth, while no resident concentration of SM was detected at any depth in the entire length of the core for both soils. The deterministic, physical equilibrium model (CXTFIT) described the peak arrival time as well as the maximum concentration of the antibiotic breakthrough curves reasonably, but showed some underestimation at the advanced stages of the leaching process. There was a significant difference in the model estimated retardation factors obtained from column study and the experimental retardation factors obtained from the conventional batch sorption experiments. [ABSTRACT FROM AUTHOR]

Abstract: Single first-order (SFO) kinetic model is often used to derive the dissipation endpoints of an organic chemical in soil. This model is used due to its simplicity and requirement by regulatory agencies. However, using the SFO model for all types of decay pattern could lead to under- or overestimation of dissipation endpoints when the deviation from first-order is significant. In this study the performance of three biphasic kinetic models — bi-exponential decay (BEXP), first-order double exponential decay (FODED), and first-order two-compartment (FOTC) models was evaluated using dissipation datasets of sulfamethoxazole (SMO) antibiotic in three different soils under varying concentration, depth, temperature, and sterile conditions. Corresponding 50% (DT50) and 90% (DT90) dissipation times for the antibiotics were numerically obtained and compared against those obtained using the SFO model. The fit of each model to the measured values was evaluated based on an array of statistical measures such as coefficient of determination (R2 adj), root mean square error (RMSE), chi-square (χ 2) test at 1% significance, Bayesian Information Criteria (BIC) and % model error. Box–whisker residual plots were also used to compare the performance of each model to the measured datasets. The antibiotic dissipation was successfully predicted by all four models. However, the nonlinear biphasic models improved the goodness-of-fit parameters for all datasets. Deviations from datasets were also often less evident with the biphasic models. The fits of FOTC and FODED models for SMO dissipation datasets were identical in most cases, and were found to be superior to the BEXP model. Among the biphasic models, the FOTC model was found to be the most suitable for obtaining the endpoints and could provide a mechanistic explanation for SMO dissipation in the soils. [Copyright &y& Elsevier]

Abstract: The dissipation of sulfamethoxazole (SMO) antibiotic in three different soils was investigated through laboratory incubation studies. The experiments were conducted under different incubation conditions such as initial chemical concentration, soil depth, temperature, and with sterilisation. The results indicate that SMO dissipated rapidly in New Zealand pasture soils, and the 50% dissipation times (DT50) in Hamilton, Te Kowhai and Horotiu soils under non-sterile conditions were 9.24, 4.3 and 13.33days respectively. During the incubation period for each sampling event the soil dehydrogenase activity (DHA) and the variation in microbial community were monitored thorough phospholipid fatty acid extraction analysis (PLFA). The DHA data correlated well with the dissipation rate constants of SMO antibiotic, an increase in the DHA activity resulted in faster antibiotic dissipation. The PLFA analysis was indicative of higher bacterial presence as compared to fungal community, highlighting the type of microbial community responsible for dissipation. The results indicate that with increasing soil depth, SMO dissipation in soil was slower (except for Horotiu) while with increase in temperature the antibiotic loss was faster, and was noticeable in all the soils. Both the degree of biological activity and the temperature of the soil influenced overall SMO dissipation. SMO is not likely to persist more than 5–6months in all three soils suggesting that natural biodegradation may be sufficient for the removal of these contaminants from the soil. Its dissipation in sterile soils indicated abiotic factors such as strong sorption onto soil components to play a role in the dissipation of SMO. [Copyright &y& Elsevier]

Abstract: The sorption potential for three sulfonamides (SAs), sulfamethoxazole (SMO), sulfachloropyridazine (SCP) and sulfamethazine (SM) and a macrolide, tylosin tartrate (TT) was assessed on six New Zealand dairy farming soils of contrasting physico-chemical properties. Kinetics studies showed that the sorption was rapid in the first few hours of the contact time (0–2h for SA and 0–4h for TT) and thereafter apparent equilibrium was achieved. Batch sorption isotherm data revealed that the degree of isotherm linearity (N) for SCP and SM varied between 0.50 and 1.08 in the six soils. Isotherms of both TT and SMO were mostly non-linear with the degree of non-linearity for TT (N =0.38–0.71) being greater than for SMO (0.42–0.75) in all soils except Manawatu (TT) and Te Kowhai (SMO) where a linear pattern was observed. Concentration-dependent effective distribution coefficient (K d eff) values for the SMO, SCP and SM antibiotics in the soils ranged from 0.85 to 16.35Lkg−1, while that for TT was 1.6 to 1042Lkg−1. The sorption affinity for all soils followed an order: TT>SCP>SM>SMO. Remarkable high sorption for tylosin in Matawhero soil as compared to other soils was attributed to the presence of oxygen containing acidic polar functional groups as evident in the FT-IR spectra of the soil. Furthermore, it was hypothesised that sorption of TT onto soils was mostly driven by metal oxide-surface mediated transformations whereas for sulfonamides it was primarily due to hydrophobic interactions. [Copyright &y& Elsevier]

Abstract: We investigated the effect of soil pH, organic carbon, ionic strength and steroid hormones on the sorption of sulfamethoxazole (SMO) and sulfachloropyridazine (SCP) in three pastoral soils of New Zealand. A model linking sorbate speciation with species-specific sorption coefficients describing the pH dependence of the apparent sorption coefficients was used to derive the fraction of each species of SMO. All soils displayed a decrease in sorption when pH was increased, with SMO exhibiting the highest sorption at pH 2. The cationic form of SMO appeared to sorb more close to pH ≥ pKa1 and, when pH ≥ pKa2 (6.5, 7.5 and 8.5) the anionic species seems to dominate, however, its sorption affinity to all soils was low. SMO sorption was affected by ionic strengths and organic carbon content, while the presence of hormones showed only a subtle decrease in SCP sorption in a selected model pasture soil. [Copyright &y& Elsevier]

A simple, yet robust analytical method was developed to detect and quantify three sulfonamides (SA), namely sulfamethoxazole (SMO), sulfachloropyridazine (SCP), and sulfamethazine (SM), and a macrolide tylosin (TT) in aqueous (calcium chloride and leachate solutions) and solid (agricultural soils) matrices using high performance liquid chromatography and ultra violet detection at 290 nm (TT) and 275 nm (SA) respectively. Chromatography was performed using a Phenomenex Onyx Monolithic C18 column for TT and a C18 Luna column for sulfonamides as single analytes eluted isocratically with a mobile phase consisting of acetonitrile: trifluoroacetic acid: tetrahydrofuran in the ratio 22.5:68:9.5 for TT, 40:55:5 for SMO, 32:63:5 for SCP and 31:64:5 for SM (v/v) at 1.0 mL min−1 and an injection volume of 20 μL. A gradient method to detect all three sulfonamides in a single run was also developed. The soil residue analysis consisted of extraction with dichloromethane and pre-concentration steps as the aqueous phase was measured directly. The limits of detection at an S/N (signal: noise) ratio of 3 were 20.0 μg L−1 and 50 μg L−1 for all sulfonamides and tylosin respectively. The average recoveries for all sulfonamides and tylosin in aqueous matrices ranged from 95 to 105% across the six concentrations investigated. Recoveries from the soils were slightly lower for sulfonamides and tylosin. The isocratic method was used to determine the sorption and degradation of sulfonamides in soils, while the gradient method was used to determine degradation kinetics and leachate concentrations in soils and aqueous systems. [ABSTRACT FROM AUTHOR]

Invasion of host cells by the malaria parasite involves recognition and interaction with cell-surface receptors. A wide variety of parasite surface proteins participate in this process, most of which are specific to the parasite's particular invasive form. Upon entry, the parasite has to dissociate itself from the host-cell receptors. One mechanism by which it does so is by shedding its surface ligands using specific enzymes. Rhomboid belongs to a family of serine proteases that cleave cellsurface proteins within their transmembrane domains. Here we identify and partially characterize a Plasmodium berghei rhomboid protease (PbROM1) that plays distinct roles during parasite development. PbROM1 localizes to the surface of sporozoites after salivary gland invasion. In blood stage merozoites, PbROM1 localizes to the apical end where proteins involved in invasion are also present. Our genetic analysis suggests that PbROM1 functions in the invasive stages of parasite development. Whereas wild-type P. berghei is lethal to mice, animals infected with PbROM1 null mutants clear the parasites efficiently and develop long-lasting protective immunity. The results indicate that P. berghei Rhomboid 1 plays a nonessential but important role during parasite development and identify rhomboid proteases as potential targets for disease control. [ABSTRACT FROM AUTHOR]

An essential requisite for transmission of Plasmodium, the causative agent of malaria, is the successful completion of a complex developmental cycle in its mosquito vector. Of hundreds of ookinetes that form in the mosquito midgut, only few transform into oocysts, a loss attributed to the action of the mosquito immune system. However, once oocysts form, they appear to be resistant to mosquito defences. During oocyst development, a thick capsule forms around the parasite and appears to function as a protective cover. Little information is available about the composition of this capsule. Here we report on the identification and partial characterization of the first Plasmodium oocyst capsule protein (PbCap380). Genetic analysis indicates that the gene is essential and that PbCap380(−) mutant parasites form oocysts in normal numbers but are gradually eliminated. As a result, mosquitoes infected with PbCap380(−) parasites do not transmit malaria. Targeting of the oocyst capsule may provide a new strategy for malaria control. [ABSTRACT FROM AUTHOR]

Understanding the life cycle of the malaria parasite in its mosquito vector is essential for developing new strategies to combat this disease. Subtractive hybridization cDNA libraries were constructed that are enriched for Plasmodium berghei and Anopheles stephensi genes expressed during oocyst differentiation on the midgut. Sequencing of 1485 random clones led to the identification of 1137 unique expressed sequence tags. Of the 608 expressed sequence tags with data base hits, 320 (53%) had significant matches to the non.redundant protein data base, whereas 288 (47%) with matches only to genomic data bases represent novel Plasmodium and Anopheles genes. Transcription of six novel parasite genes and two previously identified asexual stage genes was up-regulated during oocyst differentiation. In addition, the mRNA for an Anopheles fibrinogen domain gene was induced on day 2 after an infectious blood meal, at the time of ookinete to oocyst differentiation. The subcellular distribution of MAEBL, a sporozoite surface protein, is developmentally regulated from presumed storage organelles in day 15 oocysts to uniform distribution on the surface in day 22 oocysts. This redistribution may reflect a sporozoite maturation program in preparation for salivary gland invasion. Furthermore, apical membrane antigen 1, another parasite surface molecule, is translationally regulated late in sporozoite development, suggesting a role during infection of the vertebrate host. The present results and those of an accompanying report (Abraham, E. G., Islam, S., Srinivasan, P., Ghosh, A. K., Valenzuela, J., Ribeiro, J. M., Kafatos, F. C., Dimopoulos, G., & Jacobs-Lorena, M. (2003) J. Biol. Chem. 279, 5573-5580) provide the foundation for studies seeking to understand at the molecular level Plasmodium development and its interactions with the mosquito. [ABSTRACT FROM AUTHOR]

Malaria kills millions of people every year, yet there has been little progress in controlling this disease. For transmission to occur, the malaria parasite has to complete a complex developmental cycle in the mosquito. The mosquito is therefore a potential weak link in malaria transmission, and generating mosquito populations that are refractory to the parasite is a potential means of controlling the disease. There has been considerable progress over the last decade towards developing the tools for creating a refractory mosquito. Accomplishments include germline transformation of several important mosquito vectors, the completed genomes of the mosquito Anopheles gambiae and the malaria parasite Plasmodium falciparum, and the identification of promoters and effector genes that confer resistance in the mosquito. These tools have provided researchers with the ability to engineer a refractory mosquito vector, but there are fundamental gaps in our knowledge of how to transfer this technology safely and effectively into field populations. This review considers strategies for interfering with Plasmodium development in the mosquito, together with issues related to the transfer of laboratory-acquired knowledge to the field, such as minimization of transgene fitness load to the mosquito, driving genes through populations, avoiding the selection of resistant strains, and how to produce and release populations of males only. [ABSTRACT FROM AUTHOR]

Plasmodium, the causative agent of malaria, has to undergo sexual differentiation and development in anopheline mosquitoes for transmission to occur. To isolate genes specifically induced in both organisms during the early stages of Plasmodium differentiation in the mosquito, two cDNA libraries were constructed, one enriched for sequences expressed in differentiating Plasmodium berghei ookinetes and another enriched for sequences expressed in Anopheles stephensi guts containing invading ookinetes and early oocysts. Sequencing of 457 ookinete library clones and 652 early oocyst clones represented 175 and 346 unique expressed sequence tags, respectively. Nine of 13 Plasmodium and four of the five Anopheles novel expressed sequence tags analyzed on Northern blots were induced during ookinete differentiation and mosquito gut invasion. Ancaspase-7, an Anopheles effector caspase, is proteolytically activated during Plasmodium invasion of the midgut. WARP, a gene encoding a Plasmodium surface protein with a yon Willebrand factor A-like adhesive domain, is expressed only in ookinetes and early oocysts. An anti-WARP polyclonal antibody strongly inhibits (70-92%) Plasmodium development in the mosquito, making it a candidate antigen for transmission blocking vaccines. The present results and those of an accompanying report (Srinivasan, P., Abraham, E. G., Ghosh, A. K., Valenzuela, J., Ribeiro, J. M. C., Dimopoulos G., Kafatos, F. C., Adams, J. H., and Jacobs-Lorena, M. (2004) J. Biol. Chem. 279, 5581-5587) provide the foundation for further analysis of Plasmodium differentiation in the mosquito and of mosquito responses to the parasite. [ABSTRACT FROM AUTHOR]

Abstract: Microscale thermophoresis (MST) allows for quantitative analysis of protein interactions in free solution and with low sample consumption. The technique is based on thermophoresis, the directed motion of molecules in temperature gradients. Thermophoresis is highly sensitive to all types of binding-induced changes of molecular properties, be it in size, charge, hydration shell or conformation. In an all-optical approach, an infrared laser is used for local heating, and molecule mobility in the temperature gradient is analyzed via fluorescence. In standard MST one binding partner is fluorescently labeled. However, MST can also be performed label-free by exploiting intrinsic protein UV-fluorescence. Despite the high molecular weight ratio, the interaction of small molecules and peptides with proteins is readily accessible by MST. Furthermore, MST assays are highly adaptable to fit to the diverse requirements of different biomolecules, such as membrane proteins to be stabilized in solution. The type of buffer and additives can be chosen freely. Measuring is even possible in complex bioliquids like cell lysate allowing close to in vivo conditions without sample purification. Binding modes that are quantifiable via MST include dimerization, cooperativity and competition. Thus, its flexibility in assay design qualifies MST for analysis of biomolecular interactions in complex experimental settings, which we herein demonstrate by addressing typically challenging types of binding events from various fields of life science. [Copyright &y& Elsevier]

Abstract: Simultaneous microarray-based transcription analysis of 4987 Anopheles stephensi midgut and Plasmodium berghei infection stage specific cDNAs was done at seven successive time points: 6, 20 and 40h, and 4, 8, 14 and 20 days after ingestion of malaria infected blood. The study reveals the molecular components of several Anopheles processes relating to blood digestion, midgut expansion and response to Plasmodium-infected blood such as digestive enzymes, transporters, cytoskeletal and structural components and stress and immune responsive factors. In parallel, the analysis provide detailed expression patterns of Plasmodium genes encoding essential developmental and metabolic factors and proteins implicated in interaction with the mosquito vector and vertebrate host such as kinases, transcription and translational factors, cytoskeletal components and a variety of surface proteins, some of which are potent vaccine targets. Temporal correlation between transcription profiles of both organisms identifies putative gene clusters of interacting processes, such as Plasmodium invasion of the midgut epithelium, Anopheles immune responses to Plasmodium infection, and apoptosis and expulsion of invaded midgut cells from the epithelium. Intriguing transcription patterns for highly variable Plasmodium surface antigens may indicate parasite strategies to avoid recognition by the mosquito''s immune surveillance system. [Copyright &y& Elsevier]