Median U-turn intersection treatment (MUTIT) has been considered an alternative measure to reduce congestion and traffic conflict at intersection areas. The MUTIT is sometimes difficult to implement in the field because it requires wide median on arterials for U-turn vehicles. The objective of this study is to introduce an unconventional U-turn treatment (UUT) for intersections which requires less median space but is also effective. The UUT has a dual-bay design with different turning radiuses for small and large vehicles. The VISSIM simulation model was developed to evaluate the operational features of the UUT. The model was calibrated using data collected from intersections in China. The capacity, delay and number of stops were evaluated and compared with the direct-left-turn (DLT) for the same intersections. The results showed that the UUT significantly improved the operations at intersection areas, especially when volume/capacity ratio is small, and ratio of left-turn to through traffic is small. With the UUT, the capacity is increased by 9.81% to 10.38%, vehicle delay is decreased by 18.5% to 40.1%, and number of stops is decreased by 23.19% to 36.62%, when volume/capacity ratio is less than 0.50. The study also found that traffic efficiency could be further improved when the UUT is designed in conjunction with signal control. In the case, the UUT plus signalized control increases the capacity by 25% to 26.02%, decreases vehicle delay by 50.5% to 55.8%, and reduces number of stops by 69.5%, compared with the traditional DLT. Signal design for left-turn traffic at intersection has been long considered as a dilemma. Providing protected left-turn signal for left-turn vehicles could increase delay to through traffic , 2. Non-During the past decades, various indirect left-turn designs have been used on urban or suburban multilane arterials to eliminate problems associated with direct left-turn movements at intersections. Using non-traversable medians and/or directional median openings, direct left-turn movements from collector streets or local streets are prohibited. Left-turning vehicles will be redirected to a preselected downstream U-turn location to make U-turns. The median U-turn intersections (MUTI), superstreet intersections, crossover displaced left-turn intersections, the upstream signalized crossover schemes are common U-turn designs –15.The median U-turn intersection treatment (MUTIT), also known as the Michigan U-turn, is the most common design for U-turn vehicles. The geometric configuration of the MUTIT is shown in The MUTIT has several limitations which restrict its application around the world. One of the limitations is that the MUTIT requires sufficient median width to accommodate the design of U-turn lane. For intersections with narrow median on arterials in both directions, it is very difficult to implement the MUTIT for the U-turn design. In addition, the small vehicles and the large vehicles  have different turning radius which should be considered when designing the U-turn on arterials with narrow median and large volume of heavy vehicles.In this study, a modified MUTIT design named the unconventional U-turn treatment (UUT) was proposed to relax the unconventional of narrow median. The configuration of the UUT is shown in In the UUT, the median width of each upstream segment is enlarged only locally in a short distance in order to create an adequate turning radius for the U-turn vehicles. Though some median space is still needed for the UUT design, it is considered much less as compared to that for the MUTIT design. In each intersection arm, the UUT is designed with two U-turn bays: the inner bay (close to the intersection approach) is exclusively prepared for small vehicles, and the outer one is an exclusive U-turn of heavy vehicles, as shown in The primary objective of this study is to evaluate the operational effects of the UUT at intersections. The capacity, delay and number of stops with the UUT were evaluated for various traffic situations. For comparison purpose, the traffic operations with the direct left turn (DLT) for the same intersection were evaluated. In the following section, a review of previous studies is provided. In section 3, the model development is introduced. In section 4, data collection and model calibration are introduced. The simulation results are discussed in section 5. The paper ends with brief concluding remarks and future work in section 6.Definitions of traffic variables at UUT and their calculations are introduced in this section. The variables used in the study are summarized in C is determined by the green phases and the clearance time for both directions, which is:In the study, the green phase of one intersection arm and its opposite arm is assumed to be symmetric. The cycle length i is the sum of the demand flow of the following three movements:The demand flow in arm Besides, the demand flow of U-turn movement and through movement at median crossover is identical with the demand flow at intersection approach:P(xj) denotes the probability of a driver j selecting the former type; As stated before, left-turn vehicles can make two types of U-turns at the UUT. The selection of U-turn type where both “straight → U-turn → right-turn” and “turn right → U-turn → straight” roundabouts are determined by the binary logit model , which ii is as follows:ξi is determined as left-turn vehicles divided by total traffic volume , which is calculated byConsequently, the demand flow of each approach lane in arm Li should be large enough to prevent vehicle spillback at intersections. In this study, Li should be greater than the vehicle queue length at the intersection approach, which is calculated as:The spacing between median crossover and intersection Li could also be determined by the AASHTO Green Book  which recommends that the minimum spacing between a median crossover and the MUTIT intersection could be between 122m (400ft) and 183m (600ft), if no traffic data are available for calculating Li. Furthermore, the median width for U-turn maneuvers under UUT should be larger than the lower bound value w, as shown in In practical applications, the value of Theoretically, a before—after study in field applications is preferred to compare the operational features of the UUT and DLT design. However, in reality it is very hard to obtain the data before and after the UUT taking place on the same intersection. Thus, simulation technique was used in the study to evaluate the operations of traffic at intersections. Previously, traffic problems have been studied by models such as continuous flow models , car folTo ensure that the geometric elements of intersections are precisely modeled, the CAD layouts of typical intersections on four-lane and six-lane divided streets were imported into VISSIM. When UUT is executed without U-turn signal control, the priority rule for through and U-turn movements should be defined in VISSIM simulation. In this study, U-turning vehicles must yield to the through traffic which is also confirmed by field observations. U-turn drivers need to wait at the bay area until there is an acceptable headway gap in traffic stream . The gapThe turning radius of U-turn vehicles at the bay area can be determined by the median nose width and receiving lane width. Vehicle movements are decided for the simulation model according to field observations. The inner bay is exclusively prepared for small vehicles, and the outer one is an exclusive U-turn of heavy vehicles. In practical applications, the U-turn median width should be large enough to make sure that the vehicle turning speed is consistent to field observations for conventional U-turns, as shown in Three measures are calculated to evaluate the operational features of the U-turn design, including the delay, number of stops and capacity of intersections. Those measures are commonly used in previous studies for the evaluation of intersection performances ,28–30.DiU denotes the total delay of left-turn vehicles at intersection approach i with UUT; diU1 denotes the average stopped delay of U-turn vehicles from the entrance lanes to the target exit lanes, and diU2 denotes the average travel delay of U-turn vehicles from the entrance lanes to the target exit lanes.DiC denotes the total delay of left-turn movement vehicles at intersection approach i with DLT; diC1 denotes the average stopped delay of left-turn vehicles conventional signalized treatment vehicles from the entrance lanes to the target exit lanes, and diC2 denotes the average travel delay of left-turn vehicles operating under conventional signalized treatment from the entrance lanes to the target exit lanes. More details on the calculation of the above parameters can be found in [Delay is measure by the difference of actual travel time and desired travel time without stop. With travel time information, vehicle delay is the combination of average stop delay and travel delay . Averagefound in .SiU denotes the average count of stops of left-turn movements at intersection approach i with UUT; sijU denotes the count of stops for left-turn vehicle j at intersection approach i with UUT; Qi2 denotes total number of left-turn vehicles at intersection approach i with UUT.SiC denotes the average count of stops of left-turn movements at intersection approach i with DLT; sijC denotes the count of stops for left-turn vehicle j at intersection approach i with DLT; Qi2 denotes total number of left-turn vehicles at intersection approach i with DLT.The number of stops per vehicle is calculated by the total stop counts divided by the number of vehicles crossing the intersection, which is:i, the capacity is calculated as:CiU denotes the whole capacity at intersection approach arm i with UUT; cikU denotes the capacity of vehicles from left-turn, through and right-turn movement respectively. Note that U-turn capacity constitutes the capacity of left-turn movements at approach i with UUT.The capacity is the sum of vehicle count from all movements at intersection area with UUT. For the intersection arm i with DLT is as follows:CiC denotes the whole capacity at intersection approach arm i with DLT; cikC denotes the capacity of vehicles from left-turn, through and right-turn movement respectively. The detailed calculation of vehicles capacity of each movement is also referred to [The whole vehicle capacity at intersection approach arm erred to .Field data collection was conducted to obtain the data for model calibration. There is no specific permission required for these locations. Field data were collected from six intersections in the urban public area of Nanchang, China, other than private lands. Video cameras were used in the data collection at intersections. The field studies did not involve endangered or protected species. The city for data collection has a population of 5.6 million and an area of 7,402 square kilometers by 2013.Firstly, two intersections were selected for data collection in order to calibrate the simulation model. Because that the UUT has not been implemented in the field, our study selected intersections with geometric features similar to the UUT to calibrate the vehicle maneuver parameters in the VISSIM simulation model. The selected sites are located at intersections of Beijing East Rd—Shanghai North Rd , and Beijing East Rd—Qingshanhu Rd .Secondly, four other intersections were selected for data collection during weekdays including both peak and off-peak periods. Those sites were used to evaluate the performance of the UUT compared with the DLT. The geometric features and traffic information at each site are shown in Tables The VISSIM model was calibrated and validated against field data to ensure that it simulates accurate traffic operation. To calibrate the selected parameters in the simulation model, field data were collected including traffic data, geometric features, and drivers’ behavioral information.Initially, the logit model for predicting driver’s choice of two U-turns was calibrated. A total of 316 U-turn behaviors were identified in the field observation. The percentages of the two U-turns are 52.6% and 47.4% respectively. The green signal for the through and right-turn movement was 40 s and 30 s respectively.Some data gathered in the field were considered as the inputs in the VISSIM: 1) The percentage of private vehicles and heavy vehicles are 96% and 4% respectively; 2) The headway of private vehicles ranges from 4.0 s to 14.0 s which an average of 6.9 s; 3) The headway of heavy vehicles ranges from 7.4 s to 11.0 s with an average of 9.8 s; 4) The turning speed of private vehicles is 12.3 km/h, and the speed ranges from 7.1 km/h to 18.7 km/h; and 5) The turning speed of heavy vehicles is 9.7 km/h and the speed ranges from 7.2 km/h to 10.8 km/h.Several parameters in the VISSIM model needs to be calibrated which are the parameters in the gap-accepting model, the car-following model, and the lane-changing model. Considering that the capacity is one of the most important factors in the evaluation of U-turn movements, the capacity was considered as the measurement of goodness of fit to calibrate the predictions in the VISSIM model. This study used the Kyte’s method  to measucf denotes the capacity of U-turn movement measured in the field (veh/h); ts denotes the average service delay for each U-turning vehicle (s), which is defined as delay occurs at the first position of the U-turn queue; and tmv denotes average move-up time for each U-turning vehicle (s), which is the amount of time from when the previous U-turning vehicle exits the stop line until the subsequent queued vehicle reaches the stop line.The method proposed by Kyte in 1991 was to measure field capacity in an under-saturated condition, which is described in the following equation:n denotes the number of video sections, csi is the capacity of U-turn movement estimated by the VISSIM simulation model (veh/h), and cfi is the field capacity of U-turn movement at time interval (veh/h).Besides, capacity was selected as the goodness of fit measure for validation of the calibrated simulation model. The index used for measuring simulation error is the mean absolute percent error (MAPE), which can be estimated as:The VISSIM simulation models are developed for the same intersections to evaluate the traffic operational features, including capacity, delay, and number of stops. Actual traffic parameters such as flow and traffic components collected in the field are inputted in the simulation model.The simulation results for four intersections with both UUT and DLT design, according to traffic demands in The above results show that the UUT design has the better potential to effectively improve the operational performance at intersections than the DLT design. The UUT is more suitable for designing intersection with small the value of ratio between volume and capacity and low ratio of left-turn to through traffic volume.Field data collection does not cover all possible traffic situations which restricts the full evaluation of operational effects of the UUT design. In this section, different traffic situations are specified in the VISSIM simulation model to further investigate the differences between the UUT and DLT design. The intersection used for simulation is crossed by an arterial (6 legs) and a collector (4 legs) and the whole capacity for the intersection is 5800 pcu/h. At first, sensitivity analysis of delay and number of stops are calculated for different traffic situations. The traffic volume on the arterial street is specified to range from 600 to 2400 pcu/h, with an increase of 600 pcu/h. The traffic volume on the collector increases from 100 to 1500 pcu/h, with an increase of 200 pcu/h.Vehicle delay under different traffic situations are shown in The number of stops is also analyzed for different traffic situations. The results are shown in In summary, according to the results from sensitivity analyses, it is can be found that the intersection operates fluently if traffic demand is less than 1700 pcu/h. Furthermore, when the volume/capacity ratio of the intersections is less than 0.58, the UUT is a good design.A comparison of operational effect between UUT and DLT was also conducted. When the intersection operates with the ratio of left-turn to through traffic volume less than 0.33:1, UUT has better performance than DLT in terms of higher capacity, less delay and number of stops. The sensitivity analysis shows that when the ratio of left-turn to through traffic volume is close to or larger than 1, the advantage of the UUT design is not quite obvious as compared to the DLT. The reason for this is that as the increase of flow and decrease of headway, traffic conflicts are more severe because: (1) the U-turn vehicles cannot merge into the mainstream traffic stream easily; and (2) the right-turn vehicles cannot merge into the mainstream traffic stream easily. To further improve the operational performance, two advanced signal control strategies in conjunction with UUT are proposed.The first strategy is the UUT plus signalized control at the intersection approach (denotes as UUT plus A), as shown in The second strategy is to use the UUT pluses an integrated signalized control at both the U-turn bay and the intersection approach (denotes as UUT plus B), as show in Four intersections, as shown in UUT plus A can effectively improve the capacity of intersections and reduce the delay when compared with UUT only, especially for the intersections of large volume/capacity ratio. Capacities of intersections from site1 to 4 are increased by 13%, 9%, 13% and 19%, and delay of each site is decreased by 11%, 24%, 79% and 78%, respectively. However, the average number of stops at site1 and 2 is increased by 34% and 17% respectively, where the volume/capacity ratio was small. Actually, the average number of stops of left-turn vehicles increased due to signal control. Meanwhile, the average number of stops of site 3 and 4 significantly decreased where the volume/capacity ratio was large. In addition, the design of UUT plus B further increased the capacity and reduced the delay and number of stops at intersection areas. For example, with the UUT plus B, the capacity is increased by more than 8%, the delay is decreased by over 7%, and the average number of stops is decreased by more than 9% for the same intersections.The results showed that the UUT design with integrated signal control can effectively increase the capacity and reduce the delay and vehicle stops at intersections, as compared with either UUT only or DLT. The UUT plus B has the best operational performance. In addition, the simulation results also showed that with the integrated signal control, the UUT can still effectively improve intersection operations in the traffic situations with large volume/capacity ratio.Traditional MUTIT has the limitation in the requirement of large median width to accommodate the U-turn lane. In this study, a new U-turn design, named the unconventional U-turn treatment (UUT) is proposed. The core design of UUT is that the median is enlarged only locally to accommodate larger turning radius for U-turn vehicles but doesn’t occupy large median space. The UUT is designed with two U-turn bays with different radiuses for small vehicles and large vehicles respectively. The VISSIM simulation model is developed and calibrated to evaluate the operational features at intersections with UUT. Traditional DLT design is also evaluated for comparison.The results show that when the intersection operates with small volume/capacity ratio and low ratio of left-turn to through traffic volume, UUT has an obviously better performance than the DLT. In the case, the capacity is increased by 9.81% to 10.38%, vehicle delay is decreased by 18.5% to 40.1%, and number of stops is decreased by 23.19% to 36.62% when the volume/capacity ratio is less than 0.50. When the traffic volume of through and left-turn traffic is large, UUT has very little improvement as compared to DLT, but UUT with signal control (UUT plus A) presents obvious advantage over UUT only. Compared with DLT, the capacity is increased by 25% to 26.02%, vehicle delay is decreased by 50.5% to 55.8%, and number of stops is decreased down to 69.5% when the volume/capacity ratio is more than 1. Furthermore, the UUT in conjunction with signal control at both U-turn bay and intersection approach (UUT plus B) is the most appropriate measure to cope with high left-turn and through traffic volume, because it effectively reduced delay and number of stops.The findings of this study can provide useful information in understanding the applicability of UUT under different traffic conditions. It can be utilized as a guideline for transport policy makers and planners to determine when and where the UUT should be used. Before the UUT being used in practical applications, some issues can be further studied in the future. First, the distance between two U-turn bays in one intersection arm is fixed in this study, which can be relaxed to consider its effect on UUT. Second, this study only investigates an isolate intersection. The study scope could be extended to a broader spectrum for simulating a larger network to more precisely estimate the operational effects of UUT. In addition, the performance of DLT could be further improved by optimization of signal control. It would be interesting to compare the UUT and DLT design when both are integrated with advanced signal control techniques. The authors recommend that future studies could focus on these issues.


The honeycomb lattice structure of graphene gives rise to its exceptional electronic properties of linear dispersion relation and its chiral nature of charge carriers. The exceptional electronic properties of graphene stem from linear dispersion relation and chiral nature of charge carries, originating from its honeycomb lattice structure. Here, we address the quantum Hall effect in artificially stacked graphene bilayers and single layer graphene grown by chemical vapor deposition. The quantum Hall plateaus started to appear more than 3 T and became clearer at higher magnetic fields up to 9 T. Shubnikov-de Hass oscillations were manifestly observed in graphene bilayers texture. These unusual plateaus may have been due to the layers interaction in artificially stacked graphene bilayers. Our study initiates the understanding of interactions between artificially stacked graphene layers. The robust electronic properties of graphene stem from linear dispersion relation, containing massless Dirac fermions and chiral nature of charge carriers. This behavior of graphene originats from its honeycomb lattice structure. Bernal stacked bilayers graphene consists of massive Dirac fermions spectrum, which is demonstrated by two pairs of parabolic bands1246810111213141718The electrical transport such as quantum Hall effect in single and Bernal stacked bilayers graphene has been explored to a large extent. However, it is interesting to investigate the electronic transport properties of artificially stacked graphene layers. Here we report the electronic transport properties of artificially stacked chemical vapor deposition grown graphene bilayers. We have observed that the quantum Hall effect consisting of various plateaus with non-integer quantized values at 4.2 K, however a typical massless Dirac fermions spectrum has observed in a single layer graphene. The clear quantum Hall plateaus started to appear from 3 T in graphene bilayers and became more prominent at higher magnetic fields. The Shubnikov-de Hass (SdH) oscillations were observed as well in graphene bilayers texture.2/Si substrate. 2/Si substrate as displayed in 2/Vs, respectively; while the field effect hole and electron mobility values of the double-layer region of graphene device at 0 T appeared to be about 2058 and 1625 cm2/Vs at temperature of 4.2 K, respectively. 2D/IG) value is ~3.66 for the case of SLG and ~2.77 for ASGBL as shown in the et al.−1, respectively as shown in The optical micrograph image of complete Hall bar device structure with Au electrodes has shown in xx) and Hall conductivity (σxy) of SLG as a function of applied back-gate voltage (Vg), with magnetic field (B) of 9 T at 4.2 K. The result showed a strong evidence of quantum Hall effect (QHE) with conductivity plateaus appeared at ±2, ±6, ±10, ±14, +18, together with resistivity minima, consistent with the Landau level spectrum expected for SLG graphene2g(Vg − VDirac)/e, where Cg/e = 7.19 × 1010 cm−2V−1 and VDirac = +10 V is the offset voltage to reach charge neutralityxx) and the Hall resistivity (ρxy) as a function of magnetic field at local back gate voltage of −40 V. Interestingly, this complex system of artificially stacked graphene bilayers shows the Shubnikov-de Haas oscillation. The resulting data (ρxx vs. B) reveal SdH oscillations which starts from magnetic fields as low as B ~3 T and it is consistent with g and B data which follows the quantum Hall states randomly as a monolayer  or bilayers . The contour plot of longitudinal resistivity (ρxx) as a function of Vg and B is shown in 2526et al.et al.et al.In summary, we have studied the structural and electrical transport properties of chemical vapor deposition grown single layer graphene and artificially stacked graphene bilayers. The typical massless Dirac fermions spectrum is observed in the quantum transport of single layer graphene, however artificially stacked graphene bilayers texture follows the quantum Hall states randomly as a monolayer or bilayers at 4.2 K. The hierarchy of Dirac fermions are significantly related to charge transport due to the combination of massive and massless fermions. The unusual Dirac fermion spectrum may have possibly caused by the layers interaction in artificially stacked graphene bilayers.–4 Torr, and the temperature rose to 1010 °C with H2 gas flow (~10–2 Torr). After the temperature stabilized at 1010 °C, CH4 and H2  were injected into the furnace to synthesize the graphene for 8 min, after which the sample was cooled at a rate of 50 °C/min to room temperature4)2S2O8), and the PMMA membrane was washed with deionized water. Next, the graphene film with the PMMA membrane was transferred to the p-doped Si substrate having a top 300 nm-thick layer of SiO2. The graphene layers transferred onto the Si/SiO2 substrate were heated at 80 °C for 10 min to dry and then put in acetone for one day to completely dissolve the PMMA layer30Graphene film was grown on 25-μm-thick copper foils from Alfa Aesar (99.8% pure) via thermal CVD. A mechanically polished and electropolished copper foil was inserted into the CVD furnace. The furnace was evacuated to ~10–1, with a laser wavelength of 514.5 nm. The spot size was 1 μm and the power was kept at 1.0 mW to avoid local heating. The electrical and magneto-transport properties of graphene junctions were measured using ac lock-in techniques at frequency of 11.7 Hz with the root-mean-square current amplitude of 50 μA. Low temperature measurements were carried out in liquid helium cryostat (down to 4.2 K) with magnetic field up to 9 T. Lake Shore 331 temperature controller was utilized to modulate and control the temperature range.Raman spectra were measured with a Renishaw microspectrometer over a wavenumber range from 1100 to 3200 cmHow to cite this article: Iqbal, M. Z. et al. Interaction driven quantum Hall effect in artificially stacked graphene bilayers. Sci. Rep.6, 24815; doi: 10.1038/srep24815 (2016).


Arabidopsis thaliana. We show that it is additionally involved in splicing and facilitates the excision of two cis-spliced group II introns, nad1 intron 2 and nad2 intron 1, in Arabidopsis mitochondria. odb1 mutants lacking detectable amounts of ODB1 protein over-accumulated incompletely spliced nad1 and nad2 transcripts. The two ODB1-dependent introns were both found to splice via first-step hydrolysis and to be released as linear or circular molecules instead of lariats. Our systematic analysis of the structures of excised introns in Arabidopsis mitochondria revealed several other hydrolytically spliced group II introns in addition to nad1 intron 2 and nad2 intron 1, indicating that ODB1 is not a general determinant of the hydrolytic splicing pathway.Transcript splicing in plant mitochondria involves numerous nucleus-encoded factors, most of which are of eukaryotic origin. Some of these belong to protein families initially characterised to perform unrelated functions. The RAD52-like ODB1 protein has been reported to have roles in homologous recombination-dependent DNA repair in the nuclear and mitochondrial compartments in  Higher plant organelle genomes contain several group-II introns originating from mobile ribozymes that invaded these genomes during evolution –3. PlantArabidopsis thaliana. They can be assigned to two categories: (i) Intron-encoded RNA maturases and related maturase enzymes encoded by nuclear genes, and (ii) splicing factors proposed to have originated from host proteins and to have been recruited to additionally function in mitochondrial transcript processing  domain protein WTF9, which is specifically required for intron excision from the rpl2 and ccmFc pre-mRNAs (chloroplast RNA splicing and ribosome maturation) domain protein mCSF1 involved in the splicing of multiple mitochondrial introns (RCC1/UVR8/guanine exchange factor-like 3)  family protein mTERF15  . This pr mTERF15 . Excepti mTERF15 –26.in vitro  extending from a central hub; tertiary interactions between these domains mediate productive intron folding -dependent DNA repair in the nuclear and mitochondrial compartments; it co-localised with mitochondrial high-molecular weight complexes in a DNA- and RNA dependent manner , T-DNA insertion lines odb1–1 (Sail_25_H08) and odb1–2 (SALK_089362)  and supplemented with 1% w/v sucrose, stratified at 4°C for 3 days in the dark and then exposed to a light intensity of 100 μmol quanta m−2 s−1 in a 16-h photoperiod at 23°C._089362)  and compGenomic DNA was isolated from seedlings or flowers as previously described . RNA wasODB1 promoter, coding region and untranslated regions was amplified using Phusion DNA polymerase (Finnzymes) and primers 5′- GGGGACAAGTTTGTACAAAAAAGCAGGCTTGTTATTAAGTAACGTCCTGCCA-3′ and 5′- GGGGACCACTTTGTACAAGAAAGCTGGGTTGTTTGTTAAGTCGAATAACCACC-3′ and transferred into binary vectors pKWG (complemented line C1) or pGWB40 (complemented line C2) using gateway cloning procedures (Life Technologies). The resulting plasmids were used to transform odb1–1 plants via Agrobacterium-mediated floral dip transformation. Resulting seeds were sown on soil and transformants selected using kanamycin (pKWG) or hygromycin (pGWB40). odb1–1 plants carrying the ODB1 transgene were identified by PCR using primers amplifying the vector-insert junctions.A genomic DNA fragment comprising the Total RNA (3 μg) extracted from Arabidopsis seedlings was resolved on 1.2% w/v agarose/formaldehyde gels and transferred onto positively charged nylon membranes (Roche Applied Science), followed by membrane staining with methylene blue (0.04% (w/v) in 0.5M sodium acetate, pH 5.2). DIG-labelled DNA probes were generated using Arabidopsis genomic DNA or cDNA as template, the PCR DIG probe synthesis kit (Roche Applied Science), and primer pairs listed in Supplementary Table S1. Hybridisations were performed in DIG Easy Hyb solution (Roche Applied Science) according to the manufacturer's instructions; chemiluminescent detection of signals was performed using anti-digoxigenin-alkaline phosphatase conjugates and CSPD reagent (Roche Applied Science).18S rRNA (At3g41768) and ACT genes  were used for data normalisation.Quantitative RT-PCR (qRT-PCR) for measuring mitochondrial intron splicing efficiencies was performed using SYBR Green I Master Mix (Roche Applied Science) and primer pairs listed in Supplementary Table S2 as previously described  on a Lignad2 intron 1-specific primer (5′-GAAAGCCCGGCCAGAAC-3′) and SuperScript III Reverse Transcriptase (Life Technologies). The resulting cDNAs served as template in subsequent PCR reactions set up with primers 5′-GGACTCATGTGCCTTCCAGAAC-3′ and 5′-ATGAACCAATGGATCGTAATAAGTC-3′ and DyNAzyme DNA polymerase (Finnzymes) in order to amplify head-to-tail-ligated intron 1-containing nad2 transcripts. PCR reactions were analysed on 3% (w/v) agarose gels, and products of interest were excised, purified over NucleoSpin columns (Macherey-Nagel), and ligated into pGEM-T (Promega) for cloning and sequencing.RNA (1 μg) was incubated with 2.5 units of tobacco acid pyrophosphatase  as previously described ; controlnad5 intron 3-derived products were amplified with Phusion DNA polymerase (Finnzymes). PCR reactions were analysed on 3% (w/v) agarose gels. Products of interest were excised, purified over NucleoSpin columns (Macherey-Nagel) and ligated into pGEM-T (Promega) for cloning and sequencing. Several clones were sequenced per intron. A list of all gene-specific primers used in reverse transcription and PCR reactions is provided in Supplementary Table S3.RNA (1 μg) was treated with T4 RNA ligase (New England Biolabs) or mock-treated, extracted and precipitated as detailed above and dissolved in water. The RNA was then reverse-transcribed using gene-specific primers and SuperScript II Reverse Transcriptase (Life Technologies). The products of reverse transcription were again extracted and precipitated and subsequently amplified by PCR, using 1/10 of the RT reaction as template, intron-specifc forward and reverse primers and DyNAzyme DNA polymerase (Finnzymes). nad2 transcript that over-accumulated in odb1–1 mutant plants but was barely seen in the wild type or in odb1–2  regions. Maturation of its mRNA requires one trans- and three cis-splicing events -dependent DNA repair in mitochondria of Arabidopsis . When su2 Figure . odb1–2  of ODB1 . nad2 ing events  for both odb1–1 and wild-type seedlings. We ensured that a potential alternative primary nad2 transcript for nad2 would also be ligated by pre-treating RNA samples with tobacco acid pyrophosphatase (TAP). TAP converts primary, 5′-triphosphorylated mitochondrial transcripts into 5′-monophosphorylated transcripts  excision of this intron may proceed via first-step hydrolysis rather than lariat formation in Arabidopsis.We assumed that the nscripts . Only thnscripts . A cRT-Pe Figure . Amplifi2 3′ end  fused to1 Figure . We thuscox2, ccmFc, rpl2 and rps3 each contain a single intron, nad4 contains three introns and nad1, nad2, nad5 and nad7 each contain four introns . Introduction of an ODB1 wild-type gene copy into odb1–1 plants restored ODB1 protein expression and efficient excision of both nad2 intron 1 and nad1 intron 2 , confirming that reduced splicing of these introns in odb1–1 plants was due to ODB1 loss. ODB1 is thus required for efficient splicing of two mitochondrial introns, nad1 intron 2 and nad2 intron 1, in vivo.We performed RNA gel blot hybridisations with probes for most other mitochondrial introns and found that in agreement with qRT-PCR data, splicing of mitochondrial introns other than odb1 mutants. Both introns are classified as group II introns (http://blast.ncbi.nlm.nih.gov). Sequence identity between nad1 intron 2 and nad2 intron 1 in domains dV and dVI as well as in other domains and the intron structural core has been previously noted in the genus Oenothera, and a common evolutionary origin of the two introns has been proposed .For s Figure  or intros Figure . The strs Figure . In the nad1 intron 2, nad4 intron 2 and nad1 intron 1 lack an unpaired adenosine whereas this potential lariat branching point is present in all other Arabidopsis mitochondrial introns (Figure We subsequently applied the cRT-PCR/RT-PCR strategy to all mitochondrial introns in order to systematically determine splicing pathways used in Arabidopsis mitochondria (Figure s Figure .nad1 intron 2 and nad2 intron 1, two other introns, nad4 intron 2 and nad7 intron 1, did not yield lariat-derived RT-PCR products and appear to exclusively use a hydrolytic pathway. For nad1 intron 3, nad4 intron 1 and the single rpl2 intron, hydrolytically released molecules as well as lariats were detected; all other introns showed lariat-derived products. According to these results, ODB1 is not generally required for efficient release of hydrolytically spliced introns but has a specific role in the excision of nad1 intron 2 and nad2 intron 1. Combining our analysis of splicing pathways with current data on the specificities of known mitochondrial splicing factors shows that none of these factors is common and exclusive to introns released via a hydrolytic pathway (Figure In addition to y Figure .nad2 mRNA in Arabidopsis mitochondria (Transcript splicing in higher plant organelles involves numerous ancillary factors most of which can be classified as intron maturases, RNA helicases or members of various RNA-binding protein families ,5. A numchondria . The maichondria ,47 as wechondria  belong tchondria –50. The chondria  and showchondria ,53 incluchondria . This prchondria .nad1 intron 2 and nad2 intron 1, both of which were found to splice via a hydrolytic pathway. ODB1 is distantly related to RAD52 (Results presented here demonstrate a role for the protein ODB1 in group-II intron splicing in mitochondria of Arabidopsis. ODB1-deficient plants displayed reduced splicing efficiencies for to RAD52 , a proteto RAD52 . Accordito RAD52 . Howeverto RAD52 ,38, and to RAD52 . The duaodb1–1 plants which completely lacked ODB1 displayed defects in both HR-dependent DNA repair and nad1 and nad2 splicing, odb1–2 plants with strongly reduced but detectable amounts of ODB1 protein were impaired in DNA repair to a degree that was comparable to odb1–1 but showed no splicing defect for nad2 intron 1 and accumulated significantly less incompletely spliced nad1 transcripts than odb1–1 (nad1 and nad2 in odb1–1 shows that ODB1 is not strictly required for the splicing of these transcripts but has an accessory role. Participation of ODB1 in splicing may be more crucial under conditions that reduce splicing efficiencies, e.g. during growth in the cold (nad1 intron 2 and nad2 intron 1 in domains dV and dVI, which are highly similar between the two introns, either through interaction with other components of the splicing machinery or by direct intron binding. Organellar proteins that bind both RNA and DNA have been previously described (ODB1 is assumed to be rather abundant in Arabidopsis mitochondria . Interesthe cold . ODB1 coescribed ,57.nad1 intron 2 and nad2 intron 1 depends on first-step hydrolysis it is unlikely that ODB1 is a general determinant of the hydrolytic pathway. Two other introns, nad4 intron 2 and nad7 intron 1, were identified that spliced exclusively via a hydrolytic pathway in Arabidopsis but did not depend on ODB1. In addition, nad1 intron 1 is likely hydrolytically released (ODB1 gene inactivation. It is nevertheless expected that trans-acting factors impact on the biochemistry of mitochondrial group-II intron splicing. nad7 intron 1, which in dVI is predicted to have a bulged A flanked by G:C pairs and was thus expected to be released as lariat, appears to exclusively splice via first-step hydrolysis. On the other hand, nad2 intron 3 and nad7 intron 2 which have predicted dVI configurations similar to nad2 intron 1 were found to strictly use the lariat pathway. It thus seems that intron folding and the presence/absence of a dVI unpaired adenosine do not exclusively define the group II intron splicing pathway in higher plant mitochondria, and other factors probably influence whether intron excision proceeds via first-step transesterification or hydrolysis. As yet there is no candidate splice factor that could potentially determine the biochemistry of intron release as none of the mitochondrial splice factors identified to date is common and exclusive to introns that splice via a hydrolytic pathway.In the course of this study we performed the first systematic analysis of mitochondrial group II intron splicing pathways, which underscored unusual aspects of splicing in plant mitochondria. Although excision of both released  but its Supplementary Data are available at NAR Online.


Acute exacerbation of idiopathic pulmonary fibrosis (AE-IPF) is associated with high mortality. However, few studies have so far reviewed analyses of autopsy findings in patients with AE-IPF.We retrospectively reviewed 52 consecutive patients with AE-IPF who underwent autopsies at five university hospitals and one municipal hospital between 1999 and 2013. The following variables were abstracted from the medical records: demographic and clinical data, autopsy findings and complications during the clinical course until death.The median age at autopsy was 71 years (range 47–86 years), and the subjects included 38 (73.1%) males. High-dose corticosteroid therapy was initiated in 45 (86.5%) patients after AE-IPF. The underling fibrotic lesion was classified as having the usual interstitial pneumonia (UIP) pattern in all cases. Furthermore, 41 (78.8%) patients had diffuse alveolar damage (DAD), 15 (28.8%) exhibited pulmonary hemorrhage, nine (17.3%) developed pulmonary thromboembolism and six (11.5%) were diagnosed with lung carcinoma. In addition, six (11.5%) patients developed pneumothorax prior to death and 26 (53.1%) developed diabetes that required insulin treatment after the administration of high-dose corticosteroid therapy. In addition, 15 (28.8%) patients presented with bronchopneumonia during their clinical course and/or until death, including fungal , cytomegalovirus  and bacterial  infections.The pathological findings in patients with AE-IPF represent not only DAD, but also a variety of pathological conditions. Therefore, making a diagnosis of AE-IPF is often difficult, and the use of cautious diagnostic approaches is required for appropriate treatment. Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fibrosing form of interstitial pneumonia with a median survival after diagnosis of three to five years -4. AcuteLung tissue derived from patients with IPF shows a characteristic histopathological pattern known as usual interstitial pneumonia (UIP), which includes the presence of fibroblastic foci. The pathological findings of AE-IPF comprise diffuse alveolar damage (DAD) superimposed on underlying UIP; this is the most commonly described finding on surgical lung biopsies ,11,12. OThe current study is a retrospective review of an autopsy series designed to describe and evaluate the pathological findings, including concomitant and infectious diseases, observed during the clinical course until death in patients with AE-IPF.A total of 52 patients with AE-IPF who underwent autopsies at five university hospitals and one municipal hospital between January 1, 1999 and December 31, 2013 were identified based on medical records. The Ethics Committee of University of Occupational and Environmental Health in Kitakyushu, Japan approved this study , with a waiver for informed consent due to the retrospective study design. AE-IPF was defined according to a previous report [All autopsied lung materials were fixed in 10% formalin for more than seven days. At least one tissue block was prepared from the gross AE-IPF lesions in each lobe, and the tissue blocks were embedded in paraffin. Sections (4 μm thick) were cut and stained with hematoxylin and eosin (H&E) using standard procedures. The pathological diagnosis of UIP was made according to the current guidelines . We also2O2 in 100 ml of Tris–HCl . After counterstaining with Meyer’s hematoxylin, the sections were observed under a light microscope.Four-micrometer-thick sections were cut from the lung tissue blocks and stained with H&E for the histopathological examinations in addition to Periodic acid-Schiff (PAS) stain, Grocott's methenamine silver stain and Gram stain to identify any infectious pathogens in the presence of bronchopneumonia. Bronchopneumonia  was conclusively diagnosed based on histopathological evidence, such as neutrophil infiltration, fibrinopurulent exudate accumulation or abscess formation in the broncho-bronchioloalveolar space. For immunohistochemical detection of cytomegalovirus (CMV) antigens, the sections were incubated with a mouse monoclonal anti-CMV antibody  for 30 minutes. Secondary antibody peroxidase-linked polymers were then applied, and the sections were incubated with a solution consisting of 20 mg of 3.3’-diaminobenzidine tetrahydrochloride, 65 mg of sodium azide and 20 ml of 30% HA total of 52 patients with AE-IPF underwent autopsies during the study period. The demographics of the patients are reported in Table Nearly half of the patients had received corticosteroid therapy before AE-IPF. The therapeutic regimens for AE-IPF are shown in Table The mean duration between death and autopsy was 286 minutes (range: 60 to 990 minutes). Overall, the median weights of the right and left lungs were 658 g  and 552 g , respectively. These median weights were remarkably heavier than the standard lung weights reported for Japanese males  . The undThe pathological findings in the 11 patients with AE-IPF who met the 2007 criteria but did not have DAD were indicative of UIP alone (n = 5), alveolar hemorrhage n = 3), pulmonary thromboembolism (n = 1), OP (n = 1) and lung adenocarcinoma (n = 1)  patients Table . In thesIn this study, we retrospectively analyzed the autopsy findings of AE-IPF and clarified that AE-IPF exhibits a variety of pathological findings in addition to DAD. Moreover, the AE-IPF patients were diagnosed with various infectious diseases and complications during their clinical course after AE-IPF.DAD has been reported to be the focal pathological finding of AE-IPF . DAD alsRegarding acute respiratory distress syndrome (ARDS), in which the diagnostic criteria are based on clinical findings, in the same manner as AE-IPF, Patel S.R. et al. reported that they investigated the pathological findings of 57 cases satisfying the diagnostic criteria for ARDS via open-lung biopsy, with DAD observed in 23 patients (40.3%) . On the Although there is no established standard therapy for AE-IPF , high-doThere are several limitations associated with this study. First, only patients who died and underwent autopsy were included; therefore, the results may differ based on the status of onset of AE-IPF. In other words, no patients who survived after AE-IPF were included, and the present findings thus do not reflect all aspects of the entity of AE-IPF. Second, we did not perform endotracheal aspiration or bronchoalveolar lavage due to the presence of severe hypoxemia in all cases. However, we carefully excluded patients based on the findings of sputum, laboratory and physical examinations. In addition, a previous study reported that the features of clinically suspected acute exacerbation diagnosed based on the radiological and clinical course are similar to those of acute exacerbation diagnosed based on the results of several intensive examinations, including BAL . Third, The pathological findings of AE-IPF include DAD as well as a variety of pathological conditions, and making a definitive diagnosis of AE-IPF is difficult. Patients with AE-IPF therefore are at risk of death due to the occurrence of several complications during their clinical course, including the effects of treatment with high-dose corticosteroid therapy. In patients with AE-IPF, it is very important to treat the disease by monitoring the patient’s condition and timely intervening with appropriate treatment.


Interestingly, iso-height lines of other rough surfaces are not necessarily conformally invariant even if they have the same Hurst exponent, e.g. random Gaussian surfaces. We have found that the distribution of the modulus of the Fourier coefficients plays an important role on this property. Our results not only introduce a new universality class and place the study of suspended graphene membranes within the theory of critical phenomena, but also provide hints on the long-standing question about the origin of conformal invariance in iso-height lines of rough surfaces.Suspended graphene sheets exhibit correlated random deformations that can be studied under the framework of rough surfaces with a Hurst (roughness) exponent 0.72 ± 0.01. Here, we show that, independent of the temperature, the iso-height lines at the percolation threshold have a well-defined fractal dimension and are conformally invariant, sharing the same statistical properties as Schramm-Loewner evolution (SLE Rough surfaces are very common in nature and can be found, for instance, in real landscapes445679Graphene, consisting of literally a single carbon monolayer, represents the first instance of a truly two-dimensional material see 111213. I1112Since previous numerical studies have shown that the structure of graphene possesses self-affine propertiesL × L graphene membranes to the inner area x-y plane having small random perturbations in z-direction (to break the symmetry) and zero initial velocity (zero temperature). For each temperature and system size, we perform three independent simulations with different initial perturbations. We use an equilibration time of 0.2 ns to reach the desired temperature and verify that the respective Maxwell-Boltzmann distribution for the velocities is recovered  potentialered see . From eaS(q) determines the Hurst exponent α via the relation In order to compare graphene membranes with RGS, we generate the latter ones by using the so-called Fourier Filtering Method (FFM). In contrary to graphene sheets, where simulations are performed in real space expressing physical interactions between atoms, RGS are constructed in Fourier space, and can be designed to have a desired Hurst exponent. In the FFM method, the Fourier coefficients of RGS are constructed using the decomposition h is the height profile of the surface defined by the z component of the atoms. For self-affine rough surfaces, the function α characterises the roughness of the surface. We take the statistical averages α = 0.72 ± 0.01 is independent of both, system size and temperature, and is in agreement with the previous work by Abedpour et al.α < 0.74, even though they used different boundary conditions. The existence of a Hurst exponent confirms the self-affinity property of graphene membranes and thus sets our graphene sheets in the context of rough surfaces . This provides a strong numerical evidence that the RGS are not SLE.Therefore to test the consistency of the iso-height contour lines with SLE, one solves numerically the Loewner differential equation that describes the conformal mapping220.02 see . From th0.02 see . On the  0.8 see , which cθ between two points of the curve separated by a distance L is defined as the oriented angle between the tangential vector of the curve at the first point and the vector connecting the two points. The winding angle distribution is expected to be Gaussian of mean In order to further explore the conformal invariance of these iso-height contour lines, we study the winding angle of the curves. The winding angle df is the fractal dimension of the curves, and a a constant24df = 1.23 ± 0.03 for the fractal dimension of the curves using L, the distribution of the winding angle displays the expected Gaussian behaviour . However, by performing the same measurement in graphene, we find a distribution that can be fitted with As we have observed, not all rough self-affine surfaces satisfy SLE statistics. Therefore, we are interested in understanding the origin of this discrepancy. Since graphene sheets and RGS possess the same Hurst exponent, we have extended our study to Fourier space to seek for further information. We have taken the Fourier transform of the graphene membranes and also of the RGS. By construction, the modulus of the Fourier coefficients, ith  see . Additioith  see  and uncoith  see , which fThus, one can conclude that the distribution of the modulus of the Fourier coefficients in rough self-affine surfaces might play an important role in the conformal invariance of their iso-height lines at the percolation threshold, although it does not affect the fractal dimension. A similar connection between the distribution of the phase of the Fourier coefficients and conformal invariance has already been observed for contour lines of zero-vorticity in 2D turbulenceκ with Summarising, we have shown that the iso-height lines of graphene sheets at the percolation threshold are scale invariant and provided evidence that they might also be conformally invariant. These properties are independent of temperature (within the range of 100–600 K). Additionally, they can be described by SLEThis result provides insights on the long-standing question on which are the responsible properties for conformal invariance in self-affine surfaces, and can be used to construct self-affine surfaces with conformal invariance properties. Additionally, the fact that iso-height lines in graphene are conformally invariant place the study of graphene within the theory of critical phenomena, belonging to a new universality class.227For an experimental verification of our findings we need the height profile of suspended graphene membranes. However, it is still challenging to measure the height profile of suspended graphene with an accuracy in the sub-angstrom regime with current methods, because they are generally not well applicable to suspended regions of a two-dimensional crystal (due to its low binding rigidity). In the past few years, it has been possible to obtain the height profile of graphene grown on many different substrates, most commonly on SiOFinally, one can also explore if the scale and conformal invariance properties are typical for graphene or are present in other two-dimensional crystals. We have made tests on suspended silicene, a membrane made with silicon atoms, and unfortunately, we have found that it crumples see , preventHow to cite this article: Giordanelli, I. et al. Conformal Invariance of Graphene Sheets. Sci. Rep.6, 22949; doi: 10.1038/srep22949 (2016).


As the thinnest conductive and elastic material, graphene is expected to play a crucial role in post-Moore era. Besides applications on electronic devices, graphene has shown great potential for nano-electromechanical systems. While interlayer interactions play a key role in modifying the electronic structures of layered materials, no attention has been given to their impact on electromechanical properties. Here we report the positive piezoconductive effect observed in suspended bi- and multi-layer graphene. The effect is highly layer number dependent and shows the most pronounced response for tri-layer graphene. The effect, and its dependence on the layer number, can be understood as resulting from the strain-induced competition between interlayer coupling and intralayer transport, as confirmed by the numerical calculations based on the non-equilibrium Green's function method. Our results enrich the understanding of graphene and point to layer number as a powerful tool for tuning the electromechanical properties of graphene for future applications. The thinness and elastic properties of graphene make it an ideal candidate for nanoelectromechanical systems. Here, the authors demonstrate the positive piezoconductive effect in suspended bi- and multi-layer graphene, showing the most pronounced response for three layer samples. Graphene1238101116In situ piezoconductive measurements on graphene provide an effective approach to study the correlations between electrical properties and mechanical strains. So far, studies have been focused on mono-layer graphene, and the negative piezoconductive effect has been widely reported, independent of the differences in graphene film synthesis, transfer methods and sample substrates1819202122in situ stress with a scanning probe. We observe positive piezoconductance in bi- and multi-layer graphene, with tri-layer graphene showing the most pronounced response. This intriguing phenomenon can be explained by the model of strain-induced competition between electronic interlayer coupling and intralayer transport, and further confirmed by numerical calculations based on non-equilibrium Green's function method.Here we investigate the piezoconductive effect of suspended graphene membranes with various layer numbers by applying 2 wafers with pre-etched trenches. The number of the graphene layers is identified via colour interference, and confirmed by Raman spectroscopy. Metal electrodes (5 nm Ti/50 nm Au) are deposited through home-made shadow masks, which effectively avoid wet process-induced device performance degradationin situ piezoconductive measurements, we introduce pressure-modulated conductance microscopy25Suspended graphene membranes are mechanically exfoliated and deposited on Si/SiOg. Here g(x) is defined byG(x) is the device conductance at AFM tip position x  and G0 is the undisturbed conductance value. For mono-layer suspended graphene, the device conductance drops upon local pressure applied  with different strains (up to 1‰). The detailed results are shown in gmax . According to the geometry of our devices, the strain ɛ can be calculated by the equationTo further study the observed layer number-dependent piezoconductive effect, we perform the same measurement on various suspended graphene devices . Here h has been corrected by subtracting the height of the tip at which the force begins to rise  device shows much larger gmax than the other multi-layer devices .where The positive piezoconductive effect is difficult to explain in terms of strain-induced decrease of Fermi velocity, the model that has been applied to strained mono-layer graphene19π-orbital tight-binding model Hamiltonian, which is written as428To understand the observed positive piezoconductive effect, we numerically calculate the transport properties of strained multi-layer graphene devices, which can be calculated by applying non-equilibrium Green's function technique and using two-terminal Landauer–Büttiker formula, with details described in the Methods section. To numerically study the piezoconductive effect in the multi-layer graphene systems in the presence of an external pressure, we consider a ci are the creation and annihilation operators on the site i. The first term H0 describes the pristine multi-layer graphene sheets, with μ being the chemical potential and tintra,inter denoting the intra- and interlayer nearest-neighbour hopping strength, respectively. In our consideration, we take tintra =2.60 eV and tinter =0.34 eV, respectively. The second term HD describes the influences of external disorders and the dephasing effect that is used to recover the macroscopic behaviour from a finite-sized quantum system and can be modelled by attaching individual virtual leads at each site i. Here ɛi is the on-site Anderson disorder strength that is uniformly distributed in the interval of  with W characterizing the strength of disorder and aik are the creation and annihilation operators for the virtual lead attached at the i-th site. In our consideration, we set the disorder strength to be 0.1 eV, because the prepared multi-layer graphene sheets are rather clean and it can better smear off the room temperature-induced thermal fluctuation. The last term HS represents the applied strain-induced effects including the induced site potentials and the variation of the intra- and interlayer nearest-neighbour hopping energies in the lattice-deformed region. The AFM tip-contacted lattices should experience noticeable lattice deformations, while the lattice deformation farther away from the AFM tip gradually decreases. Thus it is reasonable to assume that the variation of the intra- and interlayer hopping energy due to the lattice deformation takes the form of r0 is the centre of deformed region and L is the system length of the disordered scattering region, and the varying site potential takes the similar form of where gmax is determined by the strain-induced competition between the intralayer hopping and interlayer interactions. In the presence of an applied pressure from the AFM tip, the direct consequences on the multi-layer graphene include: (1) the extension of the lattice constant in the lattice-deformed area that correspondingly decreases the intralayer nearest-neighbour hopping; and (2) the local compression of the interlayer separation in the lattice-deformed area that effectively modulates the interlayer interactions, that is, increasing the interlayer hopping and inducing the local site energies. By applying the non-equilibrium Green's function technique and Landauer–Büttiker formula (see details in the Methods section) on a tri-layer graphene ribbon with length of 100 nm and width of 13 nm, we theoretically study the relation between the maximum relative conductance change gmax and the applied strain ɛ for tri-layer graphene. It is noteworthy to mention that all our obtained data are used to qualitatively explain and understand the underlying physical origin of the positive piezoconductive effect, and for the better presentation we have chosen certain parameters to compare with the experimental observations. Our numerical results show that gmax is always positive and increases with increasing strain, as plotted in the blue cycles in gmax increases slower, and suggests the relative contribution from interlayer modulation becomes less dominant. This model also explains why the piezoconductive property of bi- and multi-layer graphene is distinct from mono-layer graphene. For strained mono-layer graphene, negative peizoconductance is observed because no interlayer modulation but only intralayer contribution is present.We first focus on the tri-layer graphene that shows the most pronounced positive piezoconductive effect. As illustrated in Vienna ab initio simulation packaged between the nearest two layers as a function of the layer number n in To further understand the intriguing phenomenon that tri-layer graphene shows the most pronounced positive piezoconductive effect, we have performed first-principle calculations to study the lattice configuration of different multi-layer graphene devices under strain. Our first-principle calculations are performed using the projected-augmented-wave method as implemented in the γ, to describe the piezoconductive properties of bi- and multi-layer graphene devices:We can now introduce a characteristic factor, the piezoconductive factor γ has the same sign as piezoconductive effect. The square root dependence on strain is phenomenological but describes the experimental and theoretical data well and allows us to parameterize each gmax(ɛ) curve by a single-value γ. By applying the same non-equilibrium Green's function technique as used in studying the tri-layer graphene, we calculate the gmax–ɛ relation for graphene with various layer number n (n=2 to 6)  and find they are consistent with theoretical results. For bi- and tri-layer graphene, γ increases with n, suggesting the enhancement of the contribution from strain-induced interlayer modulation. But while n increases further, γ decreases, suggesting the interlayer modulation is dominated by the top three layers and additional parallel conduction paths from the extra layers suppress the positive piezoconductive effect. Here we note that, the roles of external effects  system, where CH4 and CHF3 are used as etching gases. The typical width of the trenches is 3 μm and the depth is around 250 nm. The number of layers of graphene membranes is first identified by an optical microscopy and further confirmed by Raman spectroscopy. To avoid common wet process-induced device performance degradation and yield loss, the electronic devices are fabricated by using a home-made shadow mask methodThe suspended graphene membranes are obtained by using mechanical exfoliation method on the pre-defined trenches on 300-nm thick SiO−1. The detailed experimental setup is schematically shown in A Bruker Multimode 8 AFM is used to build up the pressure-modulated conductance microscopy setup. A non-conducting AFM tip is used to apply adjustable local pressure on the top of the suspended graphene, yielding topography/height image of the strained graphene. The conductance/resistance of the device is measured via lock-in technique and monitored simultaneously as an external input of the AFM. The comparison of the conductance/resistance image and topography image offers detailed piezoconductive information of the suspended graphene membranes. The scanning procedure is done in contact mode with a Bruker NP-S10 tip, of which the Young's modulus is 0.24 N mp  can be expressed as:Using multi-probe Landauer–Büttiker formula, the current in the lead Vp/q is the spin-independent bias in the lead p/q. The electronic transmission coefficient from lead q to lead p is calculated as Tpq = Tr[ΓpGrΓqGa], in which the line-width function Γp is defined as I is the unit matrix with the same dimension as that of H. The retarded and advanced self-energy due to the coupling to all the real leads can be obtained numericallyd is fixed to 0.01 eV. In our simulations, a small external bias is applied between the left and right lead with VL=−VR=0.5 V. For dephasing effect, electrons lose the phase memory by entering and leaving the virtual leads. Thus, for each virtual lead i, the current has the constraint that Ji=0, which ensures the current conservation. Combining the above equation of Jp together with all boundary conditions for the real and virtual leads, the voltage Vp and current Jp in each real lead can be obtained.where −1. The first Brillouin-zone integration is carried out by using the 3 × 3 × 1 Gamma-centred grids. A vacuum buffer space of 20 Å is set to prevent the interaction between adjacent slabs.The generalized gradient approximation combined the Vander Waals correction with the DFT-D2 method of Grimme is used. The kinetic energy cutoff is set to be 500 eV. During the structure relaxation, the edge atoms and the probed atoms are not allowed to relax while the others are. All parameters are chosen to converge the forces to be <0.01 eV ÅHow to cite this article: Xu, K. et al. The positive piezoconductive effect in graphene. Nat. Commun. 6:8119 doi: 10.1038/ncomms9119 (2015).Supplementary Figures 1-11, Supplementary Table 1, Supplementary Notes 1-5 and Supplementary References


Sarcoidosis is a systemic inflammatory disease characterized by the development of noncaseating granulomas in multiple organ systems. Many hematologic malignancies and solid tumors, including melanoma, have been associated with sarcoidosis. We describe the clinical and pathologic findings of a 54-year-old man with melanoma-associated sarcoidosis. In addition, we not only review the literature describing characteristics of other melanoma patients with sarcoidosis, but also the features of melanoma patients with antineoplastic therapy-associated sarcoidosis. Sarcoidosis has been described in 80 melanoma patients; sufficient information for analysis was provided in 39 of these individuals. In 43.6% of individuals (17 out of 39), sarcoidosis was directly associated with melanoma; in 56.4% of oncologic patients (22 out of 39), sarcoidosis was induced by antineoplastic therapy that had been administered for the treatment of their metastatic melanoma. The discovery of melanoma preceded the development of sarcoidosis in 12 of the 17 (70.5%) individuals who did not receive systemic treatment. Pulmonary and/or cutaneous manifestations of sarcoidosis were common among both groups of patients. Most patients did not require treatment for sarcoidosis. Melanoma patients—either following antineoplastic therapy or without systemic treatment—may be at an increased risk to develop sarcoidosis. In antineoplastic therapy naive melanoma patients, a common etiologic factor—such as exposure to ultraviolet light—may play a role in their developing melanoma and sarcoidosis. Sarcoidosis is a systemic inflammatory disease in which noncaseating granulomas develop in multiple organ systems. The lungs and intrathoracic lymph nodes are most commonly affected. However, manifestations of sarcoidosis can also involve other organs as well as the central nervous system. Hematologic malignancies and solid tumors, including melanoma, have been associated with sarcoidosis. Indeed, oncologic patients have an increased risk of developing sarcoidosis and sarcoidosis patients have an increased risk of developing a malignancy. In some individuals, sarcoidosis and cancer have been diagnosed concurrently or within 12 months of one another. In other patients, however, systemic antineoplastic therapy may have contributed to the subsequent development of sarcoidosis. in situ and cutaneous sarcoidosis. We also review the literature describing characteristics of other melanoma patients with sarcoidosis. We describe a man who presented with two skin lesions; they were biopsied and showed melanoma In March 2014, a 54-year-old man with a history of cardiomyopathy presented for evaluation with a chief complaint of intermittent tinnitus, dizziness, and nausea of one month duration. He reported that the symptoms improved with meclizine and denied any associated otalgia. In addition, he complained of intermittent numbness and shooting pain in his left leg. However, his neurological examination was normal. The patient agreed to return for nerve conduction studies and an electromyogram at a later date.In May 2014, he sought evaluation for lesions on his face, chest, and extremities. A complete head-to-toe skin examination not only revealed multiple actinic keratoses at these locations, but also a 1.5 × 0.5 centimeter red-brown colored plaque involving the right antecubital fossa that had been present for at least one year . In addiin situ arising in the context of a compound nevus. The lesion was excised without complication or recurrence.Histologic examination of the tissue from his right antecubital fossa revealed a broad proliferation of moderately atypical melanocytes arranged as both irregular-sized nests and single cells along the dermal-epidermal junction . Areas oHistologic examination of the skin from his left calf revealed sarcoidal granulomas comprised of epithelioid-appearing histiocytes within the superficial dermis. Langerhans cells were present. The overlying epidermis demonstrated areas of hyperplasia and hyperkeratosis. A lichenoid lymphocytic infiltrate was also observed at the dermal-epidermal junction. Spindle-shaped interstitial histiocytes could be seen in the upper portions of the dermis. The spindle-shaped cells as well as the epithelioid component stained positively for CD68 and negatively for cytokeratin AE1/3 . CorrelaIn July 2014, the patient returned for follow-up. Prompted by the discovery of sarcoidosis in the skin, a systemic evaluation was subsequently performed. Laboratory results for the following studies were negative or normal: angiotensin-coverting enzyme (22 mcg/L), erythrocyte sedimentation rate (7 mm/h), serum sodium (139 mEq/L), serum potassium (3.9 mEq/L), serum calcium (9.5 mg/dL), aspartate aminotransferase (16 IU/L), alanine aminotransferase (13 IU/L), and serum glucose (114 mg/dL). Pulmonary evaluation included a chest roentgenogram and a chest computed tomography scan. The former was normal. However, the scan revealed multiple scattered subpleural and pleural nodularities. Mild ground-glass opacity surrounding a bronchovascular bundle in the left upper lobe was also observed. In addition, there was reticulation and micronodularity in the left lower lobe. These findings were diagnostic of stage 1 pulmonary sarcoidosis. His neurologic symptoms of intermittent tinnitus, dizziness, nausea, and left leg numbness had not improved. Based on the diagnosis of systemic sarcoidosis (with pulmonary and cutaneous involvement), he was evaluated for neurosarcoidosis. Nerve conduction studies and an electromyogram were performed; both were normal. A lumbar puncture revealed elevated protein (65 mg/dL) and glucose (77 mg/dL) in the cerebral spinal fluid; however, no inflammatory cells were present. An orbit computed tomography scan showed a small amount of mucosal thickening along the left auditory canal extending over part of the tympanic membrane and a head computed tomography scan revealed inflammation of the external auditory canal and tympanic membrane. The central nervous system evaluation was not conclusive, but the computed tomography scans of the orbit and head were suggestive of a possible diagnosis of neurosarcoidosis.Treatment was initiated with 40 milligrams of prednisone daily; his neurological symptoms progressively improved. The concurrent discovery of skin cancer and his systemic condition was also diagnosed as melanoma-associated sarcoidosis. Patients with sarcoidosis can have disease-associated skin lesions. Skin involvement in sarcoidosis is classified as either “nonspecific” or “specific”. Nonspecific skin lesions are often associated with acute sarcoidosis and do not demonstrate sarcoidal granulomas. In contrast, disease-specific skin lesions are usually associated with chronic sarcoidosis and reveal noncaseating granulomas in the dermis, subcutaneous fat, or both . Erythema nodosum is the most common nonspecific sarcoidosis-associated skin lesion . PatientSpecific lesions associated with sarcoidosis are pleomorphic in appearance. They may present as papules, plaques, subcutaneous nodules, or hypopigmented patches. Indeed, cutaneous sarcoidosis is sometimes referred to as “the great imitator” because it can exhibit so many different lesion morphologies . Papules are the most common type of sarcoidosis-specific skin lesion . InteresLupus pernio typically presents as indurated purple facial plaques. However, the fingers may also be affected. It is most commonly seen in African American women with persistent sarcoidosis. Lupus pernio may not only mimic lupus erythematosus, but also rhinophyma if localized to the nose . Subcutaneous nodules are a rare manifestation of sarcoidosis. However, individuals with malignancy-associated sarcoidosis who present with the new onset of subcutaneous nodules should be evaluated promptly in order to exclude metastasis. Subcutaneous nodules may also mimic epidermoid cysts and lipomas . Ichthyosiform sarcoidosis is another rare cutaneous manifestation of sarcoidosis that appears identical to acquired ichthyosis—an asymptomatic scaly eruption that most commonly affects the lower extremities. Lesional biopsy not only shows pathologic changes of ichthyosis vulgaris, but also sarcoidal granulomas in the affected skin. Patients presenting with ichthyosiform sarcoidosis often develop systemic disease . Specific lesions may also develop within scars. One 49-year-old woman who had previously had a melanoma excision presented with three firm subcutaneous nodules at the border of the melanoma scar on her left arm . PatholoSarcoidosis affecting the central nervous system—neurosarcoidosis—is rare. However, neurological symptoms were observed in an oncologic patient with treatment-associated sarcoidosis . Similaret al.’s patient showed abnormal enhancing tissue in the sella turcica and adjacent to the pituitary infundibulum. Other common magnetic resonance imaging findings in patients with neurosarcoidosis include leptomeningeal enhancement, intraparenchymal mass lesions, and/or hydrocephalus [The clinical presentation of neurosarcoidosis is highly variable. For instance, our patient complained of intermittent tinnitus, dizziness, and nausea. In contrast, the 36-year-old man with melanoma and ipilimumab-induced neurosarcoidosis experienced persistent headaches . Therefocephalus . UnfortuThe coexistence of sarcoidosis and malignancy was first observed nearly a century ago. In a 1917 report, Herxheimer described sarcoid reactions in patients with carcinomas of the breast, rectum, and cystic duct . PautrieIn 1972, Brincker reviewed 1500 patients with hematologic malignancies and identified five individuals who subsequently developed systemic sarcoidosis . Intereset al. validated Brincker and Wilbek’s results and provided strong evidence for an association between sarcoidosis and cancer. Indeed, investigators concluded that “sarcoidosis and malignancy may be etiologically related in at least a quarter of cases in which both are present [Two years later, Brincker and Wilbek investigated the incidence of malignancies in patients who had previously been diagnosed with sarcoidosis. In an analysis of 2544 patients with respiratory sarcoidosis, they found that lymphoma and lung cancer occurred significantly more frequently than would be expected in the general population . A subse present ”.There are two clinical settings in which sarcoidosis may occur in melanoma patients. Individuals with melanoma who did not receive systemic antineoplastic therapy are described in There were 17 reports of MAS in which specific patient characteristics were described. A slight female predominance (65%) was observed. The age at melanoma diagnosis ranged from 22 to 81 years, with a median age of 49 years. Sarcoidosis and melanoma were diagnosed at least a year apart in ten out of 17 (58.8%) patients with MAS. However, both conditions were diagnosed simultaneously or within one year of one another in seven individuals. The interval between the diagnosis of melanoma and sarcoidosis ranged from 0 to 23 years, with a mean interval of approximately 3.5 years. Interestingly, 12 out of 17 (70.5%) patients with MAS presented first with melanoma and were later diagnosed with sarcoidosis. However, it is not uncommon for the onset of sarcoidosis to precede the development of melanoma. Indeed, some evidence suggests that there is an increased risk of melanoma among patients who were previously diagnosed with sarcoidosis .Melanoma lesions most commonly developed on the extremities, but also appeared on the face, abdomen, thorax, and ear. The Breslow thickness was reported in 16 out of 17 patients and ranged from 0.0 millimeters to 5.5 millimeters; the mean Breslow thickness was approximately 1.91 millimeters. All 17 patients had surgical excision of their primary melanoma.Pulmonary manifestations of sarcoidosis were present in 16 out of 17 patients. Five patients presented with both pulmonary sarcoidosis and sarcoidal skin lesions. In addition, one patient developed cutaneous sarcoidosis with no pulmonary involvement.In most patients, the diagnosis of pulmonary sarcoidosis was established based on chest roentgenography and/or computed tomography. Bilateral hilar lymphadenopathy was a common finding. Pulmonary infiltration and fibrosis were also present among patients who had severe disease. Several patients underwent a mediastinal lymph node biopsy in order to definitively exclude metastatic disease. Interestingly, our patient had a normal chest roentgenogram; a chest computed tomography scan was required to confirm the diagnosis of systemic sarcoidosis. Therefore, a combination of multiple diagnostic tests—including roentgenography, computed tomography, and biopsy—may sometimes be required for the assessment of suspected pulmonary sarcoidosis. All six MAS patients with cutaneous manifestations of sarcoidosis presented with specific skin lesions, including subcutaneous nodules and violaceous plaques. Sarcoidosis resolved spontaneously in the majority of individuals diagnosed with MAS. However, approximately 40% of patients required pharmacological therapy for symptomatic relief—either corticosteroids (four patients), hydroxychloroquine (one patient), or both (one patient).There were 22 reports of DAS in which specific patient characteristics were described. In contrast to patients with MAS, the female-to-male ratio was approximately equal . The age at melanoma diagnosis ranged from 16 to 70 years, with a median age of 47.5 years. Sarcoidosis and melanoma were diagnosed at least a year apart in 17 out of 22 (77.2%) patients with DAS. However, both conditions were diagnosed simultaneously or within one year of one another in five individuals. The interval between the diagnosis of melanoma and sarcoidosis ranged from 0 to 14 years, with a mean interval of approximately 3.3 years. Melanoma lesions appeared on the extremities, face, back, digits, and scalp. The primary site of melanoma was unknown in two individuals with widespread metastases. The Breslow thickness was reported in 12 out of 22 patients and ranged from 1.45 millimeters to 5.04 millimeters; at approximately 2.80 millimeters, the mean Breslow thickness was substantially greater in patients with DAS than in those with MAS.Pulmonary manifestations of sarcoidosis occurred in 19 of the 22 patients with DAS. Chest roentgenography, computed tomography, and/or positron emission tomography were used in conjunction with mediastinal lymph node biopsy in order to exclude melanoma metastasis and establish a diagnosis of pulmonary sarcoidosis. Bronchoscopy with bronchoalveolar lavage was also performed in several patients. In addition, an endobronchial biopsy was required to identify noncaseating granulomas in patients with suspected upper respiratory tract involvement. Specific sarcoidal skin lesions were present in ten patients with DAS. Seven patients had both pulmonary and cutaneous sarcoidosis; three patients presented with cutaneous manifestations only. The lesions were pleomorphic in appearance and included maculopapular eruptions and subcutaneous nodules. Notably, none of the patients developed erythema nodosum or any other nonspecific sarcoidal skin lesion.Over 60% of the patients who developed DAS were being treated with interferons. Other anti-cancer drugs that were linked to sarcoidosis included ipilimumab, vemurafenib, and vindesine. Most patients were able to continue receiving antineoplastic therapy after the onset of sarcoidosis. However, treatment was discontinued in seven patients who developed severe symptoms. In addition, three individuals required treatment for sarcoidosis—either corticosteroids (two patients) or combination therapy with corticosteroids and hydroxychloroquine (one patient). An autoimmune mechanism involving T cells—specifically TH1 cells—and tumor necrosis factor alpha plays a fundamental role in the development of sarcoidosis . HoweverBoth melanoma and nonmelanoma skin cancers are associated with sarcoidosis. Therefore, ultraviolet radiation could conceivably represent a common etiologic factor . InteresMalignancy-associated sarcoidosis has been postulated to have an immunologic origin . A studyPatients with previously diagnosed sarcoidosis have an increased risk of cancer. In addition, oncologic patients with sarcoidosis have a worse prognosis . TherefoA decreased incidence of lung cancer was observed among men who had been hospitalized for sarcoidosis . The invIn addition, researchers in this study also noted that the incidence of many malignancies—including cancers of the colon, kidney, and rectum—was significantly increased among patients who had been hospitalized for sarcoidosis . Other sChronic inflammation and impaired antigen response have been suggested to promote the occurrence of cancer in sarcoidosis patients . MyeloidAntineoplastic therapies—including interferons, cisplatin, and interleukin 2—have previously been established to be associated with sarcoidosis . ModulatThe other two antineoplastic therapies that have been linked to DAS—vemurafenib and vindesine—do not affect melanoma immunologically. Vemurafenib is a B-Raf enzyme inhibitor that interrupts the mitogen-activated protein kinase/extracellular-signal-regulated kinase (MAPK/ERK) pathway. Indeed, vemurafenib-related dysregulation of this pathway contributes to the development of sarcoidosis . VindesiIn addition to sarcoidosis, oncologic patients may also develop sarcoid-like reactions. They are also sometimes referred to as either sarcoidal reactions or sarcoid reactions. In contrast to sarcoidosis, which is a systemic condition with or without associated skin lesions, patients with sarcoid-like reactions demonstrate sarcoidal granulomas—most commonly in the lungs, skin, and/or spleen—but do not fulfill the criteria for systemic sarcoidosis. Subtypes of sarcoid-like reactions have been described. The subtypes can be distinguished by the causative agent associated with the reaction. Infectious pathogens associated with sarcoid-like reaction include hepatitis viruses, mycobacteria, and propionibacteria. Non-infectious agents that may induce sarcoid-like reaction include tumor antigens and inorganic substances ,52.Similar to sarcoidosis, sarcoid-like reactions have also been associated with malignancies. These cancers include not only hematologic malignancies, but also solid tumors . In patiIn addition to neoplastic disease, sarcoid-like reactions have also been noted in patients with infections. A 35-year-old man who presented with facial sarcoidal granulomatous plaques was suspected to have systemic sarcoidosis. Subsequent imaging studies were consistent with a diagnosis of pulmonary sarcoidosis, revealing bilateral hilar and mediastinal lymphadenopathy. However, interferon gamma releasing assay was positive for tuberculosis. Indeed, in this unique individual, both sarcoidosis and tuberculosis existed concurrently .Other infectious diseases can mimic sarcoid-like reactions. They include cutaneous leishmaniasis, leprosy, and paracoccidioidomycosis . CultureIn oncologic patients with metastatic melanoma, sarcoidosis can simultaneously occur in the lymph nodes and lungs and thereby mimic metastatic neoplastic disease . A 35-yeSarcoidosis is a systemic inflammatory disease characterized by the development of noncaseating granulomas in one or multiple organs. Pulmonary and cutaneous manifestations are most common. However, any organ system, including the central nervous system, can be affected.Patients with hematologic malignancies or solid tumors, including melanoma, may be at an increased risk of developing sarcoidosis. Moreover, evidence from epidemiologic studies indicates that malignancies are more common among patients who were previously diagnosed with sarcoidosis. Various antineoplastic therapies—including interferons and ipilimumab—may also induce sarcoidosis in some patients with melanoma.The apparent association between melanoma and sarcoidosis is suggestive of a common etiologic factor. Some investigators have hypothesized that ultraviolet light plays a key role in activating an inflammatory cascade that contributes to both sarcoidosis and melanoma. However, a definitive mechanism of pathogenesis remains to be established.