According to a basic rule of fermionic and bosonic many-body physics, known as the linked cluster theorem, physical observables are not affected by vacuum bubbles, which represent virtual particles created from vacuum and self-annihilating without interacting with real particles. Here we show that this conventional knowledge must be revised for anyons, quasiparticles that obey fractional exchange statistics intermediate between fermions and bosons. We find that a certain class of vacuum bubbles of Abelian anyons does affect physical observables. They represent virtually excited anyons that wind around real anyonic excitations. These topological bubbles result in a temperature-dependent phase shift of Fabry–Perot interference patterns in the fractional quantum Hall regime accessible in current experiments, thus providing a tool for direct and unambiguous observation of elusive fractional statistics. Quantum vacuum fluctuations can create pairs of virtual particles that annihilate each other. Here, the authors show that the conventional theory that these particles have no measurable effect on real particles does not apply to anyons, exotic quasiparticles that are intermediate between fermions and bosons. They carry a fraction e*=νe of the electron charge e and obey fractional exchange statistics, satisfying θ*=±πν. Two anyons gain a phase ±2πν from a braiding, whereby one winds around the other; ± depends on the winding direction. Although fractional charges have been detected567πν has been so far elusive. Existing theoretical proposals for the measurement involve quantities inaccessible in current experiments or suffer from unintended change of a proposed setup with external parameters91011121314151617Anyons23In many-body quantum theoryπν. We propose a realistic setup for detecting them and θ*=πν.In the following, we demonstrate that this common wisdom has to be revised for anyons: a certain class of vacuum bubbles of Abelian anyons does affect observables. These virtual particles, which we call topological vacuum bubbles, wind around a real anyonic excitation, gaining the braiding phase ±2a1 and a2 of propagation of a real particle. In a1, a virtual particle-hole pair is excited then self-annihilates after the virtual particle winds around the real particle, forming a vacuum bubble, while it is not excited in a2. The winding results in a braiding phase 2πν and an Aharonov–Bohm phase We illustrate topological vacuum bubbles. In ν=0) and fermions (ν=1) imply that this bubble diagram appears together with, and is cancelled by, a partner diagram in The limiting cases of bosons (πν)=0 in agreement with the linked cluster theorem; hence, the signal disappears. By contrast, for anyons they cancel only partially, producing the non-vanishing interference in an observable, and are topological as the braiding phase is involved.For bosons and fermions, the two diagrams fully cancel each other with sin(ν=1/(2n+1) FQH regime, coupled to an additional edge channel (Edge 1) via a quantum point contact (QPC1). At QPCi, there occurs tunnelling of a single anyon (rather than anyon bunching), fulfilledγi is the tunnelling strength and T is the temperature. Gate voltage VG is applied, to change the interferometer loop enclosing Aharonov–Bohm flux Φ. The interference part D3 is measured with bias voltage V applied to source S1; the other Si's and Di's are grounded. Together with ‘virtual'  anyon excitations in the interferometer, a voltage-biased ‘real' anyon, dilutely injected at QPC1 from Edge 1 to the interferometer, forms topological vacuum bubbles, as shown below. The bubbles contribute to In 917202122LV≡ℏvp/(e*V) of the dilutely injected anyons is much smaller than interferometer size L and the injection of hole-like anyons at QPC1 is ignored; vp is anyon velocity along the edges and e*V should be much smaller than the FQH energy gap. Because of the dilute injection and VG provides a clear signature of the topological bubbles, consequently, θ*=πν in both of the pure Aharonov–Bohm regime  and the Coulomb-dominated regime (where the interaction is strong)22We consider the regime of γ. There are four types of the processes mainly contributing to Employing the chiral Luttinger liquid theory2610LT≡ℏvp/(πνkBT) and where S1 to D3 via (i) QPC2 and (ii) QPC3, respectively. They were previously studiedType I-1 processes describe interference between two paths of an anyon moving from S1 interferes with a particle-like anyon excited at a QPC or annihilates a hole-like anyon; particle-like and hole-like anyons are pairwise excited thermally at QPCs. For example, consider the two following interfering histories: (i) an anyon is injected from S1 to D2, an anyon pair is excited at QPC3 and then the particle-like (hole-like) anyon of the pair moves to D3 (D2). (ii) An anyon is injected from S1 to D3 via QPC2 without any excitations. The hole-like anyon annihilates the injected anyon on Edge 2 in history (i) and the particle-like anyon of (i) interferes with the injected anyon of (ii) on Edge 3. The sum of such interference processes yields 2πν factor appears, because relative locations of anyons on Edge 2 or 3 differ between the processes, leading to an exchange phase ±πν, and because a process with an excitation (of a particle-like anyon moving to D2 and a hole-like one to D3) yields charge current in the opposite direction to another with its particle-hole conjugated excitation (of a particle to D3 and a hole to D2).In Type I-2, an anyon injected from S1 moves to D2 and a virtual anyon pair excited at QPC2 interferes with another at QPC3. The interference path effectively encloses the real anyon, forming a topological vacuum bubble (cf. D2 (D3). The interference of the two histories corresponds to the winding of a virtual anyon around the real one and Φ, forming a topological bubble with interference phase D2 or D3. In the interference, the winding of a virtual anyon around the real one effectively occurs through the exchanges of the positions of the anyons in each of Edges 2 and 3, as relative locations of anyons on Edges 2 and 3 differ between (i) and (ii)  see . This inS1 arrives at QPC2 and (ii) another pair is excited at QPC3 after the real one arrives at QPC3. The ensuing chronological sequence on Edge 2 is opposite to Type II: an anyon excited at QPC2 arrives at QPC3; the real one arrives at QPC3; a pair is excited at QPC3. The resulting topological bubble effectively winds around the real anyon in the direction opposite to its winding around Φ, yielding a phase L+CLT of CLT in In Type III, the two interfering histories are as follows: (i) a virtual pair is excited at QPC2 before a real anyon injected from ν=1 (fermions), due to full cancellation between partner bubbles (the linked cluster theorem). They are distinct from I-2. I-2 processes produce, for example, non-vanishing current noise ν=1, as the particle-hole conjugated excitations (mentioned before) equally contribute to the noise  of II and III, showing much weaker interference. From The phase LV/LT term represents an error in the braiding phase 2πν of the topological bubbles. It occurs when the size LV of a real anyon is not sufficiently smaller than the winding radius of a virtual anyon around the real one. It is negligible at θ→πν. Remarkably, θ depends on T, contrary to common practice in electron interferometryThe arctan 2In experimental situations of a Fabry–Perot interferometer in the FQH regime, it is expected that there exist bulk anyons localized inside the interferometer loop. There are two regimes of Fabry–Perot interferometers, the pure Aharonov–Bohm regime and the Coulomb-dominated regime. In the former regime, Coulomb interaction between the bulk anyons and the edge of the interferometer is negligible, whereas it is crucial in the latter17202122πVG/VG,0+θ) with periodicity VG,0 in the Coulomb-dominated regime and 0≡h/e rather the period θ satisfies the analytic expression in cf. For θ can be affected by possible side effects, including the external-parameter  dependence of the size, shape, QPC tunnelling and bulk anyon excitations. Below we propose how to detect θ with avoiding the side effects, using the setup in Experimental measurements of θ is experimentally measurable, by comparing D3 in the same setup under the same external parameters  with Vref/2 to S2 and −Vref/2 to S3 and keeping S1 and all Di's groundedcf. θ; θ.The phase θ→πν at A1/(1+A2/T)] with fit parameters A1 and A2 to measured data of θ(T) and extracts arctan A1=πν from the fit (cf. the second line of θ=πν or θ(T) will suggest a strong evidence of anyon braiding and topological bubbles.The fractional statistics phase is directly and unambiguously identifiable in experiments, by observing πν), (ii) that the anyon tunnelling probabilities at QPC2 and QPC3 are sufficiently small  and (iii) that anyon tunnelling (rather than electron tunnelling) occurs at the QPCs. The constraint (i) is satisfied when the anyon tunnelling probability at QPC1 is <ℏvp/(2Le*V), which is ∼0.05 under the parameters. To achieve the constraints (ii) and (iii), each tunnelling probability of QPC2 and QPC3 is typically set to be 0.4 in experiments22L/LT), which is ∼0.04 at 30 mK. With the constraints we estimate the amplitude of θ=0.9πν is reached at 30 mK, while θ=0.95πν at 40 mK under the parameters. The estimation is within a measurable range in experiments, where current The parameters in 17202122ν′=ν+ν0, in which the edge channels from the integer filling ν0 are fully transmitted through the QPCs, while the channel from the fractional filling ν forms the interferometry in θ of the ν0=0 case discussed in cf. We remark that the above strategy of detecting the fractional statistics phase is equally applicable to the more general quantum Hall regimeν=p/(2np+1), non-Abelian anyons17Certain anyonic vacuum bubbles involve topological braiding and affect physical observables surprisingly, contrary to vacuum bubbles of bosons and fermions. They can be detected with current experiment tools, which will provide an unambiguous evidence of anyonic fractional statistics. We expect that they are relevant also for other filling fractions We present the Hamiltonian for the setup. We recall the chiral Luttinger liquid theory for FQH edges.H=∑iHiedge,+Htun for the interferometer in Hiedge, for edge channel i and Htun for anyon tunnelling at QPCs. Edge channel 1 is biased by V and its Hamiltonian, employing the bosonizationThe Hamiltonian e*>0, i and φi(x) is the bosonic field of channel i at position x, which describes the plasmonic excitation of anyons. The tunnelling Hamiltonian is For the other unbiased channels, x and time t on Edge i, a is the short-length cutoff, γi is the tunnelling strength at QPC i  and the Aharonov–Bohm flux Φ enclosed by Edges i=2,3, QPC2 and QPC3 is attached to i by 1 and satisfies where φi and Fi. On Edge i, it is satisfied byThe exchange rule in Fi,The exchange rule between anyons on different edges is achieved with the commutators of cf. A conventional wayHtun as a perturbation on ∑iHiedge,. Perturbation theory is applicablee*V and kBT are higher than Cγν)1/=1 for d<x<d+L, K3(x)=1 for 0<x<L and Ki(x)=0 otherwise. The second term of NL .Supplementary Figures 1-2, Supplementary Notes 1-6, Supplementary References


In addition, a transition from positive to negative exchange bias has been observed with increasing initial magnetization field and measurement temperature. Based on a simple spin bidomain model with variable interface, two type of interfacial spin configuration formed during the initial magnetization process are proposed to interpret the observed phenomenon.Exchange bias effect obtained after zero-field cooling from unmagnetized state usually exhibits a shift of hysteresis loop negative to the direction of the initial magnetic field, known as negative zero-field cooled exchange bias. Here, positive zero-field cooled exchange bias is reported in La HEB. Although the origin of EB is still not fully understood, it is generally accepted that it is related to the interfacial ferromagnetic (FM)-antiferomagetic (AFM) exchange coupling interaction across the interface and the AFM unidirectional anisotropy hold a key role to decide the magnitude and sign of the shift678TN) of AFM subsystem, and the FM coupling results in a shift of the hysteresis loop in the opposite (‘negative’) direction to the cooling field, defined as negative EB. Positive EB effect can be observed upon strong cooling field in some cases, in which the interfacial exchange interaction is believed to be AFM10Since first reported in Co/CoO system by Meikle John and Bean234HEB was reported in bilayer systems earlyHEB up to 1300 Oe in Ni50Mn37In13 alloy by wang et al.2PtGa alloy3–Bi2Fe4O9 nanocomposite3 nanoparticles2Mn1.4Ga0.6 alloy1.5Sr0.5CoMnO6 polycrystalline ceramicsx1−CaxMnO3 nanosheet50Mn36Co4Sn10 alloy30.8Cu0.2Cr2O40.9Pr0.1CrO3 ceramics1316171819200.5Sr0.5Mn0.8Co0.2O3 ceramics, which can be ascribed to the evolution of the interfacial spin configuration during the initial magnetization process.Though ZFC-EB effect with small 0.5Sr0.5Mn0.8Co0.2O3, in which no trace of any impurity phases is discernible. The refined data obtained with the Rietveld refinement program FULLPROF are also shown in I4/mcm. The refined lattice parameters  and unit volume (V) of La0.5Sr0.5Mn0.8Co0.2O3 are a = b = 5.4475 (5) Å, c = 7.7208 (9) Å, and V = 229.12 (4) ÅM(T) measured at 50 Oe with FC and ZFC procedures. The ZFC and FC magnetization curves start to separate from each other at an irreversibility temperature around Tir ~ 280 K, and they begin to diverge strongly at low temperatures, which is a strongly pronounced typical feature of both classical spin glasses (SG) and phase-separated manganite systems with coexisting FM clusters and an AFM matrix. Two successive peaks at 133 K and 106 K, recognized as TN and freezing temperature Tf respectively, can be observed in the ZFC curve (see inset). As reported before, in canonical SG systems Tir is very close to Tf typicallyTir is far above Tf in present case, indicating the possibility of a cluster glass (CG) state at low temperatures25TG  could be associated with the occurrence of a short range FM ordering, forming FM clusters. Additionally, Tf could be considered as the freezing temperature of the FM clusters.χ′(T) of ac susceptibility measured at different frequencies with an ac magnetic field of 5 Oe. Two pronounced peaks, being close to TN and Tf respectively, can be observed. It is remarkable that the position of Tf is frequency dependent and obviously shifts toward a higher temperature with increasing frequency. Such behavior indicates a glassy character of the sample. The critical slowing-down power law, which assumes a true equilibrium phase transition with a divergence of relaxation time near the freezing point, was employed to analyze spin dynamics of the glass-like state. According to the power law, the temperature dependence of τ could be described by Tf is the peak temperature measured at frequency f, Tg is the critical temperature for SG ordering which is equivalent to Tf as f → 0, zν is a constant exponent, and τ0 is the characteristic time scale for spin dynamics (the shortest relaxation time of the system). The best fit shown in the inset of Tg = 106.5 K , zν = 4.11, τ0 = 7.92 × 10−10 s. The long spin flipping time τ0  and relative small critical exponent zν suggests a CG state nature at low temperature magnetism in the La0.5Sr0.5Mn0.8Co0.2O3 ceramics27In order to get further insight into the magnetic properties of this system, ac susceptibility under different frequencies was measured. M(H) loops of La0.5Sr0.5Mn0.8Co0.2O3 ceramics measured at 4.2 K with various magnitudes of the initial magnetization field, i.e. maximum measurement field  loops with HEB and coercivity (HC) as a function of HEB and HC were calculated using HEB = (H1 + H2)/2 and HC = |H1 − H2|/2, where H1 and H2 are the left and right fields at which the magnetization becomes zero. We can find that HEB exhibits positive value at low field, and first increases with the increase of HEB would change to negative value, reaching a minimum value of −520 Oe at HEB are strongly dependent on the magnitude of the initial magnetization field.The To explore the origin of the ZFC-EB effect, the evolution of the FM/AFM spin interface based on the simple spin bidomain model was proposed, and the schematic diagram of the spin configuration at different stages is shown in 0.5Sr0.5Mn0.8Co0.2O3 ceramics can be associated with the competition of the two ways of forming the AFM unidirectional anisotropy, highlighting the importance of the AFM spins anisotropy on the sign of ZFC-EB effect. Increasing the initial magnetization field firstly would increase the AFM unidirectional anisotropy that parallels to the direction of initial magnetization field, resulting in the strengthening of the AFM coupling at the interface and larger positive shift and HEB. Further increasing the field, the rotation of the AFM spins from negative direction to the positive direction would reduce the AFM anisotropy as well as HEB. With the increase of the interface with the AFM anisotropy antiparallel to the direction of initial magnetization field, the enhancement of the negative ZFC-EB effect with the increase of initial magnetization field can be expected. The slight increase of HEB (the decrease of |HEB|) at higher Hence, the results in the LaHEB (HEB < 0) approximately linearly decreases with increasing temperature and gradually disappears around the blocking temperature TB, which can be attributed to the decrease of the AFM anisotropy paralleling to the direction of positive magnetic field with the increase of temperature13161718192021M(H) loops at different temperatures with HEB and HC are show in HC decreases with the increase of the temperature. It is worth noting that HEB for 50 kOe decreases firstly with the increase of temperature, then changes to negative, reaching a negative peak before approaching to zero at 40 K. The temperature of 40 K can be referred as exchange blocking temperature, TB. A negative peak can also be observed for 110 kOe, indicating the AFM anisotropy parallel to the direction of positive magnetic field at low temperature. All these results are consistent with the discussions above, confirming the reasonable of the model shown in Usually in the negative FC and ZFC-EB effects, the absolute value of 0.5Sr0.5Mn1−xCoxO3 (0 ≤ x ≤ 1) series of samples were studied systematically, and only La0.5Sr0.5Mn0.8Co0.2O3 exhibits ZFC-EB. As reported before, the formation of new stable FM–SG interface with unidirectional anisotropy is usually though the percolation of isolated FM domains in the initial magnetization process171316170.5Sr0.5MnO3 exhibits FM ground state with AFM clusters in low-temperature region303+ and Mn4+ would be partially destroyed and some of the FM ground state would be transformed to a robust AFM state32Magnetic properties of La0.5Sr0.5Mn0.8Co0.2O3 ceramics, and it can transfer into negative ZFC-EB effect with the increase of magnetic field and measuring temperature. Such phenomenon can be attributed to different orientation of interfacial AFM anisotropy formed in the process of initial magnetization. The results here will enrich the type of EB effect, improve the spin bidomain model with variable interface, and benefit the deep understanding of the physical mechanism of exchange bias.In summary, we have observed positive ZFC-EB effect in La0.5Sr0.5Mn0.8Co0.2O3 ceramics was prepared by sintering corresponding powders derived from sol–gel precursors. A stoichiometric amount of La(NO3)3·6H2O, SrCO3, Mn(NO3)2·4H2O and Co(NO3)2·6H2O, and appropriate citric acid and nitric acid were used as starting materials. At the end of the process, the gel was first dried at 120 °C and then decomposed at 400 °C to result in dark brown powders, which were ground, then pressed into strip and sintered at 1300 °C for 10 h to produce the finished sample. The good crystallinity of an orthorhombic phase structure without any impurity phases was confirmed by X-ray diffraction (XRD). The magnetic properties were measured by superconducting quantum interference device  magnetometer and physical property measurement system .The LaHow to cite this article: Shang, C. et al. Positive to negative zero-field cooled exchange bias in La0.5Sr0.5Mn0.8Co0.2O3 ceramics. Sci. Rep.6, 25703; doi: 10.1038/srep25703 (2016).


Glutathionylation of Cav1.2 results in increased calcium influx and is evident in ischemic human heart. However controversy exists as to whether direct modification of Cav1.2 is responsible for altered function. We directly assessed the function of purified human Cav1.2 in proteoliposomes. Truncation of the C terminus and mutation of cysteines in the N terminal region and cytoplasmic loop III-IV linker did not alter the effects of thiol modifying agents on open probability of the channel. However mutation of cysteines in cytoplasmic loop I-II linker altered open probability and protein folding assessed by thermal shift assay. We find that C543 confers sensitivity of Cav1.2 to oxidative stress and is sufficient to modify channel function and posttranslational folding. Our data provide direct evidence for the calcium channel as a redox sensor that facilitates rapid physiological responses.Ion channels are critical to life and respond rapidly to stimuli to evoke physiological responses. Calcium influx into heart muscle occurs through the ion conducting α1C subunit (Ca It is also capable of oxidizing the thiol groups of cysteine residues to form a disulfide bond with glutathione (GSH) (glutathionylation). The functional implications of glutathionylation are not well understood but are becoming increasingly recognised in disease.Ischemic heart disease is the leading cause of death in the western world32+ influx through the L-type Ca2+ channel shapes the long plateau phase of the ventricular action potential and initiates the sequence of events that result in contraction2+ channels are heterotetrameric polypeptide complexes consisting of α1C, α2δ and β2 subunits. Cardiac muscle expresses the dihydropyridine sensitive α1C subunit, which is encoded by the CACNA1C gene and known as Cav1.2. The Cav1.2 is the main pore forming subunit and is sufficient for calcium conductance. The auxiliary β2 subunit binds to the I-II linker in the Cav1.2 subunit at the alpha interacting domain and facilitates the trafficking and insertion of the polypeptide complex into the membrane82δ and β2 subunits modify the inactivation kinetics of the current89The L-type calcium channel is critical to cardiac function and the main route for calcium influx into cardiac myocytes2+ channel can be regulated by hypoxia and oxidative stress111213v1.2 protein contains an “oxygen sensing region” encoded by exon 45v1.2 function has not been demonstrated2+ currents while thiol oxidising agents increase Ca2+ currents or attenuate the effects of hypoxia131417181920212223v1.2 protein is glutathionylated in guinea pig myocytes after ischemia reperfusion injury and in ischemic human heartv1.2 causes an increase in calcium influxIt is well recognized that the function of the native L-type Cav1.2 in liposomes for functional assessment. Under these conditions, only channel protein and lipid are present. We find that the open probability of the channel is altered when exposed to thiol oxidising or reducing agents. Truncating the C-terminal domain of the protein further increased the open probability of the channel but did not alter the response to thiol reducing and oxidising agents. We find that mutation of three critical cysteines in the loop I-II linker region of the channel prevents the functional response of the channel to thiol modification and alters protein folding. Finally we demonstrate that mutation of C543S alone is sufficient to attenuate the effect of thiol modifying agents. Our results indicate that direct modification of the Cav1.2 channel protein is responsible and sufficient for alterations in channel function during oxidative stress.The channel protein contains many cysteines that are available to be covalently modified during changes in redox state. However, it is not clear how modification of thiols alters channel function because contradictory responses have been reported, with some groups arguing that thiol reducing agents increase channel activity while others argue the opposite responsev1.2 channel is a large protein approximately 240 kDa comprised of 4 repeating domains (I-IV)  to promote channel openings. Voltage stepping the long NT isoform protein to –200 mV elicited an inward current approximately –4 pA in magnitude that decreased as voltage approached 0 mV (o) was 0.020 ± 0.003 (n = 33) and the mean open dwell time was approximately 18.6 ± 2.4 ms (n = 13), consistent with long channel openings associated with binding by BayK(–).The Cas (I-IV) . Each dohed 0 mV  reversedhed 0 mV . The opev1.2 channel is expressed predominantly as two alternatively spliced variants in human heart. Exon 1a codes for the initial 46 amino acids in the N-terminus of the long N-terminal (NT) variant and exon 1b codes for 16 amino acids in the short NT variant (o of 0.021 ± 0.001 (n = 11). When DTT was applied the Po decreased on average to 0.008 ± 0.001 and then following addition of DTNB the Po increased to 0.033 ± 0.002  for the C-terminal truncated channel. This is significantly larger than the Po recorded in the long NT isoform , when voltage was stepped to 100 mV. After application of DTNB, the Po significantly increased from 0.017 ± 0.003 to 0.026 ± 0.003 (n = 12), and in separate experiments the Po decreased from 0.020 ± 0.003 to 0.013 ± 0.002 after application of DTT (n = 19), without altering the current-voltage relationship or mean open dwell time . Application of DTNB  and attenuate the effects of GSH and GSSG on Po  recorded in the loop I-II linker mutant protein and C543S mutant protein under control conditions  decreases peak inward native L-type Ca2+ channel current (ICa-L) in cells and application of thiol oxidising agents attenuates the effect1314353637Ca-L functionThe second point that we clarify concerns whether the channel has an intrinsic ability to sense changes in oxygen tensionin vivo studies have argued against thiso , direct regulation of the Cav1.2 alpha subunit is sufficient to alter calcium influx. Since glutathionylation of Cav1.2 occurs in the ischemic human heartv1.2 is sufficient to induce hypertrophic growth23v1.2 channel protein during reperfusion may contribute to the pathology associated with ischemic heart disease.We conclude that the function of the Cav1.2 was cloned into pcDNA3.1 vector (Invitrogen), and modified to include a HIS6 tag at the N-terminus. This construct was used as the backbone for mutagenesis. Site-directed mutagenesis was undertaken to make point mutations in the LNT cDNA using the Quikchange kit according to the manufacturers protocol (Stratagene). Cysteines were mutated to serines. In some cases cysteines were mutated to alanine in order to prevent potential phosphorylation of the –OH group in vitro when preceded by an arginine at –3 position. Two oligonucleotide primers containing each desired mutation were extended by Pfu Ultra II DNA polymerase (Stratagene) during PCR. A standard PCR reaction was prepared with 1 μl of cDNA; 1μl of 10 pmol forward and reverse primers each, 5 μl of 10 XPfu buffer; 2 μl of 5 mM dNTP’s; 1 μl of Pfu Ultra II DNA polymerase and 39 μl of nuclease free water. A standard amplification protocol was performed . Mutants were confirmed by automated DNA sequencing and restriction digests using XbaI and BamHI restriction enzymes (Promega). Correct mutant DNA was then purified from an overnight culture using low copy plasmid purification protocol (Nucleobond Xtra Midi plus) kit.The cDNA of the long N terminal isoform of the Cav1.2 channel was used as a template to amplify the linker I-II region using an appropriate primer. The primer was designed to introduce BamHI and EcoRI restriction sites in the cDNA. A standard reaction mix was prepared with using KOD Hot Start DNA polymerase kit as per manufacturer’s instructions (Merck-Millipore). The linker I-II region sequence incorporating Glycine 377 to asparagine 495 was cloned into BamHI and EcoRI sites in pGEX-2T vector with a glutathione-S-transferase (GST) tag. Vector and insert were digested with BamHI and EcoRI restriction enzymes at 37 °C for 1 h. Vector and insert were gel purified and then ligated at 4:1 ratio using T4 DNA ligase (Invitrogen). Ligated product was transformed into XL-2 blue competent cells. Colony PCR was performed to identify the colonies containing the insert. Positive clones were further confirmed by restriction digests using BamHI and EcoRI as well as by Sanger DNA sequencing. The recombinant pGEX-2T vector was grown on Luria Bertini-Ampicillin-Chloramphenicol (LB-Amp-Chloramp) agar plates overnight. Several colonies were then inoculated in 5 ml LB-Amp-Chloramp broth at 225 rpm, 37 °C for approximately 3 h. Once the OD600 reached 0.6, 250 μl of 100 mM IPTG was added to the culture and then incubated for approximately 5 h at 37 °C. Following incubation, the cells were centrifuged at 20000 rcf for 5 min at room temperature. The cell pellet was resuspended in 1× phosphate buffered saline (PBS) containing 1× protease inhibitor tablet . The cell suspension was then lysed by sonication and clarified by centrifugation at 20000 rcf for 5 min at 4 °C.The cDNA of the truncated C-terminal construct of the Ca6 cells per l5 cm plate) were transfected with 15μg plasmid DNA using 35μl Lipofectamine transfection reagent according to the manufacturers protocol (Invitrogen). Cells were harvested 48 h after transfection. The cell pellet was lysed using lysis buffer . The cell suspension in the lysis buffer was sonicated and centrifuged at 10000 rpm for 10 min at 4 °C before the addition of Ni-NTA agarose beads (Qiagen). The lysate was placed on a magnetic stand and the beads were allowed to migrate towards the magnet, after which the supernatant was removed, and the beads were washed 3–4 times with 500μl wash buffer . After the last wash, 30μl of elution buffer  was added to the beads and was incubated for 1 min at room temperature The beads were allowed to migrate to the magnetic stand, following which the supernatant was removed and stored at –80 °C for later use. The purified protein was analyzed by SDS-PAGE and immunoblotting.HEK293 cells  to promote the blister formation of liposomes for 10 min at room temperature. The loading solution was then replaced by recording solution  to facilitate single channel current recording. Functional examination of Cav1.2 channel protein reconstituted in liposomes was performed using the patch clamp technique. External and pipette solution contained recording solution with 2μM BayK8644 (Sigma) to enhance channel opening. The back filled microelectrode with pipette solution had an average resistance of 16–17 MΩ. A gigaohm ohm seal was achieved upon contact between the microelectrode and a liposome blister. Single channel currents were filtered at 1 kHz, digitized at 100 kHz, and analysed using pClamp software (Molecular devices). The Cav1.2 channel was determined by the magnitude of the current, changes in open probability of the channel (Po) and sensitivity of the current to the L-type calcium channel antagonist nisoldipine. The recordings were made for a duration of 10 min. Clampfit 10.5 software was used for analysing the total number of events that the channel was open. The amplitude distribution of single channel events was analysed by Gaussian function and was fitted with Levenberg-Marquardt method  and then a goat anti-rabbit HRP conjugate secondary antibody (Abcam). The channel protein was finally, detected by .The purified channel protein (50μg) was incubated with 5× sample buffer  for 30 min at 45 °C. The channel proteins were separated by 6% sodium dodecyl sulphate – polyacrylamide gel electrophoresis (SDS-PAGE) for 1.5 h at 25 mA constant current per gel. The channel protein was electrophoretically transferred from the SDS-PAGE gel to nitrocellulose membrane using a wet transfer apparatus  in transfer buffer  for 16 h at 30 V, 400 mA and 100 W with constant voltage at 4 °C. After transfer, the membrane was incubated in blocking buffer , 0.05% Tween-20 in PBS, pH 7.4) for 1 h at room temperature. The membrane was then probed with an anti CaWild type and mutant channel proteins were expressed individually in HEK293 cells. The protein (50μg) was incubated with 5x reduced sample buffer  for 5 min at 95 °C. The channel protein was separated by 6% SDS-PAGE for 1.5 h at 25 mA constant current per gel. The 240–245 kDa protein band corresponding to the molecular weight marker was excised from the gel and was cut into fine pieces. The fine pieces of gel were immersed in 0.5 ml of elution buffer  and incubated overnight in a rotary shaker at room temperature. Following incubation, the suspension was centrifuged at 10000 × g for 10 min at 20 °C. The supernatant was transferred into a new tube. The supernatant was also tested for the presence of protein by SDS-PAGE. The purified protein (25μg) was mixed with 1:1000 dilution of 5000X Sypro Orange (Molecular Probes). All experiments were performed in triplicates. The samples were heated from 25 °C to 95 °C with a heating rate of 1 °C/min. Protein thermal unfolding curves were monitored by detection of changes in fluorescence of the Sypro Orange dye. The melting temperature (Tm) of wild type and mutant proteins was determined by converting the raw fluorescence data to the negative first derivative of the fluorescence with respect to the temperature (–dF/–dT).t-test or between groups of data using one-way ANOVA and the Tukey’s posthoc test.Results are reported as mean  ±  SEM here indicated. Statistical comparisons of responses between unpaired data were made using the student’s How to cite this article: Muralidharan, P. et al. Evidence for redox sensing by a human cardiac calcium channel. Sci. Rep. 6, 19067; doi: 10.1038/srep19067 (2016).


Selective catalytic reduction using vanadium supported TiO2 catalyst in mobile and off-road engine has been considered to be major source for N2O emission in the system. Here we have demonstrated that vanadium catalyst supported on zeolitic microporous TiO2 obtained from the hydrothermal reaction of bulk TiO2 at 400 K in the presence of LiOH suppresses significantly the N2O emission compared to conventional VOx/TiO2 catalyst, while maintaining the excellent NOx reduction, which was ascribed to the location of VOx domain in the micropore of TiO2, resulting in the strong metal support interaction. The use of zeolitic microporous TiO2 provides a new way of preparing SCR catalyst with a high thermal stability and superior catalytic performance. It can be also extended further to the other catalytic system employing TiO2-based substrate.Emission of N Most recent diesel engine emission regulation is now started to include N2O because of its high impact and stability in stratospherethe state of art technology for the reduction of NOx in most engine companies2O formation can be suppressed readily while the system has been maintained under oxidizing condition. However, the emission of N2O from diesel engine can be increased when the reducing agent for NOx is introduced in the SCR system following the reactions, such as 2NH3 + 2NO + O2 = N2O + N2 + 3H2O, 2NH3 + 2O2 = N2O + 3H2O and NH4NO3 = N2O + 2H2O3. The former two reactions were believed to be the major pathway for N2O formation in which bimolecular reaction can occur.Ever increasing demand for the reduction of greenhouse gas results in the more stringent regulation on its emission and also the corresponding research and development to capture or convert into inert moleculex abatement, VOx catalyst supported on TiO2 has been used widely in most diesel engines5672 synthesized using sol-gel method or organic or inorganic templating method91011121314x supported on TiO2 of relatively low surface area is the state of art technology catalyst. Indeed the current VOx/TiO2 catalyst emits N2O when the reducing agent is present in the stream. The current emission level of N2O is 50 mg per mile, which also depends on the catalyst composition and the system configuration such as diesel oxidation catalyst-selective catalytic reduction-diesel particulate filter2O emission characteristics of the VOx/TiO2 catalyst should be improved under reducing condition. For this purpose, the economically viable catalyst has to be developed in near future.For NO2 in the presence of alkaline hydroxide produces unique TiO2 structures differently depending on the species of alkaline hydroxide1617182, nanotube and nanorod, respectively19202 showed large surface area of 250 m2g−1 with the pore volume of 0.15–0.20 ccg−1, which was similar to those of zeolites and also suitable for catalyst preparation.It has been demonstrated that the hydrothermal conversion of commercially available TiO2 nanotube by hydrothermal synthesis in the presence of NaOH has been studied extensively but it is not clarified yet162 nanotube suggested that the nanotube is obtained with an acid washing and subsequent Na+ ion exchange after the formation of amorphous TiO2 during hydrothermal reaction between NaOH and bulk TiO2162022232 structure formed layered 2-dimensional structure and finally 1-dimensional nanotubes through sheet folding mechanism+ cation from the nanotube also deteriorates readily the thermal stability when it is heated at high temperature.The formation mechanism of TiO2 prepared from alkaline condition has not explored yet. Thus, it is interesting to know whether the zeolitic microporous TiO2 has a high thermal stability or not and also is suitable for catalytic application as a substrate. For the first time in the present work, the zeolitic microporous nanocrystalline TiO2 has been demonstrated as a catalyst support for VOx over selective catalytic reduction of NOx using ammonia in order to decrease the N2O formation.However, the use of zeolitic microporous TiO2 after the hydrothermal treatment. The morphology of the particle after the hydrothermal conversion contained sharp edges, suggesting the formation of the well-crystallized TiO2, which was consistent with the literature19202 had the typical argon adsorption-desorption isotherm consistent with the Langmuir isotherm type containing micropore mostly where the micropore area estimated from t-plot was ~200 m2g−1, corresponding to 80% of the total surface area of which the pore size was estimated to be ~7 Å. The corresponding surface area and pore volume were controlled to be 250 ± 20 m2g−1 and 0.20 ± 0.05 ccg−1, respectively, depending on the hydrothermal reaction condition. Also, the presence of the mesopore was observed above P/P0 > 0.9 but its portion can be decreased with the increase of hydrothermal reaction time. Such mesopore formation was also shown clearly in 2 frameworks of which the thickness was 3–4 nm.2 structure containing the micropore and also the mesopore of which the size was 5–7 nm, which was consistent with the result of argon and nitrogen adsorption-desorption measurement.Therefore, the results of the transmission electron micrograph observation supported the corresponding unique TiO2 into zeolitic microporous TiO2 in the presence of LiOH seems not to follow the sheet folding mechanism like TiO2 nanotube. The intercalation of Li+ ion into the TiO2 structure leads to the formation of the Li+-O-Ti bond similar to that of Na+ case, resulting in the partial delamination of the TiO2 layer where the interaction between the layers is high enough to induce the combination of the corresponding layers25The hydrothermal conversion of the bulk TiO2 under ambient condition either with the presence or absence of the saturated water was also investigated using the X-ray diffraction pattern (XRD) and N2 adsorption and desorption isotherms as a function of heating temperature. Nearly up to 773 K, the microporous structure was retained as evidenced from the results of the nitrogen adsorption-desorption isotherm for the sample shown in 2 to macroporous TiO2. The moderate thermal stability of the zeolitic microporous TiO2 due to the crystal growth into anatase in the presence of water may limit the catalytic application at higher temperature. However, many catalytic applications including photocatalysis or solar energy harvesting can adopt the present zeolitic microporous TiO2The thermal stability of the zeolitic microporous TiOx was supported onto the corresponding unique TiO2 structure following the procedure reported in the literature2728293031x catalyst up to 5 wt% resulted in the lower surface area of 105 m2g−1 with 0.19 ccg−1 because of the high calcination temperature at 773 K though the sample still contained large surface area. These textural properties were maintained before and after the catalytic reaction measurement. The TEM observation of VOx incorporated TiO2 as shown in x particle are located inside the pores of the zeolitic microporous TiO2, ~7 Å without changing the corresponding morphologies. Thereby, the VOx particle was observed clearly as a spot in 2 though the elemental analysis showed the presence of V in the same region as shown in VO2 catalyst after the calcination at different temperatures also under various reaction conditions. Increasing V content in the catalyst improved the catalytic performance of the NOx reduction comparable to that of conventional V/TiO2 catalyst containing 5 wt% V over the whole reaction temperature range while the N2O formation was much lower than that of conventional catalyst. The N2O formation from the present V/TiO2 was increased with the increase in the temperature where NH3 was combined with NOx to produce N2O. The effect of the calcination temperature was also pronounced to increase the N2O formation at high temperature but the N2O formation was still lowered than that of the conventional catalyst by more than 80%. Also, the V/TiO2 catalyst prepared from the microporous TiO2 resulted in the superior catalytic performance over the SCR reaction both in the presence of water in the reactant stream and after the aging in the presence of water at 773 K for 12 h, as shown in 3 and H2O suppressing the NH3 oxidation.2O formation is believed to be 2NH3 + 2NO + O2 = N2O + N2 + 3H2O following the literaturex inside the pore, ~7 Å was believed to have a strong metal support interaction with TiO2, resulting the smaller VOx particle size as referred from 2 and VOx led to the formation of Bronsted acid site with high strength, which is beneficial for selective catalytic reduction of NO by NH3. The N2O formation can be suppressed up to ~80% because of the increased Bronsted acidity of the VOx small particle in the microporous zeolitic TiO2 where the superior SCR activity can be maintained as illustrated in 2O formation on the V/TiO2 catalyst when the catalyst deteriorates because of the sintering3 oxidation by O2 as potential pathway for the following N2O formation reaction: 2NH3 + 2O2 = N2O + 3H2O where the catalyst deactivation was severe like the commercial V/TiO2 catalyst in the presence of water or after hydrothermal aging. One possibility to explain the superior catalytic performance V supported on zeolitic microporous TiO2 over the SCR reaction was that the growth of the vanadium oxide particle size can be limited due to the pore size, implying the encapsulation of vanadium oxide particle surrounded by TiO2 matrix.Under the present condition, the main reaction for N2 with moderate thermal stability can be prepared from the simple hydrothermal conversion from commercially available bulk TiO2 of low grade, 98% or lower in the presence of LiOH at 400–440 K, which can be scaled up easily for industrial process. The obtained zeolitic microporous nanocrystalline TiO2 contains the micropore up to 80% referred from the t-plot method. For the first time, it was proved that that the supporting VOx into such zeolitic microporous TiO2 resulted in the high NOx reduction activity with lower N2O formation, which was ascribed to the location of catalytically active VOx particles in the microporous TiO2, resulting the strong metal-support interaction and consequently the increased Bronsted acidity. Therefore, the zeolitic microporous TiO2 has potential as a substrate for the SCR reaction below 773 K while the thermal stability of the microporous TiO2 was retained.We have demonstrated that the zeolitic microporous TiO2 anatase  of 2–8 g was added to the solution containing 10 M or more LiOH in the Teflon lined autoclave for hydrothermal heating at 400–440 K for 72 hr under rotating condition at 40 rpm. After cool down to room temperature, the slurry was neutralized with 0.1 N HCl under stirring for 6 hr. The solution was filtered and washed with deionized water thoroughly. The acidification and filtration was repeated three times to remove the residual trace metal hydroxides. The obtained product was dried at 330 K in an oven and calcined under flowing oxygen at 673 K for 4 h. The inductively coupled plasma analysis of the obtained sample showed that the residual Li was ~6 ppm level, indicating the complete removal of Li+ by the neutralization and subsequent thorough washing. The scale up to ~100 g per batch was also demonstrated to give the same textural properties.TiO2 powder  was used as support to prepare the conventional catalyst containing 5 wt% V. The samples with 1 wt%, 3 wt% and 5 wt% V2O5 loading on TiO2 were prepared. After impregnation process in a rotary evaporator, catalysts were dried and then calcined at 673 K or 773 K for 4 h in air.All catalysts were prepared by applying wet impregnation of vanadium precursor solution on titania. Ammonium metavanadate  was dissolved in diluted oxalic acid solution (0.5 M) to produce the solution of vanadium precursor. Anatase TiO3, 2% O2 and balanced with N2 were introduced as reactants. In order to examine the catalytic activity in the presence of water, the reactant containing 500 ppm NO, 500 ppm NH3, 2% O2, 3% H2O balanced with N2 was used. The catalyst was further aged in the presence of 10% O2, 5% H2O balanced with N2 at 500 oC for 12 h before catalytic reaction.SCR activity was measured in a fixed-bed quartz tubular reactor. Catalysts were sieved to 300–500 μm in diameter then loaded in the reactor. 500 ppm NO, 500 ppm NH−1. We raised reaction temperature from 423 K to 673 K by 50 K. NOx concentration of outlet gas by using NOx chemiluminescence analyzer . Also, Fourier Transform Infrared (FT-IR) spectroscopy was applied to observe the N2O concentration in the gas. We used the average data of 16 scans at a resolution of 1.0 cm−1. A Nicolet 6700 (Thermo Scientific) with 2 m gas analysis cell heated to 120 °C to exclude the effect of H2O, was used for gas phase analysis.Space velocity of inlet gas was maintained to be 40,000 hHow to cite this article: Lee, S. G. et al. Suppressed N2O formation during NH3 selective catalytic reduction using vanadium on zeolitic microporous TiO2. Sci. Rep.5, 12702; doi: 10.1038/srep12702 (2015).


T) for perovskite compounds is acknowledged for evaluating crystal structures in terms of the ionic packing. However, its applicability is limited to perovskite compounds. Here, we report on extending the applicability of T to ionic compounds with arbitrary ionic arrangements and compositions. By focussing on the occupancy of constituent spherical ions in the crystal structure, we define the ionic filling fraction (IFF), which is obtained from the volumes of crystal structure and constituent ions. Ionic compounds, including perovskites, are arranged linearly by the IFF, providing consistent results with T. The linearity guides towards finding suitable unit cell and composition, thus tackling the main obstacle for determining new crystal structures. We demonstrate the utility of the IFF by solving the structure of three hydrides with new crystal structures.Crystal structure determination is essential for characterizing materials and their properties, and can be facilitated by various tools and indicators. For instance, the Goldschmidt tolerance factor ( Every compound can be identified by a unique arrangement of the atoms in the crystal structure, which determines the inter–atomic distances and the compositional ratios and thus helps understanding specific material properties. For this reason, crystal structure determination and analysis are essential not only from a mere crystallographic viewpoint, but also for numerous fields of materials research, chemistry, physics and life sciences.T ) of perovskite compounds ABX3  is widely used to assess the geometric stability and distortion of crystal structures in terms of the constituent ionic packing. The T is defined by ratios of constituent ionic radii of A, B and X as T = (RA + RX)/√2(RB + RX), where RA, RB and RX are the ionic radii of A, B and X, respectively , a geometric strain and crystal distortions arise. As the deviation from T = 1 becomes greater, the crystal adopts structures of lower symmetry than the cubic one. Thus by calculating T, the crystalline structure can be predicted and its geometric strain and stability evaluated. At the same time, the T can be used to estimate the compatibility of different ions with a crystalline structure. Because of its simplicity and practicality, the T is used extensively in a wide variety of fundamental and applied studies1234The evaluation of crystal structures can be facilitated by various tools and indicators. For instance, Goldschmidt tolerance factor  normalized by number of formula units (Z) and the total volume of constituent ions that reflects the composition ratio (Vion). Although those quantities, Vunit and Vion, have been previously used for crystal structure determinations of hydrides7161718Vunit and the Vion into various ionic compounds with hydrides, oxides and halides focusing on the relation between the Vunit normalized by Z, (Vunit/Z), and the Vion. The relation between the Vunit/Z and the Vion can be described as occupancy of constituent spherical ion in the crystal structure. We refer to it as the ionic filling fraction (IFF). The IFF surprisingly leads to well organize crystal structures of arbitrary ionic compounds. Here, we use the IFF for assessment of ionic packing of crystal structures from a geometric point of view and discuss how this can be related to the T. In addition, we demonstrate the suitability of the IFF for determining the crystal structure of three Al–based complex hydrides  with new crystal structures, LiCa(AlH4)3, LiCaAlH6 and Sr(AlH4)Cl, in which both mixed cations or anions coexist. Such compounds are attractive for fundamental understanding of crystal structures for related complex hydrides which are interests for high gravimetric hydrogen applications. It is thus important to understand their structural features for further progress9101112In this communication, we propose to extend the applicability of the T). In an ideal cubic perovskite structure with a lattice constant a, T is defined by the ratio of three kinds of ionic radii as shown a, T < 1 corresponds to a loosely packed crystal structure with small radius of A and T > 1 corresponds to a tightly packed crystal structure with large radius of A .We firstly consider the Goldschmidt tolerance factor . However, it might be more convenient to consider the relative sizes of the asymmetric unit in the crystal by evaluating the volume of the unit cell (Vunit) normalized by the number of formula units (Z): Vunit/Z. On the other hand, crystal structures are also dependent on the constituent components and their relative fraction (composition ratio). To observe this dependence, composition ratios can be represented in terms of volume using the same units as those for Vunit/Z. Assuming spherical ions with a volume governed by their Shannon radiiVion) can be calculated according to the stoichiometric ratio of the ionic compounds. Then, the ratio between Vunit/Z and Vion represents the occupancy of constituent spherical ion in the crystal structure for ionic compounds with arbitrary ionic arrangements and composition. We will refer to the concept, which is the ratio between Vunit/Z and Vion, as the ionic filling fraction (IFF). The schematic figure for the IFF to an ideal cubic perovskite compound shows in T is limited in fixed number of constituent ions with perovskite–type structure, it should be noted that the IFF concept can be flexibly responded any modifications of crystal structures and numbers of constituent ions.In order to extract occupancy of constituent spherical ions in the crystal structure from different crystal structures of various kinds of ionic compounds, we need to secondly define a standard approach for those ionic compounds. The repeating unit of a crystalline compound is determined by the unit cell given by the lattice constants . The Vunit/Z and Vion values of these compounds are listed in Vunit/Z = 1/0.69 × Vion, corresponding to IFF of 0.69. This suggests that ionic compounds adopt a constant IFF value (0.69). The constant IFF is close to the IFF of a body–centred cubic crystal structure containing a single component (IFF = 0.68). The hydrides in Using the IFF, we extend the applicability to ionic compounds including hydrides with a variety of chemically and structurally diverse compounds. 1.40 Å is used as the radius of the HVunit/Z ≧ 1/0.69 × Vion denotes crystal structures containing voids. In particular, ionic compounds with IFF ≦0.50 2 RAlH6 Vunit/Z ≦ Vion, This region should feature not optimal crystal structures or composition ratios.In the IFF plots, the region for which T = 1 for the ideal perovskite oxides ABO3 is shown in a plot of Vunit/Z vs. Vion in a is set as the sum of the diameters of B ions and oxygen ions (radius: 1.40 Å), and the ratio between RA and RB is estimated from the T. It is worth noting that the data for the crystals with T = 1.0 (blue line) is distributed in the vicinity of the Vunit/Z = 1/0.69 × Vion line (black line). In addition, considered on the relationship between the T and the IFF on the perovskite oxides listed in the T as a function of normalized IFF by the constant IFF for perovskite oxides shows in T show to increase with increasing of their IFF  as described above on an ideal cubic perovskite compound 3, LiCaAlH6 and Sr(AlH4)Cl, all showing new crystal structure and adopting complicated crystal structures where both mixed cations or anions coexist.The selection of an optimal unit cell and composition ratio is one of the most difficult steps in the determination of the crystal structures. However, this can be considerably simplified by using the linearity obtained from the IFF and following the procedure summarized in the flow chart of 2 and AlH3, and evaluating the Raman and infrared spectra (4)3. Indexing of the Bragg peaks derived from the diffraction patterns of LiCa(AlH4)3 shows that there are several possible candidates for the unit cell. This is likely due to poor crystallinity of the sample and the overlap of Bragg peaks from multiple phases or the same phase, and thus a limited number of Bragg peaks that can be used for indexing. Generally, an optimal unit cell is selected out of these candidates by means of trial and error and the space group, composition ratio and initial structure model of the lattice must be predicted. The use of the linearity obtained from the IFF minimizes this trial and error process (4)3, we can estimate the Vion by using the ionic radii for Li+, Ca2+ and [AlH4]− . This results in a value between 150 and 158 Å3. Using the relationship Vunit/Z = 1/0.69 × Vion, Vunit/Z was estimated to be in the 217–230 Å3 range. Since Z is an integer, Vunit will roughly be an integer multiple of Vunit/Z. By searching for unit cells with the appropriate indices and a Vunit value close to an integer multiple of this Vunit/Z, the range of the possible unit cells is narrowed down and the selection of the candidate unit cell is simplified. In this work, the unit cell of LiCa(AlH4)3 was chosen to be a hexagonal crystal  with Z = 2. Based on hkl extinction rules, we predicted the space group to be either P63 (No. 173), P63/m (No. 176) or P6322 (No. 182), and using this information, we then identified the CdTh(MoO4)3 structureP63/m (No. 176), a = 9.80 Å, c = 6.35 Å) as the optimal initial crystal structure model. The complete crystal structure of LiCa(AlH4)3 was then determined by combining the Rietveld refinement fit 3 crystal structure, Li+ is six–fold coordinated by [AlH4]−, Ca2+ is nine–fold coordinated by [AlH4]− and [AlH4]− is two–fold coordinated by Li+ and three–fold coordinated by Ca2+; thus, the total CN = 5 (detailed inter–atomic distances are shown in the 4]− coordination number is in between the coordination of [AlH4]− in the related compounds LiAlH4 (CN = 4)4)2 (CN = 6)In the LiCa(AlH4)3 was reported based on experiments+ is located between Ca2+ and [AlH4]−, in contrast with the previously reported model, where all ions are positioned on the same (004) plane. From Rietveld refinements it is showed that the fit of the previously reported model is worse (Rwp = 0.046) than ours (Rwp = 0.040). First–principles calculations also showed that the previously reported model is less stable than our crystal structure model, the difference being 36 kJ/mol. Furthermore, and phonon calculations show the presence of imaginary frequencies. These results suggest the model we propose is a more accurate description of the crystal structure.Recently, the crystal structure of LiCa(AlH4)3 releases hydrogen at around 400 K 3, in analogy with the related mixed cation alanate LiMg(AlH4)36. Accordingly, we can estimate the Vion by using the ionic radii for Li+, Ca2+ and [AlH6]3− . This results in a value between 75 and 84 Å3. Using the relationship Vunit/Z = 1/0.69 × Vion, the Vunit/Z was estimated to be in the 109–121 Å3 range. The PXD pattern of LiCaAlH6 is indexed using a primitive tetragonal unit cell with a ≈ 6.59 Å, c ≈ 16.78 Å, and Vunit ≈ 729 Å3. Z is estimated to be 6 or 7 from the estimated Vion, but hydrides with [AlH6]3− are plotted in the region of Vunit/Z ≦ 1/0.69 × Vion in 6]3− adopts tightly ionic packed crystal structures. Therefore, the range of Z is expanded to being in the range of 6–8. Since no prototype structure was found from the unit cell , the Z (=6–8) and the composition, the initial crystal structure model was predicted in combination with ab initio structural determination FOXP–4 (No. 81) was finally determined. The Rietveld fit is shown in Z and IFF are finally determined as 8 and 0.82 (Vion = 75 Å3 and Vunit/Z = 91 Å3), respectively. The IFF agrees reasonably with hydrides with [AlH6]3−  is 12 in the related Li3AlH69 and 6 or 7 in CaAlH56]3− in LiCaAlH6 was found to be 9, which is the mean of the CNs in Li3AlH6 and CaAlH5. This is similar to LiCa(AlH4)3.In the crystal structure, LixLiAlH4 + SrCl2 (x = 1.0–3.0), with x = 1.3 providing the highest sample purity . The space groups was predicted to be either Pmn21 (No. 31) or Pmmn (No. 59) based on the hkl extinction rules. Raman and Fourier transform infrared spectra . Since the Vunit/Z of all tetra–alanates is bigger than 69 Å3 , we expect 3 moles of [AlH4]− anions per formula unit, which must be counter-balanced by three positive charges from the cation(s) (4)3 with a hexagonal unit cell , which is however identified in the PXD pattern of the starting mixture 3.0LiAlH4 + SrCl2 3 can be clearly distinguished. Thus we have to exclude Z = 1. Z = 3 results in a too small Vunit/Z ≈ 69 Å3, which is the same value observed for LiAlH4 (Vunit/Z ≈ 69 Å3)Vunit/Z in the tetra–alanates. We must therefore also rule out Z = 3. This leaves Z = 2 as the only possible value, resulting in Vunit/Z ≈ 103 Å3. The composition ratio that fulfils the conditions of IFF < 1.0 and electro–neutrality is predicted to be Sr(AlH4)Cl , with coexisting [AlH4]− and Cl− anions. Based on this information, the optimal initial crystal structure model was found isostructural with the PbCl(ReO4) structure (space group Pmn21 (No. 31), a = 5.68 Å, b = 9.44 Å and c = 4.47 Å)4)Cl is shown in The sample was synthesized by the solid-state metathesis reaction of e purity . The Bra spectra  proved tan 69 Å3 9, the Z ation(s) . One pos + SrCl2 . In the 4)Cl compound with coexisting [AlH4]− and Cl− anions. In this structure, Sr2+ is coordinated by four Cl− and five [AlH4]− ions  and the total volume of spherical ions (Vion) estimated through stoichiometry assuming spherical constituent ion in order to extend the applicability of Goldschmidt tolerance factor (T). The ratio between Vunit/Z and Vion defined as the ionic filling fraction (IFF) was used to extract ionic packing information from ionic compounds similar to using T. The IFF for most ionic compounds are interestingly distributed around a line given by Vunit/Z = 1/0.69 × Vion. The linearity (the inverse of the slope) suggests that ionic compounds adopt a constant IFF value (0.69). The linearity was also consistent with the ideal cubic perovskite structure with the T = 1. Furthermore, the IFF showed the trend that the IFF increases with increasing T. Thus, the IFF allows uniformly classifying the data for a variety of ionic compounds and extending the applicability of the T concept for arbitrary ionic compounds.Ionic compounds with arbitrary ionic arrangements and compositional ratios were sorted using the unit cell volume per formula unit (4)3, LiCaAlH6 and Sr(AlH4)Cl, respectively, with different cations and anions coexisting within the crystal structures. Therefore, the IFF could become a new indicator for crystal structure determination.The use of this linear relationship facilitates the selection of unit cell and compositional composition ratios for novel hydrides, which has often been a bottleneck for the determination of their crystal structures. We have been able to elucidate three new crystal structures, LiCa(AlH4)3 was directly synthesized by mechanochemical milling of LiH , CaH2  and AlH3 in a molar ratios of 1:1:3. AlH3 as the starting material was synthesized in diethyl ether according to the chemical reaction of LiAlH4 and AlCl3414)3 was 10 hours. Milling time of 15 min was alternated with pauses of 5 min duration.LiCa(AlH6 was yielded from LiCa(AlH4)3 which was kept at 400 K for 1 hour in 0.10 MPa of Ar gas pressure.LiCaAlH4)Cl was synthesized by mechanochemical milling of LiAlH4  and SrCl2  in a molar ratios of x:1 (x = 1.0–3.0). The mixture was milled at 400 rpm under an Ar atmosphere of 0.10 MPa using Fritsch P7. The effective milling time was 5 h. Milling time of 15 min was alternated with pauses of 5 min duration.Sr(AlH4)Cl for powder synchrotron radiation (SR-PXD) X-ray and neutron diffraction (PND) was synthesized by mechanochemical milling of LiAlD4  and SrCl2  in a molar ratios of 1.3:1.Sr(AlD4)3 and LiCaAlH6 for PXD and the Rietveld refinements were placed in Lindemann glass capillary  and sealed with paraffin liquid for the powder X-ray diffraction measurement with a transmission geometry at room temperature.All samples were initially measured by a conventional powder X–ray diffractometer ) at room temperature. The samples of LiCa(AlH4)Cl were collected at room temperature at the Swiss-Norwegian beamlines (station BM01B) at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France. The sample was placed in a rotating 0.5 mm boron-silica glass capillary. The wavelength 0.50513 Å was obtained from a channel-cut Si (111) monochromator. Data was collected up to 40° in step of 0.0065° in 2θ.The high-resolution SR–PXD data of Sr(AlD4)Cl were collected at room temperature with the PUS instrument at the JEEP II reactor at Kjeller, Norway. The sample was placed in a cylindrical vanadium sample holder with a diameter of 6 mm. The wavelength 1.5539 Å was obtained from a Ge (511) focusing monochromator. Data was collected from 10° to 130° in step of 0.05° in 2θ.PND data of Sr(AlD4)3 and Sr(AlH4)Cl were obtained by Nicolet Almega–HD with a Nd:YVO4 laser (532 nm). The sample was placed under Ar gas in a sample holder with a glass window, and spectra were measured using the backscattering configuration with a microscope Raman spectrometer. The FTIR spectra of LiCa(AlH4)3 and Sr(AlH4)Cl were obtained by Thermo Scientific Nicolet iZ10. A thin sample, approximately a few μm thick, was prepared in an Ar–gas–filled diamond anvil cell, and transmission spectra were measured with a microscope FTIR spectrometer.The Raman spectra of LiCa(AlH4)3 was studied by thermogravimetry  with an Al sample holder heated up to 673 K at 5 K/min in a He gas flow of 150 ml/min.The hydrogen release reaction of LiCa(AlH4)3, LiCaAlH6 and Sr(AlD4)Cl were indexed by TREOR974)3 and LiCaAlH6 based on PXD data and Sr(AlD4)Cl with SR-PXD and PND data were performed combined with the ab initio structural determination program FOX (version 1.9.0.2)46The X-ray diffraction peaks of LiCa(AlH2 to prevent (hydro–) oxidation.All samples were handled in Ar or He gas filled glove boxes with a dew point below 183 K and with less than 1 ppm of O4)3 and LiCaAlH6 were studied using first-principles calculations based on density-functional theory (DFT). We used a plane-wave basis and the projector augmented wave method50514)3 and LiCaAlH6 were fully relaxed with placing the H atoms to coordinate Al in the tetrahedral and octahedral fashions, respectively. After the relaxations, phonon calculations were performed to verify if the relaxed structures were at the true minimums. When imaginary phonon frequencies were observed, we slightly displaced the atoms along the directions of eigenvectors of the imaginary modes and further relaxed the structures to eliminate them. This procedure was carried out until the ground states were reached, in the same manner as in the prediction of crystal structures for several new complex transition-metal hydrides4)3 and LiCaAlH6, respectively. The Brillouin-zone samplings were performed using the special k-point method4)3 and LiCaAlH6, respectively.The crystal structures of LiCa.


The layer controllability and large area uniformity essential for electronic and optical device applications is achieved through atomic layer deposition in what is named self-limiting layer synthesis (SLS); a process in which the number of layers is determined by temperature rather than process cycles due to the chemically inactive nature of 2D MoS2. Through spectroscopic and microscopic investigation it is demonstrated that SLS is capable of producing MoS2 with a wafer-scale (~10 cm) layer-number uniformity of more than 90%, which when used as the active layer in a top-gated field-effect transistor, produces an on/off ratio as high as 108. This process is also shown to be applicable to WSe2, with a PN diode fabricated from a MoS2/WSe2 heterostructure exhibiting gate-tunable rectifying characteristics.This work reports the self-limiting synthesis of an atomically thin, two dimensional transition metal dichalcogenides (2D TMDCs) in the form of MoS Two-dimensional 2D) materials, and the heterostructures that can be created from them, have been widely studied due to their atomic-scale thickness, flexibility and unique electrical/optical properties1D materia3462/WSe2 and MoS2/graphene) have been the most heavily studied semiconducting 2D materials34910111291416171921232526Transition metal dichalcogenides (TMDCs) and their relevant 2D heterostructures  is achieved through ALD by combining precursor exposure, purging, reactant exposure and a final purging into a single cycle. In this way, a point is reached at which the number of layers produced is determined purely by the growth temperature; a unique behavior that is directly attributable to the chemical inactivity of the 2D MoS2 surface. The characteristics and layer uniformity achieved are subsequently assessed through spectroscopic and microscopic analysis, and the universality of the process itself is tested by applying it to the fabrication of a MoS2/WSe2 heterostructure for use in a diode.In this study, the self-limiting layer synthesis (SLS) of a 2D TMDC  MoS2exc = 532 nm) in 2 exhibits E12g and A1g modes from in-plane and out-of-plane vibrations at 384.6 cm−1 and 404.8 cm−1, but these shift to 384.1 and 405.2 cm−1 with 2L MoS2, and to 383.5 and 406.8 cm−1 with 3L MoS2. The peak distance between E12g and A1g is often used to determine the number of MoS2 layers, as an increase in layers is accompanied by a softening of the E12g mode frequency and a stiffening of the A1g mode frequency30−1 for 1L, 21.1 cm−1 for 2L, and 23.3 cm−1 for 3L all agree well with previously reported values for MoS229322 layers by SLS.2 are shown in 2 spectrum exhibits a strong PL signal at 1.89 eV and a weak, wide PL signal at 2.05 eV, which correspond to the A1 and B1 direct excitonic transitions of MoS2142, and become negligible with 3L MoS2, as the increasing number of layers induces a transition from a direct to an indirect band gap. This is concordant with previous results regarding the dependence of the PL signal on the number of layers14342.The PL spectra of the synthesized MoS2 obtained on a SiO2 substrate, this was used a criterion to assess the effects of varying the number of process cycles from 40 to 250 at growth temperatures of 500, 600, 700, 800 and 900 °C . This stands in stark contrast to conventional ALD, in which the thickness does in fact increase linearly with the number of process cycles. We can therefore only conclude that the growth mechanism of the current SLS process is totally different from that of conventional ALD.Given the good correlation between the Raman peak distances and the number of layers of MoS2. Specifically, during the growth of the first layer, precursor molecules (MoCl5 in this case) chemically adsorb to the abundant adsorption sites on the SiO2 surface. However, once this initial layer is formed over the entire surface, any further chemical adsorption of precursor molecules is hindered by the absence of suitable adsorption sites on the newly created TMDC surface285 molecules on MoS2; with the adsorption/desorption of precursor molecules under this physical adsorption-dominant regime being determined by the growth temperature. This can perhaps be better explained by the framework of the Lennard-Jones potential model: i.e., at lower temperatures molecules are trapped in a potential well because their thermal energy is less than the potential depth, whereas at higher temperatures they have sufficient thermal energy to escape28This peculiar “self-limiting” behavior of ALD during the SLS process is believed to be caused by the inherently chemically inactive nature of the surface of TMDCs such as MoS2 that is induced by the positive charge between it and the SiO2 substrate also affects the potential depth of the precursor molecules; the surface potential of MoS2 decreasing with an increasing number of MoS2 layers due to their screening effect on the electric field5 molecule on MoS2 in the same way that the surface potential of physically adsorbed CH4 on h-BN decreases with an increasing number of layers2 layers has been formed at any given growth temperature, any MoCl5 molecules adsorbed onto the MoS2 basal plane can be easily desorbed due to the reduced potential depth, thereby creating a self-limiting growth behavior.The surface potential of MoS2 relies on using a sufficiently high process temperature to ensure the formation of layered 2D structure with chemically inactive surface. The lower crystallinity and non-layered 3D structure at lower growth temperatures causes deposition to proceed through chemical adsorption of precursor molecules, as is the case in the conventional ALD of MoS2 without self-limiting growth behavior215 being used in this study due to its higher thermal stability relative to other metal organic precursors.It should be noted here that the SLS of 2D MoS2 obtained through SLS was evaluated at different scales through Raman mapping of the peak distances between the A1g and E12g modes. The Raman map of the 1L SLS MoS2 in 2 onto 1.5 × 9 cm2 SiO2 substrates; the substrate size being limited in this instance by the diameter (~3 cm) and length (15 cm hot zone) of the tube furnace used. It is clear from 2 is certainly dependent on the number of layers, but to assess the cm-scale uniformity, Raman spectra were measured at nine different positions along the length of the SLS MoS2. The peak distances and full-width at half-maximum (FWHM) of the E12g and A1g modes are plotted in 12g and A1g modes with position is quite small . In addition, the Raman peak distance varies from 20.2 to 23.4 cm−1 as the number of layers is increased, confirming that good uniformity and layer control is achieved at the wafer level through the SLS of MoS2.The uniformity of the MoS2 was evaluated through high-resolution TEM (HRTEM) analysis and by using it in a top-gated field-effect transistor (FET). The low-magnification TEM image of 1L SLS MoS2 in 2; but as these represent only about 4% of the total area, the 1L SLS MoS2 can be considered to have near-perfect ( > 95%) micrometer-scale layer uniformity. This growth of 2L MoS2 on 1L MoS2 could be due to the energetically favorable adsorption of MoCl5 on defect sites such as sulfur vacancies or grain boundaries, as such adsorption of molecules is seen with graphene262 in 2 was evaluated by using it in the fabrication of a top-gated FET with Au(10 nm)/Ti(50 nm) electrodes and an ALD Al2O3 (40nm) gate insulator. The room temperature performance of this FET at 10−5 mTorr is shown in 2/V∙s field effect electron mobility in the linear regime of the transfer curve agreeing with a previous report of a MoS2 FET2 FET also has a low subthreshold swing value of ~0.36 V/dec and an excellent on/off current ratio of ~108 that is higher than anything previously achieved with 1L MoS2The crystallinity and electrical performance of the 1L SLS MoS2 flakes on a SiO2(300 nm)/Si substrate, as demonstrated by the microscopy (OM) images in 2 flake was confirmed through AFM and Raman analysis (See 2 (#2) and 12L WSe2 (#3). The AFM image of the SLS MoS2 produced on this WSe2 flake at 800 °C (2) was obtained, indicating that 1L MoS2 is deposited on both 2L WSe2 and the SiO2 substrate under these conditions. 2 on WSe2/SiO2. In the SiO2 region (#1), E12g and A1g Raman peaks for MoS2 are observed at 385.2 and 405 cm−1, respectively, with the peak distance of 20.2 indicating that 1L MoS2 was obtained as expected. Raman peaks of WSe2  are observed in the 2L WSe2 region (#2) along with peaks for MoS2 (E12g at 378.4 cm−1 and A1g at 404.8 cm−1), indicating that MoS2 was also synthesized on the WSe2 flake. Furthermore, the absence of any Raman peaks related to MoSe2 (E12g at 286 cm−1 and A1g at 244 cm−1) or WS2 (E12g at 356 cm−1 and A1g at 420 cm−1) indicates that there is no significant mixing or alloying between the two 2D materials15−1 downshift in the E12g peak of MoS2 in the 1L MoS2/WSe2 region relative to the MoS2/SiO2 region. A similar downshift has been reported in the case of an interlayer-coupled 1L MoS2/1L WSe2 heterostructure fabricated by transferring individual MoS2 and WSe2 flakes, with this being attributed to interaction between MoS2 and WSe22 Raman peaks in the 12L WSe2 (#3) region indicates that there is effectively no growth of MoS2 on 12L WSe2, further supporting the idea that the self-limiting nature of the SLS process is layer dependent.If the proposed self-limiting growth mechanism of SLS is valid, then it would be expected to apply to other 2D materials. This was therefore tested using mechanically exfoliated WSeysis See  to contat 800 °C  shows th2 on WSe2, Raman mapping of the MoS2 E12g peak intensity and position was compared against OM images of 1L SLS MoS2 grown on WSe2 flakes on a SiO2 substrate. In 2 are indicated by white arrows, with the rest being bulk WSe2. The Raman map of MoS2 E12g intensity in 2 E12g signal is observed only at 2L WSe2 regions, indicating that MoS2 was not synthesized on bulk WSe2. The Raman map of MoS2 E12g position g = 20 V. This gate-tunable characteristic could be explained by a variation in carrier density with electrical doping, as consistent with a previous report2/WSe2 junction102, 2L WSe2 and the heterostructure created from them. It is evident from this that the strong PL peak for the direct gap transition of 1L MoS2 is greatly suppressed by the WSe2 junction, which is attributed to the rapid separation of charge carriers2/WSe2 PN diode at Vg = -50 V with and without illumination by an incident optical power density of 14 W/m2. We see from this that the current increases with illumination due to the generation of optically excited carriers. The open circuit voltage of 0.2 V indicates a photovoltaic effect, with a calculated photoresponsivity of 33 mA/W at Vds = 1 V. As a result, we show the potential of SLS MoS2/WSe2 structure in photovoltaic device as well as PN diode.As the SLS process clearly allows for much greater layer control than previously reported methods10421012inset of . The for2 on a SiO2 substrate has been successfully achieved through a new self-limiting process that allows the number of layers formed to be controlled by varying the growth temperature. Though the precise mechanism requires further study, this behavior is believed to be caused by the lack of dangling bonds on the surface of MoS2 and the screening effect that MoS2 layers have on the substrate’s electric field. More importantly, this process can achieve excellent layer uniformity (up to 95%) over large areas at wafer-level scale. The resulting 2D MoS2 can produce n-type behavior and a high on/off ratio when used in a top-gated FET, and can be grown on other chemically inert 2D materials such as WSe2. Indeed, a PN diode based on a MoS2/WSe2 heterostructure is capable of a high forward/reverse current ratio, and exhibits a gate-tunable rectifying property attributable to electrical doping by gate voltage. We therefore believe that this new method could be extended to the development of other 2D TMDCs materials and 2D heterostructures.In summary, the synthesis of MoS2 directly onto SiO2 (285 nm)/Si substrates using MoCl5 and H2S as the precursor and reactant, respectively. A bubbler containing the precursor was heated to 90 °C to ensure an adequate vapor pressure for the precursor molecules to be carried into the tube by pure argon (99.999%) carrier gas. The SLS cycle consisted of four steps, each with the same with ALD procedure of: precursor exposure for 4 s, a 5s Ar purge, 3 s H2S reactant exposure, and a final 5 s Ar purge.A tube furnace reactor was used to synthesize MoS2 on the SiO2 substrate was spin coated with polymethyl methacrylate (PMMA) at 4000 rpm for 60 s. After curing the PMMA at 100 °C for 15 min, the sample was immersed in a 10% HF solution to etch away the SiO2 layer. The sample was then washed with DI water and transferred to a new SiO2/Si substrate. Finally, the PMMA was removed using acetone and the sample washed with isopropyl alcohol.The as-synthesized MoS2-based FET was fabricated from as-synthesized 1L MoS2 on a SiO2 (300 nm)/Si substrate by evaporating Au(10 nm)/Ti(50 nm) electrodes and an ALD Al2O3 (40nm) gate insulator through conventional photolithography and reactive ion (O2 plasma) etching.The MoS2 was synthesized on WSe2 flakes at 800 °C. Then, PN diode was fabricated from an SLS MoS2/WSe2 heterostructure on a SiO2 (300 nm)/Si substrate by evaporating Au(10 nm)/Ti(50 nm) electrodes. After etching, electrical contacts were formed between the drain electrode and 12L WSe2, and between the MoS2 and source electrode.The 1L SLS MoS2 nanosheets.OM (Nikon ECLIPSE LV100ND), SEM(JEOL-6701F), Raman spectroscopy , AFM , PL , XPS , TEM  and a voltage/current meter  were all used in characterizing the SLS MoSHow to cite this article: Kim, Y. et al. Self-Limiting Layer Synthesis of Transition Metal Dichalcogenides. Sci. Rep.6, 18754; doi: 10.1038/srep18754 (2016).


Translocase of IM 9 and Tim10 are essential homologue proteins of the mitochondrial intermembrane space (IMS) and form a stable hexameric Tim9–Tim10 complex there. Redox-switch of the four conserved cysteine residues plays a key role during the biogenesis of these proteins and, in turn, the Tim proteins play a vital chaperone-like role during import of mitochondrial membrane proteins. However, the functional mechanism of the small Tim chaperones is far from solved and it is unclear whether the individual proteins play specific roles or the complex functions as a single unit. In the present study, we examined the requirement and role for the individual disulfide bonds of Tim9 on cell viability, complex formation and stability using yeast genetic, biochemical and biophysical methods. Loss of the Tim9 inner disulfide bond led to a temperature-sensitive phenotype and degradation of both Tim9 and Tim10. The growth phenotype could be suppressed by deletion of the mitochondrial i-AAA (ATPases associated with diverse cellular activities) protease Yme1, and this correlates strongly with stabilization of the Tim10 protein regardless of Tim9 levels. Formation of both disulfide bonds is not essential for Tim9 function, but it can facilitate the formation and improve the stability of the hexameric Tim9–Tim10 complex. Furthermore, our results suggest that the primary function of Tim9 is to protect Tim10 from degradation by Yme1 via assembly into the Tim9–Tim10 complex. We propose that Tim10, rather than the hexameric Tim9–Tim10 complex, is the functional form of these proteins. Tim9 and Tim10 are essential homologue proteins forming a stable complex in mitochondria. The present study suggests that primary function of Tim9 is to protect Tim10 from degradation by Yme1 via assembly into the Tim9–Tim10 complex. These hexameric complexes are considered to be the functional forms of these proteins  using the primers 9loxF and 9loxR containing regions homologous to the upstream and downstream sequences of TIM9. This PCR product was then transformed into BY4742 + pLG025 and transformants were selected on 100 μg/ml phleomycin. Recombination into the TIM9 locus was confirmed by PCR and to distinguish between recombination into the genomic TIM9 locus as opposed to the plasmid copy, transformants were streaked on to 5-fluoroorotic acid (5-FOA), with those with the genomic copy knocked out requiring the presence of the URA3 plasmid and therefore unable to survive on 5-FOA. This strain (MPS4) was then used for a further round of PCR-based homologous recombination to knockout the YME1 gene. This PCR used the plasmid pFa6a–KanMX4 as template to amplify the KanMX4 gene that confers G418 resistance using the primers Yme1F1 and Yme1R1. The PCR product was transformed into the MPS4 strain with the tim9 gene knocked out and transformants were selected on 200 μg/ml G418 and confirmed by PCR to generate strain MPS19. To generate the tim9 mutant strains in the in the YME1 and yme1Δ backgrounds, the WT and mutant TIM9 LEU2 plasmids pLG040, pLG041 or pLG042 were transformed into MPS4 and MPS19 respectively. WT TIM9 URA3 plasmid was removed by streaking on 5-FOA to generate MPS21 , MPS22 , MPS28  and MPS29 .A Selected individual colonies were grown YPD (yeast extract protein dextrose) overnight before spot-testing on YPD or YPEG (yeast extract peptone ethanol glycerol) plates at 30°C or 37°C.Mitochondrial preparations were performed as per ,35. For Cells were grown in synthetic selective media lacking methionine overnight, then diluted to pre-log phase in fresh media and grown for 6 h. Cells were pelleted at 3000 g and resuspended in 500 μl of medium and incubated at 30°C for 5 min in a shaking waterbath. Pulse-labelling was performed by the addition of 5μl (50 μCi) of 35S Express Protein labeling mix (Perkin–Elmer) followed by a further 5 min incubation. Chase was initiated by addition of unlabelled methionine to 20 mM and 10 μg/ml cycloheximide. Samples were taken at regular time points by freezing cells in 10% TCA (trichloroacetic acid). For immunoprecipitations (IPs), cells were thawed and pelleted, then resuspended in urea lysis buffer  and broken by glass bead lysis. Proteins were denatured by heating at 95°C for 5 min and diluted in 1ml of IP buffer . Samples were cleared by centrifugation at 14000 g for 40 min at 4°C. Antibody was added at 1 μl/unit 600 of cells and incubated for 1 h at room temperature (RT), followed by addition of Protein A sepharose and incubation for 1 h. Antibody was denatured by the addition of 1× SDS sample buffer + DTT and heating at 95°C for 5 min. Proteins were separated by SDS/PAGE and visualized by autoradiography and images were captured using a Typhoon phosporimager and quantified using AIDA densitometry software.Escherichia coli Rosetta-gami cells as a GST–Tim fusion protein (with pGEX-4T plasmid) and purified using GST affinity-binding beads followed by thrombin cleavage to release the proteins as described previously . Thi. Thiin vA further quality control system for the IMS has been proposed to exist in the cytosol, where the ubiquitin proteasome has been shown to degrade un-imported IMS protein precursors . Bragosz