Manilkara subsericea against in vivo (hemorrhagic and edema) and in vitro  activities caused by Lachesis muta venom. All the tested activities were totally or at least partially reduced by M. subsericea. However, when L. muta venom was injected into mice 15 min first or after the materials, hemorrhage and edema were not inhibited. Thus, M. subsericea could be used as antivenom in snakebites of L. muta. And, this work also highlights Brazilian flora as a rich source of molecules with antivenom properties.Snake venom is composed of a mixture of substances that caused in victims a variety of pathophysiological effects. Besides antivenom, literature has described plants able to inhibit injuries and lethal activities induced by snake venoms. This work describes the inhibitory potential of ethanol, hexane, ethyl acetate, or dichloromethane extracts and fractions from stem and leaves of  Within their range, they are often abundant and are an important cause of snakebites [L. muta snakebites are mainly characterized by systemic  and local effects  [B. jararaca has a higher incidence of accidents (95%) than L. muta (2%), but, on the other hand, L. muta bites led to more severe symptoms (listed above) and have lethality indexes three times higher than B. jararaca [Snakebites are a public health problem that affects mainly poor people who live in rural areas of Africa, Asia, Latin America, and Oceania , 2. Accod others , 4. Lachakebites . L. mutaecrosis) , 7. In Sjararaca . Thus, rAlthough antivenoms are effective against systemic effects, they do not inhibit the local effects. And, a few countries produce antivenom of adequate quality as well. Moreover, antivenoms may produce side effects and their cost of production may be expensive. In fact, worldwide the production of antivenoms is a global challenge. Due to all these limitations, the search for alternative or complementary treatment for snakebites deserves attention.Manilkara subsericea (Mart.) Dubard (Sapotaceae), popularly known in Brazil as guracica, is an endemic species with edible fruits from the Brazilian Atlantic forest and grows in sandy coastal plains [Manilkara has shown the presence of triterpenes [Manilkara displayed antimicrobial [Plants are a rich source of pharmacologically active molecules that have been used for native people to treat several diseases, including snakebites. Several molecules have been isolated and tested against some lethal activities induced by snake venoms –11. Manil plains . A chemiterpenes , 14, sapterpenes , and flaterpenes . The exticrobial , 17, 18,icrobial , 20, anticrobial , againsticrobial , or antiicrobial  propertiM. subsericea and solvent-partitioned fractions to neutralize some in vitro and in vivo biological activities induced by L. muta snake venom.The present work investigates the ability of different ethanolic extracts of leaves and stem of All solvents were of the best analytical grade. Dimethylsulfoxide (DMSO) and azocasein were obtained from Sigma Chemical Co. .Manilkara subsericea (Mart.) Dubard (Sapotaceae) was collected in January 2009 at the Jurubatiba Sandbank National Park, located in the Brazilian state of Rio de Janeiro (220° 16′ 4.93′′ S–41° 38′ 8.50′′ W), under license number 13659-2 from Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis—BAMA/SISBIO. The material was identified by Professor Dr. Marcelo Guerra Santos, a member of Department of Botany of State University of Rio de Janeiro (UERJ) and a voucher specimen was deposited in the Herbarium of UERJ and assigned under the number 13416.M. subsericea were dried at 40°C for two days, ground, and macerated with ethanol 96% (v/v) at room temperature until exhaustion. Then, both materials were filtered and solvent evaporated using a rotary evaporator, yielding 530 g of ethanolic extract of leaves (ETL) and 169.3 g ethanolic extract of stems (ETS). These extracts were sequentially washed with solvents of increasing polarities (hexane and ethyl acetate). After complete removal of solvent under reduced pressure, using a rotary evaporator, 21.6 g hexane-soluble (HEL) and 11.5 g ethyl acetate-soluble (EAL) fractions from leaves and 12.1 g hexane (HES) and 13.7 g ethyl acetate (EAS) fractions from stems were obtained. All materials were dissolved in DMSO (30% v/v) to perform the biological assays.The leaves (1.930 kg) and stems (0.960 kg) of Lachesis muta lyophilized venom was kindly supplied from Fundação Ezequiel Dias (FUNED), Brazil. Venom was diluted in physiological saline (1 mg/mL) and stored at −20°C until experiments. Male Swiss mice (18–20 g) were obtained from the Center of Laboratory Animals (NAL) of the Federal Fluminense University (UFF). All experiments were approved by the UFF Institutional Committee for Ethics in Animal Experimentation (protocol number 212) that were in accordance with the guidelines of the Brazilian Committee for Animal Experimentation (COBEA).L. muta venom was determined according to the method of Garcia et al. [2, and pH 8.8). Aliquots of L. muta venom (5–50 μg/mL) were incubated with 0.4 mL azocasein at 37°C for 90 min in a total volume of 1.2 mL. The enzymatic reaction was stopped by adding trichloroacetic acid . The tubes were centrifuged at 15,000 ×g for 3 min. Then, the supernatant was removed and mixed with 2 N NaOH, and the tubes were read at A 420 nm (spectrophotometer Hitachi U-5100) to measure the release of azo dye and recorded from the samples. An effective concentration (EC) was defined as the amount of venom (μg/mL) able to produce a variation of 0.2 units at A 420 nm. For the inhibitory experiments, one EC of L. muta venom (8.75 μg/mL) was incubated at room temperature with the extracts or fractions of M. subsericea at 1 : 10 venom : plant (w/w). Then, proteolytic activity was determined accordingly. Positive control experiments were performed by incubating L. muta venom with saline or DMSO (1% v/v).Proteolytic activity of a et al. , using aL. muta snake venom (2.5–70 μg/mL) and the clotting time was monitored using an Amelung coagulometer, model KC4A . The concentration of venom (μg/mL) that clotted plasma in 60 seconds was considered as the minimum coagulant dose (MCD). To evaluate the inhibitory effect, the extracts or fractions of M. subsericea were incubated at room temperature for 30 minutes with one MCD of venom (25 μg/mL) at 1 : 10 venom : plant (w/w), and then the mixture was added to plasma and clotting time was recorded. Negative control experiments were performed in parallel by mixing extracts or DMSO (0.5% v/v) with plasma in the absence of venom.A pool of citrated normal human plasma  was obtained from the local blood bank  and was mixed with L. muta venom was determined by the indirect hemolytic test using human erythrocytes and hen's egg yolk emulsion as substrate [L. muta venom (μg/mL) able to produce 100% hemolysis. To verify the inhibitory action of M. subsericea, one MIDH (17.6 µg/mL) was incubated at room temperature for 30 minutes with M. subsericea extracts or fractions at 1 : 10 venom : plant (w/w) ratio. Then, the hemolytic assay was performed. One hundred percent of hemolysis was obtained after lysing erythrocytes with distilled water. Negative control experiments were performed in parallel by mixing extracts or DMSO (1% v/v) with erythrocytes in the absence of venom.Hemolytic activity of ubstrate . One minL. muta crude venom were quantified using a procedure described by Kondo et al. [µL) were injected intradermally (i.d.) into abdominal skin of mice. Two hours later, animals were euthanized and the skin was removed, stretched, and inspected for visual changes in the inner surface or subcutaneous layers to localize hemorrhagic spots. Hemorrhage was quantified as the minimum hemorrhagic dose (MHD), defined as the amount of venom (μg/g) able to produce a hemorrhagic halo of 10 millimeters (mm) and that was the positive control. To evaluate the inhibitory effect, the extracts or fractions were incubated with two MHD (1.56 μg/g) of venom at room temperature for 30 minutes at 1 : 10 venom : plant ratio (w/w). This mixture was injected i.d. into animals and hemorrhagic activity evaluated, as described above. In another set of experiments, L. muta venom was injected i.d. 15 min first or after injection of M. subsericea extract or fractions. Hemorrhage was expressed as the mean diameter (in millimeters) of the hemorrhagic halo induced by venom in the absence and presence of the extract. Negative control experiments were performed in parallel by injecting i.d. extracts or DMSO (1% v/v) in the absence of venom.Hemorrhagic lesions produced by o et al. , with miL. muta venom was determined according to Yamakawa et al. [μL of L. muta venom (0.74 μg/g) in the right paw, while the left paw received 50 μL of saline, DMSO (1% v/v), or extracts. One hour after injection, edema was evaluated as the percentage increase in weight of the right paw compared to the left one. Antiedematogenic activity was performed by incubating the extracts or fractions of M. subsericea with L. muta venom for 30 min at room temperature at 1 : 10 venom : plant ratio (w/w), and then mixture was injected s.c. into mice. Also, venom was also injected s.c. prior to or after the extract. Positive control experiments were performed by incubating L. muta venom with saline or DMSO (1% v/v) prior to injection.Edema-inducing activity of a et al. , with mot-test was used and P values of ≤ 0.05 were considered statistically significant.Results are expressed as means ± SEM of indicated number of animals or experiments performed. Student's M. subsericea (87.5 μg/mL) inhibited 96% and 93%, respectively, the proteolytic activity of L. muta venom (8.75 μg/mL)  inhibited 4%. And, ethyl acetate-soluble (EAL) and hexane-soluble (HEL) fractions of the leaves inhibited 84% and 9% proteolytic activity, respectively (The crude ethanolic extract of stem (ETS) and leaves (ETL) of 5 μg/mL) . Furtherectively .μg/mL) fully prevented L-muta-induced clotting and ETS delayed clotting so that it took around eight times longer, when compared to control value  that clotted plasma in around 60 seconds (L. muta venom (ETL (250  seconds . When ve seconds . On the ta venom .μg/mL) inhibited 100% hemolysis induced by L. muta venom, while ETS or HES inhibited circa of 95% and HEL and ETL inhibited 75% and 60% hemolysis, respectively (The extracts ETL or EAS (176 ectively .L. muta venom (1.56 μg/g) injected into mice induced a hemorrhage halo of 20 mm that represents two MHD. But, in mice injected with venom mixed with ETL, ETS, EAS, or EAL (15.6 μg/g), the mice were fully protected from hemorrhage induced by the venom .he venom . HEL inhmorrhage . Howeverμg/g) fully inhibited the formation of edema induced by L. muta venom (0.73 μg/g), and ETS inhibited around 70%  to inhibit some toxic and harmful activities induced by L. muta venom. Overall, extracts of the stem and leaves of M. subsericea as well as its fractions inhibited such activities. Their inhibition occurred around 90% the proteolytic activity of L. muta venom that has been associated with some symptoms that follow snakebites, as hemorrhage and/or clotting. As seen, extracts of stem or leaves inhibited clotting and hemorrhage induced by L. muta venom and thus would prevent bleeding or vascular disturbances caused by L. muta bites. ETL inhibited clotting more efficiently than ETS, but EAS was the most powerful to prevent clotting. In addition, ETS, ETL, EAS, and EAL inhibited 100% hemorrhage induced by L. muta. Hemorrhage induced by snake venoms may occur through activating blood clotting and/or degrading specific proteins (as fibrinogen) of the blood cascade system. Metalloproteases and serine proteases are responsible for such harmful effects. Data from the literature state that plants or their products, mainly those enriched in phenolic compounds [Hemidesmus indicus [Vitex negundo and Emblica officinalis [2 promote several harmful activities, including hemolysis, platelet aggregation, edema, and myotoxicity [M. subsericea interfered on some of these effects. ETS, ETL, HES, and HEL of M. subsericea inhibited 100% hemolysis, but only ETL fully protected mice from edema. ETS or EAL inhibited edema above 60% and EAS inhibited it by 40%. Polar and nonpolar secondary metabolites have been described as responsible for the inhibition of PLA2 in snake venom [2 enzymes, such as diterpenes from the marine brown algae Spatoglossum schröederi [Canistrocarpus cervicornis [L. muta venom [M. subsericea (unpublished data). It should be mentioned that these substances displayed antivenom activity [Thus, in this work, we tested the ability of ethanolic crude extracts from stem and leaves of ompounds , inhibitompounds , 31. Othompounds , 33. Pre indicus  or from icinalis  able to icinalis . Phosphotoxicity , 37, 38,ke venom . Besidesta venom , 22 thatM. subsericea extracts inhibited in vivo (hemorrhagic and edematogenic) and in vitro  harmful activities induced by L. muta venom, they may have a potential to the development of an alternative or complementary treatment for snakebites caused by L. muta.Since


This electrically driven sandwich-type monolayer molecular rotor device showed an on/off ratio of approximately 104, a read window of about 2.5 V, and a retention time of greater than 104 s. The rotation speed of this type of molecular rotor has been reported to be in the picosecond timescale, which provides a potential of high switching speed applications. Current-voltage spectroscopy (I-V) revealed a temperature-dependent negative differential resistance (NDR) associated with the device. The analysis of the device I–V characteristics suggests the source of the observed switching effects to be the result of the redox-induced ligand rotation around the copper metal center and this attribution of switching is consistent with the observed temperature dependence of the switching behavior as well as the proposed energy diagram of the device. The observed resistance switching shows the potential for future non-volatile memories and logic devices applications. This review will discuss the progress and provide a perspective of molecular motion for nanoelectronics and other applications.The use of a functional molecular unit acting as a state variable provides an attractive alternative for the next generations of nanoscale electronics. It may help overcome the limits of conventional MOSFETd due to their potential scalability, low-cost, low variability, and highly integratable characteristics as well as the capability to exploit bottom-up self-assembly processes. This bottom-up construction and the operation of nanoscale machines/devices, in which the molecular motion can be controlled to perform functions, have been studied for their functionalities. Being triggered by external stimuli such as light, electricity or chemical reagents, these devices have shown various functions including those of diodes, rectifiers, memories, resonant tunnel junctions and single settable molecular switches that can be electronically configured for logic gates. Molecule-specific electronic switching has also been reported for several of these device structures, including nanopores containing oligo(phenylene ethynylene) monolayers, and planar junctions incorporating rotaxane and catenane monolayers for the construction and operation of complex molecular machines. A specific electrically driven surface mounted molecular rotor is described in detail in this review. The rotor is comprised of a monolayer of redox-active ligated copper compounds sandwiched between a gold electrode and a highly-doped P However, as the physical limitation comes closer, further scaling of the basic components of the microelectronic circuit such as memory and switch are now being questioned. The anticipation of this limitation has led researchers in academia and industry alike to consider many conceptually new materials and structures from the molecular scale point of view. The above scenario provides an opportunity for exploring molecular machines and nanomachines which can be functionalized as nanoscale electronic devices. These devices based on functional molecular units acting as state variables  are attractive due to their potential ultimate small size, low-cost, low variability, highly integratable characteristics and the ability to self-assemble themselves .etc. The focus of discussion here is on potential devices which could be integrated with conventional semiconductor technologies.A specific kind of molecular machine can be a particular molecular device designed to perform mechanical movements under control of appropriate energy inputs ,8. TriggIn this paper, we limit our discussion to the rotational motion and we first describe the different types of rotary motors generally. Then we will focus on a specific electrically driven surface mounted molecular rotor because of its potential capability of integration with standard semiconductor fabrication technologies, which provides the realization possibility in the near future. In particular, we will discuss their molecular structures, mechanisms and operation principles, respectively. Finally we will provide a perspective of scalability, challenges and potential applications. This review will concentrate on a real prospect of integrating a bottom-up approach, based on molecular design and micro-fabrication, in order to construct molecular electronic devices that can store information with very high densities.1.1.Besides the linear motion, as mentioned in the previous section, the rotary motion is another category of molecular machines. Like the linear motion of molecules, these molecules can be activated to make rotation movement chemically, optically, or electrically as a switch, and the recognition of the change of positions of the chemical elements by the charge transport through the layer results in the off and on states.The molecular rotor usually consists of two parts that can easily rotate relative to each other. The different types of rotors can generate different rotation angles due to the specific rotating ligands. Normally the part with a larger moment of inertia is defined as the stator and the part with a smaller moment of inertia as the rotator, while the term rotor refers to the whole molecule. The distinction is somewhat arbitrary except the stationary part is fixed on or sits within a much more massive object, or on a solid substrate. In the absence of a fixed mounting, the rotator and the stator both turn around the same axle.Showing proper electrical characteristics, some molecule rotors foray into the new field of molecular electronics. The idea is the use of molecular building blocks for the construction of electronic components, both passive  and active . The concept of molecular electronics is attractive and excites scientists and engineers alike due to its prospect of size reduction in electronics offered by molecular-level control of assembly. Hence it provides means to go beyond the anticipated physical size of the conventional CMOS in view of fundamental limiting processes  and cost of top down micro-fabrication technology. However, to realize logic functions using molecular devices, we may face the challenges that the dynamics of molecules in contact with electrodes is far from easy grasp in determining its mechanism as the effects of electrodes and interfaces and others tend to complicate the characterization of the behaviors. In particular, the effect of different switchable and nonswitchable compounds on the observable electrical signals, such as current flow and voltage thresholds, complicate the problem and thus has to be carefully studied to obtain a microscopic understanding of what is really happening inside a particular molecular electronic device. Consequently, appropriate control experiments need to be performed and great care must be exercised in order both to understand the principle of operation and to attribute specific models to the observed switching effects. Although in many cases, results obtained and conclusions drawn may not be as definitive as those of traditional semiconductor physics, where analytical techniques have been relatively mature, hypothesis testing and detailed investigation can help further fundamental understandings.Molecule-specific electronic switching has been reported for several device structures, including nanopores containing oligo(phenylene ethynylene) monolayers ,12 and pi.e., macroscopic gold electrodes) to achieve its different states. In particular, transition metals offer useful features in controlling these state variables due to their accessible oxidation states. This process can results in low power consumption. The variables which can be controlled in the transition metals include coordination number, color, and geometry [i.e., the metal in the center can be used to drive intra-rotational motion. The next section will address rotational molecular switches.Molecular switches use a state variable for which information storage relies on physical changes of the molecule. For example, a bistable molecular switch may be immobilized onto a bottom electrode and the molecular layer is then sandwiched by a top metal electrode  on a solid support; and the surface mounted molecular rotor is that is attached or bonded to the surface of a solid support. As discussed above, for solution phase rotors, the distinction between the rotator and the stator may be sometimes ambiguous, as both parts of the molecule rotator are related to each other. For solid phase rotors, the molecule close to the solid support is normally the stator. And, for a surface-mounted molecule, one can clear specify the stator, which is rigidly attached to the surface, and forms a part of the molecular structure. In addition, the axle of rotation about which the rotator turns can be perpendicular  or parallel  to the surface . For solWithin each category of solution phase, solid phase and surface mounted molecular rotors; a subdivision may be made based on the chemical nature of the axle, such as a single bond, a triple bond, or a metallic atom. This chemical nature of the bond strongly influences one of the fundamental energy terms in the system, the intrinsic barrier energy and width of rotation.In addition to the above structural classification, the other method to categorize rotors is by their nature of dynamics under a given set of environmental conditions. Two extreme cases of thermally activated systems are: (i) the rotor with a small rotational potential energy barrier such that it is easy for the rotor to reorient at a sufficient temperature; and (ii) those having a stator lying in the lowest energy state and thus a large barrier. Switching will normally not occur unless the system is perturbed by an external force. It is obvious that the interplay of the different energy terms in a system determines the rotor dynamics; the environment temperature also plays an important role in determining the energy barrier of rotation.2.Previously reported rotary motions such as a rotational actuator based on multi-walled carbon nanotubes , fluorestransoid sandwich with two pairs of carbon vertices reflected through a center of symmetry, but that of the Ni(IV) species is cisoid  around a nickel axle . The Ni [The schematic structure of this molecular device is shown in  Ni(III)  and Ni(I Ni(III)    with a N Ni(III) ,74.trans configuration [commo-bis-7,8-dicarbollyl metallacarboranes, while some forms the cisoid configuration (such as in Ni(IV)) with its pairs of carbon vertices on the same side of the molecule [versus the standard calomel electrode (SCE)). The authors also showed experimentally and theoretically that the interconversion acts as a rotating molecular machine in their paper. In their model, the photoexcitation induced geometrical changes were simulated to compare with the results caused by electrochemical reduction. Therefore reduction places an electron in the lowest unoccupied molecular orbital (LUMO); photoexcitation removes an electron from the highest occupied molecular orbital (HOMO) and places it in the LUMO. There are other solution phase molecular rotors. For example, in suitably designed rotaxanes, the swinging movement of the ring around the axle can be electrochemically driven as well.The interconversion of the geometries of Ni(III) and Ni(IV) controlled by the change of the nickel oxidation state provides the basis for the controlled, rotational, oscillatory motion, and can be achieved electrochemically by redox reactions or photochemically. It is based on the presence of two adjacent CH vertices, which introduces localized regions of reduction negative charge . The resguration ,72,76  molecules of around 1.5 nm in diameter  on a solid support. A good example is that of single-molecule rotors, and it was found that the rotor, from a study of scanning tunneling microscopy (STM) in ultrahigh vacuum , was surdiameter . It showdiameter . Interesdiameter . It shouSupported by experiments of this kind and, by the existence of a wealth of nanometer scale natural molecular machines ,80, it s4.As we mentioned in the section of solution phase molecular rotors, previously reported rotary motions including a rotational actuator ,83, fluoi.e., eVs per molecule, where Vs is the voltage for which the redox event occurs. Because information storage is based on the nanoscopic movement of atomic nuclei about the copper center, the maximum rate of switching process is fundamentally limited by the frequencies of the relevant rotational vibrational modes of the compound, giving a minimum switching speed on the order of a picosecond [The use of transition metal ions as templates to construct multicomponent chemical systems with interlock or knotted topologies has been extensively exploited . Some ofcosecond . The het+ Si) and a Cu axle), the metal axle (Cu), and a diimine rigid rotator  on the doped Si  Cu(I) and bis Cu(II) complexes in methylene chloride. An absorption peak, corresponding to a metal-to-ligand charge transfer (MLCT) indicative of tetrahedral geometry, was observed near 450 nm for the Cu(I) complex but not for the Cu(II) complex. Two additional peaks observed in both Cu(I) and Cu(II) complexes and centered at approximately 280 nm and 340 nm are attributed to ligand-to-ligand transitions, 2 with a peak-to-valley ratio (PVR) near 5. This RT-PVR exceeds that observed in typical heterostructure resonant tunneling diodes. The author also performed temperature dependent I-V measurements of the device from 77 K to room temperature as illustrated in I-V characteristics of the monolayer device are shown in a is the activation energy, ΔI is the peak minus background current, which is related to the rate constant of chemical reactions, T is the temperature and R is the gas constant. Curve fitting of the data for the temperature range of T > 244 K shown in The results have been further analyzed to extract the activation energy. From the well-known Arrhenius equation below:+ Si substrate shifts the energy position of the molecular state relative to both gold electrode and P+ Si band-edges. As the rotary motion around the molecular axle is controlled by electron transfer, a portion of the applied potential is used to initiate the redox process within the molecular layer. First, calculations of the band gaps for the Cu(I) and Cu(II) complexes by TD-DFT yielded values of approximately 2.7 eV and 3.4 eV, respectively. Additional calculations of the molecular redox energy produced a value of 1.05 eV, which is of the same magnitude as the 0.7 eV difference of the band gaps between the Cu(I) and Cu(II) complexes. The 0.35 eV difference between these two values may be attributed to the rotational energy contribution. Finally, the electron affinity of the Cu(I) complex is estimated to be 3.7 eV, which anchors the LUMO energy state with respective to the gold Fermi level .The switching process begins with the low conductivity state of the Cu(II) complex under a positive bias as shown in + Si substrate was estimated. Since this molecular rotor is sufficiently large, the rotation speed can be estimated classically based on the kinetic energy of rotation and rotational inertia. As the angle between the Cu(I) and Cu(II) systems is 90 degrees, the rotation speed can be estimated as 1.4 ps, ignoring acceleration components of rotation during the beginning and the end of the movement. This value is reasonable as compared to previous simulation results of rotation in solution giving a speed of 0.5 ps [An additional attractive property of functional molecules based on rotational motion is their fast operational speed. In most cases, previous studies have focused on solution-based molecular rotors. With the perspective of exploiting the switching speed in mind, the rotation speed of the molecular rotor on Pf 0.5 ps .To achieve logic functions, the molecular switches have to be able to be compatible with conventional CMOS manufactory technology. From this perspective, the surface mounted molecular device is the most likely candidate for the molecular electronics.5.As we have described above, this review deals with molecular rotors from the point of view of their potential utility for molecular switches and logic devices. In gases, liquids, and certain solids, entire molecules can rotate as a whole around independent axes freely, but they cannot be used as a stable electrical device and furthermore certain process flows have to be developed to be compatible with the traditional CMOS technology; so we restrict the scope of this section to the surface mounted molecular rotors, in which groundwork was laid down for the current developments –93. As s5.1.In the advent of nanoelectronics, the question of inherent physical limits as well as possible technological barriers to scaling is among the most important. In this section, we will start with discussions on the fundamental physical limits of scaling; then we focus on the scalability of molecular rotor devices based on the redox assisted electron tunneling transport.−25 kg, while that of free electron is 9.1 × 10−31 kg, four orders of magnitude higher than a free electron. Therefore the molecular rotor device can in principle be scaled down to below 5 nm. In fact, the designed monolayer molecular rotor device discussed in the last section is on the order of 3 nm scale. However the transport of this device is still controlled by electron tunneling instead of the atomic molecular tunneling if an electrical field is applied even though the tunneling barrier height may be larger than Si, for example. Nevertheless as to be discussed later, the redox energy adds additional tunneling barrier height.It was pointed out that the logic device of the size below 5 nm may preferably use a state variable that has a heavier effective mass than that of an electron . In prinAs we discussed in the last section, the surface mounted molecular rotor is based on a redox-assisted electron tunneling transport model for the device operation. An applied electric field can induce the rotation associated with the redox reaction, thus change the energy barrier height and thus alters the availability of the states to be tunneled to. When the device operates at the Cu(II) state, the energy barrier is increased and electron transport only happens through a barrier to access the available states. Conversely when the device operates at the Cu(I) state, the energy barrier becomes lower and, the accessible states are readily available; thus electrons can easily tunnel through it. In general, the current at the Cu(II) case is lower and strongly unidirectional since the barrier behaves like a Schottky barrier due to the use of Si substrate; while the current at the Cu(I) case is larger and bidirectional, showing close to ohmic behavior.The transition between the Schottky and tunneling modes is achieved by simply changing the polarity of the bias, which activates the redox reaction associated with the rotation movement. Thus the I-V data shows a nonlinear behavior with hysteresis as well as giving rise to negative differential resistance as described in It is important to note that the operation of this device is based on both electron transport and Redox reaction. Although the ultimate limiting factor of scalability of this device is still the leakage current as a result of the quantum tunneling of electron, the energy required by the redox reaction adds an additional energy barrier to the total tunneling barrier so that the probability of the tunneling becomes smaller. Due to the redox reaction, the scalability of this redox-assisted tunneling device should be better than the pure electron tunneling devices, in term of minimizing the leakage.The suggestion that heavier particles might be preferred for nanoscale devices has attracted the attention of several research groups –96. A nuFurther improvement of the scalability of this surface mounted molecular rotor can be achieved by replacing the metal electrode with a solid electrolyte. The idea is to build a pure ion transport system. It is important to carefully choose the solid electrolyte which determines the energy barrier as well as transport ions that are responsible for the current flow where a low bias is applied to move ions while electron tunneling does not occur. A mimic system may be human brain . In huma5.2.Although specific organic molecules have their advantageous electronic properties, the reliability is always a challenge. The problem may be due to poor material uniformity because of inherent impurities, material interfaces, and electrode contacts. Therefore, the inherent characteristics of these molecules are easily masked by effects from the electrode materials and experimental conditions. Great cares must be exercised in attributing mechanisms and in constructing models to the observed switching behaviors and thus critical control experiments are required to obtain fundamental physical understanding. Once we fully understand the particular switching mechanism, studies of reliability, including a thorough investigation of all conceivable failure mechanisms, and subsequent development of optimization steps to correct them can follow.The heat dissipation is another issue to be considered. The specific heat capacity and thermal conductivity of materials affect the device performance and limit the scalability of the molecular device. Proper selections of materials for their favorable heat diffusion rates and for controlled material interfaces on the atomic scale may circumvent the heat dissipation problem to the point to allow for scaling beyond the ultimate tunneling limit.5.3.To date, most of resistive memory devices are based on the change of resistance caused by an applied external electrical field or current . The bas3) [A number of organic MIM devices have been reported to show resistive switching. Among the earliest work, Gregor ,102 used3) ,106, N,N3) , 2-amino3) , several3) ,110, and3) ,112. RepThe redox-based nanoionic memory becomes attractive to the semiconductor industry because experimental demonstrations of scalability, retention and endurance are encouraging ,114. TheAlthough the devices we discussed above are all two terminal devices. In principle, a three terminal device could be designed with a barrier height controlled by a gate similar to conventional Field Effect Transistor (FET) devices. Such approach could reduce the leakage current due to the presence of an additional energy barrier as well as the use of heavier particles. Proper selections of materials for their electron affinity and controlled material interface on the atomic scale may also allow this device for going beyond the ultimate tunneling limit of electron and offers a potential to realize future non-volatile memory or logic devices for information processing applications.To achieve these devices comparable to molecular devices of the size scale of <5 nm, the cost of conventional top down lithography may become prohibitively high for manufactory of these devices of integrated circuits. The patterning size variability due to the presence of subatomic particles and processing fluctuations at this scale increases the rate of faults. Beside to these fabrication challenges, nanoscale circuits also likely contain defects so numerous that some forms of defect tolerance will be necessary to achieve acceptable yields.The above challenges of nanoscale devices in general and the yield issues associated with the molecular devices described above lead us to address on configurable crossbar architectures –123, wheThe crossbar structure possesses many attractive features as it offers the highest possible device density and the simplest interconnect configuration that still allows for external access to each nanodevice . It is aHybrid nanocrossbar/CMOS architectures have also been proposed to complement the limited functionalities of two-terminal switches by integrating with CMOS components to expand their functionalities –130. It If a crossbar structure is referred to as a passive matrix architecture, the two-terminal molecular device, however, can also be integrated to become active matrix architecture by introducing a select transistor at each node by which the storage cells can be decoupled for those not addressed. This concept significantly reduces crosstalks, interferences and disturbing signals in the conventional crossbar system.Overall, the concept of molecular memory emphasizes extreme scaling; in principle, one bit of information can be stored in the space of a single molecule. Because of their small size, very dense circuits could be built, and bottom-up self-assembly process could be applied to augment top-down lithography fabrication techniques. Computing with molecules as circuit building blocks is an exciting concept with several desirable advantages over conventional circuit elements. However, referring to the 2011 ITRS on emerging research devices, the molecular memory device still stands at a long term research state because of our limited understanding of the phenomena accompanying molecular switching, where currently many questions remain. In addition, the variability and reliability will need to be established.In summary, there are a lot of interesting works in this field under progress such as ferroelectric molecular rotors , design


Karenia brevis  which are evolutionarily related to Type I PKS but were apparently expressed as monofunctional proteins, a feature typical of Type II PKS. Here, we investigated expression units of PKS I-like sequences in Alexandrium ostenfeldii  and Heterocapsa triquetra  at the transcript and protein level. The five full length transcripts we obtained were all characterized by polyadenylation, a 3′ UTR and the dinoflagellate specific spliced leader sequence at the 5′end. Each of the five transcripts encoded a single ketoacylsynthase (KS) domain showing high similarity to K. brevis KS sequences. The monofunctional structure was also confirmed using dinoflagellate specific KS antibodies in Western Blots. In a maximum likelihood phylogenetic analysis of KS domains from diverse PKSs, dinoflagellate KSs formed a clade placed well within the protist Type I PKS clade between apicomplexa, haptophytes and chlorophytes. These findings indicate that the atypical PKS I structure, i.e., expression as putative monofunctional units, might be a dinoflagellate specific feature. In addition, the sequenced transcripts harbored a previously unknown, apparently dinoflagellate specific conserved N-terminal domain. We discuss the implications of this novel region with regard to the putative monofunctional organization of Type I PKS in dinoflagellates.Marine dinoflagellates  are microalgae of which some cause harmful algal blooms and produce a broad variety of most likely polyketide synthesis derived phycotoxins. Recently, novel polyketide synthesase (PKS) transcripts have been described from the Florida red tide dinoflagellate  Dinoflagellates are among the most important primary producers and therefore are important drivers of marine food webs. They also play a crucial role in structuring microbial plankton communities due to their auto- and heterotrophic or even parasitic life styles. Apart from that, some of them are the major causative agents for red tides and harmful algal blooms (HAB) in marine environments. Dinoflagellates produce a wide variety of most likely polyketide synthesis derived secondary metabolites among which several well described phycotoxins can elicit life-threating symptoms in humans Polyketides are a structurally diverse class of secondary metabolites with various potential biomedical or pharmaceutical applications as antibiotics, insecticides, immunosurpressive and anti-tumor agents PKS are traditionally classified into three types. Type I PKS are large multifunctional proteins which combine several domains in one protein. Two subclasses are known for Type I PKS. Fungal iterative Type I PKSs use the same set of catalytic domains on one protein several times for chain extension, analogously to vertebrate FAS. In contrast, modular Type I PKS function in a conveyor belt like manner in that the different catalytic domains are organized in modules comprising all required enzymatic functions. Each module is used only once during polyketide assembly, thus the architecture of each module influences the overall polyketide structure. For Type II PKSs, the catalytic domains are spread on iteratively used monofunctional proteins which form complexes for the assembly of polyketides. The Type III PKS form monofunctional, homodimeric proteins and do not require ACP but act directly on acetyl-Coenzyme A during synthesis Type I PKS genes have been identified in both prokaryotes and eukaryotes. They have been reported from fungi and bacteria Karenia brevisK. brevis (ACP-KS) Heterocapsa circularisquama (KS-KR) K. brevis, and an evolutionary classification of a comprehensive dinoflagellate KS data set encompassing representatives from different dinoflagellate orders has not been done.In 2008, eight full length PKS transcripts showing phylogenetic affinities to apicomplexan modular Type I PKS were sequenced from the toxic dinoflagellate Alexandrium ostenfeldii  and Heterocapsa triqueta , we address the question concerning the number of PKS related domains per expression unit in dinoflagellates. With respect to PKS evolution, we performed maximum likelihood analysis in order to test the hypothesis that dinoflagellate PKS derive from Type I PKS and form their own phylogenetic clade indicating a common evolutionary origin. Our findings support the hypothesis that the monofunctional Type I PKS organization is widespread in dinoflagellates.Here, we aimed to gain further insights into the organization of PKS genes in dinoflagellates and to resolve the phylogenetic relationships of dinoflagellate PKS with respect to other available protistan representatives. By sequencing full length transcripts from two further species representing other major groups within dinoflagellates, K. brevis KS domain sequence as a query, we identified five similar sequences in publicly available EST data from A. ostenfeldiiH. triquetraA. ostenfeldii KS  and two H. triquetra KS   . All traone case .In silico translation of the transcripts resulted in an average protein size of approximately 100 kDa. The presence of a KS domain was confirmed for all five candidates by a HMMER comparison with PFAM profiles. Notably, none of the transcripts contained more than one PKS related domain. We verified that the characteristic functionally important amino acid (aa) residues are present in the active sites A. ostenfeldii and H. triquetra showing a distinct band for both species at predicted size (approximately 100 kDa) for a single KS protein  as well as those 5 sequences described here have a mean size of 389±21 aa  and are located in the center of the respective protein sequences. The complete length of these putative proteins was estimated as 915±54 . The central KS domain is in each case flanked by a longer N-terminal  and a shorter C-terminal sequence segment . Neither BLAST nor PFAM domain search revealed any significant similarity to previously characterized proteins/domains. Nevertheless, these amino acid sequences showed similarities to the respective region of K. brevis KS sequences from public databases. The sequence identities for the A. ostenfeldii and H. triquetra N-terminal regions to K. brevis KB2006 ranged from 25% to 53%. A maximum likelihood phylogeny  of the tA. ostenfeldii and H. triquetra. An origin from bacterial contamination of these KS candidates can be excluded as the obtained sequences all include 3′-UTRs and poly(A+) tails as typical eukaryotic feature. The presence of the DinoSL at the 5′end further substantiates their dinoflagellate origin and the integrity of the 5′end K. brevis within a protistan clade K. brevisWe sequenced and analysed five novel full length KS transcripts from K. brevisThis is the second report of full length PKS transcripts from dinoflagellates. Previously, full length PKS transcripts have only been identified and characterized in Cryptosporidium parvum (Apicomplexa) or Emiliana huxleyi (haptophytes), display classic modular Type I PKS proteins with several different enzymatic domains organized into distinct modules within what appears to be a single transcriptional and translational unit We demonstrated both at the transcript and protein level that these putative PKS constituents are expressed as monofunctional units. This feature seems to be unique to dinoflagellates. Even other chromalveolates, like This poses the question why and how the most probably multifunctional PKS proteins have evolved into the observed monofunctional organization in dinoflagellates. The complexity, the enormous size of the dinoflagellate genome and the low gene density might play a role in this context A. ostenfeldii and H. triquetra to the KS phylogenetic dataset provides strong evidence for a unique dinoflagellate KS clade within PKS gene evolution. Also other phylogenetic groups  that diverged early on in eukaryote evolution retained evolutionary signal in their KS domains and remained in well supported monophyletic classes within the tree. This is consistent with earlier findings regarding the evolution of protistian PKS K. brevis KS and Apicomplexan PKS K. brevis which show alterations in their active sites The addition of our novel sequences from The dinoflagellate KS proteins identified so far exhibit less conserved flanking regions on usually both sides of the conserved KS domain. In the N-terminal region, at least one highly conserved amino acid motif was identified. The function of this motif cannot be determined based on homology, as it shows no significant similarity to any other currently available sequences and therefore, this signature N-terminal motif seems to be unique to dinoflagellates thus far. The C-terminal region does not show conserved aa structure in the alignment and harbors rather different phylogenetic signal from both the KS domain and the N-terminal flanking region. One could speculate that the function of the N-terminal region might be related to the monofunctional organization of these KS. We propose three possible functions. First, the N-terminal region might be required for structural purposes. If the PKS in dinoflagellates derives from modular Type I PKS, the loss of adjacent protein domains might have led to structural rearrangements of the 3D protein structure affecting the enzymatic activity. In this case, the N-terminal region could be a structural replacement for absent adjacent PKS domains and the conserved motifs might be crucial for enabling a correct folding of the polypeptides. Second, the N-terminal region could have a substrate docking function in either delivering the growing polyketide or fatty acid to the KS domain like an ACP or AT. And third, the N-terminal region might be a protein-protein interaction site in which case the motifs could be required to recognize binding partners during the assembly of the different enzymes involved in assembly of a polyketide.Saccharopolyspora erythraea, the N-terminal docking domains encompass approximately 120 amino acids in length upstream of the first KS domain Analysis of N- and C- terminal docking domains of modular Type I PKS and non-ribosomal polypeptide synthases has shown that such linker structures do exist for multifunctional enzymes. However, the linker regions observed there are much shorter (below 100 amino acids) than our N- and C- terminal regions A. ostenfeldii and H. triquetra. We provide evidence that 1) they are evolutionarily correctly classified as Type I PKS; 2) untypical for this class of PKS, but similarly to the situation previously reported from K. brevis, they are expressed as monofunctional units. Thus, Type I PKS expressed as monofunctional units seems to be a feature widespread in dinoflagellates, providing one more example of the limitations of the traditional type I, II and III paradigm for classifying PKS We characterized five novel full length transcripts encoding putative monofunctional PKS KS proteins from the dinoflagellates Alexandrium ostenfeldii  and Heterocapsa triquetra (SCCAP strain K-0481) strains were cultured at 15°C under cool-white fluorescent light at a photon flux density of 40 µmol photons m−2 s−1 and a 16 h:8 h light:dark photocycle. The dinoflagellate cultures were grown in 500 mL Erlenmeyer flasks in K-medium The Total RNA and protein fraction was isolated from dinoflagellate cultures using TriReagent®  according to the manufacture’s protocol. Algal cells were harvested by separation over 10 µm gauze to reduce bacterial contaminants and collected by centrifugation for 15 min at 3000×g . The cells were resuspended in 1 mL TriReagent® and disrupted using a beadmill  in presence of glassbeads (Ø∼200 µm). Phase separation took place after addition of chloroform; RNA, DNA and proteins were precipitated stepwise from aqueous and organic phase by addition of isopropanol, ethanol and acetone, respectively. Protein and RNA was washed according to manufacturer’s protocol. Protein pellets were resuspended in 20 mM Tris-HCl buffer, pH 7.4 containing 1% SDS. RNA was resuspended in RNAse-free water . Subsequently, the protein content was estimated with the BCA (bichinoacid assay)- method  and the samples were stored at −20°C for later application. RNA purity, yield and integrity were assessed using Nanodrop ND-1000  and 2100 Bioanalyzer  and stored for later application at −80°C.Karenia brevis KS was blasted using tblastx implementation at the NCBI website against the total set of dinoflagellate ESTs in the NCBI sequence collection. A total number of three A. ostenfeldii KS  and two H. triquetra KS ESTs  were collected and used to design gene specific Primers within the CLC main workbench  for rapid amplification of cDNA PCR to obtain full length transcript contigs.In order to identify novel PKS candidates in dinoflagellates, the protein sequence of the previously found A. ostenfeldii AOSH 2 and H. triquetra K-0481 in presence of either 1µl random hexamer primers (50 ng/µL) or a 1 µL special adapter ligated oligo dT primer (50 µM) (GCTGTCAACGATACGCTACGTAACGGCATGACAGTGTTTTTTTTTTTTTTTTTTTTTTTT) according to the manufacturer’s recommendations . Random hexamer primers or the oligo dT primer were chosen when the later experiment was rapid amplification of 5′ or 3′ end, respectively.cDNA synthesis was performed with 1 µL Superscript III reverse transcriptase (200 U/µL) and 500 ng total RNA from For 3′ RACE PCR, gene specific primers (GSP) were designed according to the GeneRacer manual’s suggestions  .. The PCCCGTAGCCATTTTGGCTCTTG, 21 nt, Tm = 64°C) was used as the forward primer and the respective reverse GSP has been designed accordingly   . The PCRE. coli cells  following the manufacturer’s protocols. The obtained clones were checked for the expected insert in a 50 µL PCR reaction with 1 µL cell lysate, 1 µL M13 F primer, 1 µL 10 µM M13 R, 1 µL cDNA, 1 µL dNTP solution, 1 µL (5 U/µL) Platinium Hot Master Taq polymerase nuclease free water and 5 µL10×PCR buffer  with the following cycling parameters: 94°C for 2 min and 25 cycles with 94°C for 1 min, 68°C for 1 min and 55°C for 3 min and a final round of extension at 68°C for 10 min. The PKS candidates were then sequenced on a 3130xl genetic analyzer  according to the manufacturer’s instructions. Internal sequencing primers were designed in order to close the contigs  and transformed into One Shot® TOP10 electrocompetent  contigs  and applIn silico translation was done with the open reading frame finder plug-in and the virtual ribosome implemented into the program. Amino acid sequences were then annotated using the PFAM domain search The obtained nucleotide sequences were aligned with the referring PKS-EST to construct full length transcript contigs by using the CLC main workbench . K. brevis and the dinoflagellate KS sequences described here. Multiple amino acid alignment was performed with the MUSCLE algorithm Amino acid sequences of KS domains were obtained from NCBI Genbank and John et al (2008) K. brevis KS was obtained after BLASTx analysis. Multiple alignment and phylogenies of the truncated sequences were calculated as described above for the KS domains.In order to identify the origin of the N- and C- terminal regions of dinoflagellate KS sequences, the respective regions were truncated from the adjacent KS domain and blasted against the NCBI and PFAM database. No significant hit beside the known Karenia brevis KS KB 2006 1:5000) were diluted in 5% skimmed milk in TBS-T and the membranes were incubated at 4°C overnight. Subsequently, the membranes were washed three times with TBS-T and incubated with the appropriate secondary antibody dilution (Goat anti-rabbit 1∶20000 Peroxidase-conjugated)  in 5% skimmed milk/TBS-T for 2 h at room temperature.10 µg total protein was separated on 10% SDS/polyacrylamide gel and transferred onto polyvinylidene fluoride membranes by Western Blot. Membranes were blocked with 5% skimmed milk in Tris buffered saline with 0.5% Tween 20 (TBS-T) for 1 h. Primary antibodies  were kindly provided by Dr. Frances Van Dolah .Western Blots: Custom peptide polyclonal antibodies specific for Sequence accession numbers: The PKS transcripts identified in this manuscript have been submitted to GenBank under the following accession numbers: Ac0019 JX068836, Ac0038 JX068837, 10-x_J14 JX068838, HTE5908 JX068839 and HTE6310 JX068840.Figure S1Presence of KS proteins in A. ostenfeldii and Heterocapsa triquetra. Western blot analysis of protein extracts from A. ostenfeldii AOSH 2 and Heterocapsa triquetra SCCAP strain K-0481 using a polyclonal rabbit anti- K. brevis KS antibody. Note that the size of the labelled polypeptide is consistent with the molecular weight predicted based on the transcript sequences, confirming expression as monofunctional units.(TIF)Click here for additional data file.Figure S2Extended phylogenetic tree of Type I and Type II KS domains from prokaryotic and eukaryotic PKS and FAS. Sixty-eight taxa representing Type I and Type II KS domains were retrieved from NCBI genbank, John et al., 2008 and Lopèz-Legentil et al., 2010 and analyzed by a maximum likelihood approach. Multiple amino acid alignment was performed with the MUSCLE algorithm included in MEGA 5.0. Subsequently, a maximum likelihood phylogenetic tree was generated and processed with MEGA 5.0 using a Jones-Taylor-Thornton amino acid substitution matrix and 100 bootstrap analyses were performed as a measure of credibility for each branch. The Type II KS and the acyl carrier protein synthases (ACPS) were used as outgroup. Bootstrap values ≥50% are marked on appropriate branches. Overall tree topology is in accord with previous reports Karenia brevis sequences which were added upon the reviewer’s request cluster mainly with modular Type I PKS trans AT but neither with KS reported here nor with Monroe & van Dolah K. brevis KS sequences.(TIFF)Click here for additional data file.Figure S3Multiple alignment of Type I and Type II KS domains from prokaryotic and eukaryotic PKS and FAS. Fifty-nine taxa representing Type I and Type II KS domains were in silico translated and aligned using the MUSCLE algoritm included into MEGA 5.0. If no GenBank accession numbers provided information can be get from *John U, Beszteri B, Derelle E, Van de Peer Y, Read B, et al. (2008) Novel insights into evolution of protistan polyketide synthases through phylogenomic analysis. Protist 159: 21–30. doi:10.1016/j.protis.2007.08.001.(TXT)Click here for additional data file.Table S1Primer collection used to obtain the 5 full length KS transcripts. Method abbreviations: S –Sanger Sequencing; 5′RACE-Rapid amplification of 5′ cDNA ends; 3′RACE-Rapid amplification of 3′ cDNA ends.(DOCX)Click here for additional data file.


The development of diet-induced obesity (DIO) can potently alter multiple aspects of dopamine signaling, including dopamine transporter (DAT) expression and dopamine reuptake. However, the time-course of diet-induced changes in DAT expression and function and whether such changes are dependent upon the development of DIO remains unresolved. Here, we fed rats a high (HFD) or low (LFD) fat diet for 2 or 6 weeks. Following diet exposure, rats were anesthetized with urethane and striatal DAT function was assessed by electrically stimulating the dopamine cell bodies in the ventral tegmental area (VTA) and recording resultant changes in dopamine concentration in the ventral striatum using fast-scan cyclic voltammetry. We also quantified the effect of HFD on membrane associated DAT in striatal cell fractions from a separate group of rats following exposure to the same diet protocol. Notably, none of our treatment groups differed in body weight. We found a deficit in the rate of dopamine reuptake in HFD rats relative to LFD rats after 6 but not 2 weeks of diet exposure. Additionally, the increase in evoked dopamine following a pharmacological challenge of cocaine was significantly attenuated in HFD relative to LFD rats. Western blot analysis revealed that there was no effect of diet on total DAT protein. However, 6 weeks of HFD exposure significantly reduced the 50 kDa DAT isoform in a synaptosomal membrane-associated fraction, but not in a fraction associated with recycling endosomes. Our data provide further evidence for diet-induced alterations in dopamine reuptake independent of changes in DAT production and demonstrates that such changes can manifest without the development of DIO. The overweight and obese represent an increasingly larger percentage of the United States and worldwide populations Striatal dopamine signaling is regulated by several factors including dopamine production by the enzyme tyrosine hydroxylase, pre- and postsynaptic dopamine receptors, and presynaptic dopamine transporters (DATs), all of which have been implicated in obesity Recently, correlations between obesity and DAT availability as well as diet-induced alterations of DAT function have been explored. Body mass index (BMI) is negatively correlated with DAT availability in the human striatum This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Animal Care Committee at the University of Illinois, Chicago. All surgery was performed under urethane anesthesia, and all efforts were made to minimize suffering.ad libitum access to standard lab chow and water.Standard male Sprague–Dawley rats (n = 67), approximately 2 months old and weighing 225–275 g upon arrival were used. Animals were individually housed in plastic cages (26.5×50×20 cm) in a temperature- (22°C) and humidity- (30%) controlled environment on a 12∶12 h light:dark cycle (lights on at 07∶00 h). Rats acclimated to the facility for one week with ad libitum access to water. Food intake and body weight measurements were made three times/wk and data are reported separately for rats undergoing voltammetric recordings or DAT protein/message analysis.After acclimation, rats were weighed and randomly assigned to 1 of 4 groups that were counterbalanced for initial body weight. Two groups were maintained on low fat diet ). The other 2 groups were maintained on HFD ). For each diet, rats were maintained for either 2 or 6 weeks (wks). Thus, the 4 groups were: LFD-2 wk (n = 18), HFD-2 wk (n = 16), LFD-6 wk (n = 16) and HFD-6 wk (n = 17). All groups had Following diet exposure, a subset of rats that did not differ in body weight were prepared for voltammetric recordings (LFD-2 wk (n = 8), HFD-2 wk (n = 6), LFD-6 wk (n = 6), and HFD-6 wk (n = 7)) under urethane (1.5 g/kg) anesthesia . A guide cannula  was positioned above the ventral striatum , a chlorinated silver wire (Ag/AgCl) reference electrode was implanted in the contralateral cortex and both were secured to the skull with stainless steel screws and dental cement. A micromanipulator containing a carbon-fiber electrode (CFE) was inserted into the guide cannula and the electrode was lowered into the ventral striatum. The CFE and reference electrode were connected to a headstage and the potential of the CFE was scanned from −0.4 to +1.3 V (vs. Ag/AgCl) and back . A bipolar stimulating electrode  was then gradually lowered into the ventral tegmental area/substantia nigra pars compacta  in 0.2 mm increments. At each increment, a train of current pulses  was delivered. When the stimulating electrode is positioned in the VTA/SNpc and the CFE is in the striatum, stimulation reliably evokes dopamine release - extracted from voltammetric data using principal component analysis m, the apparent affinity of dopamine for the DAT Dopamine reuptake was modeled using Demon Voltammetry Analysis Software . Here we report the decay constant tau as our measure of the rate of dopamine reuptake. Tau is derived from an exponential curve fit that encompasses the majority of the dopamine clearance curve and is highly correlated (r = .9899) with KAfter each recording, a stainless steel electrode  was lowered to the same depth as the CFE and a lesion  was made to mark the recording location. Brains were removed and stored in 10% formalin. Light microscopy was used to identify the lesion location on coronal sections (50 µm) through the striatum. All recordings reported here were made in the ventral striatum 3 punches through the ventral striatum (average tissue weight: 15.2 mg) were homogenized for 20 s in 0.8 ml ice-cold TEVP  +320 mM sucrose buffer. A 100 µl aliquot of total homogenate (H) was saved. The remainder of H was centrifuged at 800×g for 10 min at 4°C. The pellet  was resuspended in 0.2 ml TEVP buffer and saved. The supernatant (S1) was removed and placed in a clean tube on ice. S1 was centrifuged at 9200×g for 15 min at 4°C to generate a pellet  and a supernatant (S2). P2 was rinsed once in TEVP +35.6 mM sucrose buffer and then resuspended in 0.25 ml of TEVP +35.6 mM sucrose buffer, vortexed gently for 3 s and hypo-osmotically lysed by keeping the sample on ice for 30 min. Supernatant (S2) was collected and spun at 165,000×g for 2 h to generate a pellet  that was resuspended in TEVP (0.1 ml) and saved. All samples were kept at −80°C until polyacrylamide gel electrophoresis.Rats  were killed by decapitation. Biochemical fractionation was performed using the protocol described in Protein content was determined using the Bio-Rad DC Protein Assay kit , and the concentration of each sample was adjusted to 0.3 mg/ml protein. NuPAGE LDS (lithium dodecyl sulfate) sample buffer  and 50 mM dithiothreitol were added to each sample prior to heating at 70°C for 10 min. To load equivalent amounts of protein for each fraction, 3 µg of each sample were loaded into NuPAGE Novex 4–12% Bis-Tris gels (Invitrogen) for separation by gel electrophoresis. Proteins were subsequently transferred to polyvinylidene fluoride membrane (PVDF) . Nonspecific binding sites were blocked for 2 hr at room temperature in blocking buffer (5% nonfat dry milk in PBS and 0.02% Tween 20 [PBS-T]). Blots were then incubated in primary antibody  . Blots were cut into 3 parts: high (>97 kDa), medium (46–97 kDa), and low (<46 kDa) weights and each part probed with an antibody that recognized a protein within that weight range. Apparent molecular weights for the antibodies used are: NR2B, 180 kDa; DAT, 75, 64, and 50 kDa; TrfR, 95 kDa. After probing medium weight range blots for DAT, antibodies were stripped by incubation with stripping buffer  for 15 min at 50°C. Blots were subsequently re-blocked and probed with anti-TfR. SeeBlue Plus 2 (Invitrogen) pre-stained standards were run for molecular weight estimation.Protein immunoblots were analyzed using Carestream Molecular Imaging Software 5.0. Net intensity (the sum of the pixels within the band of interest minus the sum of the background pixels) was determined for each band. To permit comparisons between blots, data were normalized to the LFD controls at 2 and 6 wks. Data are expressed as the mean fold induction compared to LFD ± SEM.3 punches of VTA and SN tissue (average tissue weight = 15.0 mg) were made and RNA was extracted using PureLink RNA Mini Kit (Invitrogen). RNA quality and quantity were assessed using an RNA 6000 Nano Chip  on an Agilent Bioanalyzer 2100. RNA integrity number (RIN) exceeded 7 for all samples, indicating high quality. One microgram of total RNA was used to synthesize cDNA using iScript cDNA Synthesis Kit (BioRad) in a ThermoHybaid iCycler (Thermo Scientific). Primers specific for DAT , β-actin , and TATA box binding protein  genes  were designed using NCBI Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) and purchased from Integrated DNA Technologies . Melt curve analysis and polyacrylamide gel electrophoresis confirmed the specificity of the primers. The DAT, β-actin, and Tbp amplicons are 266, 182, and 136 base pairs in length, respectively.Following collection of striatal punches for western blot analysis, frozen brains were coronally sectioned on the microtome until reaching the VTA/SN. Bilateral 1-mm10 of the dilution values was plotted against the threshold cycle values for the standard curves. MyiQ Optical System Software (BioRad) was used to analyze the data. Samples containing no cDNA template and samples from cDNA reactions containing no reverse transcriptase were run as controls for contamination and amplification of genomic DNA, respectively. Reported values were normalized to the average values of the internal standards ß-actin and Tbp for each sample. Data are expressed as mean relative levels of DAT/internal standards mRNA±SEM.A Q-PCR kit  was used. The reaction was carried out on a MyiQ Single Color Real-Time PCR Detection System (BioRad) in a volume of 20 µl, with 2 µL of 3 µM forward and reverse primers and 4 µL cDNA sample diluted 1∶10. PCR cycling conditions were 95°C for 5 min; 40 cycles at 94°C for 15 s, 60° for 15 s, 72°C for 15 s. Data were collected at a read temperature of 84°C for 15 s based on the amplicon melt temperatures. Standard dilution curves were generated for each primer set by serially diluting  a master cDNA stock comprising an equal mix of cDNA from all treatment groups. The logDAT expression dynamically changes during the lifecycle in both humans p = 0.37), or 6 wk  groups. Despite consuming diets of drastically different composition, we found no differences in body weight between diet groups following either 2 or 6 wks (p<0.0001 for both diet durations).Prior to the onset of diet exposure there were no differences in initial body weight in the 2 wk . Thus, 6 wks of HFD reduces the rate of dopamine clearance in the ventral striatum compared to animals that consumed a LFD.Voltammetric recordings were made in the ventral striatum . Figure p<0.01). Our results suggest that 6, but not 2 wks, of HFD exposure reduces the dopamine response to cocaine.To further probe for diet-induced alterations in DAT, we injected rats with the DAT blocker cocaine. Peak dopamine concentration following electrical stimulation is caused by dopamine release but is also limited by simultaneous removal of dopamine by the DAT N-linked glycoprotein with an apparent molecular weight of between 50 and 80 kDa due to increasing levels of glycosylation as the protein matures F = 8.361, p<0.01); To determine if effects of prolonged HFD were due to changes in DAT number, DAT protein levels were quantified in total tissue homogenates (H fraction), synaptosomal membranes (P2 fraction) and intracellular recycling endosomes (P3 fraction). DAT is an To determine if decreased levels of DAT protein in the P2 fraction was due, in part, to a reduction in DAT transcription, VTA/SNc DAT mRNA levels were measured in the same rats as above (Prolonged HFD consumption can lead to DIO and plasticity within the central nervous system. Dopamine neurons and striatal dopamine receptors appear to be one set of CNS targets that are affected by a HFD and in obese individuals Obesity is correlated with multiple aspects of striatal dopamine signaling, including DAT availability in both humans To our knowledge, this is the first study demonstrating that a HFD reduces the dopamine response to cocaine. Given dopamine’s role in drug reward, our results are consistent with previous work demonstrating that rats fed a HFD for approximately 6 weeks are slower to acquire cocaine self-administration than animals fed a control diet Our western blot analysis suggests that prolonged consumption of a HFD does not affect total striatal DAT protein, but instead reduces the integration of the non-glycosylated 50 kDa DAT isoform into synaptosomal membranes. While DAT glycosylation improves the rate of dopamine transport and increases membrane surface stability The lack of differences in VTA/SNpc DAT mRNA levels after either 2 or 6 wks of HFD exposure further supports the notion that overall DAT levels were unaffected by our diet manipulations. This result contrasts with a previous report showing reduced DAT mRNA in the mouse VTA following 17 weeks of HFD consumption Our data add to a growing literature implicating diet in the regulation of dopamine function, and provide further evidence that diet induced changes in DAT expression leads to functionally relevant changes in dopamine signaling. Diet-induced alterations in the dynamics of striatal dopamine signaling via the DAT are likely to have consequences for feeding behavior. Food-related stimuli evoke phasic increases in striatal dopamine


Molecular pathology is now an integral part of diagnostic pathology. This has brought excitement and responsibilities to us as pathologists and molecular biologists in pathology—excitement, due to the many new possibilities we have for diagnosis, prediction of outcome, and response to therapy of disease, and responsibilities, since we need to perform the tests with high accuracy, which requires expertise. But is this different conventional histology and immunohistochemistry? At the time of Virchow, histopathology brought about enormous excitement, and as we are all aware, the responsibilities became rapidly clear. The first attempt to diagnose a tumor in 1882, on a biopsy of a Crown Prince, resulted in a failure, which led to many publications on this topic. Many of us remember the early days of immunohistochemistry, again enormous excitement, many publications (still ongoing) on its use, and obviously, great responsibilities since without immunohistochemistry, many diagnoses are impossible or, at the least, unreliable. But the pathologists looking down the microscope remained the final decision makers. And we all know that a good pathologist can make a correct diagnosis on a poor slide and/or a poor immunostained slide, but without knowledge and experience, even the best slide does not suffice. For molecular pathology, it is different.For molecular tests , the tissue is ground, DNA or RNA is extracted and processed, and bands, or colors or letters, appear on a screen, blot, or paper. These need to be interpreted in the context of the histological and immunohistochemical findings in order for a final conclusion to be drawn. This needs teamwork between a molecular biologist and pathologist, and both need to understand each other's trade. For pathologists, it is important to realize that there are factors that may influence the molecular results, factors such as fixation, tissue processing, and DNA extraction to name just a few. For molecular biologists, these issues are well known, but they also need to be informed about the exact diagnosis and the absolute and relative quantity of tumor cells and other potential test confounders. Not only that, both need to be aware of the test characteristics specific to the questions asked. That this is crucial can be learned from the KRAS story in colorectal cancer.EGFR targeting agents have a modest effect on metastasized colorectal cancer. As it turned out, they are only effective on tumors with a wild-type KRAS gene, but in patients with a KRAS mutated tumor, the drug even has a negative effect. It is therefore mandatory that the test is performed with high accuracy. Therefore, the European Society of Pathology has initiated a European external quality assurance system, which is now in its fourth year -3. ThrouIn this issue, Lee et al. describe a novel PCR-based method for KRAS testing and compare the results with the most commonly used test, Sanger sequencing . This la


Anopheles gambiae sensu stricto (s.s.) is a primary vector of Plasmodium falciparum in sub-Saharan Africa. Although some physiological differences among molecular and chromosomal forms of this species have been demonstrated, the relative susceptibility to malaria parasite infection among them has not been unequivocally shown. The objective of this study was to investigate P. falciparum circumsporozoite protein infection (CSP) positivity among An. gambiae s.s. chromosomal and molecular forms.An. gambiae from two sites Kela (n = 464) and Sidarebougou (n = 266) in Mali were screened for the presence of P. falciparum CSP using an enzyme-linked immunosorbent assay (ELISA). Samples were then identified to molecular form using multiple PCR diagnostics (n = 713) and chromosomal form using chromosomal karyotyping (n = 419).Wild An. gambiae sensu lato (s.l.) mosquitoes, 89 (12.2%) were CSP ELISA positive. The percentage of positive mosquitoes varied by site: 52 (11.2%) in Kela and 37 (13.9%) in Sidarebougou. Eighty-seven of the positive mosquitoes were identified to molecular form and they consisted of nine Anopheles arabiensis (21.4%), 46 S (10.9%), 31 M (12.8%), and one MS hybrid (14.3%). Sixty of the positive mosquitoes were identified to chromosomal form and they consisted of five An. arabiensis (20.0%), 21 Savanna (15.1%), 21 Mopti (30.4%), 11 Bamako (9.2%), and two hybrids (20.0%).Of 730 P. falciparum infection in the M form was equivalent to infection in the S form . There was a significant differential infection by chromosomal form such that, P. falciparum infection was more prevalent in the Mopti chromosomal forms than in the Bamako or Savanna forms; the Mopti form was also the most underrepresented in the collection. Continued research on the differential P. falciparum infection of An. gambiae s.s. chromosomal and molecular forms may suggest that Plasmodium – An. gambiae interactions play a role in malaria transmission.In this collection, the prevalence of  Anopheles gambiae complex are vectors of human malaria parasites, including Plasmodium falciparum, the species of greatest public health importance in Sub-Saharan Africa. The An. gambiae complex consists of at least seven member species and subspecies [Anopheles gambiae sensu stricto (s.s.) is further divided into chromosomal and molecular forms [An. gambiae that is the basis of the sub-division into the M and S molecular forms [Anopheles gambiae are also divided into chromosomal forms based on the arrangements of 5 paracentric inversions on 2R  and one on 2 L (a), which define Mopti from Savanna from Bamako forms [Several members of the bspecies -3. Anophar forms . The M aar forms -7 that aar forms -10. Anopko forms . In Maliko forms . In someko forms ,13-16.An. gambiae have been associated with adaptation to different local environments [Plasmodium falciparum circumsporozoite protein (CSP) positivity in An. gambiae was significantly greater in mosquitoes with isozyme allotypes Mpi130/130 and Acp110/100[-/a-[APL1[TEP1r[An. gambiae have identified transcript expression patterns that are significantly associated with malaria parasite and bacterial infection [An. gambiae laboratory and field-collected M and S molecular forms [An. gambiae were significantly associated with natural P. falciparum infection [P. falciparum infection [P. falciparum infection, indicating potential differential immune responses of the two molecular forms to parasite infection [An. gambiae was identified in the Sudan-Savanna zone as susceptible to P. falciparum[Genetic polymorphisms within ronments -21. For ronments . There icp110/100. In addi/100[-/a-. Quantit/100[-/a-. Chromos-/a-[APL1, and chrPL1[TEP1r both gennfection -31. Furtar forms ,33. Threalciparum.Reports of genetic, proteomic, and genomic differences in An. gambiae s.s. suggest, by extension, that susceptibility to P. falciparum infection varies among different molecular forms and populations of An. gambiae[. This study examined in more detail the hypothesis that P. falciparum infection prevalence of An. gambiae s.s. differs among molecular and chromosomal forms in Mali. Natural P. falciparum infection levels were compared among An. gambiae molecular and chromosomal forms in two villages in Mali where these forms occur in sympatry. Inversion-specific correlations between P. falciparum infection and standard, heterozygous, and homozygous chromosomal arrangements [. gambiae,27,33,35An. gambiae mosquitoes were collected in October 2009 from the villages of Kela , and Sidarebougou  joined with Kolayerebougou  in Mali. Resting mosquitoes were collected in the morning via mouth aspirators from inside homes. Mosquitoes were held in cups until they had reached the half-gravid stage. Mosquitoes morphologically identified as An. gambiae s.l. were dissected and separated into head/thorax for P. falciparum CSP ELISA, abdomen/wings/legs for molecular form identification via polymerase chain reaction (PCR), and ovaries for chromosomal form identification via karyotyping. Head/thorax samples were stored in 100% ethanol, while abdomens/legs/wings were stored in 70% ethanol and half-gravid extracted ovaries were stored in modified Carnoy’s solution .Adult . falciparum CSP ELISA [P. falciparum protozoan had disseminated across the midgut. The head and thorax, stored in 100% ethanol, were dried prior to tissue lysis. For each ELISA plate, a minimum of two colony-reared An. gambiae mosquitoes  and serial dilutions of P. falciparum monoclonal antibodies  were used. The positive control P. falciparum CSP was serially diluted  to quantify CSP in field-collected mosquitoes. Samples with absorbance values greater than three times the standard deviations from the mean of the negative control samples on each ELISA plate were designated as “positive” for P. falciparum infection [P. falciparum to ensure the protozoan had disseminated the midgut and that the protozoan was in the ‘infective’ sporozoite phase.Lysates of head/thorax samples were assayed using a PSP ELISA ,37 accornfection . CSP ELIAn. gambiae s.l. were identified to species [An. gambiae molecular form identifications were performed on each mosquito [Abdomen, legs and wings stored in ethanol were ground using a TissueLyser , after which DNA was extracted using the BioSprint 96 Bloodkit and automated workstation . Mosquitoes morphologically identified as  species  and An. mosquito -42.An. gambiae complex and chromosomal forms [Polytene chromosome spreads were prepared from ovarian nurse cells , except al forms ,11.Summary statistics, relative abundance of forms, Fisher’s exact tests and two-tailed T-tests were performed in Excel 2007 to determine differences within populations . Where aAn. gambiae s.l. were analysed, of which 42 (5.8%) were identified as An. arabiensis[Anopheles arabiensis were CSP ELISA positive and 20% (2/10) were CSP ELISA positive in Sidarebougou. Seventeen of the mosquitoes were not identified to molecular form or karyotyped, and two were positive, both from Kela. The remaining 671 mosquitoes were identified as An. gambiae s.s., of which 78 (11.6%) were P. falciparum CSP ELISA positive. Infection prevalence differed for the two collection sites, from 13.9% (35/251) in Sidarebougou and 10.2% (43/420) in Kela, but they were not significantly different .In total, 730 rabiensis. Data frAn. gambiae s.s are presented in Figure An. gambiae s.s. molecular form (94.1%) and 372 were identified to An. gambiae s.s. chromosomal form (52.1%). Both molecular and chromosomal form identifications were performed for 395 mosquitoes of which 370 were An. gambiae s.s. and 25 were An. arabiensis, separate analyses were conducted for each.The frequencies of molecular and chromosomal forms of An. gambiae s.s. This sample was comprised of 62.9% S, 36.1% M and 1.0% M/S molecular form hybrids, but the relative abundance of each molecular form and infection prevalence varied between sites  were P. falciparum CSP ELISA positive, of which, 31 were M, 46 were S, and 1 was a M/S molecular hybrid. Contingency tests on the number of positive to negative samples for each An. gambiae molecular form were not significantly different   or M molecular forms  were not significantly different at these collection sites. At Sidarebougou  and Kela , infection prevalence among molecular forms was not significantly different.Infectivity and molecular form association analyses were conducted on 671 s Figure , Table 1An. gambiae s.s., 37.4% were Savanna, 32.3% were Bamako, 18.5% were Mopti, 9.1% were Mopti-Savanna, and 2.7% were Bamako-Savanna. Of the 207 samples karyotyped from Kela, 57.0% were Bamako, 22.7% were Mopti, 7.2% were Savanna, 10.1% were Mopti-Savanna, and 2.9% were Bamako-Savanna  , Savanna , or Mopti chromosomal forms . Within Kela, Savanna and Mopti chromosomal forms were significantly more likely to be positive than the Bamako form , but not in Sidarebougou . On average across villages, 19.3% (11/57) of Mopti M forms were CSP positive, 16.9% (20/118) of Savanna S forms were CSP positive, and 9.2% (11/119) Bamako S forms were positive and these patterns were not significant .Among mosquitoes identified as Mopti chromosomal and M molecular forms, 15.8% (6/38) and 26.3% (5/19) were CSP positive in Kela and Sidarebougou, respectively. Among mosquitoes identified as Bamako chromosomal and S molecular forms in Kela, 9.4% (11/106) were CSP positive, whereas none (0/2) with this chromosomal and molecular form combination were CSP positive in Sidarebougou. Among mosquitoes identified as Savanna chromosomal and S molecular forms in Kela, 50.0% (5/10) were CSP positive, while 13.9% (15/108) of this form combination were CSP positive in Sidarebougou. These differences were significant in Kela  were positive than were the Savanna (15.1%) and Bamako (9.2%) chromosomal forms. As the Mopti chromosomal form corresponds to M molecular form in Mali in most cases [P. falciparum infection is not surprising. Site-specific differences in the number of CSP positive chromosomal form infection between Kela and Sidarebougou were also observed. In particular, the most common chromosomal form in each village, Bamako in Kela and Savanna in Sidarebougou, was least likely to be CSP positive.An average of 12.2% of P. falciparum infection between M and S molecular forms [P. falciparum susceptibility assay among An. gambiae s.s. molecular forms from Senegal found significantly higher numbers of P. falciparum oocysts and sporozoites in the S molecular form than in the M form [P. falciparum, whereas the Senegal study [P. falciparum to standardize age and potential for infection. These conflicting findings may result from the origin of field collected samples , to different techniques , or to a differences in the age structure of the samples. Field studies from multiple sites and over multiple sampling periods are necessary to confirm the observed patterns.The insignificant infection prevalence in the M molecular form in southern Mali corroborates with other studies from Cameroon and Senegal that reported no differences in ar forms ,46. A ree M form . This stal study  collecteAn. gambiae s.s. among sample sites can also limit comparisons among the previous studies [An. gambiae s.s. populations were observed in Mali: the M molecular form, the S molecular form (S1), and a subdivided S Pimperena form (S2) [P. falciparum infection were differentially distributed among M, S1, and S2 populations [et al[An. gambiae, indistinguishable in molecular form but distinguishable via microsatellites amplified from chromosome 3, was susceptible to P. falciparum[Cryptic genetic differences in  studies ,46 and t studies ,21. In aorm (S2) . Furtherulations . Of intens [et al where a alciparum.An. gambiae forms and P. falciparum infection. For example, Dolo et al[et al[et al[An. gambiae. Collectively, these studies highlight the importance of both genetic and environmental determinants of susceptibility to infection.Differences in the local environment may likewise affect associations between olo et al demonstr al[et al hypothes al[et al,50. The  al[et al hypothesP. falciparum differential infection rates among chromosomal forms and trends among chromosome inversions. Mopti had the highest CSP positivity (30.4%), followed by Savanna (15.1%) and Bamako (9.2%) forms. The data presented here indicated that Mopti chromosomal forms (2Rbc/u) were more likely to be positive for P. falciparum CSP. A previous study in Kenya identified a significant association between the total number of inversions and a decreased likelihood for P. falciparum infection [There were statistically significant nfection . Data prsitivity 0.4%, folP. falciparum genotype [APL1 gene, which encodes for natural resistance to P. falciparum, exhibited extremely low genetic diversity within the M molecular form, but high diversity in the S molecular form that may have arisen from larval infection [. Alternatively, higher diversity at the APL1 locus in the S molecular form may be associated with a more diverse array of responses to P. falciparum and reduced susceptibility to infection. Similar results were identified within the anti-parasite and anti-bacterial gene TEP1r[TEP1r was diverged between the M and S molecular forms and one variant showed a strong association with resistance to infection with a rodent malaria parasite and with P. falciparum[An. gambiae molecular and chromosomal forms with P. falciparum infection that incorporate An. gambiae speciation, genetic diversity at immune loci  as well as larger temporal and spatial scales may help to extend findings reported in this study.A number of studies have examined associations of chromosomal polymorphisms with malaria infection. A small region of chromosome 2 L has been associated with infection susceptibility, regardless of genotype . Genes wgenotype . Within nfection ,35. Alteene TEP1r. Specifialciparum. AdditioP. falciparum infection prevalence at geographic locations with multiple molecular and chromosomal forms are likely due to discrepancies in the relative abundance of forms, genetic diversity in immune signaling genes within different forms, age structure of field collections, and local environmental variations that influence infection and transmission success.Whilst the correlation between M and Mopti forms was expected, higher infection prevalences in the Mopti chromosomal form have not been demonstrated previously. In general, significant differences in The authors declare we have no competing interests with the work presented in this manuscript.AJC, GCL, SL, and YL conceived the study and designed the experiments. YL, AJC, AF, and SFT conducted the work in Mali. YL, CSN, and RTF conducted the laboratory work. RTF analysed the data and wrote the manuscript. All authors have read and approved the final manuscript.