By optimizing the annealing conditions, superior electromagnetic properties with enhanced saturation magnetization (>1.05 T), high resistivity (>1200 μΩ∙cm) and broadened Δf (>3.0 GHz) are obtained. Phase separation with Al2O3 aggregating as inclusions in crystallized Fe(Al) matrix is observed after annealing at 673 K, resulting in a metallic-like resistivity. We provide a feasible way to achieve both high resistivity and large saturation magnetization for the FeAlO films with dominating metallic component and show that the microstructure can be tuned for desirable performance.Metal-insulator granular film is technologically important for microwave applications. It has been challenging to obtain simultaneous high electrical resistivity and large saturation magnetization due to the balance of insulating non-magnetic and metallic magnetic components. FeAlO granular films satisfying both requirements have been prepared by pulsed laser deposition. The as-deposited film exhibits a high resistivity of 3700 μΩ∙cm with a negative temperature coefficient despite that Fe content (0.77) exceeds the percolation threshold. This originates from its unique microstructure containing amorphous Fe nanoparticles embedded in Al Electrons percolate through the interconnected metallic channels, giving rise to a low ρ with positive temperature coefficient (TCR). If the film contains excessive dielectric component (p < 0.4), it falls in the dielectric regime where metallic grains are embedded in the continuous insulator matrix. In this case, electrons transport via thermally activated tunneling and an insulator-like electrical resistivity with negative TCR is expected. The transition regime from metallic to dielectric is obtained wherep < 0.6.Integration of magnetic components, such as inductors, noise suppressors and microwave absorbers on a single chip is critical for the miniaturization of electromagnetic devices and high-speed data transmission12345ρ of the dielectric regime is desirable13Ms and broad Δf are also required for device minimization and wide-band applications. It has been challenging to use conventional methods including co-sputtering and reactive sputtering to achieve simultaneous large Ms, high ρ and broad Δf, which require careful control of the ratio for the metallic and insulating components. The domination of insulator in the dielectric regime significantly decreases the Ms and may even transform the film from ferromagnetic to super paramagnetic15Ms, high ρ and broad Δf. The as-deposited FeAlO film exhibits a granular structure with Fe nanoparticles dispersed in the Al2O3 matrix even with a volume fraction of Fe above the percolation threshold (p = 0.77). The structural transition upon annealing can be tuned for superior electromagnetic properties with enhanced saturation magnetization, high electrical resistivity and broadened applicable frequency range, which is particularly desirable for microwave absorption.For soft magnetic films used at microwave frequencies, high electrical resistivity is essential to achieve minimum eddy current and impedance matching, where the intrinsic high 2 (Z is the atomic number), the bright region and dark region in HADDF image are attributed to Fe and Al2O3 respectively. The corresponding elemental distribution and chemical state of Fe and Al are also confirmed by EDS mapping and XPS measurements  for electron percolation. Note that the interconnected Fe regions are still separated from other Fe nanoparticles by Al2O3. Selected area electron diffraction (SAED) pattern and Fast Fourier transform (FFT) image as the insets in 2O3 network accounts for a volume fraction of approx. 23% by measuring the corresponding area in multiple TEM images. Due to the non-equilibrium growth process and the immiscibility of Fe and Al2O3, the as-deposited FeAlO film contains Al2O3 precipitating along the Fe particle boundaries despite that the volume fraction of the metallic phase (~0.77) is above the percolation threshold (0.6). The exchange length K1 = 48 kJ m−3 denoting the magneto crystalline anisotropy2O3 continuum. The anisotropy field of the FeAlO film is decreased by taking the average of exchange interaction over several nanoparticles, which is beneficial to achieve excellent soft magnetic propertieslnρ ~ 1/Lex is much larger than the granular size and intergranular distance. The negative TCR can be fitted with the equation lnρ ~ 400/(T + 125), as shown in the inset of lnρT1/(T + T2) in disordered materials characterized by long conducting pathways separated by small insulating barrierT1, T2, are related to the tunneling constant x and tunneling distant ω by T1/T2 = πxω/2. Assuming the localization length 1/x to be about 2–3 Å which is the order of the extension of electron wave functions out of metal into vacuum2O3 layer observed by TEM , the resistivity decreases slightly as the temperature increasing to 485 K. The HRTEM image of film subjected to 473 K annealing . At stage ρ experiences faster decreasing as the temperature increases to 578 K. The corresponding HRTEM image of the film annealed at 573 K  with the normalized resistivity ρ/ρ0 falls to nearly 0.1. This is similar to the resistivity of the granular film in the metallic regime.In order to determine thermal stability of the FeAlO film, its electrical resistivity has been measured under Nnnealing  reveals at 573 K  shows pa2O3 are two immiscible phases, the as-deposited granular structure with Fe(Al) nanoparticles embedded in the Al2O3 network possesses larger interfacial energy which is metastable. Post annealing promotes the phase separation of Fe(Al) and Al2O3, reducing the interfacial areas of these two phases via atom diffusion. The crystallized Fe(Al) particles interconnects with each other to form larger size of ~20 nm, while the insulating Al2O3 becomes isolated and aggregates as inclusions in metallic matrix after annealing. Such drastic structural transition leads to a typical metallic conduction behavior of the film in the temperature range of 4 K–300 K . The coexistence of amorphous and crystallized Fe(Al) particle gives rise to double resonance peaks for broadened FWHM Δf up to 3.0 GHz in the permeability spectra  can be obtained after annealing at 473 K and 573 K. The FeAlO films reported here exhibit excellent comprehensive performance, which serve as promising candidates for microwave absorptionThe static and dynamic magnetic properties of the film are also of significant importance for microwave applications. AlO film . Post an50 μΩ∙cm . The Ms  spectra . The ima spectra . Table 1Ms and high ρ. The granular structure can be modulated by post annealing at different temperatures, leading to excellent microwave absorption performance with enhanced Ms (>1.05 T), high ρ (>1200 μΩ∙cm) and broadened Δf (>3.0 GHz). The structural transition upon annealing of the metastable FeAlO granular film reveals a feasible way to understand and tune the performance of granular films.In summary, metastable FeAlO granular film which falls into the metallic regime has been fabricated by PLD as a non-equilibrium method to achieve both large 0.8Al0.2 alloy disk with a diameter of 1 inch was used as the target. A static magnetic field of 250 Oe was applied parallel to the substrate surface during deposition to induce in-plane uniaxial magnetic anisotropy in the film. The deposition chamber was evacuated to a base pressure of 6 × 10−6 torr. The wavelength of the laser is 248 nm. The incident laser energy is 250 mJ with a repetition of 10 Hz and a deposition time of 4.5 h. The FeAlO films have been grown repeatedly and similar granular structure has been obtained which confirms the reproducibility. The as-deposited sample was cut into four pieces, three of which were subjected to vacuum annealing at temperatures ranging from 473 K to 773 K for 2 h. The film was also deposited on a NaCl substrate from which the film can be separated for transmission electron microscopy (TEM) investigation by dissolving the substrate in deionized water.FeAlO films were fabricated on Si (100) single crystal substrates by PLD. A Fe2 atmosphere (DZL-100). The low temperature dependence of resistivity was measured by Physical Property Measurement System (PPMS). The permeability spectra were measured by shorted microstrip transmission-line perturbation method with a vector network analyzer The microstructure of the film was examined by a high resolution TEM (JEM 2100 F) with the film deposited on NaCl substrate. HADDF images and EDS mapping were also taken using the STEM mode. A superconducting quantum interference device (SQUID) was used to measure the static magnetic properties of the films. The room temperature resistivity was measured by four-point method (RTS-8). The chemical state of the film was investigated with XPS . The high temperature dependence of resistivity of the sample was obtained using four-point method under NHow to cite this article: Bai, G. et al. Structural, electron transportation and magnetic behavior transition of metastable FeAlO granular films. Sci. Rep.6, 24410; doi: 10.1038/srep24410 (2016).


The empirical research on the public goods game (PGG) indicates that both institutional rewards and institutional punishment can curb free-riding and that the punishment effect is stronger than the reward effect. Self-regarding models that are based on Nash equilibrium (NE) strategies or evolutionary game dynamics correctly predict which incentives are best at promoting cooperation, but individuals do not play these rational strategies overall. The goal of our study is to investigate the dynamics of human decision making in the repeated PGG with institutional incentives. We consider that an individual’s contribution is affected by four factors, which are self-interest, the behavior of others, the reaction to rewards, and the reaction to punishment. We find that people on average do not react to rewards and punishment, and that self-interest and the behavior of others sufficiently explain the dynamics of human behavior. Further analysis suggests that institutional incentives promote cooperation by affecting the self-regarding preference and that the other-regarding preference seems to be independent of incentive schemes. Because individuals do not change their behavioral patterns even if they were not rewarded or punished, the mere potential to punish defectors and reward cooperators can lead to considerable increases in the level of cooperation. In the public goods game (PGG), the only Nash equilibrium (NE) that is based on monetary considerations is for all players to free ride. However, in PGG experiments, most individuals contributed approximately half of their endowment to the public pool, and this contribution tends to decrease as individuals play the game repeatedly12356891089101112131415Much of the empirical and theoretical research on PGG indicates that rewards and/or punishment can curb free-riding. One class of literature has addressed the so-called peer  incentives, where players can impose fines or bonuses on others at a cost to themselves21819202122232425262728131530313233343536373839404142437182425262728293134353637384041424318242527313435363738411825262829353738151920212215182528353715215The above discussion suggests that it is important to recognize that actual people are not perfectly rational, and models that are based on self-interest may fail to predict the effectiveness of incentives at promoting cooperation. However, it is unclear how people make decisions when confronted with institutional incentives and why subjects have different attitudes on reward and punishment. In this study, we analyze the observed outcomes in PGG experiments with institutional incentivesThe experimental setups and primary experimental results were reported in our previous studyet al.et al.The primary experimental results are shown in t asFollowing Fischbacher and GächterC(t −1) and OC(t −1) denote the contribution of the player and the average contribution of his/her three other group members, respectively, in round t −1. Thus, b1 and b2 measure the effects of the self-regarding preference and the other-regarding preference, respectively (see SI Section 2 for a detailed discussion), where b2 = 0 represents individuals who consider only their self-interests, and b1 = 0 represents individuals who care only about others’ behavior. In contrast, R(t −1) and P(t −1) are the amounts of reward and punishment, respectively, that are received in round t −1, and R(t −1) (or P(t −1)) equals 0 if the player was not rewarded (or punished). Therefore, b3 and b4 describe the reactions of being rewarded or punished, respectively, where a player tends to increase his/her contribution after being rewarded (or punished) if b3 > 0 (or b4 > 0).In b1, b2, b3, b4), where free-riders, unconditional contributors and conditional cooperators . This finding means that people on average do not react to reward or punishment.Based on the regression results, we first examine the reactions to reward and punishment. Surprisingly, b3 and b4 are small and their impacts on contributions are not significant, we drop them from Because b3 and b4 from b1 and the other-regarding parameter b2. In addition, the regression results that are based on In b1 (or b2) in Up, Const and Down. b1 (or b2) in the three incentive intensities, i.e., small changes in the amount of reward or punishment do not affect b1 and b2. We therefore combine the data in Up, Const and Down and investigate b1 and b2 in the four schemes of Control, IR, IP and IRP.Based on b2 are between 0.52 and 0.55 in all four schemes, and the differences among them are not significant  smaller than in the last 25 rounds. These findings are consistent with the observation of Fischbacher and GächtersAs shown in ents see . This reb1 in IRP and IP is larger than in IR and Control and why b2 in different incentive schemes is similar. We answer these questions by investigating the correlations between the group average payoff and the group average, b1, b2 and b1 + b2. The main results are shown in b2 and the payoff is not significant, whereas we observe a strong positive correlation between b1 and the payoff in IP and IRP. Thus, a larger b1 is preferred in IRP and IP because it can lead to a higher payoff. In Control, there is a strong positive correlation between b2 and the group average payoff, i.e., the level of group average contribution increases in b2. This finding is consistent with the previous study that conformity promotes the evolution of cooperation in PGGThese regression results raise two additional questions, namely, why b1 + b2. Furthermore, a high level of contributions can be maintained in a PGG if the group average of b1 + b2 ≈ 1 and b2 are not too small  and conditional cooperators  are the two largest types. Thus, cooperation can be maintained in groups that consist of conditional cooperators, and contributions drop if there are free-riders or imperfect conditional cooperators in the group  and PGG46474849505152535456575859616261A total of 792 university students participated in our PGG experiments at the School of Mathematical Sciences Computer Lab at Beijing Normal University. All participants provided written informed consent after the nature and possible consequences of the studies were explained. All experimental methods were conducted according to the approved guidelines. All experimental protocols were approved by the Ethics Review Committee of the Institute of Zoology.The subjects interacted anonymously through computer screens for 50 rounds of the repeated game among the same four players. The control experiment  is a standard four-player repeated PGG. In the treatment experiments, each round of PGG is followed by an incentive stage, which corresponds to an institutional punishment (IP), an institutional reward (IR), or both institutional reward and punishment (IRP). Furthermore, for each IR, IP and IRP, there are three different types of incentive intensities that are called Const, Up and Down. In IP, there are 80 subjects (20 groups) in Const, 76 subjects (19 groups) in Up and 84 subjects (21 groups) in Down. In IR, there are 80 subjects (20 groups) in Const, 72 subjects (18 groups) in Up and 84 subjects (21 groups) in Down. In IRP, there are 80 subjects (20 groups) in Const, 80 subjects (20 groups) in Up and 80 subjects (20 groups) in Down.C and the average contribution of the group is πIR = πC + PIRA in IR, πIP = πC −PIPA in IP, and πIRP = πC + (PIR−PIP)A in IRP, where A denotes the amount of incentives  is the probability that the individual is rewarded in IR (or punished in IP). Specifically, et al.In Control, an individual’s single round expected payoff is How to cite this article: Dong, Y. et al. The dynamics of human behavior in the public goods game with institutional incentives. Sci. Rep. 6, 28809; doi: 10.1038/srep28809 (2016).


The local Schottky barrier height is found to vary between 0 and 1.26 eV depending on the position of the dopant. The band bending is caused by a dopant induced dipole field between the interface and the dopant site, whereas the pristine Au/TiO2 interface does not show any band bending. These findings open the possibility for atomic scale optimisation of the Schottky barrier and light harvesting in metal-semiconductor nanostructures.The formation of a Schottky barrier at the metal-semiconductor interface is widely utilised in semiconductor devices. With the emerging of novel Schottky barrier based nanoelectronics, a further microscopic understanding of this interface is in high demand. Here we provide an atomistic insight into potential barrier formation and band bending by  The presence of a Schottky barrier (SB) between a semiconductor and a metal is of paramount importance to numerous application fields, including electronics24578510121314151619 cm−3). However, recent advances in nanotechnology122022The quantum transmission of electrons or holes across the SB is determined by two quantities: the barrier height and, more importantly, the decay length of the band bending. Together, these quantities determine the probability of transmission and the energy distribution of hot carriers across the metal-semiconductor interface. The conventional SB model assumes a uniform charge depletion region on the semiconductor side and a charge accumulation layer localised at the interface18i.e., the Au/TiO2 interface. A substitutional niobium dopant (NbTi) was introduced to model discrete defects in the n-type semiconductor. The concentration and spatial distribution of the NbTi can be controlled experimentally2425262 interface has a relatively large barrier height, but shows no band bending. The band bending and decay length is instead determined by the precise locations of the dopant. Our results also show that the band bending is inhomogeneous and highly localised to the defect region. We calculated the dopant position dependent barrier height and show that it can be qualitatively understood by the deep level (DL) barrier modelHere, we report first-principles calculations of a prototype Schottky diode, 22[001] direction with a lattice mismatch as small as 0.4%. Different epitaxial layers are obtained by rotating around the [110] axis. In the following, we focus on the Au(112)/TiO2(110) interface, which has been observed after high temperature depositionc5 in the rutile TiO2(110) surface (ϕm) and the electron affinity of the TiO2 slab (χs) were calculated to be 5.13 eV and 4.27 eV, which is in good agreement with experimental values (5.2 eVNbTi). In addition, the effect of changing the concentration of the Nb-dopants was checked by increasing the surface unitcell to (1×2) and (2×3) using a 5-layer TiO2 slab.In order to explore the effect of chemical composition on the SB, a large number of different atomic structures were calculated based on the lattice alignment and orientation of Au nanoparticles on thin film rutile (110) TiO surface 31. We mo surface , and dip2 slabs (left) and the interface formed upon contact (right). The interface band structure reveals a rigid upward shift of about 0.5 eV for both the valence and conduction bands of TiO2, while the Fermi level and Au bands change by less than 0.1 eV. This difference is caused by charge polarisation, as shown in panel (b). We analysed this effect using the contact induced charge density The electronic structure of the pristine interface is shown in ρMS and ρD-MS are the charge densities of the undoped and doped Au/TiO2 interfaces, respectively, ρM and ρS are those of the isolated metal and semiconductor slabs, while ρTi and ρNb are the charge densities of the free atoms. The areal density of the interface induced dipole 2 to Au  was chosen at the electronic potential maximum between the TiO2 and Au layers. Importantly, the conduction band edge is found to be flat throughout the semiconductor slab  is defined as the energy difference between the Fermi level (EF) and the conduction band minimum (CBM) of the TiO2 layer at the interface. We found that both the potential profile and the SBH critically depend on the position of the dopant. When the NbTi is located at the contacting layer, the defect state density is high enough to pin the Fermi level, which is very close to the bottom of the conduction band, as found in previous studiesdD = 5.9 Å) and becomes more prominent as the dopant moves to the other end of the slab at 12th layer (dD = 39.2 Å). Thus, our results clearly show that the local dopant confines the SBH and dictates the band bending.The results above show that the pristine TiOdD > 16 Å. The magnitude of the induced interface dipole is found to be inversely proportional to the dopant-interface distance, 2, while the induced dipole around the dopant points in the opposite direction. The charge transfer between the metal and the semiconductor slabs was found to be negligible for the pristine interface as well as for the doped cases . The excess electron contributed by the Nb-dopant is instead mainly distributed on the Nb atom and the neighbouring Ti atoms along the [001] direction. This localized charge distribution is consistent with previous DFT+U calculationsEF + ζ eV, where ζ is in the range of 0.2 ∼ 0.5 eV and varies with dD. The same shift was observed for the Ti 4 s semi-core states  cell, we have ND = 1.3×1021 cm−3. The depletion layer width is found to be 10 Å (25 Å) with εS = 10 (εS = 60). The SBH reduction is then 0.58 eV for εS = 10 (0.15 eV for εS = 60) (see where  60) see , which d 60) see , and doe 60) see . This quEDL) below the CBM. The SBH reduction depends on the charge donor-interface distance (dDL) and the areal dopant density (σDL) according to29We now compare the DFT+U results with the alternative deep level (DL) modela = 5 Å is the characteristic width of the interface, EDL = 0.2 ev is the DL energy below the CBM, and we set εS = 10 and T = 1000 K. The resulting SBH obtained from the DL model is plotted in where ulations :  is the2 interfaces show that the SBH is tuned by interface dipoles induced by contact and dopant. The local barrier profile, i.e. the band bending, shift almost linearly between the interface and the dopant layer. The barrier width is a-few-layer thick and depends on the dopant position. The reported experimental SBH for Au/TiO2 interfaces lies in the range 0.9 ~1.2 eV837In conclusion, first-principles calculations of Au/TiO2 rutile (110) interface supercell. A more condensed k-sampling (9×21×1) was adopted when extracting the density of states (DOS). The geometry optimisations were done on the GGA level. All of the atoms were relaxed in the geometry optimisation processes, and were considered to be relaxed when the maximum forces on each atom was smaller than 0.01 eV/Å. The calculated lattice constants of TiO2 (rutile) and Au (fcc) are a = 4.64 Å, c = 2.96 Å, u = 0.305 and a = 4.17 Å, respectively, in good agreement with experimental valuesAll the calculation in this work was done using the Vienna Ab initio Simulation Packages (VASP)40414244462 and comparing with results from both G0W0 calculations and experimental measurements. The calculations were performed using a cell with 6 atoms and a 9×9×11 Monkhorst-Pack k-point sampling. The parameter was determined to be 2 rutile slab was found to be 4.27 eV, which also agrees well with the experimental value of 4.33 eVIn the DFT+U calculations, the on-site Coulomb repulsion U parameter for Ti 3d orbital was carefully calibrated by calculating the bandgap of pristine rutile TiO  eV see . FurtherU(Nb4d) was then determined by keeping the U(Ti3d) fixed at 10 eV and calculating the dopant state with U(Nb4d) varied from 0 eV to 5 eV. A supercell of 13.13×8.88×6.57 Å3 along  direction. The calculated DOS below the CBM was found compatible with x-ray photoemission spectra (XPS)U(Nb4d) = 0 is sufficient in this work.The tion see  and stattion see  were notHow to cite this article: Jiao, Y. et al. Schottky barrier formation and band bending revealed by first-principles calculations. Sci. Rep.5, 11374; doi: 10.1038/srep11374 (2015).


After initial dissolution in dHFIP Aβ aggregates sediment with a major component of pure monomers showing a hydrodynamic radius of 1.8±0.3 nm for Aβ1–40 and 3.2±0.4 nm for Aβ1–42 including a surface layer of dHFIP solvent molecules.Small proteins like amyloid beta (Aβ) monomers are related to neurodegenerative disorders by aggregation to insoluble fibrils. Small angle neutron scattering (SANS) is a nondestructive method to observe the aggregation process in solution. We show that SANS is able to resolve monomers of small molecular weight like Aβ for aggregation studies. We examine Aβ monomers after prolonged storing in d-hexafluoroisopropanol (dHFIP) by using SANS and dynamic light scattering (DLS). We determined the radius of gyration from SANS as 1.0±0.1 nm for Aβ The residues Leu17-Ala21 constitute the hydrophobic core of Aβ. The residues Lys28-Ala42 and Gly9-Ala21 are capable to form α-helical or β-sheet structure, where the β-sheet is the priority for Lys28-Ala42. The. The1-42ayer see . For Aβ1ures see  we obserRS of a sphere to the radius of gyration by Rs of 2.06 nm for Aβ1–42 and 1.3 nm for Aβ1–40. If we assume that the monomer is surrounded by a solvent layer contributing to the apparent hydrodynamic radius  the hydrodynamic radius of Aβ1–42 and Aβ1–40 monomer is about 2.7 nm and 2.0 nm respectively, which correspond approximately to the measured RH from DLS experiment. In a different approach based on measurements of Rg and RH for proteins between 50–400 residues in denaturing solvents the relation Rg/RH = 1.06 is found[The question arises how to compare the radius of gyration and the hydrodynamic radius as both are influenced by a solvent surface layer, but in different ways. A simple approach for globular proteins relates the radius  is found. Even ta1–42 and Aβ1–40 in water is according to Nag et al. 0.9 nm[1–40 the more compact structure reduces the surface to the solvent and a lower number of solvent molecules may contribute to the complex resulting in the smaller hydrodynamic radius compared to Aβ1–42. The strong hydrogen bonding of dHFIP allows solvation of monomeric peptides without aggregation for Aβ and seems to increase the apparent hydrodynamic radius.The hydrodynamic radii of Aβl. 0.9 nm. For bot1–40 compared to Aβ1–42 showing that low resolution information can give valuable insight about the general structure. The difference in structure between Aβ1–40 and Aβ1–42 regarding the distribution of α-helices and disordered regions seems to be preserved in pure dHFIP. Additionally the results show the probable existence of a surface layer with increased density of dHFIP solvent similar to the hydration layer of proteins in water, which was not observed by other methods. The solvent surface layer increases the hydrodynamic radius considerably. Still the application of SANS onto low concentration samples of a protein as small as Aβ would benefit from higher intensity and it is at the limit of the nowadays possibilities.In this study we investigated the applicability of SANS to the study of the Alzheimer’s disease associated amyloid beta peptide (Aβ). Several properties of this 4 to 5 kDa small peptide make structural studies with high-resolution methods of the monomer as well as of the early assembly states extremely difficult. Therefore, other methods giving less detailed structural information become more important. Small angle neutron scattering and dynamic light scattering at relatively high concentration (for Aβ aggregation experiments) was used to probe the application of SANS as a scattering method without radiation damage for Aβ solutions as a prerequisite for further kinetic measurements of Aβ aggregation and fibril formation. We found that the solvation of Aβ in dHFIP is a fast process, where after a short incubation of less than 1 h the remaining aggregates mainly sediment. Therefore preparation time can be shortened by centrifugation to sediment the small amount of aggregates. We showed that SANS is able to perform measurements with monomeric Aβ solutions to determine the radius of gyration. Although we cannot differentiate between conformations as in high-resolution techniques, we find a more compact structure of AβS1 Dataset(ZIP)Click here for additional data file.S1 Figτ → 0 (intercept) is due to the increase in signal to noise ratio. With more large aggregates, respectively higher intensity, the signal has less noise and the intercept is closer to one. Additionally the signal to noise ratio depends on the laser attenuation chosen automatically by the instrument adjusting the mean intensity to values between 200000 and 500000 counts/s dependent on the total scattered intensity of a sample.The sample was measured between 0.5 h and 12 days after initial incubation at 20°C. The strong change in the absolute value of the correlation function at (EPS)Click here for additional data file.S2 FigThe sample was measured between 0.5 h and 14 days after initial incubation at 20°C in HFIP. See (EPS)Click here for additional data file.S3 FigRg is the average of the different structures in the PDB file as evaluated from the scattering in the Guinier region. The differences in the forward scattering for the same protein arise due to the changed protein volume if the configuration is changed.Black and green lines take only the contrast to the dHFIP solvent into account, while for red and blue lines additionally a solvent surface layer with 0.3 nm thickness and 5% higher density compared to bulk dHFIP is used. The given radius of gyration (EPS)Click here for additional data file.


Early diagnosis of agenesis of the mandibular second premolar (P2) enhances management of the dental arch in the growing child. The aim of this study was to explore the relationship in the development of the mandibular first molar (M1) and first premolar (P1) at early stages of P2 (second premolar). Specifically, we ask if the likelihood of P2 agenesis can be predicted from adjacent developing teeth. We selected archived dental panoramic radiographs with P2 at crown formation stages (N = 212) and calculated the likelihood of P2 at initial mineralisation stage ‘Ci’ given the tooth stage of adjacent teeth. Our results show that the probability of observing mandibular P2 at initial mineralisation stage ‘Ci’ decreased as both the adjacent P1 and M1 matured. The modal stage at P2 ‘Ci’ was P1 ‘Coc’ (cusp outline complete) and M1 ‘Crc’ (crown complete). Initial mineralisation of P2 was observed up to P1 ‘Crc’ and M1 stage ‘R½’ . The chance of observing P2 at least ‘Coc’  was considerably greater prior to these threshold stages compared to later stages of P1 and M1. These findings suggest that P2 is highly unlikely to develop if P1 is beyond ‘Crc’ and M1 is beyond ‘R½’. An understanding of dental development is crucial for clinicians, especially those treating children and young adults. Recognising the normal timing of developing teeth during growth is fundamental. Distinguishing between late development and agenesis of a tooth can impact significantly on treatment choice. Agenesis of teeth affects 4–6% of the population with mandibular second premolars reported to be the most commonly missing teeth, after third molars ,2. ImporThe first evidence of cusp tip formation of the mandibular second premolar P2) has been described as occurring during the second to fourth years ,5. Very  has beenThe aim of this study was to describe tooth development of the permanent mandibular first premolar (P1) and first molar (M1) at early radiographic stages of the permanent mandibular second premolar (P2). This information may contribute to assessing the likelihood of P2 formation in cases of late P2 formation or agenesis of P2. Such information can be gained from panoramic dental radiographs taken for diagnosis and treatment of routine dental care in young children.This was a retrospective study of archived panoramic radiographs taken at the Institute of Dentistry, Bart’s and the London School of Medicine and Dentistry, London. Ethical approval was obtained from Queen Mary Research Ethics Committee. Dental panoramic radiographs (N = 213) were selected from archived dental radiographs taken during diagnosis and treatment of local patients (White and Bangladeshi) attending the Institute of Dentistry. Age and sex of the sample is detailed in et al. [https://atlas.dentistry.qmul.ac.uk/) were used to identify tooth stages. At each stage, in addition to the criteria for that stage, the criteria for the previous stage were satisfied. In borderline cases the earlier stage was always assigned. Reliability of tooth stage assessment was done by repeat assessment of 14 radiographs after a period of two weeks. Cohen’s Kappa was calculated to measure the strength of agreement for intra-observer reliability.The first author recorded tooth formation stages of M1, P1 and P2 from panoramic radiographs. These teeth on the left side were examined without magnification using a standard light box. If this region of the radiograph was unclear on the left side, teeth on the right side were assessed. The modified description of Moorrees et al.  stages oet al.   were calculated for the sample, as well as individuals with P2 at stage ‘Ci’. Tooth stages of P1 and M1 were cross tabulated relative to P2 tooth stages. The earliest tooth stage of P2 at ‘Ci’ was of particular interest and the distribution of P1 and M1 stages conditional on P2 at ‘Ci’ was described (N = 89). Modal stage of development of P1 and M1 conditional on P2 at ‘Ci’ was described. The odds ratio (OR) of P2 being observed as stage ‘Cco’  or later was calculated indicating the chance of P1 up to ‘Crc’ stage divided by the chance of P1 root stages. This was done by dividing the data into four groups. The first group was the number of individuals with P1 up to and including stage ‘Crc’ and P2 at stage ‘Ci’. The second group included P1 stages later than ‘Crc’ and P2 at stage ‘Ci’. The third group was individuals with P1 stages up to and including ‘Crc’ and P2 stage ‘Cco’ and later. The fourth group included P1 stages later than ‘Crc’ and P2 stage ‘Cco’ and later. Similarly, the OR of P2 being observed as stage ‘Cco’ or later was calculated indicating the chance of M1 up to ‘R½’ stage divided by the chance of M1 ‘R¾’ or later root stages.Kappa value for intra-observer reliability of tooth stage assessment was 0.77 showing excellent agreement.The sample of selected radiographs consisted of 112 males and 101 females with a mean age of 4.20 years  mostly made up of three and four year old children. Four children did not have recorded age. The mean age of P2 stage ‘Ci’ was 3.56 years .Tooth stages were cross-tabulated to demonstrate the relationship of P2 development against M1 and P1 Tables . M1 and A radiographic example of the modal stages of P1 ‘Coc’ and M1 ‘Crc’ conditional on P2 at ‘Ci’ and P2 relatively delayed are shown in The chance of P2 being observed at ‘Cco’ or later stages was (15/0)/(109/89) = 25.3 times greater if P1 was in stage ‘Ri’ or earlier, compared to P1 later than ‘Ri’ . The chance of P2 being observed at ‘Cco’ or later stages was (19/0)/(105/89) = 33.1 times greater if M1 was in stage ‘R½’ or earlier, compared to P1 later than ‘R¾’ .Our results show that the age of P2 initiation is highly variable and P2 stage ‘Ci’ can be considerably delayed in timing as well as relative development. Stage ‘Ci’ of P2 can be expressed in chronological age but assessing if a child is dentally advanced or delayed is better expressed in developmental terms. Stage ‘Ci’ of P2 was most frequently seen during the first half of P1 crown formation stages. In dentally advanced children P2 initiates soon after P1 and in dentally delayed children, P2 initiates considerably later. Stage P2 ‘Ci’ was observed one to five stages delayed relative to P1. A one stage difference between P1 and P2 could be considered as a dentally advanced child. A difference of four or five stages between P1 and P2, where P2 ‘Ci’ is observed towards the end of P1 crown formation, could be considered as a dentally delayed child where the sequence of tooth formation has a longer duration. Once a few millimetres of root are visible on P1, agenesis of P2 is likely. Similarly, P2 ‘Ci’ was most frequently seen during late crown stages and early root stages of M1. A dentally advanced child may present with P2 ‘Ci’ prior to completion of M1 crown, while M1 ‘R¼’ or ‘R½’ would be regarded as relatively delayed. Once root length is considerably longer than crown height, agenesis of P2 is likely. These threshold stages of M1 and P1 could be tested on another sample ideally on a longitudinal radiographic collection that includes young children.The bar charts  are not et al. [th birthday. Another study from birth reports the median age of attainment of cusp initiation of P2 at 3.32 years in boys and 3.20 years in girls [The age of initiation of P2 at 2½ years of age from an early anatomical study is probably based on at most two individuals . Severalet al.  and thesin girls . A recenin girls . These sin girls .et al., [et al. [et al., ) illustr [et al. ) mean agLate onset of P2 mineralisation could give false-positive diagnosis of agenesis on radiographs  and diagOne of the strengths of this study is the age range of the sample, however a limitation is the cross-sectional nature of the radiographs, the non-random selection of radiographs and the lack of full medical history from each patient. A further limitation is the difficulty visualising early premolar tooth stages on dental radiographs. The crypt of P2 is not always clearly defined and a visible cusp tip can be seen prior to the inferior border of the crypt see .Our results have some clinical importance. A panoramic radiographs prior to eruption of permanent teeth can be of value. Early diagnosis of P2 agenesis can influence a clinical decision whether to save or extract a deciduous second molar or if extracted, whether to maintain or close the space. Early planning minimises the likelihood of a developing malocclusion and will influence the orthodontic treatment plan. Early diagnosis of P2 agenesis also influence the treatment plan for the first permanent molar in an older patient. If P2 cannot be observed once M1 has developed beyond ‘R½’ and P1 beyond ‘Crc’, a heavily restored or carious M1, in a borderline extraction case might be saved, rather than run the risk of two missing units in a single quadrant making subsequent management increasingly complex. Furthermore, the findings can be used to limit emotional and functional problems associated with agenesis of mandibular second premolars and its associated dental anomalies particularly during adolescence, via allowing for early diagnosis and the development of a definitive treatment plan through interdisciplinary management and patient counselling. Treatment can be less complicated and more cost effective as a consequence of early diagnosis and treatment planning, impacting on public health departments and health insurance companies as well as the individual and family levels.In summary, the probability of observing P2 at ‘Ci’ stage decreases as both the adjacent P1 and M1 mature and their development can help predict the likelihood of mandibular P2 agenesis. By the time M1 had reached stage ‘R¾’ and P1 stage ‘Ri’ no individual in this sample showed evidence of initial cusp formation of P2. These results suggest that once M1 has developed beyond ‘R½’ and P1 ‘Crc’ and P2 is not observed at ‘Ci’ stage, P2 is unlikely to develop.S1 File(PDF)Click here for additional data file.


These receptors share a similar conformation, with seven transmembrane segments, three intracellular loops, a C‐terminus tail, and three extracellular loops. However, the amino acid sequences of these two receptors bear no resemblance to each other. Coincidently the two ligands, α‐ and a‐factor, have different sequences. Both receptors activate the same G protein. To identify amino acid residues that are important for signal transduction, the STE2 and STE3 genes were mutagenized by a random PCR‐based method. Mutant receptors were analyzed in MATα cells mutated in the ITC1 gene, whose product represses transcription of a‐specific genes in MATα. Expression of STE2 or STE3 in these cells results in autocrine activation of the mating pathway, since this strain produces the Ste2 receptor in addition to its specific ligand, α‐factor. It also produces a‐factor in addition to its specific receptor, Ste3. Therefore, this strain provides a convenient model to analyze mutants of both receptors in the same background. Many hyperactive mutations were found in STE3, whereas none was detected in STE2. This result is consistent with the different strategies that the two genes have adopted to be expressed.The G protein‐coupled receptors Ste2 and Ste3 bind α‐ and  G protein‐coupled receptors (GPCRs) constitute the major class of signal transducing proteins, present in all eukaryotic organisms from yeasts to mammals. Several hundred different GPCRs are encoded by the human genome, responding to a variety of stimuli, such as hormones, neurotransmitters, odorants, and light signals  secrete a peptide pheromone  that acts on the other cell type to promote conjugation, resulting in the formation of MATa/α diploid cells. The a‐ and α‐mating factors bind to cell surface GPCRs, encoded, respectively, by the STE3 and STE2 genes. In response to pheromone binding the receptors, the G protein α subunit replaces bound GDP with GTP and dissociates from the G protein βγ complex, which in turn triggers a cascade of events leading to transcriptional induction of specific genes, stimulation of morphological changes, and inhibition of growth. The deletion of the gene encoding the yeast Gα subunit (GPA1) results in lethality in haploid cells because the free Gβγ complex constitutively activates the pathway leading to growth inhibition. The Ste2 and Ste3 receptors share a similar general conformation, common to all GPCRs, but their amino acid sequences bear no resemblance to each other. This is consistent with the fact that the two pheromone peptides, a‐ and α‐factor, have different sequences. In contrast, both receptors activate the same heterotrimeric G protein.The yeast Several studies were performed to characterize the yeast receptors through the identification of mutations affecting their function. The Ste2 receptor was highly studied  derived from strain RM27  derived from strain RM27; M18  derived from strain DDS4   containing the CEN sequence, the HIS3 gene, and the GAL promoter. A unique MluI site in the 5′‐UTR of STE2 was used to create pYX123‐STE2‐HA. Plasmid pSTE2‐Δ297 was derived from the latter and contains the STE2 coding region without the C‐terminal region from position 297 onward. Plasmid pSM1 is derived from pYX123‐STE2‐HA where the coding region of STE2‐HA is placed under the control of the TPI promoter instead of the GAL promoter. Plasmid pSM2‐Δ307 contains the STE3 gene and was constructed by replacing the STE2 sequence of pSM1 with the STE3‐HA sequence without the C‐terminal region from position 307 onward, obtained by PCR using primers P3 and P4 and yeast genomic DNA as the template. Plasmid pSM3 bears the full‐length coding region of STE3‐HA. We used a modified version of STE3 where a MluI restriction site was created by mutating the ACGAGA sequence into ACGCGT, changing A184 into C and A186 into T avoiding changes in the protein sequence.The The primers used for PCR, plasmid constructs, and sequencing are listed in Table S3.STE2 or STE3  together with plasmid pSTE2‐Δ297 or pSM2‐Δ307 that were linearized at a single site by digestion, respectively, with ClaI or PmeI. Growth on medium lacking histidine selected for cells carrying circular plasmids incorporated a mutagenized PCR fragment via homologous recombination.To generate a large collection of receptor mutants, plasmids containing portions of Pfu DNA‐polymerase (Stratagene) or Accuprime Taq DNA Polymerase System . In the STE2 subcloning, if the mutation was positioned at the 5′ of the ClaI site, the mutant plasmid was amplified with primers 8 and 6. The PCR product was cut with EcoRI and ClaI, and cloned into pSM1 cut with the same enzymes. On the other hand, if the mutation was placed at the 3′ of the ClaI site, the mutated plasmid was amplified with primers 9 and 10. The PCR product was cut with ClaI and SacI and cloned into pSM1 cut in the same way. In some cases, two PCRs were done followed by a third one. The product was then cut with EcoRI and ClaI or EcoRI and SacI, and inserted into pSM1 cut in the same way (see Table S1).Mutated plasmids were used as a template for PCR amplifications using STE3, if the mutation was placed at the 5′ of the PmeI site, the mutant plasmid was amplified with primers 11 and 12. The PCR product was cut with MluI and PmeI and cloned into pSM3 cut in the same way. If the mutation was positioned at the 3′ of the PmeI site, specific sets of primers were used (Table S1). The products containing the mutation were then digested with MluI and SalI, and cloned into pSM3 digested the same way.In the case of STE2 and STE3, PCRs were done using specific set of primers carrying the mutation on plasmids pSM1 or pSM3 (Table S1).To separate some multiple mutations of Plasmids from colonies were rescued by the rapid isolation method (Treco and Lundblad STE2 and STE3 mutants were quantitatively assayed for the ability to induce FUS1‐lacZ expression. Cells from a mid‐log phase culture were diluted to A600 = 0.2 and incubated for 4 h (for STE2) or 3 h (for STE3) at 30°C without an external source of mating factors in the case of autocrine activation, or using a cell‐free broth of an overnight culture of cells of the opposite mating type when activation must be induced exogenously. Cells were then harvested and suspended in 1 mL of Z‐buffer , and two drops of chloroform were added. The cell suspension was incubated at 30°C for 15 min, and then 200 μL (4 mg/mL) of chlorophenol‐red‐β‐D‐galactopyranoside  or o‐nitrophenyl β‐D‐galactopyranoside  were added as substrate. At appropriate times, the absorbance was measured, respectively, at 574 nm or 420 nm, and the relative β‐galactosidase activity was calculated with respect to wild‐type receptors.μL of buffer containing 200 mmol/L Tris‐HCl (pH 8), 1 mmol/L EDTA, 150 mmol/L (NH4)2 SO4, 10% glycerol, 2 mmol/L DTT, and Protease Inhibitor Complete 1X . The lysate was cleared twice by centrifugation. Protein concentration was measured by the dye‐binding assay using protein assay reagent  according to the manufacturer's instructions. A total of 20 or 30 μg of protein were loaded onto SDS/PAGE gels. After transfer, the membrane was probed with rat monoclonal horseradish peroxidase‐conjugated anti‐HA antibodies . The blots were developed using the ECL western blotting detection reagents (Amersham Biosciences). Bio‐Rad Precision Plus Protein WesternC Standards were used as markers.Cells from an overnight culture were harvested and lysed by agitation with glass beads in 200 STE2‐GFP was generated with primers P50 and P51 and pGFP(S65T)‐CAM plasmid (a gift from G. Papoff) as a template. The PCR product was digested by HindIII and XhoI, and ligated to full‐length STE2 cut using the same enzymes. Plasmid pSM3‐STE3‐GFP was obtained with a three‐step ligation. A PCR reaction with primers P52 and P51 generated GFP(S65T) coding sequences flanked by XhoI and PstI restriction sites. The PCR product was digested with XhoI and PstI, and ligated together with a PCR product obtained from pSM3 with primers P10 and P11, after digestion with PstI and MluI. The ligated product was cloned into pSM3 previously excised with XhoI and MluI.To localize the receptors in living cells, we constructed plasmids which express the green fluorescent protein (GFP) fused to receptors Ste2 or Ste3 at their C‐termini. Plasmid pSM1‐600 = 1 × 107, resuspended in 1 mL of broth, and then incubated for 4 h at 30°C. To label acidic organelles such as vacuoles, samples were resuspended in 250 μL 10 mmol/L HEPES buffer pH 7.4 containing 5% glucose with 100 μmol/L Cell Tracker Blue CMAC (Life Technologies), and incubated for 30 min at room temperature. Samples were spun for 3 min at 6700 g and the pellet resuspended in 1 mL of low melting agarose (Bio‐Rad) dissolved in the HEPES buffer. Aliquotes of 10 μL for each sample were seeded on glass bottom dishes  and covered with a 13 mm ø glass cover slip. Cells were observed with a laser scanning confocal microscope, TCS SP5  using a Plan Apo 63×(NA = 1.20) water‐immersion lens with optical pinhole at 1 AU. An Argon multiline laser operating at 488 nm (GFP), and a UV Diode laser at 405 nm (blue vacuole CMAC marker) were used as excitation sources. Confocal Z‐stacks were collected with 0.29 mm interval in a 5 mm total optical depth. Images for direct comparison were collected under the same parameters and representative images were chosen among 20 cells in multiple assays.Samples (5 mL) were grown over night up ASTE2 and STE3 was performed on 3′‐end‐truncated genes, given that pheromone receptors devoid of the C‐terminal region elicit a more elevated response in signal transduction . This strain is also mutated in the SST2 gene, whose mutants have a supersensitive phenotype.The mutagenesis was carried out on a substantial part of the receptors, in particular, the region encompassing four transmembrane segments (for Ste2) or five (for Ste3) and their connecting loops, a region which according to much previous work is the most important for signal transduction. Random mutagenesis was accomplished using PCR under error‐prone conditions. PCR fragments were then mixed with a ITC1, a gene whose product together with the product of ISW2, is known to repress transcriptional initiation of STE2 and other a‐specific genes in MATα cells, such as the a‐factor gene and the BAR1 protease  assay. Figure TPI gene, encoding triose phosphate isomerase (Schiestl and Gietz MATα cells as α‐factor source (strain GDS30). We used this source of α‐factor instead of the commercial one in order to compare the results to the analysis of the Ste3 receptor, for which there is no commercial a‐factor. In order to standardize the experimental conditions, MATα cells used as α‐factor source were at the same level of time incubation and optical density in each experiment, and we repeated the assays several times. Moreover, in each assay, we compared the mutant receptors against the wild‐type, as an internal standard.We continued the analysis on 56 mutants bearing a single or a double mutation. To confirm that the mutant phenotype was plasmid dependent, the rescued plasmids were retransformed into the M18 strain and analyzed with a liquid, quantitative Twenty‐seven mutants showed activity lower than 60% of that of the wild‐type Ste2 receptor , comparing the results to those obtained with RM6, which is neither autocrine nor supersensitive. For 14 amino acid residues, we found that in the autocrine strain, the activity of the mutants was increased with respect to the nonautocrine strain Fig. .β‐Gal activity was at least 4–5 times. This increase cannot be solely attributed to different extents of growth between FAR1+ RM6 cells, which are inhibited in responsive conditions, and the autocrine, far1‐deleted DDS4 cells, which are not. A likely explanation of the increase in the response in an autocrine strain with respect to nonautocrine cells is that the defect in these mutants causes a lower affinity to the pheromone , the amino acid change produced a null mutation in all strains we checked.For the other 13 mutations, the autocrine feature alone did not rescue the We verified the presence of the Ste2 protein in the null mutants in RM6 by Western analysis using anti‐HA antibodies. The protein is indeed abundant in null mutants S207P and Y266C, and at a lower level in C252R and I261K Fig. B. We alsSTE2 to the gene encoding the GFP. Although it is possible that such a fusion is not properly folded, we determined that there is no difference in the response between receptors with or without the GFP . Therefore, valuable indications may be obtained by these constructs concerning the localization of the different mutant receptors . All the mutants in the second and third extracellular loop, as well as those in the second and third intracellular loop, were also analyzed in DDS2, which is supersensitive but not autocrine for the Ste3 receptor. Pheromone response activation in the nonautocrine strains was achieved using a cell‐free broth of a culture of MATa cells as a source of a‐factor. As we did for the Ste2 receptor, we standardized the experimental conditions, making sure that MATa cells used as a source of a‐factor were at the same level of time incubation and optical density in each experiment, repeating the assays several times. Moreover, in each assay, we compared mutant receptors against the wild‐type receptor, as an internal standard.Each of the mutations displaying an activity lower than 60% of the activity of the wild‐type Ste3 receptor were transferred into the full‐length receptor gene by PCR methods. Multiple mutations were separated from each other (see Table S1). The activity of the mutants was analyzed by a Figure sst2) up to a level comparable to the wild‐type one, indicating that the down‐phenotype originally displayed in the first screening .For some mutants in the extracellular loops of the Ste3 receptor , the activity was improved in the M18 strain  the receptor protein is being synthesized, a Western analysis was performed. Mutant Ste3 receptors are present at a level comparable to that of wild type, indicating that our null mutations do not affect the synthesis or the stability of the receptor , neither the supersensitive nor the autocrine trait, nor the two together, suppressed the down mutation; therefore, these can be considered null mutants. For other mutants (positions 146 and 148), when the autocrine and the tor Fig. B. We alsWild‐type and mutant receptors were observed furthermore by confocal microscopy using a receptor‐GFP fusion protein. Several strains were analyzed: DDS4, DDS2, and M18. Wild‐type Ste3‐GFP receptor molecules are located at the cell membrane and around the vacuolar lumen in all these strains. In the Ste3‐C147Y mutant, the GFP signal localizes throughout the cytoplasm and at the perivacuolar area, but apparently not at the cell membrane, in any of the strains that were tested Fig. .STE3 mutations, we found several mutants that displayed an interesting characteristic, that is, they caused a superactive phenotype, a feature not generally observed. Although the original screening of random mutants was meant to identify “down” mutations, we obtained eight STE3 mutants that exhibit a higher activity in the mating‐response pathway compared to the wild‐type receptor. Two of the down mutants originally found harbored multiple mutations, and when we separated the amino acid changes from each other and analyzed them, we observed that some mutations strongly decreased the activity, but others caused an increase in the mating response. In such cases, the down mutations were clearly cis‐dominant with regard to the “up” mutations (Table S2). In one case, the double mutant was more hyperactive than the sum of the two single “up” mutations . Among the “up” mutations found in the Ste3 receptor, one is localized in the second cytoplasmic loop, one at the border between the second intracellular loop and the fourth transmembrane segment, four in the third cytoplasmic loop, one in the second extracellular loop, and one in the last transmembrane segment .Although in the screening described here, we found eight hyperactive mutations out of 58 mutants of STE2 with the endogenous promoter is scarcely expressed in the M18 strain, it is possible to screen for up mutants using X‐Gal plates that produce colonies with a deep blue color. Full‐length STE2 DNA was mutagenized with the same PCR‐method used before, transferred into M18, and plated on X‐Gal plates. Ten blue colonies were found out of about 2000. Plasmids were rescued and sequenced. Two candidates harbored the N271I mutation, one candidate had a single change, R234S, and one candidate was mutated in two sites, K225N and A229T. Six candidates had wild‐type STE2. We inserted the three mutants into the plasmid pSM1, containing the TPI promoter, and tested them in RM6, which is, for the STE2 gene, the analogous strain to DDS4 for STE3, where the “up” phenotype was observed. No hyperactivity was found for any of these STE2 mutants in RM6 .We wondered if hyperactive mutants of the Ste2 receptor could be obtained using a different approach. Since STE2 and STE3 genes can lead to a better understanding of the mechanisms that regulate signal transduction involving G protein‐coupled receptors.The analysis of mutations in the yeast In the present work, we detected dozens of different mutations in several regions of both receptors. Our approach was not to perform an extensive characterization of every mutant we detected, but instead, to compare the results obtained for these two receptors, which are different in their sequence but bind the same G protein, and draw inferences about their general nature.A number of mutations found in this study were detected previously by other groups. Table Some mutant features are common in both the Ste2 and Ste3 receptor. For several cases, defective mutants recover their activity when they are assayed in an autocrine strain see Fig.  for Ste3It is remarkable that the second extracellular loop of Ste3 is a sort of hot spot of mutations, with a change or even two in almost every position. In the corresponding loop of the Ste2 receptor, mutations were much fewer . One of the two residues is located in the middle of the second intracellular loop, and the other one is placed at the junction between this loop and the fourth transmembrane segment. It is possible that both residues are involved in the binding of the Gα subunit or that they interact with each other producing a more efficient receptor molecule.STE2 gene must be expressed in MATa cells but repressed in MATα cells, and conversely in the case of STE3. For this kind of regulation to be effective, the repression of STE2 in MATα cells should be very rigorous. In fact, if MATα cells, due to leakiness in the system, produced even a small amount of the opposite receptor, that is, Ste2, they would be autocrinely activated by the α‐factor that they secrete. The activation of the mating pathway, in turn, would lead to growth arrest and therefore to lethality.The main difference between the Ste2 and Ste3 receptor that we observed in this work was that while hyperactive mutations were found for Ste3, we did not observe any gain‐of‐function mutant for the Ste2 receptor. We interpret this finding as an outcome of the different strategies the two genes have adopted for their expression  site in the 3′ untranslated end is present. The two cell types, MATa and MATα, produce an incomplete STE2 transcript, but only MATa cells generate a full‐length STE2 mRNA.Support for this interpretation is found in our previous work, where we found that the STE2 expression. This means that even low amounts of the Ste2 receptor, such as those derived by escaping the repression of transcription, could have harmful effects.Several previous studies, especially that of Shah and Marsh , reporteSTE2 and STE3 genes appear to be very different, not only at the level of their DNA and the derived amino acid sequences but also regarding their transcriptional and post‐transcriptional regulation. Here, we have shown that they are also distant from each other regarding the characteristics of the proteins encoded by them. These receptors are a good example of convergent evolution, shedding new insight on signal transduction and receptor function.The None declared.Figure S1. Western blot.Click here for additional data file.Figure S2. Quantitative β‐Gal assay of wild‐type Ste2 receptor fused to the GFP or not, espresse in the M18 strain.Click here for additional data file.Figure S3. Confocal microscopy analysis on Ste2‐GFP (WT) and mutant Ste2‐S207P‐GFP in DDS4, DDS2 and M18 strains.Click here for additional data file.Figure S4. Activity of a double‐mutant Ste3 receptor is higher than that of each single mutant. The position of the two mutations of Ste3 is indicated.Click here for additional data file.Figure S5. Analysis of mutations in the Ste2 receptor.Click here for additional data file.Table S1. Primers for the subcloning of mutations from C‐truncated forms (short) of the STE2 and STE3 genes to full‐length (long) TPI‐STE2 and TPI‐STE3.Click here for additional data file.Table S2. Mating response induced by C‐truncated (short) and full‐length (long) forms of single and multiple STE2 and STE3 mutants, respectively, in RM6 and DDS4 strain.Click here for additional data file.Table S3. List of the oligodeoxyribonucleotide primers used for sequencing and PCR.Click here for additional data file.


Since the re-emergence of Ebola in West Africa in 2014, comprehensive and stringent interventions have been implemented to decelerate the spread of the disease. The effectiveness of interventions still remains unclear. In this paper, we develop an epidemiological model that includes various controlling measures to systematically evaluate their effects on the disease transmission dynamics. By fitting the model to reported cumulative cases and deaths in Guinea, Sierra Leone and Liberia until March 22, 2015, we estimate the basic reproduction number in these countries as 1.2552, 1.6093 and 1.7994, respectively. Model analysis shows that there exists a threshold of the effectiveness of isolation, below which increasing the fraction of latent individuals diagnosed prior to symptoms onset or shortening the duration between symptoms onset and isolation may lead to more Ebola infection. This challenges an existing view. Media coverage plays a substantial role in reducing the final epidemic size. The response to reported cumulative infected cases and deaths may have a different effect on the epidemic spread in different countries. Among all the interventions, we find that shortening the duration between death and burial and improving the effectiveness of isolation are two effective interventions for controlling the outbreak of Ebola virus infection. The Ebola virus disease (EVD) affecting multiple countries in West Africa in 2014 has an unprecedented magnitude, which is far larger than all previous EVD outbreaks combined. By March 22, 2015, a total of 24,872 cases  of EVD, as well as 10,311 deaths from the infection, have been reported in Guinea, Sierra Leone, Liberia34et al.Chowell Media report on the numbers of infected cases and deaths can greatly affect social behavior and play an important role in defining health issues in the EVD epidemic678101113141516Recently, the first large-scale trials that aim to assess the safety and efficacy of two Ebola vaccines (cAd3-EBOZ and VSV-ZEBOV) were initiated in Liberia on February 2, 2015S , V , E1 , E2 , I , J , D , and R . Let N denote the total population size, i.e., N = S + V + E1 + E2 + I + J + R.We develop a mathematical model Eq.  to studyβ is the mean human-to-human transmission rate per day, βD is the transmission rate during funerals. In the model, CI and CD are the cumulative infected cases and deaths, respectively. m1 and m2 are non-negative parameters that measure the effect of media reported cumulative numbers of infected cases and deaths on the contact transmission. When mi  is 0 or relatively small, the transmission rate β is equal or close to the constant β1. For mi > 0, the public is more aware of the disease so that the transmission rate could be decreased to β0 (<β1) as the number of accumulated infected cases CI or deaths CD increases. Similarly, the transmission rate βD0 is assumed to be less than βD1 during funerals.Susceptible individuals become infected through contact with infectious individuals and enter into the latent class at rate ξ. The effectiveness of immunization is assumed to be η where 0 ≤ η ≤ 1 with η = 0 meaning that the vaccine is perfectly effective and η = 1 meaning that the vaccine has no effect. Latent undetectable individuals (E1) progress to the latent detectable class E2 at a rate k1, and subsequently move to the infectious symptomatic class I at a rate k2. Let fT be the rate at which latent detectable individuals are detected and isolated. Thus, θ = fT/(fT + k2) represents the fraction of isolated patients among latent detectable individuals exiting the class. Infectious individuals are isolated at a rate α, or removed from the class at a rate γ due to recovery (with probability 1 − δ) or disease-induced death (with probability δ). Similarly, isolated individuals are removed at a rate γr because of recovery (with probability 1 − δ) or disease-induced death (with probability δ). A flow diagram of the model  by applying the solutions of Eq. It should be noted that we keep track of the cumulative number of infected cases ξ = η = 0), we obtain the basic reproduction number R0 which is the spectral radius of the matrix In the absence of effective vaccine  is the fraction of latent detectable individuals becoming symptomatic among those exiting the E2 class. 1/(α + γ) is the mean infectious period of symptomatic cases (not isolated). α/(α + γ) is the fraction of symptomatic cases that are isolated among those exiting the I class. 1/γr is the mean infectious period of isolated cases and 1/γD is the mean duration of the infectious period between death and burial. RI0, RJ0 and RD0 reflect the contribution to new infection from the symptomatic class (I), the isolated class (J), and the dead class (D), respectively. When vaccine is included, i.e., ξ > 0, η > 0, the reproduction number can be calculated as RV0 = ηR0, where R0 is given by (3).Note that each term of the aforementioned expression for t0  for Guinea, Sierra Leone, Liberia from the World Health Organization (WHO)t0 see  as the sk1 + 1/k2 = 7) with 4 days from the latent undetectable class to latent detectable class (1/k1 = 4) and 3 days from the latent detectable class to symptomatic class (1/k2 = 3)3α = 3). The mean time that infectious individuals are removed from the class after recovery or disease-induced death is 6 days (1/γ = 6)3γD = 2)N, we used the number of 11745189 for Guinea, 6092075 for Sierra Leone, and 4294077 for Liberia, provided by the World BankMatlab. The estimated parameter values and their 95% confidence intervals are listed in Based on previous studies, the mean incubation time for EVD is 7 daysR0 in Guinea, Sierra Leone and Liberia as 1.2552, 1.6093 and 1.7994, respectively. They are all within the range of estimated values in the literature (see R0 accounts for 0.1844 (14.7%), the isolated component for 0.7515 (59.9%) and the dead component for 0.3193 (25.4%). For the epidemic in Sierra Leone, transmission by the symptomatic, isolated and dead class accounts for 0.2668 (16.6%), 1.2375 (76.9%), and 0.1049 (6.5%), respectively, in the value of R0. For Liberia, these three components account for 0.2835 (15.8%), 1.2314 (68.4%), and 0.2846 (15.8%), respectively.We first consider the situation without vaccination because of the unavailability of vaccine before February 2, 2015, and then explore the effect of vaccination in Liberia where the first large-scale vaccine trials were implemented since thenture see . In GuinWe plotted the simulated newly infected individuals in 252627The solution of model (1) is shown in R0, we calculate the derivative of R0 with respect to the parameter θ  as followsTo explore the effect of early and broad diagnosis on the basic reproduction number θ of detection of pre-symptomatic cases can increase the basic reproduction number R0. This is harmful to the control of the disease. If θ does not have any effect on R0. Only when isolation is effective  = CD(t1) + R(t1)t1 is min{t > 0,E1(t) + E2(t) + I(t) + J(t) + D(t) = 0}. Although the explicit formula between the final size Z and θ cannot be obtained, there exists a threshold which decides whether the final epidemic size increases as θ increases. The relationship between Z and θ is similar to that between R0 and θ. For example, it follows from θ increases. This suggests that strict measures must be taken to limit the contact of isolated individuals. Otherwise, low effectiveness of isolation would lead to more infected individuals because isolated people live longer due to improved medical care and have more chances to infect other people. The benefit of having a reduced transmission rate would be counteracted by a longer infectious period of the isolated class. θ increases.Next, we study the effect of the fraction θ , the simulated final size is 3907, 13360 and 10439 in these three countries respectively  between symptom onset and isolation  on the basic reproduction number R0, we plot R0 and θ when the the relative transmissibility R0 with respect to α isSimilarly, to investigate the effect of shortening the time  in Sierra Leone and Liberia. In this case α) is in Guinea.It has the same threshold m1 (response to the reported cumulative number of infected cases) and m2 (response to the reported cumulative deaths) on the final epidemic size. Let q (0 ≤ q ≤ 1) be the percentage of increase of m1 and m2 from its baseline estimate (q in m1 (or m2) by 30% from its baseline value (while keeping other parameters fixed) can reduce the final epidemic size by 7.62% (or 17.81%) in Guinea, 22.79% (or 0.31%) in Sierra Leone, and 1.68% (or 21.95%) in Liberia, respectively. This indicates that the response to the reported cumulative deaths (m2) may be stronger than the response to reported cumulative cases (m1) in Guinea and Liberia. A possible explanation of this result is that the case fatality rate in these two countries is relative high ) for various durations of the traditional burial 1/γD in R0 in all three countries. In particular, the epidemic in Guinea could be controlled by following very restrict burial procedures (a large zD). For example, when 1/γD = 2  and Liberia (RD0/R0 = 15.8%) than in Guinea (RD0/R0 = 25.4%). R0 decreases as the duration of funeral decreases. This suggests that the duration of funeral should be as short as possible for the control of the disease.To assess the effect of reducing post-death transmission on the basic reproduction number ge zD is . This isR0 and the final epidemic size are most sensitive to, we perform sensitivity analysis using the Latin Hypercube Sampling (LHS) technique and calculate the partial rank correlation coefficients (PRCCs)31R0 and the final epidemic size). It follows from R0 are the relative transmissibility γD. A smaller relative transmissibility R0, which is consistent with the results shown in γD) also have the most significant impact on the final epidemic size, as shown in γD) are of crucial importance to reduce the EVD infection. The effect of media (m1 or m2) has no effect on R0 (see the formula of R0 in (3)), which is in agreement with the findings in refs. In order to identify which parameters the basic reproductive number ξ and the efficacy of vaccine η  is longer than that for non-isolated individuals because of improved clinical care. This is consistent with the parameter estimates shown in θ) are isolated but the isolation may not be effective in reducing the transmission of the disease. Ebola virus is very contagious and the transmission is also rapid, which makes isolation, as a containment strategy, usually inefficientA few reasons may explain why the isolated class contributes more to the basic reproductive number E2) and from the infectious class with symptoms (I). Isolated cases from the E2 class may have a different duration (1/γr) staying in the isolated class from the isolated cases coming from the I class. However, the above conclusion that the isolated class contributes more to R0 should still be valid as long as the infectious period of isolated individuals is longer than that of their corresponding preceding class. This is usually true in view of the improved health care received for isolated individuals.In our model, we did not discriminate isolated individuals from the latent detectable class (δ in Guinea (0.6728) is almost as twice as that in Sierra Leone  is more sensitive than the response to the reported cumulative cases (m1) in Guinea and Liberia. It can be explained by a higher case fatality rate in these two countries .


There are errors in the values for LARS for thyroid cancer throughout the paper. Please see below for a description of the errors and their corrections.−6 (Case 1) and 5.6–79×10−6 (Case 2) in Fukushima City, 0.73–13×10−6 in Tokyo, and 0.04–0.49×10−6 in Osaka. The contributions of LARs of thyroid cancers due to ingestion were 5.4%–11% of all exposure (Case 1) and 11%–25% (Case 2) in Fukushima City.There are errors in the last two sentences of the Abstract. The correct sentences are: Lifetime attributable risks (LARs) of thyroid cancers due to ingestion were 1.7–37×10−6 in Fukushima City (Case 1), 5.6–79×10−6 in Fukushima City (Case 2), 0.73–13×10−6 in Tokyo, and 0.04–0.49×10−6 in Osaka, and the maximum LAR in Fukushima City (Case 1) was found in females <1 y old.There are errors in the seventh sentence of the “Lars of cancer incidence” section of the Results and Discussion. The correct sentence is: The LARs of thyroid cancers were 1.7–37×10131I to the lifetime-exposure thyroid doses were 88%-95% in Fukushima City (Case 1), 80%-91% in Fukushima City (Case 2), 89%–95% in Tokyo, and 42%–67% in Osaka.There are errors in the eleventh sentence of the “Lars of cancer incidence” section of the Results and Discussion. The correct sentence is: The contributions of −6 in Fukushima City (Case 1) and 1400–4200×10−6 in Fukushima City (Case 2); those of thyroid cancer were 29–490×10−6 in Fukushima City (Case 1) and 33–510×10−6 in Fukushima City (Case 2)  . The con131I to the lifetime-exposure thyroid doses were >80% for Fukushima City, and the contributions of LARs from foods in the second and subsequent years were negligible.There are errors in the first sentence of the last paragraph of the “Lars of cancer incidence” section of the Results and Discussion. The correct sentence is: As described above, the contributions of There are errors in Tables


Heterochromatin protein 1 (HP1) is a central factor in establishing and maintaining the heterochromatin state. As consequence of playing a structural role in heterochromatin, HP1 proteins can have both an activating as well as repressive function in gene expression. Here we probe how oligomerisation of the HP1-α isoform modulates interaction with chromatin, by spatially resolved fluorescence correlation spectroscopy (FCS). We find from fluctuation analysis of HP1-α dynamics that this isoform exists as a dimer around the periphery of heterochromatin foci and these foci locally rotate with characteristic turn rates that range from 5–100ms. From inhibition of HP1-α homo-oligomerization we find the slow turn rates (20–100 ms) are dimer dependent. From treatment with drugs that disrupt or promote chromatin compaction, we find that HP1-α dimers spatially redistribute to favor fast (5–10 ms) or slow (20–100 ms) turn rates. Collectively our results demonstrate HP1-α oligomerization is critical to the maintenance of heterochromatin and the tunable dynamics of this HP1 isoform. More in detail, we preliminarily probed, for the first time, the actual existence and precise localization of HP1α homo-dimers in the nuclei of live cells by the Number and Brightness (N&B) analysisIn previous works we measured chromatin dynamics apparent = ε + 1) in each pixel of the image into a brightness map (background = 1), the molecular brightness of monomeric EGFP is thus 0.3 counts/dwelltime/s. From this value we can extrapolate that a HP1-α dimer would have an apparent brightness of 1.60 (given that Bdimer = (2 × 0.3) = 0.6), a HP1-α trimer would have an apparent brightness of 1.90 (given that Btrimer = (3 × 0.3) = 0.9) and so on.Here we use NIH3T3 cells transiently transfected with HP1α-EGFP and stained with Hoechst 33342 (as a reference of DNA) to measure the spatiotemporal regulation of HP1-α oligomerization within the nuclear compartment and how this modulates HP1-α dynamics. To investigate HP1-α oligomerization we use the well-established N&B approachness map , we founin vitro study that reported HP1-α dimers to induce the nucleation of a higher order chromatin structure that is incompatible with transcription. To prove that the detected increase of molecular brightness observed at the periphery of heterochromatin foci is in fact due to HP1-α-EGFP oligomerization and not solely HP1-α binding interaction, we then performed the same type of experiment in a cell transiently transfected with HP1-α-I165E-EGFP . In both instances the rotation of heterochromatin foci is modulated, to favor either slow or fast turn rates, and thus it appears that the tunable dynamics of HP1-α is achieved by changes in its oligomerization state. In general, two major findings here are somewhat surprising and deserve a more detailed discussion. First, the HP1-α oligomers (dimers or tetramers) are found only at the heterochromatin periphery (irrespective of the size of the heterochromatin focus), while the monomeric form is found at the heterochromatin center and throughout the nucleoplasm. This in turn suggests that a precise spatial organization of HP1-α oligomers is crucial for the regulation of heterochromatin organization. We may speculate that HP1-α dimers at the periphery of heterochromatin foci form a dimerization interface that recruits specific ligands, presumably involved in heterochromatin spreading, although the mechanism of spreading is still to be clarified30Proper understanding of how heterochromatin is established and maintained will ultimately enhance our understanding of stable gene repression. Here, we detected for the first time pericentric heterochromatin, a major repressive chromatin domain, mainly composed of major and minor satellite repeats . Different functions have been assigned to this repressive chromatin locus, the most commonly reported being its participation in kinetochore attachment in mitosis and subsequent chromosome segregation. However, pericentric heterochromatin seems to play also important roles in organizing the repressive compartments of the nucleus, probably by recruitment of silenced genes to the pericentric region, as shown for imprinting or allelic exclusion during B cell developmentTo conclude, we may speculate that an intranuclear heterochromatin target area of particular interest for future investigations might be 2. Freshly split cells were plated onto 35-mm glass bottom dishes coated with fibronectin and then, after twenty four hours, transiently transfected with EGFP, H2B-EGFP (Plasmid #11680 purchased from Addgene), HP1α-EGFP (Plasmid #17652 purchased from Addgene) or HP1α-I165E-EGFP . Freshly split cells were plated onto MatTek 35-mm glass bottom dishes coated with 3 μg/mL fibronectin and then transiently transfected with one of the EGFP tagged plasmids using Lipofectamine 2000 according to manufacturer’s protocol. Induction of chromatin compaction was carried out by treating the NIH3T3 cells transiently transfected with HP1α with Actinomycin D  for 5–10 minutes. Induction of chromatin loosening was carried out by treating the NIH3T3 cells transiently transfected with HP1α with sodium butyrate  for 1–4 hoursNIH3T3 cells were grown in high glucose medium from Invitrogen, supplemented with 10% Fetal Bovine Serum, 5 ml of Pen-Strep and HEPES at 37°C and in 5% CO5 times in the EGFP channel at maximum speed , with fluorescence being sampled every 100 nm (32 pixels to a line). The average laser power at the sample for both frame and line scan acquisitions was maintained at the 1–2 mW. The data acquired were processed by the SimFCS software developed at the Laboratory for Fluorescence Dynamics (www.lfd.uci.edu).The microscopy measurements were performed on a Zeiss LSM710 Quasar laser scanning microscope, using a 40X water immersion objective 1.2 N.A. . EGFP was excited with the 488 nm emission line of an Argon laser. Hoechst 33342 was excited with the 405 nm emission line of a diode laser. EGFP and Hoechst were measured sequentially using the 510–560 nm and 410–490 nm emission ranges, respectively. For each channel the pinhole was set to 1 Airy Unit. Image acquisition for Number and Brightness analysis involved selecting a region of interest within a NIH3T3 cell nucleus at an electronic zoom that resulted in a pixel size of 50–100 nm for a 256 × 256 pixel frame size. A time series of 100 frames was then collected in the EGFP channel at this zoom, with the pixel dwell time set to 12.61 μs/pixel, which resulted in a line time of 7.56 ms and a frame time of 1.15 s. Calibration of the monomeric brightness of the EGFP based constructs was performed by measurement of cells transfected with free EGFP under identical experimental conditions. Line scan acquisition for pair correlation analysis involved selecting a 3 μm line within the NB acquisition ROI that traversed heterochromatin foci (as indicated by the Hoechst 33342 or HP1α fluorescence intensity). The selected line was then rapidly scanned 2 × 10www.lfd.uci.edu, as described in previously published papers272 (second moment). These two properties describe the apparent number (N) and brightness (B) of the molecules that give rise to the intensity fluctuation, according to equations Calculation of the Number and Brightness of molecules in each pixel of an image was performed using the SimFCS software available at The true molecular brightness (ε) of the molecules that give rise to the measured apparent brightness (B) in the case of a photon counting detector are related according to equation 2detector) should equal the average intensity of the detector noise (‹k›detector). In the case of an analogue detector (as was used in the results presented here) this is not true due to characteristics of the analog amplifier and the settings of the analog-to-digital converter. Thus the detector’s brightness contribution needs to be accounted for by a term we call the S factor, which returns the background brightness to 1 so that the molecular brightness of the molecules can be extracted. By analysis of pixels that did not contain fluorescence from the sample, we found the brightness of the background to be between 1.3–1.6 depending on the laser power required. Taking into account this S factor we then calibrated the monomeric brightness of free EGFP so that in the instance oligomerization was observed when tagged to H2B or HP1α we could extract the stoichiometry of the oligomer. The effect of cell movement was subtracted by the moving average algorithm, as previously describedwhere 1 is the brightness contribution of the detector given that the photon counting detector variance (σwww.lfd.uci.edu), as described in the 567x-coordinate corresponds to the point along the line (pixels) and the y-coordinate corresponds to the time of acquisition. The pair correlation function (pCF(pixel distance)) is displayed in pseudo colors in an image in which the x-coordinate corresponds to the point along the line and the y-coordinate corresponds to the autocorrelation time in a log scale. The distances at which pCF analysis was carried out were not fixed across all experiments, but instead determined on an individual basis by the chromatin density variation along each line measured. In general a distance of 4–8 pixels (which corresponds to 400–800 nm) was employed. In general for each experiment, 2 × 105 consecutive lines  were acquired. Time regions within each experiment  with no average change in fluorescence intensity (e.g. due to photobleaching) were then selected for the correlation analysis, as also described elsewhere6Calculation of the pair-correlation functions was done using the SimFCS software developed at the Laboratory for Fluorescence Dynamics .


While electrical and thermal conductivities both indubitably decrease with the diameter reduction, the two physical properties clearly exhibit different diameter dependent behaviors. For 750 and 490 nm NWs, much lower thermal conductivities (0.72 and 0.69 W/m-K respectively) were observed as compared with the theoretical prediction of Callaway model. The consequence indicates that in addition to the size effect, extra phonon scattering of defects created by Ga ion irradiation was attributed to the reduction of thermal conductivities. As the NW was further trimmed down to 285 nm, both the electrical and thermal conductivities exhibited a dramatic reduction which was ascribed to the formation of amorphous structure due to Ga ion irradiation. The size dependence of Seebeck coefficient and figure of merit (ZT) show the maximum at 750 nm, then decrease linearly with size decrease. The study not only provides the thoroughly understanding of the size and defect effects on the thermoelectric properties but also proposes a possible method to manipulate the thermal conductivity of NWs via ion irradiation.In this report, the thermoelectric properties of a Bi Although both electrical and thermal conductivities indubitably decrease with the diameter reduction is expected due to the confinement effect, their detailed mechanism of phonon and electron scatterings has not been fully understood. If this mechanism can be understood further, then the material engineering for practical applications can be realized. One typical example of these is the searching of high performance thermoelectric materials for waste heat recovery. Owing to the energy crisis and global warming, reducing the energy consumption by energy recycling is demanded. The efficiency of TE material is deduced by the dimensionless figure of merit (ZT), written bySize effects on thermal and transport properties have been extensively studied on the thickness dependence in two dimensional systems, however there are limited literatures and reports on the 1D nanowires (NWs). Due to the vast surfaces and easier oxidation in air, the intrinsic properties of the NWs are hardly examined. To overcome these problems, an et al.et al.2Te3 NWs array grown by electrodeposition displayed a significant low thermal conductivity 0.75 W/m-K at 300 K, which is about only one-third of its bulk value. A similar result also reported in p-type Bi0.5Sb1.5Te3 nanowires of 60–280 nm in diameter2-xSbxTe3-y nanowires are selected as the specimens in the work.where α, σ, κ, and T represent the Seebeck coefficient, electrical conductivity, thermal conductivity, and absolute temperature respectively. Obviously electrical and thermal conductivities are two crucial parameters to be tuned for the enhancement of the figure of merit (ZT). Low-dimensional nanostructures are the potential candidates to achieve the goal in terms of their vast phonon-interface scatterings24570.5Sb1.5Te3 thin film by thermal annealingin-situ in the FIB chamber.In this work, the highly crystalline BST NWs were grown directly from Bi2Te3 NWThe selected area electron diffraction (SAED) and the high resolution transmission electron microscopy (HRTEM) of the pristine 750 nm NW and trimmed 285 nm NW are shown in in-situ characterization of stoichiometric composition of NW was performed by SEM-EDS and listed in The In order to monitor the condition of NWs, the electrical resistance of BST NW was simultaneously measured through the whole FIB trimming processes. The time dependence of electrical resistance is shown in 17−3 to 3.4 × 10−3 K−1 and then to 2.7 × 10−3 K−1 for the bulk, 750 and 490 nm NW respectively. It then drastically decreases to 0.7 × 10−3 K−1 for 285 nm NW  calculated from R(T) for 300–310 K first slowly decreases from 5.2 × 1085 nm NW . This isThe diameter-dependent thermal conductivities of NW were carried out by a self-heating 3ω technique1120κ is contributed both by κe of electronic carries and κp of lattice phonons. Since the electronic part can be estimated from electrical conductivity through the Wiedemann-Franz law, κe = LσT, where L is Lorenz number. Thus the net contribution of lattice phonons can be obtained from total thermal conductivity by subtracting electronic portion. The Lorenz number for the bulk is The size dependence of thermal conductivity can be understood by quantitative analysis. The total thermal conductivity D is the Debye temperature and x is the dimensionless parameter including reduced Plank constant ħ, angular frequency ω and Boltzmann constant kB. The combined relaxation rate τc−1 can be obtained by Matthiessen’s rule from three sources which are written aswhere υ is the average sound velocity, θυ is the phonon group velocity and D is the diameter of NW. where In 0.75Sb1.25Te2.86 NW was prepared. 7In order to study the temperature dependence of phonon thermal conductivity, a 280 nm pristine BiThe Seebeck coefficient data of the bulk and NWs are shown in 2-xSbxTe3-y NW has been in-situ studied in FIB trimming. As the diameter was reduced from the bulk to 750 and 490 nm, a drastic decrease of thermal conductivity but electrical conductivity was distinctly seen.The diameter dependence of thermoelectric properties of single BiThe consequence implies the size and defect effects have more influence on phonon scattering than that of electron carriers in this diameter region. When the NW was further trimmed to 285 nm, both the electrical and thermal conductivities started a dramatic drop due to the formation of a large number of defects and the amorphous-like structure by severe Ga ion irradiation which was confirmed by HRTEM images in FIB trimming. As compared with the bulk counterpart, the novel physical properties of thermal/electrical conductivity and Seebeck coefficient in NWs had been revealed. In addition to the renowned size effect, the ion irradiation induced amorphous-like structure also plays a significant role in thermal conductivity reduction. The study not only provides the thoroughly understanding of the size and ion irradiation effects on the electrical and thermal transport properties but also provides a possible method to manipulate the physical properties in nanostructures for the extensive applications in engineering high ZT thermoelectric materials.0.5Sb1.5Te3 films were deposited on SiO2/Si substrates by pulse laser deposition under high vacuum (~2 × 10−6 torr). Then, the as grown BST films were sealed in the quartz tube under 5 × 10−6 torr and annealed at 340 °C for 5 days. According to the thermal expansion coefficient difference between SiO2 (~2.4 × 10−6/°C)/Si (~0.5 × 10−6/°C) substrate and BST (~13.4 × 10−6/°C) thin film, the highly crystalline NWs with diameter range of 200 to 1000 nm were obtained. 18 cm−2 at 30 kV with spot size 12.5 nm) was used to trim the pristine NW from 750 nm to 490 and 285 nm sequentially. Since the cross section of NW is not completely circular, thus the corresponding diameter of NW was derived from the formula πr2 = A, where 2r is the diameter, and A is the cross section area.The thermal annealing methodThe electrical conductivity was measured by standard four- probes method to avoid the influence from leading wire and contact resistance. The applied current was also tested and monitored to ensure no noticeable joule heat in the resistance measurements. The sample holder was designed for easy sample mounting and temperature control in the range of 300–320 K. The AC current source  and the lock-in amplifier  were then connected to the template through electrical feedthrough for data characterization. The alternating Seebeck coefficient measurement technique14−5 torr in FIB chamber. The thermal conductivity (κ) was calculated from the third harmonic voltage signal (V3ω) through the formula listed belowThe self-heating 3ω method has been extensively used to characterize the thermal conductivity of 1D wires290sinωt was applied to generate appropriate temperature fluctuation on the NW for measurement purpose. γ ≡ L2/π2α, where α is thermal diffusivity, represents the characteristic thermal time constant of the NW. To verify the reliability of measurement results, three crucial relations among V3ω, I, and ω: (1) V3ω ∝ I3 (2)φ ∝ ω had been carefully measured and confirmedwhere R and R′ are the resistance and its derivative at the corresponding temperature. L and S are the length and cross section of the NW. The alternating current I = IC) obtained from 3ω method. Experimental results from these two methods are in consistent with each other and the accuracy of the electrical and thermal conductivities are insured. The measurement errors of thermal conductivity are mainly from the uncertainty of third harmonic voltage signal (V3ω) which is estimated to be ~5–10%.The cross-section area of trimmed NWs is critical in the derivation of thermal conductivity and electrical conductivity. In general, the diameter of sample was directly measured by SEM image, however the uncertainty caused by the variation of electromagnetic lensHow to cite this article: Chien, C.-H. et al. In-situ Observation of Size and Irradiation Effects on Thermoelectric Properties of Bi-Sb-Te Nanowire in FIB Trimming. Sci. Rep. 6, 23672; doi: 10.1038/srep23672 (2016).