Mapping the Catalytic Cycle of Schistosoma mansoni Thioredoxin Glutathione Reductase by X ray Crystallography

Schistosomiasis is the second most widespread human parasitic disease. It is principally treated with one drug, praziquantel, that is administered to 100 million people each year; less sensitive strains of schistosomes are emerging. One of the most appealing drug targets against schistosomiasis is thioredoxin glutathione reductase (TGR). This natural chimeric enzyme is a peculiar fusion of a glutaredoxin domain with a thioredoxin selenocysteine (U)-containing reductase domain. Selenocysteine is located on a flexible C-terminal arm that is usually disordered in the available structures of the protein and is essential for the full catalytic activity of TGR. In this study, we dissect the catalytic cycle of Schistosoma mansoni TGR by structural and functional analysis of the U597C mutant. The crystallographic data presented herein include the following: the oxidized form (at 1.9 Å resolution); the NADPH- and GSH-bound forms (2.3 and 1.9 Å, respectively); and a different crystal form of the (partially) reduced enzyme (3.1 Å), showing the physiological dimer and the entire C terminus of one subunit. Whenever possible, we determined the rate constants for the interconversion between the different oxidation states of TGR by kinetic methods. By combining the crystallographic analysis with computer modeling, we were able to throw further light on the mechanism of action of S. mansoni TGR. In particular, we hereby propose the putative functionally relevant conformational change of the C terminus after the transfer of reducing equivalents from NADPH to the redox sites of the enzyme

Performances and Emissions of a Small Size Diesel Powered Generator Fueled with Vegetable Oil and its Water-in Emulsions

Diesel engines are worldwide used in the transport, electricity generation and shaft power and they burn oil products which can be partially or totally replaced by alternative fuels, such as vegetable oils. Vegetable oils have physical characteristics close to those of diesel oil and as they are of plant origin, they have the potential to significantly reduce CO2 emissions into the atmosphere. Nowadays fuelling compression ignition engine with vegetable oil is a feasible solution, with a relevant number of working plants, mainly for medium size electric generators; not the same can be told about small size diesel generators. Few marine propulsion examples are also reported. In this paper, a rapeseed oil and some its water-in emulsions, in different percentage 5, 10 and 15% v/v, have been fuelled in a small size Diesel engine used as power generator in a co-generation system. Tests were conducted in a naturally aspirated Scania DC09 direct injection four-stroke 250kW, 5-cylinders, 9.3 dm3 displacement Diesel engine from low load to 120kW as load conditions. Emulsions were produced on site by a device that adds steam to vegetable oil, so continuously feeds the engine with emulsified oil at about 60°C; tests were also carried out with sample emulsions made offline with an ultrasonic device. Gaseous (CO, HC, NO/NOx, CO2) and particulate emissions have been characterized in steady state conditions. CO, HC and particulate emissions are strongly reduced by fuelling vegetable oil emulsions compared to commercial diesel fuel. NOx emission are not significantly affected, rather a small increase is observed in some conditions; authors believe that an engine optimized for reducing NOx with diesel oil requires a different injection tuning, not feasible during the experiments

Design of a test platform for miniaturized electric propulsion

Nanosatellites represent an emerging opportunity to pursue a broad set of mission goals, including remote sensing, technology demonstration, and communications, at low cost and fast delivery. These new opportunities require a technological improvement to increase capabilities such as orbit change and transfer, formation flying, and constellation build up, close proximity operations and deorbiting. In this sense, miniaturized electrical propulsion systems increase the range of missions performed with multi-unit CubeSats. At subsystem level, many concepts have been recently developed but their level of readiness is limited. Moreover, the integration of propulsion poses new challenges at system level that influence heavily the spacecraft design and its verification. The present research, developed by Politecnico di Torino and supported by ESA, intends to build a flexible test platform and define effective procedures to support the evaluation of suitable propulsion systems for future nanosatellites. The main objectives are to investigate the interaction of propulsion systems with CubeSat-technology from different perspectives (mechanical, electrical, magnetic, and chemical) and to evaluate the performance of the integrated platform. The test platform is a 6U CubeSat able to host electric propulsion systems selected among European solutions, providing mechanical, electrical and data interfaces. A flexible and robust structure holds and protects the propulsion system and avionics, and externally interfaces the test platform with the facility of ESA/ESTEC propulsion laboratory. The on-board Electrical Power System adapts the voltage and power provided by batteries to serve loads up to 2A @ 28V. Different protocols are included in the on-board computer to exchange data and commands with a range of propulsion systems. The test platform is equipped with a wide range of sensors (e.g. temperature sensors, accelerometers, and magnetometers) to measure and acquire parameters both of the propulsion system and of on-board avionics. Data are stored on-board and sent to the ground support system via wired and/or RF links. The test platform operations are controlled through commands sent by the operator and by autonomous onboard routines in charge of managing transitions between operative modes and for detection, identification and recovery of failures. The test platform will be ready in December 2018, and it will represent the first important step for the evaluation of electrical propulsion systems integrated in small satellites. The paper describes into the detail the design and development of the platform and the plan for the test campaign

Comparative metabolic profiling by 1H-NMR spectroscopy analysis reveals the adaptation of S. mansoni from its host to in vitro culture conditions: a pilot study with ex vivo and GSH-supplemented medium-cultured parasites

Schistosomiasis is a neglected tropical disease caused by parasitic flatworms (blood fluke) of the genus Schistosoma. Parasites acquire most nutrients for their development and sustainment within the definitive host either by ingestion into the gut or across the body surface. Over the years, the best conditions for long-term maintenance of parasites in vitro have been thoroughly established. In our hands, H-1-NMR spectroscopy represents a powerful tool to characterize the metabolic changes in S. mansoni in response to culturing condition perturbations. In order to compare the metabolic fingerprint of ex vivo and parasites cultured in vitro with or without the supplement of reduced glutathione, we conducted a pilot study applying the H-1-NMR spectroscopy-based metabolomics. We obtained new insight into specific metabolic pathways modulated under these different experimental conditions

Development of Green Hydrogen Peroxide Monopropellant Rocket Engines and Testing of Advanced Catalytic Beds

With the financial support of the European Space Agency, Alta S.p.A. in Italy and DELTACAT Ltd. in the United Kingdom are conducting a joint study on the development of two hydrogen peroxide monopropellant thruster prototypes (5 and 25 N vacuum thrust) using advanced catalytic beds. The present article illustrates the results of recent tests on the developed catalysts and the main aspects of the thruster design as well as of the technique used for quantitative determination of the observed decomposition rates. Preliminary results indicate that platinum deposited on a γ−alumina substrate yields the best performance with respect to the other solutions under consideration

Verification campaign of small test platform hosting electric propulsion systems

The growing interest of the market in nano-satellites is pushing the technology associated with development of these small platforms Among the enabling technologies, miniaturized electrical propulsion (EP)systems deeply increase the range of missions performed with multi-unit CubeSats (6U+). Actually, the level of readiness of these technologies is still low, due to the lack of effective and safe processes and facilities to verify and validate them before in-orbit demonstration. One of the major issue sis to understand what the mutual impact of an electric propulsion system and the spacecraft technology is, in order to avoid loss of onboard functionalities and anomalies and misbehaviours. The present paper deals with the verification campaign of a versatile test platform based on CubeSat-like technology, able to host a wide range of EP system favouring an effective verification campaign thanks to precise procedures and a proper setup of the facility (the Small Plasma Facility /SPF) of ESA-ESTEC). The platform is equipped with specific sensors and tools, i.e. temperature sensors (NTC), current and voltage sensing circuits, strips of open resistors, magnetometers, silica wafers mounted on the CubeSat faces, and a very precise (24-bit resolution) acquisition system. The objective is to obtain unprecedented information that, integrated with external devices normally used for the verification campaign of propulsion system, e.g. faraday cups and Magnetic Field Mapper, allows to assess the functional behaviour of the integrated system (platform and EP system). The test platform has already been integrated with SPF and the functional test campaign has been successfully performed, demonstrating the test platform is ready for final tests

Switching between the Alternative Structures and Functions of a 2 Cys Peroxiredoxin, by Site Directed Mutagenesis

Members of the typical 2-Cys peroxiredoxin (Prx) subfamily represent an intriguing example of protein moonlighting behavior since this enzyme shifts function: indeed, upon chemical stimuli, such as oxidative stress, Prx undergoes a switch from peroxidase to molecular chaperone, associated to a change in quaternary structure from dimers/decamers to higher-molecular-weight (HMW) species. In order to detail the structural mechanism of this switch at molecular level, we have designed and expressed mutants of peroxiredoxin I from Schistosoma mansoni (SmPrxI) with constitutive HMW assembly and molecular chaperone activity. By a combination of X-ray crystallography, transmission electron microscopy and functional experiments, we defined the structural events responsible for the moonlighting behavior of 2-Cys Prx and we demonstrated that acidification is coupled to local structural variations localized at the active site and a change in oligomerization to HMW forms, similar to those induced by oxidative stress. Moreover, we suggest that the binding site of the unfolded polypeptide is at least in part contributed by the hydrophobic surface exposed by the unfolding of the active site. We also find an inverse correlation between the extent of ring stacking and molecular chaperone activity that is explained assuming that the binding occurs at the extremities of the nanotube, and the longer the nanotube is, the lesser the ratio binding sites/molecular mass is

Moonlighting by Different Stressors Crystal Structure of the Chaperone Species of a 2 Cys Peroxiredoxin

2-Cys peroxiredoxins (Prxs) play two different roles depending on the physiological status of the cell. They are thioredoxin-dependent peroxidases under low oxidative stress and ATP-independent chaperones upon exposure to high peroxide concentrations. These alternative functions have been associated with changes in the oligomerization state from low-(LMW) to high-molecular-weight (HMW) species. Here we present the structures of Schistosoma mansoni Prx1 in both states: the LMW decamer and the HMW 20-mer formed by two stacked decamers. The latter is the structure of a 2-Cys Prx chaperonic form. Comparison of the structures sheds light on the mechanism by which chemical stressors, such as high H2O2 concentration and acidic pH, are sensed and translated into a functional switch in this protein family.. We also propose a model to account for the in vivo formation of long filaments of stacked Prx rings