Cloning, expression and functional activity of deoxyhypusine synthase from Plasmodium vivax

BACKGROUND: Plasmodium vivax is the most widespread human malaria parasite. However, genetic information about its pathogenesis is limited at present, due to the lack of a reproducible in vitro cultivation method. Sequencing of the Plasmodium vivax genome suggested the presence of a homolog of deoxyhypusine synthase (DHS) from P. falciparum, the key regulatory enzyme in the first committed step of hypusine biosynthesis. DHS is involved in cell proliferation, and thus a valuable drug target for the human malaria parasite P. falciparum. A comparison of the enzymatic properties of the DHS enzymes between the benign and severe Plasmodium species should contribute to our understanding of the differences in pathogenicity and phylogeny of both malaria parasites. RESULTS: We describe the cloning of a 1368 bp putative deoxyhypusine synthase gene (dhs) sequence from genomic DNA of P. vivax PEST strain Salvador I (Accession number AJ549098) after touchdown PCR. The corresponding protein was expressed and functionally characterized as deoxyhypusine synthase by determination of its specific activity and cross-reactivity to human DHS on a Western blot. The putative DHS protein from P. vivax displays a FASTA score of 75 relative to DHS from rodent malaria parasite, P. yoelii, and 74 relative to that from the human parasite, P. falciparum strain 3D7. The ORF encoding 456 amino acids was expressed under control of IPTG-inducible T7 promoter, and expressed as a protein of approximately 50 kDa (theoretically 52.7 kDa) in E. coli BL21 DE3 cells. The N-terminal histidine-tagged protein was purified by Nickel-chelate affinity chromatography under denaturing conditions. DHS with a theoretical pI of 6.0 was present in both eluate fractions. The specific enzymatic activity of DHS was determined as 1268 U/mg protein. The inhibitor, N-guanyl-1, 7-diaminoheptane (GC7), suppressed specific activity by 36-fold. Western blot analysis performed with a polyclonal anti-human DHS antibody revealed cross-reactivity to DHS from P. vivax, despite an amino acid identity of 44% between the proteins. CONCLUSION: We identify a novel DHS protein in the more benign malaria parasite,P. vivax, on the basis of specific enzymatic activity, cross-reactivity with a polyclonal antibody against human DHS, and amino acid identity with DHS homologs from the rodent malaria parasite, P. yoelii, and human P. falciparum strains

Efficacy of Bacillus thuringiensis var. israelensis against malaria mosquitoes in northwestern Burkina Faso

Background: In Sub Saharan Africa malaria remains one of the major health problems and its control represents an important public health measure. Integrated malaria control comprises the use of impregnated mosquito nets and indoor residual spraying. The use of drugs to treat patients can create additional pressure on the equation of malaria transmission. Vector control may target the adult mosquitoes or their aquatic larval stages. Biological larvicides such as Bacillus thuringiensis israelensis (Bti) represent a promising approach to support malaria control programs by creating additional pressure on the equation of malaria transmission. Methods: In this study we examined the efficacy of a water-dispersible granule formulation (WDG) of the biological larvicide Bti (VectoBac®) against wild Anopheles spp. larvae. Different concentrations of the larvicide were tested in standardized plastic tubs in the field against untreated controls. In weekly intervals tubs were treated with fixed concentrations of larvicide and the percentage reduction of larvae and pupae was calculated. Results: All used concentrations successfully killed 100 percent of the larvae within 24 hours, while the higher concentrations showed a slightly prolonged residual effect. Natural reconolization of larvae took place after two and three days respectively, late instar larvae were not found before 5 days after treatment. For the higher concentrations, up to three days no new larvae were found, implicating that the residual effect of WDG in tropical conditions is approximately one to two days. The overall pupae reduction in treated tubs was 98.5%. Conclusions: Biological larviciding with Bti can be a promising, additional tool in the fight against malaria in Africa. Environmental particularities in tropical Africa, first and foremost the rapid development of mosquitoes from oviposition to imago have to be taken into account before implementing such counter measures in national or international vector control programs. Nonetheless biological larviciding seems to be an appropriate measure for selected conditions, offering a significant contribution to the future of malaria control

In Vitro and In Vivo Antimalarial Activity Assays of Seeds from Balanites aegyptiaca: Compounds of the Extract Show Growth Inhibition and Activity against Plasmodial Aminopeptidase

Balanites aegyptiaca (Balanitaceae) is a widely grown desert plant with multiuse potential. In the present paper, a crude extract from B. aegyptiaca seeds equivalent to a ratio of 1 : 2000 seeds to the extract was screened for antiplasmodial activity. The determined IC50 value for the chloroquine-susceptible Plasmodium falciparum NF54 strain was 68.26 μg/μL ± 3.5. Analysis of the extract by gas chromatography-mass spectrometry detected 6-phenyl-2(H)-1,2,4-triazin-5-one oxime, an inhibitor of the parasitic M18 Aspartyl Aminopeptidase as one of the compounds which is responsible for the in vitro antiplasmodial activity. The crude plant extract had a Ki of 2.35 μg/μL and showed a dose-dependent response. After depletion of the compound, a significantly lower inhibition was determined with a Ki of 4.8 μg/μL. Moreover, two phenolic compounds, that is, 2,6-di-tert-butyl-phenol and 2,4-di-tert-butyl-phenol, with determined IC50 values of 50.29 μM ± 3 and 47.82 μM ± 2.5, respectively, were detected. These compounds may contribute to the in vitro antimalarial activity due to their antioxidative properties. In an in vivo experiment, treatment of BALB/c mice with the aqueous Balanite extract did not lead to eradication of the parasites, although a reduced parasitemia at day 12 p.i. was observed

Challenges of implementing a large scale larviciding campaign against malaria in rural Burkina Faso – lessons learned and recommendations derived from the EMIRA project

Background: Recent malaria control and elimination attempts show remarkable success in several parts of sub-Saharan Africa. Vector control via larval source management represents a new and to date underrepresented approach in low income countries to further reduce malaria transmission. Although the positive impact of such campaigns on malaria incidence has been researched, there is a lack of data on which prerequisites are needed for implementing such programs on a routine basis on large scale. Our objectives are to point out important steps in implementing an anti-malaria larviciding campaign in a resource and infrastructure restraint setting and share the lessons learned from our experience during a three-year intervention study in rural Burkina Faso. Methods: We describe the approaches we followed and the challenges that have been encountered during the EMIRA project, a three-year study on the impact of environmental larviciding on vector ecology and human health. An inventory of all performed work packages and associated problems and peculiarities was assembled. Results: Key to the successful implementation of the larviciding program within a health district was the support and infrastructure from the local research center run by the government. This included availability of trained scientific personnel for local project management, data collection and analysis by medical personnel, entomologists and demographers and teams of fieldworkers for the larviciding intervention. A detailed a priori assessment of the environment and vector breeding site ecology was essential to calculate personnel requirements and the need for larvicide and application apparel. In our case of a three-year project, solid funding for the whole duration was an important issue, which restricted the number of possible donors. We found the acquisition of qualified field personnel in fair numbers not to be always easy and training in application techniques and basic entomologic knowledge required several weeks of theoretical and practical formation. A further crucial point was to establish an effective quality control system that ensured the timely verification of larviciding success and facilitated in time data handling. While the experiences of running a larviciding campaign may vary globally, the experiences gained and the methods used in the Nouna health district may be employed in similar settings. Conclusions: Our observations highlight important components and strategies that should be taken into account when planning and running a similar larviciding program against malaria in a resource limited setting. A strong local partnership, meticulous planning with the possibility of ad-hoc adaption of project components and a reliable source of funding turned out to be crucial factors to successfully accomplish such a project

Routine implementation costs of larviciding with Bacillus thuringiensis israelensis against malaria vectors in a district in rural Burkina Faso

Background: The key tools in malaria control are early diagnosis and treatment of cases as well as vector control. Current strategies for malaria vector control in sub-Saharan Africa are largely based on long-lasting insecticide-treated nets (LLINs) and to a much smaller extent on indoor residual spraying (IRS). An additional tool in the fight against malaria vectors, larval source management (LSM), has not been used in sub-Saharan Africa on a wider scale since the abandonment of environmental spraying of DDT. Increasing concerns about limitations of LLINs and IRS and encouraging results from large larvicide-based LSM trials make a strong case for using biological larviciding as a complementary tool to existing control measures. Arguments that are often quoted against such a combined approach are the alleged high implementation costs of LSM. This study makes the first step to test this argument. The implementation costs of larval source management based on Bacillus thuringiensis israelensis (Bti) (strain AM65-52) spraying under different implementation scenarios were analysed in a rural health district in Burkina Faso. Methods: The analysis draws on detailed cost data gathered during a large-scale LSM intervention between 2013 and 2015. All 127 villages in the study setup were assigned to two treatment arms and one control group. Treatment either implied exhaustive spraying of all available water collections or targeted spraying of the 50 % most productive larval sources via remote-sensing derived and entomologically validated risk maps. Based on the cost reports from both intervention arms, the per capita programme costs were calculated under the assumption of covering the whole district with either intervention scenario. Cost calculations have been generalized by providing an adaptable cost formula. In addition, this study assesses the sensitivity of per capita programme costs with respect to changes in the underlying cost components. Results: The average annual per capita costs of exhaustive larviciding with Bti during the main malaria transmission period (June–October) in the Nouna health district were calculated to be US$1.05. When targeted spraying of the 50$ 0.77. Additionally, a high sensitivity of per capita programme costs against changes in total surface of potential larval sources and the number of spraying repetitions was found. Discussion: The per capita costs for larval source management interventions with Bti are roughly a third of the annual per capita expenditures for anti-malarial drugs and those for LLINs in Burkina Faso which are US$ 3.80 and 3.00, respectively. The average LSM costs compare to those of IRS and LLINs for sub-Saharan Africa. The authors argue that in such a setting LSM based on Bti spraying is within the range of affordable anti-malarial strategies and, consequently, should deserve more attention in practice. Future research includes a cost-benefit calculation, based on entomological and epidemiological data collected during the research project

Normal right- and left ventricular volumes and myocardial mass in children measured by steady state free precession cardiovascular magnetic resonance

BACKGROUND: Quantification of ventricular volume by steady state free precession (SSFP) cardiovascular magnetic resonance is accurate and reproducible. Normal values exist for adults, but are lacking for children.We sought to establish normal values for left and right ventricular volumes, mass and function in healthy children by using SSFP. METHODS AND RESULTS: Fifty children (27 females, 23 males) without cardiovascular disease were evaluated. Median age was 11 years (range 7 months - 18 years), weight 35 kg (range 7-77 kg), height 146 cm (range 66-181 cm). Thirty-six examinations were performed with breath holding, 14 in freely breathing sedated children.Ventricular volumes and mass were measured in the end systolic and end diastolic phase on SSFP cine images acquired in a short axis plane as a stack of 12 contiguous slices covering full length of both ventricles. Regression analysis showed an exponential relationship between body surface area (BSA) and ventricular volumes and mass (normal value = a*BSAb). Normative curves for males and females are presented in relation to BSA for the end-diastolic volume, end-systolic volume and mass of both ventricles. Intra- and interobserver variability of the measurements was within the limits of 2% and 7% respectively, except for right ventricular mass (10%). CONCLUSION: The exponential equation for calculation of normal values for each ventricular parameter and graphical display of normative curves for data acquired in healthy children by SSFP cardiovascular magnetic resonance are provided

Risk factors for epilepsy in Bas-Uélé Province, Democratic Republic of the Congo: a case–control study

Background: The reason for the high prevalence of epilepsy in onchocerciasis endemic areas remains unknown. The aim of this study was to detect risk factors associated with epilepsy in a region endemic for onchocerciasis. Methods: In June 2014, a case–control study was performed in Titule, Bas-Uélé Province in the Democratic Republic of the Congo. Individuals with unprovoked convulsive epilepsy of unknown aetiology were enrolled as cases (n = 59). Healthy members of families without cases of epilepsy in the same village were recruited as controls (n = 61). A multivariate binomial logistic regression analysis was performed to identify potential risk factors associated with epilepsy. To evaluate the potential protective effect of ivermectin treatment on the development of epilepsy, a nested age-matched case–control study was performed including only those who were eligible for ivermectin treatment in the year before they developed epilepsy. Results: Suspected onchocerciasis skin lesions were more often present in cases than in controls: 12/41 (29%) vs. 1/56 (2%), respectively (odds ratio (OR) 20.26, 95% confidence interval (CI) 2.42–170; p < 0.01). Ivermectin had been taken 7 months earlier in 29/59 (49%) cases and 29/61 (48%) controls. Onchocerca volvulus (OV) DNA was detected by PCR in skin snips in 26/34 cases (76%) and 10/14 controls (71%) (p = 0.7), and there was presence of OV IgG4 antibodies in 35/48 (73%) cases and 15/18 (83%) controls (p = 0.5). OV DNA was not detected in the cerebrospinal fluid of cases (controls not tested). Both cases and controls reported frequent bites by blackflies (Diptera, Simuliidae). Bathing daily as opposed to less often (OR 16.7, 95% CI 2.2–125.8; p < 0.01), bathing between 11 a.m. and 4 p.m. (OR 12.7, 95% CI 1.6–103.7; p = 0.02), and washing clothes between 11 a.m. and 4 p.m. (OR 10.9, 95% CI 1.5–77.3; p = 0.02) were all independently associated with epilepsy. Blood screening by specific PCR tests for Toxoplasma and Wuchereria bancrofti was negative in all cases and controls. A Loa loa infestation was found in only one case and one control by PCR and Giemsa smear. Antibodies to Taenia solium, Toxocara, and Trypanosoma sp were not detected in any of the participants. In an age-matched case–control analysis, 16/18 (89%) cases had not taken ivermectin the year before they developed epilepsy, compared to 7/18 (39%) controls that same year (p = 0.002). Conclusions: These data suggest that frequent activities at rivers known to be blackfly breeding sites and a historical lack of ivermectin treatment were risk factors for epilepsy in this onchocerciasis endemic area

Long-Term Soil Structure Observatory for Monitoring Post-Compaction Evolution of Soil Structure

The projected intensification of agriculture to meet food targets of a rapidly growing world population are likely to accentuate already acute problems of soil compaction and deteriorating soil structure in many regions of the world. The key role of soil structure for soil functions, the sensitivity of soil structure to agronomic management practices, and the lack of reliable observations and metrics for soil structure recovery rates after compaction motivated the establishment of a long-term Soil Structure Observatory (SSO) at the Agroscope research institute in Zürich, Switzerland. The primary objective of the SSO is to provide long-term observation data on soil structure evolution after disturbance by compaction, enabling quantification of compaction recovery rates and times. The SSO was designed to provide information on recovery of compacted soil under different post-compaction soil management regimes, including natural recovery of bare and vegetated soil as well as recovery with and without soil tillage. This study focused on the design of the SSO and the characterization of the pre- and post-compaction state of the field. We deployed a monitoring network for continuous observation of soil state variables related to hydrologic and biophysical functions (soil water content, matric potential, temperature, soil air O2 and CO2 concentrations, O2 diffusion rates, and redox states) as well as periodic sampling and in situ measurements of infiltration, mechanical impedance, soil porosity, gas and water transport properties, crop yields, earthworm populations, and plot-scale geophysical measurements. Besides enabling quantification of recovery rates of compacted soil, we expect that data provided by the SSO will help improve our general understanding of soil structure dynamics

The IFN-γ-Inducible GTPase, Irga6, Protects Mice against Toxoplasma gondii but Not against Plasmodium berghei and Some Other Intracellular Pathogens

Clearance of infection with intracellular pathogens in mice involves interferon-regulated GTPases of the IRG protein family. Experiments with mice genetically deficient in members of this family such as Irgm1(LRG-47), Irgm3(IGTP), and Irgd(IRG-47) has revealed a critical role in microbial clearance, especially for Toxoplasma gondii. The in vivo role of another member of this family, Irga6 (IIGP, IIGP1) has been studied in less detail. We investigated the susceptibility of two independently generated mouse strains deficient in Irga6 to in vivo infection with T. gondii, Mycobacterium tuberculosis, Leishmania mexicana, L. major, Listeria monocytogenes, Anaplasma phagocytophilum and Plasmodium berghei. Compared with wild-type mice, mice deficient in Irga6 showed increased susceptibility to oral and intraperitoneal infection with T. gondii but not to infection with the other organisms. Surprisingly, infection of Irga6-deficient mice with the related apicomplexan parasite, P. berghei, did not result in increased replication in the liver stage and no Irga6 (or any other IRG protein) was detected at the parasitophorous vacuole membrane in IFN-γ-induced wild-type cells infected with P. berghei in vitro. Susceptibility to infection with T. gondii was associated with increased mortality and reduced time to death, increased numbers of inflammatory foci in the brains and elevated parasite loads in brains of infected Irga6-deficient mice. In vitro, Irga6-deficient macrophages and fibroblasts stimulated with IFN-γ were defective in controlling parasite replication. Taken together, our results implicate Irga6 in the control of infection with T. gondii and further highlight the importance of the IRG system for resistance to this pathogen

Somatosensory System Deficits in Schizophrenia Revealed by MEG during a Median-Nerve Oddball Task

Although impairments related to somatosensory perception are common in schizophrenia, they have rarely been examined in functional imaging studies. In the present study, magnetoencephalography (MEG) was used to identify neural networks that support attention to somatosensory stimuli in healthy adults and abnormalities in these networks in patient with schizophrenia. A median-nerve oddball task was used to probe attention to somatosensory stimuli, and an advanced, high-resolution MEG source-imaging method was applied to assess activity throughout the brain. In nineteen healthy subjects, attention-related activation was seen in a sensorimotor network involving primary somatosensory (S1), secondary somatosensory (S2), primary motor (M1), pre-motor (PMA), and paracentral lobule (PCL) areas. A frontal–parietal–temporal “attention network”, containing dorsal- and ventral–lateral prefrontal cortex (DLPFC and VLPFC), orbitofrontal cortex (OFC), anterior cingulate cortex (ACC), superior parietal lobule (SPL), inferior parietal lobule (IPL)/supramarginal gyrus (SMG), and temporal lobe areas, was also activated. Seventeen individuals with schizophrenia showed early attention-related hyperactivations in S1 and M1 but hypo-activation in S1, S2, M1, and PMA at later latency in the sensorimotor network. Within this attention network, hypoactivation was found in SPL, DLPFC, orbitofrontal cortex, and the dorsal aspect of ACC. Hyperactivation was seen in SMG/IPL, frontal pole, and the ventral aspect of ACC in patients. These findings link attention-related somatosensory deficits to dysfunction in both sensorimotor and frontal–parietal–temporal networks in schizophrenia