Upon re-submersion, an additional rapid rise in DMSgas was observed, reflecting increased production by the coral and/or dissolution of DMS-rich mucus formed by the coral during air exposure. Depletion in DMS following re-submersion was likely due to biologically-driven conversion of DMS to dimethylsulfoxide (DMSO). Fast Repetition Rate fluorometry showed downregulated photosynthesis during air exposure but rapid recovery upon re-submersion, suggesting that DMS enhances coral tolerance to oxidative stress during a process that can induce photoinhibition. We estimate that DMS emission from exposed coral reefs may be comparable in magnitude to emissions from other marine DMS hotspots. Coral DMS emission likely comprises a regular and significant source of sulfur to the tropical marine atmosphere, which is currently unrecognised in global DMS emission estimates and Earth System Models.Corals are prolific producers of dimethylsulfoniopropionate (DMSP). High atmospheric concentrations of the DMSP breakdown product dimethylsulfide (DMS) have been linked to coral reefs during low tides. DMS is a potentially key sulfur source to the tropical atmosphere, but DMS emission from corals during tidal exposure is not well quantified. Here we show that gas phase DMS concentrations (DMS Symbiodinium47896Reef-building corals are prolific producers of dimethylsulfoniopropionate (DMSP), an important precursor to the climate-relevant gas dimethylsulfide, DMS12Symbiodinum sppCO2) and elevated DMSP, DMS and DMSO in pelagic microalgae12131516178202022Dimethylated sulfur compounds, particularly DMS and DMSO, readily scavenge harmful hydroxyl radicals and other reactive oxygen species8Acropora cf. horrida, Seriatopora hystrix, Porites cylindrica) to air for short periods and subsequent re-submersion. Measurements of atmospheric DMS (DMSgas), seawater DMS, DMSP, DMSO and Fast Repetition Rate fluorometry (FRRf) were made before, during and after each exposure. Together, our results revealed a marked response in coral sulfur cycling and photophysiological condition that carry important implications both for estimates of sulfur emissions to the tropical/sub-tropical atmosphere and for our understanding of how corals respond to environmental fluctuations.Air exposure experiments are a potentially useful tool to evaluate how reef-forming corals contribute to local atmospheric DMS concentrations and regulate dimethylated sulfur compounds during environmental stress. We describe a series of novel laboratory experiments under controlled temperature and light conditions exposing three common Indo-Pacific scleractinian coral species , originally from the same parent colony from the Indo-Pacific, were used to generate between 1 and 25 (mean 11 ± 9) equal-sized nubbins (ramets), 2–5 cm tall, and distributed equally on holding racks receiving one of two light acclimation levels  for 8–10 weeks before experiments commenced. Ramets were followed throughout experimental design and selected from different genets to ensure biological replication for each treatment. At the beginning of experiments, coral nubbins (ramets) were transferred within a few seconds from the acclimation tank to 3 × 870 mL glass flasks (n = 3 or 4 nubbins per flask) with gas-tight lids and fully covered  in 500 mL seawater from a 1000 L tropical aquarium. The number of nubbins used was dependent on the number that could be placed inside the flask without the nubbins touching each other or the flask walls. Each flask was positioned within a temperature-controlled water bath at 26 °C illuminated by 54 W fluorescent tubes  delivering ~300 μmol photons/m2/s within each flask.Full details of the analytical methods and coral experimental facility, including coral husbandry, are given in the air exposure; Stage II) by removing seawater from the flasks. The mean exposure time for all experiments (32.7 ± 11.7 min) falls within the range of tidal exposure times of 15–105 min experienced in a natural reef environmentre-submersed; Stage III). When the coral nubbins were (re-)submersed, rising bubbles fully equilibrated with seawater DMS (see Discussion) and exited in the waste air stream. During the air exposure phase the air flow from the base directly displaced the headspace in the flask.Seawater in the flasks was continuously and gently bubbled with compressed air from tubing inserted to the base of the flask. Set using metering valves and digitally measured , the mean (±SD) flow for all experiments through the flasks was 59.3 ± 6.5 mL/min. It was ensured that neither the tubing nor the bubble stream came into contact with the coral nubbins. Flasks were left for 1 h to approach constant DMS concentrations where net biological production by the coral holobiont was balanced by removal of DMS due to bubbling. Coral nubbins were subsequently exposed to air (gas) were monitored in the waste air stream from the flasks before, during and after each period of exposure. In experiments with A. cf. horrida, DMSgas was quantified every 4 s using atmospheric pressure ionisation–chemical ionisation mass spectrometry, API-CIMS  to make up the total flow required by the API-CIMS instrumentgas measurements from API-CIMS during the first 2 minutes after each stage change  are not completely representative of the gas phase DMS within the flasks, and are excluded from analyses. The API-CIMS D.L. within this experimental setup was calculated as 3 standard deviations above the mean of the control ‘no coral’ flasks (0.03 ppm for 5-minute averaged data).During replicate incubations , the was−1. The trap was heated rapidly to inject onto the GC column and DMS quantifiedWaste gas outflow from experimental flasks was collected for 10 min in 5 L Tedlar bags pre-rinsed with oxygen-free nitrogen . The bag contents were pre-concentrated on a cryogenic trap at 60 mL mint) and dissolved DMSO (DMSOd) samples were taken before and after coral exposure. For DMS, 5 mL samples of pre-filtered seawater media were analysed via purge and cryogenic trap followed by GC-FPD54 to seawater samples, and purge and cryogenic trap followed by GC-FPD29In a separate experiment without bubbling with compressed air, seawater DMS, total aqueous phase DMSP  data was collected from coral nubbins during parallel air exposure experiments. FRRf inductions (0.2 Hz) parameterised the photosynthetic efficiency , effective absorption cross sectional area , whilst the other species were within a lower range of 0.05–0.2 ppm (0.01-10.4 pmol/cm2)  was 900–1600 nmol/cm2 for A. cf. horrida but 40–80 nmol/cm2 for the remaining three species 13819During initial submersion, DMSmol/cm2) . It is icies see . Our A. gas from a flask containing a colony of A. cf. horrida increased by about an order of magnitude from 0.3 ± 0.2 ppm (mean ± SE) during submersion up to a maximum of 5.8 ppm within the first 2 minutes after air exposure  is 11.4 at 26 °Cgas within the first minutes after air exposure. After the initial spike, DMSgas steadily decreased during the subsequent 12 minutes and returned to near baseline levels. We estimate the exponential decay in DMSgas due to flushing with DMS-free compressed air . We note that this observation does not imply zero DMS production from the coral holobiont upon air exposure. DMS production likely continues leading to the initial spike in DMSgas, but is then trapped by the mucus layer that quickly forms on the coral surface upon exposure (see Discussion).This initial spike in DMSA. cf. horrida , while the other species yielded less than 1 ppm (<100 pmol/cm2). Both raw DMSgas concentrations and zooxanthellae cell density-normalised DMS concentrations yielded the same trends (2/s)A. cf. horrida100 and two flasks holding A. cf. horrida400 (n = 3 nubbins per flask). The same response was seen during air exposure, with DMSgas reaching maxima that ranged from 0.8–4 ppm across four experimental flasks, compared to 0.1–1.5 ppm during submersion  from the coral, we started from the mass balance equation of gas phase DMS:gas = 700 mL, the gas flow Flow = 60 mL/min. To make the coral emissions comparable to other marine DMS flux hotspots, we converted our flux estimates into equivalent sea surface areas (SSA); we take SSA for each fragment in our experiments to be the birds-eye-view water surface area above the coral (estimated as 25 cm2 for each fragment x 3 nubbins = 75 cm2). gas increase (dDMSgas/dt), which we could not accurately quantify due to the measurement system design. For simplicity, we therefore assume the averaged DMSgas shown in A. cf. horrida was 3–11 mmol/m2/day. Emissions from other coral species were lower, at 1.7 mmol/m2/day for P. cylindrica and 2.3 mmol/m2/day for S. hystrix.Here the headspace volume during air exposure VA. cf. horrida at 24 minutes into the experiment resulted in a second rapid increase of DMSgas up to 6 ppm in the outflow of the flask, followed by a near-exponential decrease in concentration , re-submersing the coral in seawater could result in dissolution and liberation of the DMS trapped in mucus. The subsequent decrease in DMSgas is driven by biological processes associated with the presence of coral, with water-to-air emission accounting for only a quarter of the DMS loss (see Discussion).Re-submersion of ntration . Unlike Fq’/Fm’) in conjunction with an increased photosynthetic electron turnover time (τ´)  periods of exposure during spring tides38Acroporids may possess physiological mechanisms that instil tolerance to a range of environmental conditions.Continuous FRRf measurements provided a diagnostic of photosynthetic performance before, during, and after air exposure. Photosynthetic downregulation was identified by a temporary decrease in the effective absorption cross-section (σ´)  and the ime (τ´) . This iso-damage . P. cyliexposure . The fuld) and total aqueous phase DMSP (DMSPt) at regular intervals after re-submersion of A. cf. horrida400 in a separate experiment . Abiotic photochemical loss of DMS, inferred from previous measurements from the Sargasso SeaTo explore the roles of dimethylated sulfur compounds as anti-oxidants, we measured the seawater concentrations of DMS, dissolved DMSO . Simultaneously, DMSOd increased by 175 nM (1.4 nmol/cm2). We see that the net increase in DMSO concentration  was 55 nM higher than the net decrease in DMS concentration. Using a simplistic mass balance where no DMSO is directly released by the coral, 100% of DMS is converted to DMSO, and DMSO loss is negligible, 42After 20 minutes, DMS levels had decreased by 120 nM  in a separate experiment  and monitored the rate of decrease due to bubbling (60 mL/min) in a separate test. The observed reduction , which shows excellent agreement with the modelled DMS equilibrium outgassing (red line). Based on this, we convert the API-CIMS gas phase DMS mixing ratio to a waterside concentration  via the ideal gas law and the Henry’s solubility.We developed a simple box model in order to explore the plausible bounds of biological DMS production and consumption. As an independent measure, we used the biological consumption rate derived from the no-bubbling experiment  to modelperiment . The corperiment . DMS in sw a few minutes after re-submersion. Similar to previous experiments, elevated DMSsw (~1 μM) was observed immediately after the coral was re-submersed followed by a gradual decline in concentration to ~0.01 μM after 60 minutes. The model shows that equilibrium outgassing can only explain about 25% of the total DMS loss within the first 20 minutes after re-submersion. This indicates additional coral-associated loss of DMSsw, similar to sw relative to observations. A consumption-only model run  concentrations that are orders of magnitude higher than in seawaterA. cf. horrida that had been exposed to air for 15 minutes (a separate colony to those used in the main experiments - see SI methods). Concentrations ranged from 279–577 nM in mucus, with a value of 373 nM in a sample of mucus rope It is worth mentioning that even a high DMS production of 2 μM/h see  does notsw] upon dissolution of DMS-rich mucus into the water phase:Using this information, we can estimate the resultant [DMSA. milleporaA. cf. horrida nubbins used in our study is 125.7 cm2 (See mucus) of 0.006 L. Seawater volume in the flask (volwater) was 0.5 L. Taking the maximum value of [DMSmucus] we measured for A. cf. horrida (577 nM) would result in [DMSsw] of only 7.3 nM, far from the ~1 μM we measured . The extent of exposed coral reef during low tide events is not well documented but typically includes the reef crestAcropora colonies and is exposed on average 12 hours per month (~0.5 days per 30 days = ~0.02)252/day (i.e. 3–11 mmol/m2/day *(0.158)*0.02*1000) by assuming that Acropora on the reef rim are the sole source of DMS and only emit to the atmosphere when exposed to air. This simplistic calculation does not account for the complexity of the reef environment . Furthermore, the experimental exposure times used here (32.7 ± 11.7 min) fall short of maximum exposure times in a natural reef environment (up to >100 min)47Our experiments suggest that coral reefs represent an as-yet unquantified all-year-round direct DMS flux into the tropical atmosphere (~23°N to 23°S), which is not currently accounted for in existing climatologies and Earth System models56572/day2/day were measured over a coccolithophore-dominated bloom in the North Atlantic2/day was seen over a dinoflagellate bloom in the Southern OceanNevertheless, DMS emissions from corals may be in the same order of magnitude as other marine DMS hot spots, which tend to be more transient. For example, DMSP-rich surface pack ice in Antarctica is estimated to emit DMS to the atmosphere at a few tens of μmol/mAcropora dominate coral communities. Acropora is a highly successful genus contributing to rich species diversity and productivity on coral reefs via their complex architecture and rapid growthAcropora is also particularly susceptible to major environmental and climatic stressors, which are expected to increase in frequency in the futureAcropora cover could impact upon future biodiversity and productivity associated with coral reefsIn situ measurements of atmospheric DMS concentrations and coral-to-air DMS fluxes in reef environments at a range of temporal and spatial scales are required to better quantify the importance of DMS in the tropical marine atmospheric chemistry.Our experiments show that when water is removed and corals are exposed to air, a solubility effect results in an order of magnitude increase in DMS concentrations in the overlying air in the first few minutes after exposure. This is consistent with observations of elevated DMS in the air above coral reefs following low tides222364How to cite this article: Hopkins, F. E. et al. Air exposure of coral is a significant source of dimethylsulfide (DMS) to the atmosphere. Sci. Rep.6, 36031; doi: 10.1038/srep36031 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Surprisingly, the passing ion does not tear nanometre-sized holes into the single layer graphene. We use time-dependent density functional theory to gain insight into the multielectron dynamics.The way conduction electrons respond to ultrafast external perturbations in low dimensional materials is at the core of the design of future devices for (opto)electronics, photodetection and spintronics. Highly charged ions provide a tool for probing the electronic response of solids to extremely strong electric fields localized down to nanometre-sized areas. With ion transmission times in the order of femtoseconds, we can directly probe the local electronic dynamics of an ultrathin foil on this timescale. Here we report on the ability of freestanding single layer graphene to provide tens of electrons for charge neutralization of a slow highly charged ion within a few femtoseconds. With values higher than 10 Graphene has so far demonstrated remarkable properties, making it increasingly interesting for ultrafast electronic applications. Here, the authors show that, when probed by a highly charged ion, freestanding graphene is able to provide dozens of electrons for ion neutralization within a few femtoseconds. A charge state of  fs refs , 33, 34.qin=30 captures ∼25 electrons on a timescale of a few fs. Based on the experimental data, a lower bound for the current densities reached locally within a nm2 area is at least 1012 A cm−2 exceeding the breakdown current densities reported so far567In this contribution, we take the final step and present the results for the ultimately thin carbon target, a freestanding single layer of graphene. Measurements of the charge state and energy of the transmitted ions and time-dependent density functional theory (TDDFT) calculations show that large number of electrons are extracted from a very small surface area, which implies a high local surface current density. We identify a multielectron process and estimate the relevant timescales for charge transfer along the graphene layer. For example, while passing through SLG, the HCI with initial charge state qout and the energy of highly charged Xe ions after transmission through SLG , The amount of charge transferred to the HCI can be estimated by measuring the distribution of exit charge states analyser . Typicalanalyser  show a dτ=deff/v, where v is the ion velocity. The effective interaction length deff corresponds to the projectile-surface distance where the electron transfer processes between the HCI and graphene take place. The values of deff can be obtained from the TDDFT calculations which yield for qin=20 a value of Depending on the initial charge state the HCI captures and stabilizes between 20 and 30 electrons during its transmission through graphene . The expThe number of captured and stabilized electrons (they are not reemitted due to autoionization processes) as function of interaction time is shown in deff=9 Å we obtain qin=35, and qin=20, respectively. The performed TDDFT study  into the SLG, as we have observed for carbon nanomembranes35qin=20 projectile incident at graphene with velocity v=0.87 nm fs−1. Results of the TDDFT calculations are presented for four different HCI–graphene distances zion along the ingoing trajectory path. We use cylindrical  coordinates, with the z axis set along the projectile trajectory assuming normal incidence geometry. Note that because of the cylindrical symmetry in our calculations  can be observed.To illustrate the way the electronic processes take place, in Jz determines the charge exchange and energy loss of the HCI (see the section Energy loss), while the current density along the transverse direction Jρ shows the fast local response of graphene to the strong HCI perturbation. In fact, our calculations reproduce our experimental estimate since the transverse current densities in the proximity of the graphene layer reaches values as high as 1012 A cm−2. This means that, transiently and locally, in the femtosecond and nanometre scale, graphene is able to sustain extremely high current densities. The positive charges created by electron capture and electron emission are spread over the entire layerThe current density along the direction of motion qout=2 and qout=4 increases quadratically with the incident charge state  all the missing electrons in the projectile. Our experiments and TDDFT calculations, both suggest local current densities in the graphene plane exceeding 10q keV corresponding to velocities between 0.13 and 0.5 nm fs−1. The pressure in the experimental chamber is kept below 5 × 10−9 mbar during measurements to prevent charge-exchange processes of the ions before interaction with the target.The measurements are performed at the Ion Beam Center of the Helmholtz-Zentrum Dresden-Rossendorf. Highly charged Xe ions are produced in a room temperature electron beam ion trap, charge state separated by an analysing magnet and then guided by several electrostatic lenses into the target chamber. Due to an electrostatic deceleration system, the kinetic energy of the extracted Xe ions can be varied between 0.1 and 4.4E/E≈1.5 × 10−3. The analyser's maximum operation voltage of 5,000 V limits the range of measurable charge states for projectiles with larger kinetic energies.The freestanding SLG sheets, which span over a regular array of holes in a TEM grid, are produced at the University Duisburg-Essen and transferred without the use of polymer coating see . The eleR=0.26 Å, Coulomb tail Q/r) and a planar jellium disk with the correct work function value (4.6 eV)13r=R. Finite size effects have been checked using jellium disks of different sizes containing 500, 1,004 and 2,000 electrons .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Notes and Supplementary References.in=20 at v=0.87 nm fs-1. The polarization of the surface of graphene due to the approaching HCI, the charge transfer from graphene to the projectile and the excitation of the graphene layer while and after the crossing of the ion are clearly visible.The video illustrates the induced charge density for an incoming ion with q


Metallic gate electrodes are often employed to control the conductivity of graphene based field effect devices. The lack of transparency of such electrodes in many optical applications is a key limiting factor. We demonstrate a working concept of a double layer graphene field effect device that utilizes a thin film of sputtered aluminum nitride as dielectric gate material. For this system, we show that the graphene resistance can be modified by a voltage between the two graphene layers. We study how a second gate voltage applied to the silicon back gate modifies the measured microwave transport data at around 8.7 GHz. As confirmed by numerical simulations based on the Boltzmann equation, this system resembles a parallel circuit of two graphene layers with different intrinsic doping levels. The obtained experimental results indicate that the graphene-aluminum nitride-graphene device concept presents a promising technology platform for terahertz- to- optical devices as well as radio-frequency acoustic devices where piezoelectricity in aluminum nitride can also be exploited. Flexible and transparent FET devices may lead to novel device integration concepts such as wearable electronics and solar cells, integrated RFID tags in packaging material, and would therefore contribute to the ongoing electronic revolutionGraphene is a promising material for terahertz-to-optical devices, in particular voltage controlled attenuators and modulators for terahertz components to be used for wireless communication with high data rate1569101112132 layer. The device is fabricated in three steps as demonstrated by 2. This is followed by magnetron- sputtering of 50 nm AlN over the entire area at 400 °C substrate temperature  layer sandwiched between two graphene layers. Previous attempts to fabricate double-graphene structures include implementation of poly (methyl methacrylate), PMMA, in between two graphene layersperature  and thenperature . This arperature . As a corature 19. The gra01δ, mode of the dielectric puck is the lowest fundamental mode that is excited via two coupling loop antennas in the cavity and the resulting resonance curve is measured using a Rohde-Schwarz ZVA vector network analyzer (VNA). This mode exhibits only azimuthal electric field component and therefore the field remains continuous on the surface of the ceramic resonator and high confinements of the field within the puck is achieved. The resulting evanescent field leaks through the aperture of the cavity and excites circular high-frequency currents in the graphene layers. As such, it represents an ideal measurement methodology for highly accurate and contact-free electrical measurements of 2D materialsThe transverse electric, TE01δ mode21Q-factor. Prior to presenting the double-gated structure, we first verify this characterization technique on a simpler commonly employed single gated silicon based system. For this system, we transferred graphene on both nSiO2/Si and AlN/Si substrates. This was then followed by the microwave field-effect measurements using the dielectric-loaded cavity method. Q, which determines the sheet resistance via equation (1)The entire GFET device is semi-transparent for the electromagnetic fields of the TEQ-factor values. Here, the characteristic impedance of the cavity, ZC, is determined by a substrate calibration based on the measured shift of the resonant frequency due to the graphene-free substrateQS denotes the measured quality factor with the uncoated HRS substrate and Q(V) is the measured quality factor at a given gate voltage for a graphene covered substrate. The ratio between the resistance R(V) at a given gate voltage V  and the minimum resistance Rmin, (representing maximum field induced n- or p-type doping), given by Qmin represents the smallest measured quality factor corresponding to the minimum sheet resistance. Note that this ratio is independent of the cavity calibration factor ZC. It is important to note that the achieved modulation depth of 2.1 dB is much lower than the maximum achievable modulation depth for a graphene-modulated microwave cavity. This is merely because the silicon substrate, in spite of its high resistivity, contributes significantly to the cavity losses.Here, We compare the voltage dependent conductivity to the theoretical Boltzmann equation results given by equation (3)kB and μ denote the Boltzmann constant and the chemical potential, respectively, T the temperature, and e denotes the electron charge and ħ is reduced Planck’s constant. At microwave frequencies the Boltzmann equation represents an accurate approximation to the more general Kubo formula for the conductivity, because the interband contribution to σ is negligible for frequencies below 100 GHzτ+) and negative (denoted τ−) energies. The chemical potential, μ, is determined by the carrier density, which is controlled via the gate voltage VGS. For a single graphene layer, the charge density is given by n = n0 + CVGS/e, where n0. denotes the intrinsic charge density and C is the areal capacitance associated with the gate dielectric between graphene and Si. For the double layer (see e2/πh), the widely accepted minimum optical conductivity of graphenewhere ayer see  we obtai2 and 5 nm thick AlN respectively with the simulations based on the Boltzmann equation result (τ+ = 17 fs a τ− = 14 fs give the best fits for nSiO2 and τ− = 14.5 fs for AlN as well as n0 = 8.6 × 10111/cm2 for the intrinsic charge density of graphene are used to obtain the fit (see 9252627n result . Electro fit see . We note2) double graphene-FET as a function of the applied voltage between the two graphene layers. The different curves correspond to different voltages applied to the HRS back gate. Note that the partial transparency of graphene and AlN to the electromagnetic cavity fields enables the measurements of the parallel resistance of the two graphene layers. In order to understand the variation of the sheet resistance with the voltage applied to the Si back-gate and the measured asymmetry of the curves, simulations based on the Boltzmann equation were performed. τ+ = τ− = 3.6 fs and n0 = 10.8 × 10111/cm2 for the top graphene sheet and τ+ = 10.9 fs, τ−= 5.8 fs and n0 = −4.0 × 10111/cm2 for the bottom graphene sheet. Areal capacitances of 0.3 mF/m2 and 0.17 mF/m2 were used for the top capacitor (50 nm AlN) and the bottom capacitor (nSiO2), respectively.The simulations reproduce some of the key features of the experimental result, including the shift of the Dirac point of the bottom graphene layer for changes of the back gate voltage and the strong asymmetry of the conductivity curves, which arises from the presence of the top graphene layer whose Dirac point is at a large negative bias voltage which lies outside the range of leakage-free voltages between the two graphene layers. It is important to note that the induced current in the bottom graphene layer is slightly higher than the current in the top graphene layer due to partial reflection of the magnetic field from the bottom graphene layer. The results demonstrate a switching ratio as high as 4.5 for the parallel resistance due to alteration of the gate voltage between the two graphene layers, i.e. without any change of the back-gate voltage and without any metallic top gate. As such, this structure represents an all-graphene voltage-controlled attenuator for microwave frequencies. The attenuation of the microwave signal results from an Ohmic current through the graphene layers which is induced by the electromagnetic wave. Any variation of the sheet-resistance changes the amount of partial reflection and transmission. As a result, the on-off ratio of the sheet resistance is directly proportional to the on-off ratio of the attenuator. Moreover, we find that the minimum conductivity in the theoretical results increases upon increasing the back gate voltage, while its value is almost constant in the experimental measurement. We attribute this discrepancy to the assumption of a constant scattering time in the computed conductivity, which cannot reproduce the experimentally observed saturation of the conductivity at large gate voltages. Furthermore, the spacing between the resistance curves in In conclusion, we fabricated an all graphene field-effect device that operates at microwave frequencies and established a microwave cavity technique as a powerful tool for the characterization of such a field effect device. A structure composed of a 50 nm thick aluminum nitride layer between two graphene layers exhibits variation of the stack sheet resistance with a pronounced Dirac point and an on-off ratio of 4.5 by applying a DC voltage between the two graphene layers only. Use of native oxide gate dielectrics in combination with annealing at moderate temperatures results in reproducible transfer curves with charge neutrality points around zero volts, which are in very good agreement with the expectation from our theoretical model. This structure possesses a great potential for microwave-to-THz applications, such as in voltage-controlled attenuators and modulators for high data-rate wireless communication. Moreover, this technology may be used to build integrated electrodes for piezoelectric devices enabling excitation of mechanical oscillations in AlN based thin film bulk acoustic devices (FBAR) and thin film microbridges at RF and microwave frequencies. It is expected that the outstanding mechanical performance of AlN is not negatively affected by the presence of ultrathin graphene layers, which possess outstanding mechanical properties themselves. The possible combination of electric biosensing by monitoring conductivity changes of functionalized graphene and mass detection by an AlN microbridge at the same time within one device can pave the way to a new generation of silicon-integrated dual-mode biosensors with two orthogonal sensor channels.via a PMMA method, which has been described in our previous work2/CH4 gas flow ratio of 4. The growth temperature is set to 1050 °C for the duration of 30 minutes. In fabrication of the DG-FET structures, graphene films were grown on a 7 mm by 5 mm copper substrates to facilitate the transfer process.Large area single layer graphene was fabricated using a vertical CVD technique and transferred −4 at 10 GHz) and a high relative permittivity (29) is placed inside a copper cavity. The puck is fixed on top of a quartz holder approximately 1 mm below a circular aperture of 5 mm diameter, which is located at the center of the top wall of the cavity and allows the resonant field to interact with the graphene sample ) with a diameter of 7 mm, a very low loss-tangent at microwave frequencies  magnetron sputtering with thickness of 50 nm. The RF sputtering process was conducted in a Mantis combined sputtering/thermal evaporation system with a base pressure of 5 × 10The schematic in How to cite this article: Adabi, M. et al. Microwave Study of Field-Effect Devices Based on Graphene/Aluminum Nitride/Graphene Structures. Sci. Rep.7, 44202; doi: 10.1038/srep44202 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


This barrier can be mitigated by considering graphene-MoS2 heterostructure, as graphene possesses strong mechanical properties. We have developed efficient closed-form expressions for the equivalent elastic properties of such multi-layer hexagonal nano-hetrostructures. Based on these physics-based analytical formulae, mechanical properties are investigated for different heterostructures such as graphene-MoS2, graphene-hBN, graphene-stanene and stanene-MoS2. The proposed formulae will enable efficient characterization of mechanical properties in developing a wide range of application-specific nano-heterostructures.Two-dimensional and quasi-two-dimensional materials are important nanostructures because of their exciting electronic, optical, thermal, chemical and mechanical properties. However, a single-layer nanomaterial may not possess a particular property adequately, or multiple desired properties simultaneously. Recently a new trend has emerged to develop nano-heterostructures by assembling multiple monolayers of different nanostructures to achieve various tunable desired properties simultaneously. For example, transition metal dichalcogenides such as MoS The interest in such hexagonal two-dimensional materials has expanded over the last decade from hBN, BCN, graphene oxides to Chalcogenides like MoS2, MoSe2 and other forms of two-dimensional materials like stanene, silicene, sermanene, phosphorene, borophene etc.6. Among these two-dimensional materials, hexagonal honeycomb-like nano-structure is a prevalent structural form3. Four different classes of single-layer materials with hexagonal nano-structure exist from a geometrical point of view, as shown in Fig. 7 consists of a single type of atom (carbon) to form a honeycomb-like hexagonal lattice structure in a single plane, while there is a different class of materials that possess hexagonal monoplanar nanostructure with different constituent atoms such as hBN8, BCN9 etc. Unlike these monoplanar hexagonal nanostructures, there are plenty of other materials that have the atoms placed in multiple planes to form a hexagonal top view. Such multiplanar hexagonal nanostructures may be consisted of either a single type of atom , or different atoms . Even though these two-dimensional materials show promising electronic, optical, thermal, chemical and mechanical characteristics for exciting future applications, a single nanomaterial may not possess a particular property adequately, or multiple desired properties simultaneously. To mitigate this lacuna, recently a new trend has emerged to develop nano-heterostructures by assembling multiple monolayers of different nanostructures for achieving various tunable desired properties simultaneously.A generalized analytical approach is presented to derive closed-form formulae for the effective in-plane elastic moduli of hexagonal multiplanar nano-structures and heterostructures. Hexagonal nano-structural forms are common in various two-dimensional and quasi-two-dimensional materials. The fascinating properties of graphene2 show exciting electronic and piezoelectric properties, but their low in-plane mechanical strength is a constraint for any practical application. In contrast, graphene possesses strong in-plane mechanical properties. Moreover, graphene is extremely soft in the out-of-plane direction with a very low bending modulus, whereas the bending modulus of MoS2 is comparatively much higher, depending on their respective single-layer thickness19. Having noticed that graphene and MoS2 possess such complementary physical properties, it is a quite rational attempt to combine these two materials in the form of a graphene-MoS2 heterostructure, which could exhibit the desired level of electronic properties and in-plane as well as out-of-plane strengths. Besides intense research on different two dimensional hexagonal nano-structural forms, recently the development of novel application-specific heterostructures has started receiving considerable attention from the scientific community due to the tremendous prospect of combining different single layer materials in intelligent and intuitive ways to achieve several such desired physical and chemical properties26.Although the single-layer of two-dimensional materials have hexagonal lattice nano-structure (top-view) in common, their out-of-plane lattice characteristics are quite different, as discussed in the preceding paragraph. Subsequently, these materials exhibit significantly different mechanical and electronic properties. For example, transition metal dichalcogenides such as MoS27, heterostructure containing both mono-planar and multi-planar nanostructures  and heterostructure containing only multi-planar nanostructures . Recently different forms of multi-layer heterostructures have started receiving immense attention from the scientific community for showing interesting chemical, thermal, optical, electronic and transport properties30. Even though the heterostructures show various exciting physical and chemical characteristics, effective mechanical properties such as Young’s moduli and Poisson’s ratios are of utmost importance for accessing the viability in application of such nano-heterostructures in various nanoelectromechanical systems. The research in this field is still in a very nascent stage and investigations on elastic properties of these built-up structural forms are very scarce to find in literature21.The hexagonal nano-heterostructures can be broadly classified into three categories based on structural configuration, as shown in Fig. 2 etc.33. This approach of mechanical property characterization for single-layer nanostructures is computationally very efficient, yet accurate and physically insightful. However, the analytical models concerning two-dimesional hexagonal nano-structures developed so far are limited to single-layer structural forms; development of efficient analytical approaches has not been attempted yet for nano-heterostructures. Considering the future prospect of research in this field, it is essential to develop computationally efficient closed-form formulae for the elastic moduli of nano-hetrostructures that can serve as a ready reference for the researchers without the need of conducting expensive and time consuming molecular dynamics simulations or laboratory experiments. This will accelerate the process of novel material development based on the application-specific need of achieving multiple tunable properties simultaneously to a desirable extent.The common practises to investigate these nanostructures are first principle studies/ab-initio and molecular dynamics, which can reproduce the results of experimental analysis with the cost of computationally expensive and time consuming supercomputing facilities. Moreover, availability of interatomic potentials can be a practical barrier in carrying out molecular dynamics simulation for nano-heterostructures, which are consisted of multiple materials. The accuracy of molecular dynamics simulation depends on the interatomic potentials and the situation can become worse in case of nano-heterostructures due to the possibility of having lesser accuracy for built-up structural forms. Molecular mechanics based analytical closed form formulae are presented by many researchers for materials having hexagonal nano-structures in a single layer such as graphene, hBN, stanene, MoS2, graphene-stanene and stanene-MoS2), belonging to all the three classes as discussed in the preceding paragraphs, are investigated considering various stacking sequences. The analytical formulae for elastic moduli of heterostructures are applicable to any number of different constituent single-layer materials with multi-planar or mono-planar hexagonal nanostructures.In this article, we aim to address the strong rationale for developing a generalized compact analytical model leading to closed-form and high fidelity expressions for characterizing the mechanical properties of a wide range of hexagonal nano-heterostructures. Elastic properties of four different heterostructures  and thereby presenting new results for various stacking sequence of different nano-heterostructures belonging to the three different classes as described in the preceding section .In this section, the closed-form analytical expressions of elastic moduli for generalized multiplaner hexagonal nano-heterostructures are presented. The molecular mechanics based approach for obtaining the equivalent elastic properties of atomic bonds is well-documented in scientific literature34. Total strain energy (E) is expressed as the sum of energy contributions from bending of bonds (Eb), bond stretching (Es), torsion of bonds (Et) and energies associated with non-bonded terms (Enb) such as the van der Waals attraction, the core repulsions and the coulombic energy , the strain energy caused by bending consists of two components, in-plane component (EbI) and out-of-plane component (EbO). The out-of-plane component becomes zero for monoplanar nanostructures such as graphane and hBN. Thus the total interatomic potential energy (E) can be expressed asl, Δθ and Δα denote the change in bond length, in-plane and out-of-plane angle respectively. The quantities kr and kθ represents the force constants for bond stretching and bending respectively. The molecular mechanics parameters (kr and kθ) and structural mechanics parameters (EA and EI) of a uniform circular beam with cross-sectional area A, length l, Young’s modulus E, and second moment of area I, are related as: 35. Based on this relationship, the closed form expressions for the effective elastic moduli of multilayer hexagonal nano-heterostructures are derived following a multi-stage idealization scheme using force equilibrium and deformation compatibility conditions. The closed form expressions for the two in-plane Young’s moduli of nano-heterostructures are derived asHowever, among all the energy components, effect of bending and stretching are predominant in case of small deformationi in the above expressions indicates the molecular mechanics and geometrical properties , bond length (l), in-plane bond angle (ψ) and out-of-plane angle (α), which are well-documented in the molecular mechanics literature. The analytical formulae are valid for small deformation of the structure (i.e. the linear region of stress-strain curve). The effect of inter-layer stiffness contribution due Lennard-Jones potentials are found to be negligible for the in-plane elastic moduli considered in this study and therefore, neglected in the analytical derivation .Here 2, graphene-hBN, graphene-stanene and stanene-MoS2 (belonging to the three categories as depicted in the introduction section). Though all these four heterostructures have received attention from the concerned scientific community for different physical and chemical properties recently, only the graphene-MoS2 heterostructure has been investigated using molecular dynamics simulation for the Young’s modulus among all other elastic moduli21. Thus we have validated the proposed analytical formulae for Young’s moduli of graphene-MoS2 heterostructure with available results from literature. New results are presented for the two in-plane Poisson’s ratios of graphene-MoS2 heterostructure using the analytical formulae, which are validated by carrying out separate molecular dynamics simulations. Having the developed analytical formulae validated for the two Young’s moduli and Poisson’s ratios, new results are provided for the other three considered heterostructures accounting for the effect of stacking sequence. Moreover, it can be noted that for single layer of the heterostructure (i.e. for n = 1), the proposed analytical formulae can be used to predict the effective elastic moduli of monoplanar (i.e. α = 0) and multiplanar (i.e. α ≠ 0) materials. The analytical predictions for the Young’s moduli and Poisson’s ratios of such single-layer materials are further validated with reference results from literature, as available.Results are presented for the effective elastic moduli of hexagonal multi-layer nano-heterostructures based on the formulae proposed in the preceding section. As investigations on nano-heterostructures is a new and emerging field of research, the results available for the elastic moduli of different forms of heterostructures is very scarce in scientific literature. We have considered four different nano-heterostructures to present the results: graphene-MoSn = 1) belonging to all the four classes as described in the preceding section , the in-plane Young’s moduli obtained using the proposed analytical formulae are in good agreement with reported values in literature for graphene, hBN, stanene and MoS2. These observations corroborate the validity of the proposed analytical formulae in case of a single-layer. However, in case of Poisson’s ratios, the reported values in scientific literature for graphene and hBN show wide range of variability, while the reference values of Poisson’s ratios for stanene and MoS2 are very scarce in available literature. The results predicted by the proposed formulae agree well with most of the reported values for Poisson’s ratios.As shown in Tables 2. The results are compared with the numerical values reported in scientific literature. It can be noted that the difference between E1 and E2 is not recognized in most of the previous investigations and the results presented as E1 = E2. The Young’s moduli E1 and E2 are found to be different for multiplanar single-layer nanostructural forms (such as stanene and MoS2). A similar trend has been reported before by Li41 for MoS2. Thus the effective Young’s moduli of the heterostructures with at least one layer of multiplanar structural form is expected to exhibit different E1 and E2 values. In Table E1 = E2, while for single and bi-layer of MoS2E1 ≠ E2. In case of heterostructures consisting of both graphene and MoS2 the value of E2 is observed to be higher than E1. However, the numerical values of E1 for different stacking sequences are found to be in good agreement with the values of Young’s modulus reported in literature (presumably obtained for direction-1) corroborating the validity of the developed closed-form expressions. We have carried out separate molecular dynamics simulations for graphene – MoS2 heterostructures to validate the analytical predictions of Poisson’s ratios, as Poisson’s ratios have not been reported for graphene–MoS2 heterostructures in literature. The analytical predictions of Poisson’s ratios reported in Table 2 heterostructure, the two in-plane Poisson’s ratios (ν12 and ν21) are found to have different values when at least one multi-planar structural form is present in the heterostructure. Thus having the analytical formulae for all the elastic moduli validated, we have provided new results for three other nano-heterostructures in the following paragraphs based on Equations Table E1 = E2 and ν12 = ν21) in case of graphene-hBN heterostructure as these are consisted of only mono-planar structural forms. Table E1 ≠ E2 and ν12 ≠ ν21) when at least one of the constituent layers of the heterostructure is stanene. Table 2 heterostructure considering different stacking sequences. As stanene and MoS2 both have multi-planar structural form, the two Young’s moduli and two in-plane Poisson’s ratios show considerably different values (i.e. E1 ≠ E2 and ν12 ≠ ν21). The results of different elastic moduli corresponding to various stacking sequences are noticed to have an intermediate value between the respective elastic modulus for single layer of the constituent materials, as expected on a logical basis.Table 2 and graphene-stanene heterostructures with little difference in the actual numerical values. The variation of elastic moduli for graphene-hBN heterostructure are presented for E1 and ν12 as the numerical values are exactly same for the two Young’s moduli and two in-plane Poisson’s ratios, respectively. The plots furnished in this section can readily provide an idea about the trend of variation of elastic moduli with stacking sequence of multi-layer nano-heterostructures in a comprehensive manner; exact values of the elastic moduli corresponding to various stacking sequences can be easily obtained using the proposed computationally efficient closed-form formulae.The physics based analytical formulae for nano-heterostructures presented in this article are capable of obtaining the elastic moduli corresponding to any stacking sequence of the constituent layer of nano-materials. However, from the expressions it can be discerned that the numerical values of elastic moduli actually depend on the number of layers of different constituent materials rather than their exact stacking sequences. From a mechanics view-point, this is because of the fact that the in-plane properties are not a function of the distance of individual constituent layers from the neutral plane of the entire heterostructure. Figures α ≠ 0) or monoplanar (i.e. α = 0) configurations. It is interesting to notice that the generalized analytical formulae developed for the Young’s moduli of heterostructures can be reduced to the closed-form expressions provided by Shokrieh and Rafiee31 for graphene considering single-layer (i.e. n = 1), α = 0 and ψ = 30°.We have presented computationally efficient analytical closed-form expressions for the effective elastic moduli of multi-layer nano-heterostructures, wherein individual layers may have multiplanar (i.e. E1 = E2 and ν12 = ν21). However, for single-layer materials with multiplanar nanostructure, the elastic modulus for direction-2 is more than that of direction-1, even though the difference is not significant. Similar observation is found to be reported in literature41. For single-layer of materials, the formulae of elastic moduli deduced from Equations n = 1, perfectly obeys the Reciprocal theorem (i.e. E1ν21 = E2ν12)42. In case of nano-heterostructures, the Young’s moduli and Poisson’s ratios possess different values if at least any one of the layers have a material with multiplanar hexagonal nano-structure (i.e. E1 ≠ E2 and ν12 ≠ ν21). An advantage of the proposed bottom-up approach of considering layer-wise equivalent material property is that it allows us to neglect the effect of lattice mismatch in evaluating the effective elastic moduli for multi-layer heterostructures consisting of different materials. In the derivation for effective elastic moduli of such heterostructues, the deformation compatibility conditions of the adjacent layers are satisfied. This is expected to give rise to some strain energy locally at the interfaces, which is noted in previous studies21. From the derived expressions it can be discerned that the numerical values of elastic moduli actually depend on the number of layers of different constituent materials rather than their stacking sequences. In case of multi-layer nanostructures constituted of the layers of same material , it can be expected from Equations t.It can be noted from the presented results that the single-layer materials having regular monoplanar hexagonal nano-structures (such as graphene and hBN) have equal value of elastic modulus in two perpendicular directions . Even though the results are presented in this article considering only two different constituent materials in a single heterostructure , the proposed formulae can be used for heterostructures containing any number of different materials26. The physics-based analytical formulae are capable of providing a comprehensive in-depth insight on the behaviour of such multilayer heterostructures. Noteworthy feature of the present analytical approach is the computational efficiency and cost-effectiveness compared to conducting nano-scale experiments or molecular dynamics simulations. Thus, besides deterministic analysis of elastic moduli, as presented in this paper, the efficient closed-form formulae could be an attractive option for carrying out uncertainty analysis50 based on a Monte Carlo simulation based approach . The bottom-up approach based concept to develop expressions for hexagonal nano-heterostructures can be extended to other forms of nanostrcutures in future.Effective mechanical properties such as Young’s moduli and Poisson’s ratios are of utmost importance to access the viability for the use of nano-heterostructures in various nanoelectromechanical applications. The major contribution of this work is to develop the generalized closed-form analytical formulae for multi-layer nano-heterostructures. Thses formulae are also applicable to single-layer of materials with monoplanar as well as multiplanar nanostructures. Thus the developed analytical formulae for elastic moduli can be used as an efficient reference for the entire spectrum of materials with lattice-like structural form and the heterostructures obtained by combining multiple layers of different such materials with any stacking sequence. Such generalization in the derived formulae, with the advantage of being computationally efficient and easy to implement, opens up a tremendous potential scope in the field of novel application-specific heterostructure development. We have validated the proposed expressions considering multiple stacking sequences with existing results of literature and separate molecular dynamics simulations for the Young’s moduli and Poisson’s ratios of graphene-MoS23, as the interest in graphene did few years ago. The attentiveness is expected to expand further in coming years with the possibility to consider different tunable nanoelectromechanical properties of the prospective combination (single and multi-layer structures with different stacking sequences) of so many two dimensional materials. This, in turn introduces the possibility of opening a new dimension of application-specific material development that is analogous to metamaterials52 in nano-scale. The present article can contribute significantly in this exciting endeavour.After several years of intensive investigation, research concerning graphene has logically reached to a rather mature stage. Thus investigation of other two dimensional and quasi-two dimensional materials have started receiving the due attention recently. However, the possibility of combining single layers of different two dimensional materials (heterostructures) has expanded this field of research dramatically; well beyond the scope of considering a simple single layer graphene or other 2D material. The interest in such heterostructures is growing very rapidly with the advancement of synthesizing such materials in laboratory2 heterostructures by carrying out separate molecular dynamics simulations and available results from literature. New results are presented for graphene–hBN, graphene–stanene and stanene–MoS2 heterostructures using the developed analytical framework. As the proposed closed-form formulae are general in nature and applicable to wide range of materials and their combinations with hexagonal nano-structures, the present article can serve as a ready reference for characterizing the material properties in future nano-materials development.In summary, we have developed computationally efficient physics-based analytical expressions for predicting the equivalent elastic moduli of multi-layer nano-heterostructures. The proposed expressions are validated for graphene–MoSkr and kθ), bond length and bond angles for different materials, which are used to obtain numerical results based on Equations A concise description of the basic philosophy behind the developed analytical framework is explained in this section . A multi-stage bottom-up idealization scheme is adopted for deriving the closed-form expressions, as depicted in Fig. kr and kθ can be obtained from literature using AMBER force filed53 as kr = 938 kcal mol−1 nm−2 = 6.52 × 10−7 Nnm−1 and kθ = 126 kcal mol−1 rad−2 = 8.76 × 10−10 Nnm rad−2. The out-of-plane angle for graphene is α = 0 and the bond angle is θ = 120° (i.e. ψ = 30°), while bond length and thickness of single-layer graphene can be obtained from literature as 0.142 nm and 0.34 nm respectively7. In case of hBN, the molecular mechanics parameters kr and kθ can be obtained from literature using DREIDING force model54 as kr = 4.865 × 10−7 Nnm−1 and kθ = 6.952 × 10−10 Nnm rad−2 55. The out-of-plane angle for hBN is α = 0 and the bond angle is θ = 120° (i.e. ψ = 30°), while bond length and thickness of single-layer hBN can be obtained from literature as 0.145 nm and 0.098 nm respectively8. In case of stanene, the molecular mechanics parameters kr and kθ can be obtained from literature as kr = 0.85 × 10−7 Nnm−1 and kθ = 1.121 × 10−9 Nnm rad−2 57. The out-of-plane angle for stanene is α = 17.5° and the bond angle is θ = 109° (i.e. ψ = 35.5°), while bond length and thickness of single layer stanene can be obtained from literature as 0.283 nm and 0.172 nm respectively59. In case of MoS2, the molecular mechanics parameters kr and kθ can be obtained from literature as kr = 1.646 × 10−7 Nnm−1 and kθ = 1.677 × 10−9 Nnm rad−2, while the out-of-plane angle, bond angle, bond length and thickness of single layer MoS2 are α = 48.15°, θ = 82.92° (i.e. ψ = 48.54°), 0.242 nm and 0.6033 nm respectively62.The molecular mechanics parameters and geometric properties of the bonds are well-documented in scientific literature. In case of graphene, the molecular mechanics parameters 64 for calculating the Poisson’s ratios of graphene–MoS2 bilayers and heterostructures through molecular dynamics simulation. The interatomic potential used for carbon-carbon, molybdenum-sulfur interactions are the second-generation Brenner interatomic potential66. We have stabilized the heterostructures following the same method as described in literature21. The MoS2 and graphene layers of the heterostructures are coupled by van der Waals interactions, as described by the Lennard-Jones potential. The adopted cut-off is 10.0A° for M/G/M and 5.0A° for G/M/G heterostructures. These cut-off values are determined by stabilizing and minimizing the M/G/M and G/M/G heterostructures21.We have followed a similar method as reported in literatureSupplementary Information


Anopheles funestus is threatening recent gains in the fight against malaria. Here, we established the role of an over-expressed P450, A. funestus CYP6AA1 in insecticides resistance. Transcription profiling of CYP6AA1 across Africa using microarray and quantitative reverse transcription polymerase chain reaction (qRT-PCR) revealed that it is significantly more over-expressed in southern African populations compared to West (Benin) and East African (Uganda). Heterologous expression in Escherichia coli coupled with metabolism assays demonstrated that CYP6AA1 metabolises type I (permethrin) and type II (deltamethrin) pyrethroids, as well as bendiocarb (a carbamate). Transgenic Drosophila melanogaster flies over-expressing CYP6AA1 were significantly more resistant to pyrethroid insecticides, permethrin and deltamethrin compared with control flies not expressing the gene, validating the role of this gene in pyrethroid resistance. In silico modelling and docking simulations predicted the intermolecular receptor-ligand interactions which allow this P450 to metabolise the pyrethroids and bendiocarb. Validation of CYP6AA1 as a pyrethroid resistance gene makes it possible to monitor the spread of resistance in the field where this P450 is over-expressed. Its potential cross-resistance role makes it necessary to monitor the gene closely to inform control programs on molecular basis of multiple resistance in the field.Resistance to pyrethroids (the ingredients in bed net insecticides) in the major malaria vector  Anopheles gambiae Complex and the Anopheles funestus Group are increasingly common  compared with the negative control, –NADPH) and a t-test used for statistical analysis. Metabolism assays were conducted with permethrin (representative type I) and deltamethrin (type II) pyrethroids, DDT (an organochlorine), the carbamates-bendiocarb and propoxur, as well as malathion (an organophosphate). Protocols for incubations and high-performance liquid chromatography (HPLC) analyses for the above insecticides followed procedures previously published ,34. Brieications . All reamK and maxV were established from the plot of substrate concentrations against the initial velocities and fitting of the data to the Michaelis-Menten module using the least squares non-linear regression in the GraphPad Prism 6.03 Software .Steady state kinetic parameters were determined with permethrin and deltamethrin by measuring the rate of reaction for 20 min while varying the substrate concentrations (3–24 µM) in presence of 22.5 pmol recombinant CYP6AA1. Reactions were performed in triplicates both for +NADPH and –NADPH for each concentration. CYP6AA1 alone can confer resistance to the pyrethroid insecticides, transgenic D. melanogaster flies expressing this gene were generated using the GAL4/UAS system. The preparation of the transgenic flies followed the protocols described previously  by Genetic Services  to generate the transgenic line UAS-CYP6AA1. Ubiquitous expression of the transgene in adult F1 progeny  was obtained after crossing virgin females from the driver strain Act5C-GAL4 ), with polar metabolites eluting at the beginning of the chromatogram of incubation with NADPH+. These kind of putative metabolites have been described from in vitro assays of recombinant A. funestus CYP6Z1 with bendiocarb [HPLC analyses established that CYP6AA1 metabolises permethrin and deltamethrin respectively, with high depletion of 83.5% ± 4.6 (cubation A. No actndiocarb . p = 0.2) with no polar metabolites eluting at the beginning of chromatogram. Also, less than 5% of DDT (even following addition of solubilising agent sodium cholate) and malathion were depleted by the recombinant CYP6AA1, indicating lack of enzymatic activity toward the organochlorine and organophosphate insecticides. This is in line with the DDT and malathion susceptibility observed in southern Africa, Mozambique, for example [In contrast, very low activity was observed with propoxur  of 11.99 min−1 ± 2.17 and 15.65 min−1 ± 2.642, respectively for permethrin and deltamethrin (Km) for permethrin and deltamethrin were also comparable, 33.62 µM ± 9.180 and 30.01 µM ± 7.915, respectively. Though these Km values are within the ranges described for binding and metabolism by insect cDNA-expressed P450s [A. funestus CYP6P6P9a and CYP6P9b [Km values obtained from recombinant A. minimus CYP6AA3 and CYP6P7 with pyrethroids [−1 µM−1 ± 0.12 and 0.51 min−1 µM−1 ± 0.16 respectively, for permethrin and deltamethrin. Metabolism of pyrethroids follows the canonical Michaelis-Menten pattern with high maximal catalytic rate   at all the eight different exposure times compared with the control groups  A. p < 0.05) at 12 h and 36 h of exposure. These results confirmed that over-expression of CYP6AA1 alone is sufficient to confer resistance to permethrin and deltamethrin. A similar pattern was observed using deltamethrin B with a CYP6AA1 (1518 bp) from different regions of Africa revealed relative homogeneity within each geographic region, with haplotypes from each country forming a cluster in the maximum likelihood phylogenetic tree , from 10 haplotypes out of 22 sequences from four different countries). The very low Hd in the sequences, especially from Malawi and Ghana  suggests a directional selection (selective pressure) acting on CYP6AA1 in populations from these two regions. A neutrality test of all sequences revealed Li and Fu’s D* as positive but not statistically significant. When all sequences were analysed according to country of origin, statistics was only positive with FANG sequences, suggesting rare polymorphism and lack of background selection. Haplotype diversity is high , West (Ghana) and East Africa (Uganda and FANG) populations. High genetic differentiation estimates were observed between Ghana (0.82 < KST < 1), East African (0.53 < KST < 0.68) and the other two populations, whereas the two southern African populations exhibited a null level of genetic differentiation (KST = 0.0)   and clusT = 0.0) C.A. funestus CYP6AA1 to other closely related sequences reveals that it is 89% identical to its ortholog A. minimus CYP6AA3 (GenBank: AAN05727.1), 87% identical to A. gambiae CYP6AA1 (AGAP002862) and 57% identical to Culex pipiens pallens CYP6AA9 (GenBank: AKA45037.1) (Culex CYP6AA9 (515 amino acids) all the other three P450s are composed of 505 amino acids.Comparison of 45037.1) . Apart fAnopheles sequences , and SRS-6. Sequence-to-sequence mapping reveals that the WxxxR motif, the signatory oxygen-binding pocket (AGFETS)/proton transfer groove, the ExxR motif which stabilises the heme structural core, the cysteine pocket/heme-binding region (PFxxGxxxCxG), which forms the fifth axial ligand to the heme iron ,51 were equences . Major sA. funestus CYP6AA1 able to metabolise pyrethroid insecticides, a docking simulation was carried out using the homology models of A. funestus CYP6AA1 and A. gambiae CYP6AA1 with insecticides from three of the four classes used in public health control of malaria vectors. The binding parameters for each insecticide are provided in A. funestus CYP6AA1 model, deltamethrin exhibited the highest affinity (lowest binding energy) with high contribution from hydrophobic interactions and intermolecular hydrogen bonding. The insecticide docked into the active site of CYP6AA1 with the 4′ spot of phenoxy ring oriented above the heme at a distance of 3.6Å from heme iron, suggesting ring hydroxylation (A. gambiae [To understand the underlying mechanism which makes xylation B. 4′-hyd gambiae  and in o gambiae . A. gambiae CYP6AA1 model consistent with the pose of deltamethrin in the active site of this P450 (2 position of benzyl ring positioned 6.4Å from heme iron)  . Very hiectively . The traA. funestus CYP6AA1. The binding energy of −7.8 kcal/mol indicates moderate affinity to CYP6AA1, compared with A. gambiae CYP6AA1 with lower affinity (higher binding energy) of −7.2 kcal/mol. This is consistent with the moderate scores obtained from hydrophobic interactions, which is less than half the values obtained from the pyrethroids. The carbamate insecticide docked productively with the carbamic ester group above the heme catalytic centre, at 3.7Å  from heme catalytic centre  with permethrin, deltamethrin, and bendiocarb were compared from docking simulations of models from A. funestus and A. gambiae, respectively. For both permethrin and deltamethrin, Phe309 was predicted to enhance catalysis through π-stacking with the phenoxy ring in the case of permethrin, and with both phenoxy- and benzyl rings in the case of deltamethrin, in A. funestus model (307Phe308Phe309) from the substrate recognition site-4 (SRS-4) within the αI helix (109 (SRS-1) were predicted to enhance catalysis via π-stacking with the aromatic rings of both permethrin and deltamethrin in A. gambiae CYP6AA1 active site  was predicted to be involved in hydrophobic interactions with aromatic rings of deltamethrin and bendiocarb in the A. funestus CYP6AA1 models  to the ester oxygen of the carbamate moiety (A. gambiae: (i) donated by alcohol side chain of Tyr109 to benzodioxol moiety; (ii) donated by His120 to the amide nitrogen (-NH) of the methylcarbamate moiety. No hydrogen bonding between permethrin and redicted . In contredicted , both doe moiety . This co106 of A. gambiae CYP6AA1 with deltamethrin , and even down-regulated in East Africa (Uganda). Overall, the expression pattern of CYP6AA1 resembles that of the duplicated P450 genes CYP6P9a and CYP6P9b which have so far been found to be highly over-expressed in southern Africa and only moderately over-expressed in West (Benin) and East Africa (Uganda and Kenya) [CYP6AA1 is located on the chromosome 2R, together with CYP6P9a and CYP6P9b, on the same cluster of cytochrome P450s spanning the rp1 pyrethroid resistance quantitative trait locus (QTL) [d Kenya) ,53. CYP6us (QTL) . CYP6AA1 exhibited pyrethroid activities comparable to values observed from other P450s implicated in pyrethroid resistance in A. funestus, notably CYP6P9a and CYP6M7 [CYP6Z1 [A. gambiae P450s such as CYP6M2 [A. funestus, A. minimus, and A. gambiae particularly suggests that its detoxification function was retained even after speciation. Indeed, the ortholog from A. minimus was shown to metabolise type I and type II pyrethroids in vitro, with higher activity towards deltamethrin than permethrin [A. funestus and A. gambiae CYP6AA1 suggests possibility of multiple metabolites. Of course, the ortholog A. minimus CYP6AA3 is known to possess a very large substrate access channel [A. funestus CYP6AA1, the ortholog from A. minimus displayed no activity towards malathion and propoxur. The inability of CYP6AA1 to metabolise malathion further explains the field susceptibility to these insecticides in A. funestus [d CYP6M7 , and CYP [CYP6Z1  or others CYP6M2 . The conrmethrin . The dif channel  which ac channel . Like A.funestus ,56.A. funestus with potential metabolic activity towards bendiocarb, though with very low activity compared with A. funestus CYP6Z1 [A. funestus CYP6AA1 is not polymorphic like the A. funestus CYP6M7 characterised by [CYP6P9a and -b has been shown to be the major driver of resistance to pyrethroid insecticides across Africa in the same A. funestus species [It is important to monitor the spread and evolution of this potential cross resistance gene, as it is the second P450 found in s CYP6Z1 . A. funerised by , and is rised by . For exa species . CYP6AA1 exhibited lower activity in vitro and confers lower resistance to permethrin and deltamethrin in transgenic flies compared with the major pyrethroid resistance gene CYP6P9b [Though  CYP6P9b  it is anCYP6AA1 without hydrogen bonding contributions was as observed with A. arabiensis CYP6P4 model [A. funestus CYP6AA1. Metabolism of permethrin is possibly driven through hydrophobic interactions, as further supported by the presence of Trp219 of SRS-2 within its catalytic hotspot. This is the opposite of the situation with deltamethrin, in which hydrogen bonding plays a significant role in energetic contribution. Indeed, differences in the composition of amino acids lining catalytic hotspot are known to dictate presence or absence of intermolecular hydrogen bonding interactions with resulting differences in choice of substrates even among closely related proteins. For example, polar cages composed of hydrophilic residues in the active site of specialist CYP6B1 from Papilio polyxenes contribute to the P450’s hydrogen bonding capabilities which contribute to its ability to metabolise only two furanocoumarins [CYP6B8 from Helicoverpa zea is devoid of polar cages (hydrogen bonding network) in its active site and was established to metabolise diverse allelochemicals, and even a pyrethroid cypermethrin. In silico predictions using modelling and docking simulations have become important tools used to study insecticide resistance genes and their heterogeneities in the metabolism of insecticides ,34,57,58oumarins . In contA. funestus CYP6AA1 to metabolise DDT is consistent with docking predictions, with the organochloride insecticide binding unproductively. Indeed, the productive pose of DDT has been established as C-1 of the trichloromethyl group docking above the heme in A. gambiae CYP6Z1 [The inability of e CYP6Z1 .CYP6AA1 contributes to the cocktail of P450s responsible for pyrethroids resistance in southern African populations of A. funestus. But, unlike the major pyrethroid resistance genes CYP6P9a and –b, this P450 exhibited activity toward indoor residual spray insecticide bendiocarb as well, making it a potential cross-resistance gene which should be monitored closely in the field.Knowledge of insecticide resistance in malaria mosquitoes and the mechanisms driving it in the field is essential for design of appropriate malaria control tool and for management of the resistance. However, this requires a thorough understanding of the molecular basis of the resistance to inform control programs and influence policy decisions. Here, we established that


Control of both the regularity of a material ensemble and nanoscale architecture provides unique opportunities to develop novel thermoelectric applications based on 2D materials. As an example, the authors explore the electronic and thermal properties of functionalized graphene nanoribbons (GNRs) in the single‐sheet and helical architectures using multiscale simulations. The results suggest that appropriate functionalization enables precise tuning of the doping density in a planar donor/acceptor GNR ensemble without the need to introduce an explicit dopant, which is critical to the optimization of power factor. In addition, the self‐interaction between turns of a GNR may induce long‐range disorder along the helical axis, which suppresses the thermal contribution from phonons with long wavelengths, leading to anomalous length independent phonon thermal transport in the quasi‐1D system. With regard to ensemble regularity, both verticalAs an example of exploring material architecture, a recent theoretical study predicted that nanopatterning via chemical functionalization can enhance the optimized ZT of graphene significantly, due to the increased Seebeck coefficient and reduced thermal conductivity.In this work, we explore the impact of the material ensemble and architecture on TE properties, with emphasis on understanding the tunability of doping and thermal conductivity. Among the numerous derivatives of 2D materials, GNRs are chosen as our prototype systems because their electronic structure can be tailored by both geometry and passivation, which makes them especially attractive to provide desirable properties for electronic and spintronic applications.T = 300 K). Our results suggest that appropriate design of the GNRs leads to precise tuning of the electronic structure and modification of vibrational channels via self‐interactions. For example, with the single‐sheet architecture, type‐III electronic interfaces can be established to achieve controllable ground state doping while avoiding effects that can arise from an extrinsic dopant  of the donor and the lowest conduction band (CB) of the acceptor near the Γ point Figure a–d. Whil12 to 2.1 × 1013 cm−2 becomes attainable  has been observed in graphene encapsulated by hexagonal boron nitride,4–105 cm2 V−1 s−1 predicted by deformation potential theory assuming acoustic phonon is the dominant scattering mechanism.In order to tune the potential profile and attain a range of doping densities, two different groups of GNRs with various widths were used, with the end groups of their ligands switched between H, CN, and OH. Consistent with the previous study on graphene nanoroads,6 S cm−1, more than twice that of the optimal σ in graphene. Only the conductivity in the y‐direction  was computed as carrier transport should be restricted to the 1D domain as revealed from the flat bandstructure in the x‐direction , up to a value of 1.8 × 10S) and electrical conductivity  of the GNRs was calculated based on the fluctuation–dissipation theorem and rescaled to account for the long‐wavelength phonon contributions. The simulations were conducted on functionalized graphene sheets composed of a single type of GNR with the H end group, which accounts for the major impact of adjacent GNRs on κp while taking advantage of the efficient force field that is available only for C and H atoms. Consistent with the literature,p of GNRs are reduced by 5–6 times compared to that of pristine graphene (1810 W m−1 K−1)T = 300 K, with which the device ZT is estimated to be 0.55, much higher than the ZT of graphene (0.09) at its optimal doping level, mainly due to the reduction of κp . To further illustrate the importance of accurate control of the doping level, we computed the S2σ and ZT at various doping levels for graphene and the functionalized GNRs  can either be suppressed by the scattering of long‐wavelength phonons which account for a large proportion of κp, or enhanced by the additional transport paths through the side chain ligands in the helix architecture as illustrated by the cartoon in Figure Since the radius of a helical GNR is much larger than the sizes of the relevant atomic orbitals (≈2 Å), as perhaps expected the electronic properties of the helical GNR are similar to those of the corresponding planar GNR, with a slight drop in the computed conductivity due to the scattering induced by surface curvature  of the GNR—a nearly invariant κp, in contrast to the power‐law relation for the planar GNR 2) as defined in Section A in the Supporting Information) of the GNR helix with L = 54 nm is 40% lower than the κp of its planar counterpart . As expected, the reduction decreases with increasing spacing between turns due to the smaller curvature, while κp is still independent of L in most cases , implemented in the Vienna Ab Initio Simulation package (v5.3),p) of the functionalized graphene sheets and the planar/helical GNRs were calculated using equilibrium molecular dynamics (EMD) and nonequilibrium MD (NEMD) simulations, respectively, as implemented in the Large‐scale Atomic/Molecular Massively Parallel Simulator package (v2014).The phonon contributions to the thermal conductivities (κ7 MD steps (1 ns) within the NVE ensemble after a 100 ps equilibration period within the NVT ensemble. All interactions were described by the adaptive intermolecular reactive empirical bond order (AIREBO) potential.All simulations were carried out at 300 K with a time step of 0.05 fs. For the functionalized graphene sheets, the EMD approach based on the fluctuation–dissipation theorem was chosen to reduce finite‐size effects. The Einstein relation was applied,p, which reveals interesting physics for this architecture, could not be obtained by the EMD approach. Therefore, the reverse NEMD approach with the Muller–Plathe algorithmFor the helical GNRs, the EMD method is inappropriate because the direction of the microscopic heat flux is ill‐defined. In addition, the length dependence of κWe note that a number of approximations have been applied in this work in order to make the estimation of the thermoelectric figure of merit possible, which inevitably introduce uncertainties, for example, in the accuracy of the DFT energy levels, the phonon thermal conductivity based on finite‐size classical MD, and the absence of defects and boundary disorder. Thus, rather than interpreted in a quantitative way, this study is more appropriate to be considered as an attempt to qualitatively explore the relative potential benefits of architecture design in 2D materials for thermoelectric applications.SupplementaryClick here for additional data file.


GRN gene cause frontotemporal lobar degeneration (FTLD). The affected neurons display distinctive TAR DNA binding protein 43 (TDP-43) inclusions. How partial loss of PGRN causes TDP-43 neuropathology is poorly understood. TDP-43 inclusions are also found in affected neurons of patients with other neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and Alzheimer's disease. In ALS, TDP-43 inclusions are typically also immunoreactive for fused in sarcoma (FUS). Mutations within TDP-43 or FUS are themselves neuropathogenic in ALS and some cases of FTLD. We used the outgrowth of caudal primary motor neurons (MNs) in zebrafish embryos to investigate the interaction of PGRN with TDP-43 and FUS in vivo. As reported previously, depletion of zebrafish PGRN-A (zfPGRN-A) is associated with truncated primary MNs and impaired motor function. Here we found that depletion of zfPGRN-A results in primary MNs outgrowth stalling at the horizontal myoseptum, a line of demarcation separating the myotome into dorsal and ventral compartments that is where the final destination of primary motor is assigned. Successful axonal outgrowth beyond the horizontal myoseptum depends in part upon formation of acetylcholine receptor clusters and this was found to be disorganized upon depletion of zfPGRN-A. PGRN reversed the effects of zfPGRN-A knockdown, but a related gene, zfPGRN-1, was without effect. Both knockdown of TDP-43 or FUS, as well as expression of humanTDP-43 and FUS mutants results in MN abnormalities that are reversed by co-expression of hPGRN mRNA. Neither TDP-43 nor FUS reversed MN phenotypes caused by the depletion of PGRN. Thus TDP-43 and FUS lie upstream of PGRN in a gene complementation pathway. The ability of PGRN to override TDP-43 and FUS neurotoxicity due to partial loss of function or mutation in the corresponding genes may have therapeutic relevance.Progranulin (PGRN) is a glycoprotein with multiple roles in normal and disease states. Mutations within the  GRN suppresses the development of disease-like phenotypes in murine models of Parkinson’s disease (PD) [GRN, the gene encoding PGRN, results in 50% depletion of PGRN protein levels, and causes frontotemporal dementia (FTD), an early onset form of dementia [GRN have been reported (www.molgen.ua.ac.be/FTDmutations/) the majority of which result in nonsense mediated decay of the GRN mRNA transcript [MAPT), GRN and hexanucleotide repeat expansions in chromosome 9 open reading frame 72 (C9orf72) [GRN mutations are responsible for 5–10% of all cases of FTLD and 13–25% of familial cases [MAPT [GRN or C9orf72 [TARDBP, the gene encoding TDP-43 or FUS can cause either ALS [Progranulin (PGRN) is a secreted glycoprotein that regulates many processes including cell proliferation, survival and motility . In mammase (PD)  and Alzhase (PD) . Haploindementia , 5. To danscript –6. FTD ianscript , 8. AbouC9orf72) . GRN mutes [MAPT , ubiquit C9orf72 , 16. TDP C9orf72 , and in  C9orf72 . In some C9orf72 . FUS-con C9orf72 . Mutatiother ALS , 21, 22 ther ALS  confirmihuntingtin gene. Both TDP-43 and FUS are required for full expression of polyQ toxicity in mammalian striatal neurons [C. elegans model of MN toxicity caused by huntingtin exon-1 polyQ toxicity. These results support a genetic interaction between TDP-43 and PGRN in evoked neuronal cell death. This conclusion is based upon the fact that PGRN blocks polyQ toxicity and TDP-43 blocks the cytoprotective action of PGRN. However, no corresponding interaction between PGRN and FUS was observed.Both TDP-43 and FUS are RNA-binding proteins. Other neurodegenerative conditions including spinal muscular atrophy are also caused by mutations within genes for RNA-binding proteins . Underst neurons . PGRN alC. elegans, can be readily performed in order to address such questions. The zebrafish genome carries four GRN genes [zfPGRN-A is the orthologue of mammalian GRN, and is a long-form PGRN [zfPGRN-B is also a long-form PGRN, whereas zfPGRN-1 and zfPGRN-2 are short-form PGRNs, with one and one half granulin motifs, zfPGRN-A [zfPGRN-A or zfPGRN-B alone or double mutants produced neither truncated CaP MN nor other neuropathological phenotypes suggesting the activation of compensatory mechanisms upon loss of GRN genes in the germline [It is unclear, whether the depletion of PGRN is by itself a direct cause of TDP-43 toxicity, or whether TDP-43 related neurotoxicity is a secondary response to a more generalized cellular dysfunction caused by PGRN depletion. Zebrafish provide a vertebrate model system in which gene complementation studies, similar to those undertaken in simpler invertebrate animals such as RN genes . Of thesorm PGRN , encodinzfPGRN-A  and whenzfPGRN-A . EctopiczfPGRN-A . In contgermline . Similargermline .zfPGRN-1 is able to rescue motor defects resulting from the depletion of long form zfPGRN-A.Here we take advantage of the zebrafish model of motor dysfunction to investigate the genetic interactions of PGRN with TDP-43 and FUS in a vertebrate system. We also determined whether the short form Wild type zebrafish were purchased from Aquatica Tropicals (Florida) and housed on a 14h/10h light/dark cycle at 28.5°C in a laboratory aquarium . The McGill University Institutional Animal Care and Use Committee (IACUC) specifically approved this study and Zebrafish were maintained according to Protocol Number 3935. Fish were fed twice daily. In the late afternoon of the day before embryos are required (approximately 3:00 p.m.), fish were transferred to a breeding tanks with a divider to maintain male and female separately. In the morning, immediately after the light cycle begins, divider removed to facilitate spawning. After spawning has stopped, the eggs that have fallen through the breeding tanks to the outer tank were collected from the bottom of the tank. Embryos to be used for experimental studies were collected and staged by hours post fertilization (hpf)  and stor5'GAGCAGGTGGATTTGTGAACAGCGG3' [Tardbp (GTACATCTCGGCCATCTTTCCTCAG3’5’) and Fus (5’GGCCATAATCATTTGACGCCATGTT3’) and standard control (5’CCTCTTACC TCAGTTACAATTTATA3’) were also used. For Morpholino injection the optimal concentration of gene- specific AMOs were determined initially and 10 ng zfPGRN-A, 10 ng of Fus, 1.5 ng of Tardbp or 10ng of standard control were used.Anti-Sense Morpholino oligonucleotides (AMO)  were obtCAGCGG3' . The seqFUS (WT or mutant R521H) or 25ng/ μl of Tardbp (WT or mutant G348C) or PGRN-1 100 ng/ μl mRNA was used. The optimal concentration of gene specific mRNAs was determined to avoid toxicity related phenotype.For zfPGRN-A mRNA over-expression and rescue experiments a full-length zfPGRN-A/pcDNA3 vector was generated . BrieflyThe znp1 immuno-labelled wild-type embryos were carried out according to previously described protocol . BrieflyTo analyse the rescue effect of PGRN in reversing MN defects due to altered expression of TDP43 or FUS at a functional level, we tested the embryo touch response and avoidance swimming behaviour at 52 hrs post injection , 57. TheCaudal primary motor axons in whole-mounted 48 hpf wild-type embryos labelled with Znp1 monoclonal antibody. Only the trunk CaP MNs (12 pairs) were scored . Embryosin vivo. Images were captured at 5x magnification and the hatched box was further subject to 4-5x zoom. MN axonal length from the specified areas in the spinal cord region were measured using imageJ by tracing the labelled MN axons. The spinal cord length or embryo length were also measured by tracing spinal cord or embryo using imageJ. For each treatment at least three experiments were performed and at least 50 MNs were scored per determination. Values were expressed as mean +/- standard error of the mean.Embryos obtained from the HB9:GFP transgenic fish expressing GFP within the MNs following microinjection of embryos with AMOs or mRNAs or co-injection of both were visualized and photographed Statistical significance among experimental groups was determined by one-way ANOVA followed by Student-Newman-keuls Multiple Comparisons Test  using GraphPad software  or one-way ANOVA followed by Holm-Sidak Multiple Comparisons Test (p < 0.05-*) using Sigma plot software . Error bars represent s.e.m.Zebrafish embryos were lysed using Laemmli buffer and extracts were boiled for 5 minutes. Embryo extracts were resolved using 10% acrylamide gels and transferred to nitrocellulose membrane. SDS/PAGE western blotting analyses were performed as described previously using a p5'-TGATGGAACCACATGCTGTAA-3'/ 5'-CAGATGACCTCTGACCTGCTC-3’ or 5'-CTCAACTCACTCACATCCGC3'/5'-GTTTATAGAGTTAGGGCTC-3’as described in [5'-CCCCATGTCTAAATGCTCTCA-3'/5'-CTTCTCACTCTTCGCCATCAC-3' or 5'-CGTCACCTTCGCAGACGATCAGGTT-3'/5'-GCCCACGATCCATCATTTGCCTACTATT-3' and 5'-CGTCACCTTCGCAGACGATCAGG TT-3'/5'-GCCTAAGCACAATAATATTCATCACCTCTTTTCCAATT-3', respectively as described in [5'- ATGGATGATGAAATTGCCGC-3' and 5'-TGTCATCTTTTCCCTGTTGG-3' primers and zfPGRN-A mRNA observed employing 5'-TGATGGAACCACATGCTGTAA-3'/ 5'-CAGATGACCTCTGACCTGCTC-3’ as described in [Zebrafish embryos were lysed in trizol and the extracted RNA was quantified. cDNA synthesis was carried out according to the manufacturer’s protocol (Fermentas) and used for gene specific PCR reactions (Invitrogen). Species-specific primer sets designed to detect zfPGRN-A mRNA employing ribed in . TDP-43 ribed in . Beta-acribed in . PolymerIn previous work, we demonstrated that administration of the antisense morpholinos against zfPGRN-A but not control morpholinos, knock down zfPGRN-A protein expression. The protein suppression is specific for zfPGRN-A since there was no effect on levels of zf-PGRN-B, and the zfPGRN-A phenotype, namely impaired touch-sensitive motor responses, truncation of MN development and aberrant branching of MNs, is reversed by co-injection of zfPGRN-A mRNA or its human ortholog, hPGRN mRNA, but not by GFP mRNA when co-injected together with the zfPGRN-A morpholinos . Here weWe have further characterized the phenotype of zfPGRN-A knockdown animals. They display reduced overall body length and a corresponding reduction in the length of the spinal cord . This inTardbp, which is the ortholog of the mammalian TARDBP gene and its paralog, Tardbpl (TAR DNA binding protein-like). Tardbpl lacks the glycine-rich RNA binding domain where the ALS and FTLD-U mutations occur [Tardbp can result in alternate splicing of Tardpbl, resulting in restoration of the glycine-rich domain allowing the alternately spliced tardbpl protein product to potentially compensate for the loss of Tardbp [Tardbpl  or FUS(R521H) mutants, resulted in motor neuron abnormality using HB9:GFP embryos as well as WT embryos. We have observed at the morphological level, TDP43  and FUS We have quantified the axonal length among different groups. Reduced expression of either TDP-43 or FUS resulted in reduction of axonal length while co-injection of either TDP-43 AMO or FUS AMO together with PGRN significantly increased axonal length . In addiG348C) or FUS (R521H) mRNA  mRNA  resultedTo determine whether there is complementarity between TDP-43 and FUS with GRN, we investigated whether enhanced expression of TDP-43 or FUS is able to compensate for the depletion of zfPGRN-A. Expression of WT TDP-43 or FUS was unable to reverse the touch evoked swim response or the MN structural abnormalities in the zfPGRN-A knockdown in both HB9:GFP embryos  as well GRN results in a form of FTLD-U that is characterized by the accumulation of cellular inclusions of ubiquitinated-TDP-43 in affected neurons [GRN. There is considerable potential for therapies based upon PGRN. In contrast, PGRN does not suppress the development of disease-like phenotypes in models of ALS that were caused by mutations of SOD1 (Superoxide dismutase-1) [The development of CaP MNs in zebrafish embryos provides a valuable tool with which to investigate the interaction of genes that influence vertebrate MN development and function , 67. In  neurons , 68, 69. neurons  and AD [utase-1) , 70. It TARDBP represent 1–4% of familial ALS cases while mutations in FUS represent 4% of familial ALS cases [TARDBP [Mutations in LS cases . FUS andLS cases , raising [TARDBP . We haveunplugged that interfere with AchR clustering, also disrupt the outgrowth of primary MNs beyond the horizontal myoseptum [Depletion of zfPGRN-A in zebrafish embryos resulted in delayed development, as indicated by shorter body length . In addiyoseptum . AchR clyoseptum . We founGRN genes Click here for additional data file.S2 FigS2B Fig TDP-43 knockdown and partial rescue with over-expression of PGRN mRNA in WT embryos. Average number of Truncated (B1) and Branched (B2) CaP MNs per group. S2C Fig. FUS knockdown and partial rescue with over-expression of PGRN. Average number of Branched (C1) and Truncated (C2) CaP MNs per group.S2A Top panel. PGRN rescues motor axon defects produced by TDP-43 Knockdown in WT embryos. TDP-43 knockdown produced shorter axons that were rescued by co-injection of hPGRN mRNA. Lateral views  of embryos labelled with znp1 mAb at 27 hpf in wild-type embryos, embryos injected with TDP-43 AMO, embryos co-injected with hPGRN mRNA (TDP-43AMO+hPGRN). Embryos co-injected with hPGRN mRNA (TDP-43AMO+hPGRN) partially reversed the truncation phenotype. Observed phenotypes were normal MN development (WT), increase in truncated and branched axons (TDP43 MO) and partial rescue of truncated MNs (TDP43 MO+PGRN). Dashed lines represent the horizontal myoseptum. S2A Fig bottom panel. PGRN rescues motor axon defects induced by FUS knockdown. FUS knockdown produced shorter axons that were rescued by hPGRN mRNA. Lateral views  of embryos labelled with znp1 mAb at 27 hpf in wild type embryos, embryos injected with FUS AMO, embryos co-injected with hPGRN mRNA (FUS AMO +hPGRN). Embryos co-injected with hPGRN mRNA (FUS AMO+hPGRN) partially reversed truncation phenotype. Observed phenotypes were normal MN development (WT), increase in truncated and branched axons (FUS MO) and partial rescue of truncated MNs (FUS MO+PGRN). Dashed lines represent horizontal myoseptum. Images were captured at 20X magnification and the hatched box was further subject to 4-5X Zoom. (TIF)Click here for additional data file.S3 FigS3A Fig PGRN rescues motor defects due to TDP-43 knockdown (A1) but not vice versa (A2) in WT embryos. Touch-evoked swimming is greatly impaired in embryos injected with TDP43 Antisense MO. The motility defect was partially but significantly rescued when the embryos were co-injected with hPGRN together with the TDP-43 antisense MO (A1). S3B Fig PGRN rescues motor defects due to FUS knockdown (B1) but not vice versa (B2) in WT embryos. Touch-evoked swimming is impaired in embryos injected with FUS antisense MO. The motility defect was partially but significantly rescued when the embryos were co-injected with hPGRN mRNA together with the FUS antisense MO (B1). S3C Fig PGRN rescues motor defects due to toxic gain of function induced by TDP-43(G348C) (C1) /FUS (R521H) (C2) in WT embryos. Touch-evoked swimming is greatly impaired in embryos injected with TDP43 (G348C) or FUS (R512H). The motility defect was partially but significantly rescued when the embryos were co-injected with hPGRN mRNA together with TDP43 (G348C) (C1) or FUS (R521H) (C2).(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.