Lateral graphene p–n junctions are important since they constitute the core components in a variety of electronic/photonic systems. However, formation of lateral graphene p–n junctions with a controllable doping levels is still a great challenge due to the monolayer feature of graphene. Herein, by performing selective ion implantation and in situ growth by dynamic chemical vapor deposition, direct formation of seamless lateral graphene p–n junctions with spatial control and tunable doping is demonstrated. Uniform lattice substitution with heteroatoms is achieved in both the boron-doped and nitrogen-doped regions and photoelectrical assessment reveals that the seamless lateral p–n junctions exhibit a distinct photocurrent response under ambient conditions. As ion implantation is a standard technique in microelectronics, our study suggests a simple and effective strategy for mass production of graphene p–n junctions with batch capability and spatial controllability, which can be readily integrated into the production of graphene-based electronics and photonics. Fabricating lateral graphene p–n junctions with controlled doping levels is instrumental to realize ultrafast and efficient optoelectronic devices. Here, the authors report a seamless graphene based photodetector doped by selective ion implantation and in-situ chemical vapour deposition. To realize the full potential of graphene, fabrication of graphene p–n junctions is vital as they are the basic building blocks of modern semiconductor devices such as diodes, bipolar transistors, photodetectors, light-emitting diodes, and photovoltaic cells6. However, current techniques to produce graphene p–n junctions mainly based on multiple electrostatic gates11, charge transfer from adsorbed chemical dopants15, and electrostatic substrate engineering16, all are difficult to implement in practical applications. In contrast, substitutional doping by introducing dopants into the graphene lattice during chemical vapor deposition (CVD) is an effective means to mass produce doped graphene with the desirable carrier types 20 and tunable Fermi energy levels 23. Using CVD doping, vertical graphene p–n junctions can be created by transfer and stacking of graphene with the desired doping type. However, the fabrication process has severe limitations including contamination and stacking orientation control which must be addressed25. Alternatively, a modulation-doped CVD strategy has been proposed to create lateral graphene heterostructures including i–n junctions26 and p–n junctions6. As the lateral graphene p–n junctions are formed by grafted growth of N-doped graphene from randomly nucleated B-doped graphene, it is challenging to obtain p–n junctions with good spatial control. In this respect, ion implantation which is routinely used in silicon processing to precisely dope bulk semiconductors offers excellent spatial and dose control. Recently, ion implantation has been extended to two-dimensional (2-D) materials including graphene for chemical doping and incorporation of N and B has been demonstrated29. However, the ion energy  much lower than the standard condition for IC industry is required to suppress the formation of implantation-induced defects. Owing to the monolayer or bilayer nature of graphene, the carbon atoms displaced during ion implantation with moderate energy cannot remain as interstitials within graphene basal plane and its immediate vicinities to participate in subsequent damage recovery through recombination of Frenkel pairs30. Therefore, the damage-curing annealing process routinely used in microelectronics processing cannot be applied directly to two-dimensional graphene.Graphene with unique properties has large potential in atomic-level electronic/photonic systems12 cmHz1/2 W−1) and responsivity (1.4~4.7 AW−1). As the ion implantation technology has been widely adopted in the current IC manufacturing, the fabrication of seamless lateral graphene p–n junctions with good spatial controllability, using the combination of ion implantation and in situ CVD, may pave the way for the industrial application of graphene in electronic and photonic devices.Herein, we describe a well-controlled approach to fabricate seamless lateral graphene p–n junctions by selected-area pre-implantation of heteroatoms into the catalytic substrate followed by in situ growth of doped graphene by dynamic CVD. The doping type and dopant concentration are strictly determined by the pre-implanted heteroatom species and pre-designed fluence, respectively. Since the doping process is analogous to in situ doping of epitaxial semiconductor layers in the integrated circuits (IC) industry, both the p-type and n-type regions in the graphene p–n junction are expected to possess an excellent crystalline quality comparable to that of pristine graphene grown under identical conditions. Our strategy combines the substitutional doping advantage rendered by CVD and the spatial and dose control provided by ion implantation. The obtained lateral graphene p–n junctions exhibit a typical rectification behavior together with a superior photo-response rate. The photodetector fabricated on the lateral graphene p–n junction shows excellent performance in the range of 532–1550 nm with high detectivity  to the B-doped graphene (orange). The micro-Raman spectra acquired along the p–n junction confirm that N shifts the 2D band to a smaller wavenumber, whereas B shifts it to a larger wavenumber relative to the pristine undoped graphene. At the junction between the N-doped and B-doped graphene, two characteristic bands corresponding to N doping and B doping emerge simultaneously, as shown in Fig. s of the B-doped graphene and the slightly asymmetric B-1s peak suggests that the predominant bonding for B is B-C33. The deconvoluted high-resolution N-1s spectrum , the B-K edge at 190 eV36 and N-K edge at 403 eV37 are observed from the B-doped and N-doped graphene, respectively. The EELS elemental maps show that either B or N is uniformly distributed throughout graphene as described in Supplementary Figure 38.Figure ray Fig. . The corrum Fig.  shows th15 to 4 × 1016 atoms/cm2, the 2D band of the B-doped graphene shifts gradually from ~2708 to ~2720 cm−1, whereas a monotonic decrease from ~2680 to ~2666 cm−1 is observed from the N-doped graphene , both N-1s and B-1s signals begin to rise at ~ 400 eV and ~ 190 eV, respectively. As the ion implantation fluence is increased from 8 × 1015 to 4 × 1016 atoms/cm2, N-1s, and B−1s peaks become resolved and their intensity increase accordingly. Based on the XPS intensity, Fig. In ion implantation, the dopant concentration can be controlled accurately. Figure ene Fig. . Electro41 as shown in the inset in Fig. * and 1s to σ* transitions, respectively. The sharp excitonic features at 291.5 eV indicate the long-range order in the electronic structure in the B-doped and N-doped graphene. The angular dependence in the C K-edge NEXAFS spectra in Fig. 43. Moreover, the B and N K-edge NEXAFS spectra are similar to the C K-edge NEXAFS spectra showing the 1s → π* and 1s → σ* transitions 44. The two representative STM topographic images obtained from B-doped and N-doped graphene show that the clear honeycomb lattice is interspersed with bright protrusions, which correspond to the substitutional dopants or the intrinsic defects are present as shown in Fig. 45, appears. As the STM tip moves towards the B dopant, the second minimum, i.e., a depression, emerges at the positive sample bias, which is ascribed to the Dirac point (ED = eVD) of graphene. The Dirac point is located at the positive sample bias, which is above the Fermi level, suggesting that the graphene film is doped with holes46. In addition, a noticeable shift in the Dirac point towards a more positive sample bias with enhanced depletion is observed as the STM tip gets closer to the B dopant due to electronic coupling between the B dopant and its neighboring C atom and the maximum shift is observed at exactly the center of the B atom. The recurrence of the Dirac point shift as the STM tip moves away from the B dopant verifies that the Dirac point is associated with B and the shift in the Dirac point is induced by hole charge doping due to B. As for the N-doped graphene, besides the Fermi level (0 eV), the depression associated with the Dirac point is also observed but at a negative sample bias, suggesting that the graphene film is electron doped. Furthermore, due to the distinct STS spectra obtained from B-doped and N-doped graphene, it is concluded that the observed protrusions originate from the dopants, not the suspected defects. Because of the similar electronic coupling effect, the circuitous shift of the Dirac point location is also observed when the STM tip moves across the N-doped region47. The same dependence has been previously reported for graphene supported by individual water clusters on Au (111), where nanoscale water clusters introduce the strong doping of electrons in graphene and the doping level is controlled by the cluster size48.To gain insights into the incorporation of dopants as well as the local electronic structure of the doped graphene, scanning tunneling microscopy (STM) and scanning tunneling spectroscopy (STS) are performed at 77 K on the doped graphene grown on Cu-like alloyIDS−VG curve reveals a typical symmetry in the electron and hole conductance with the charge neutrality at nearly zero volt  and compared with pristine graphene as shown in Fig. 53, as shown in Figure 19 and Supplementary Note 55 ) and near-infrared light  with a fixed power density of 15 mW/cm2 are chosen to excite the p–n junction region. Figure Ids−Vds characteristics of the lateral graphene p–n junction in the dark and under light illumination with different wavelengths. Importantly, a clear current rectification behavior, which is a typical feature for semiconductor p–n junction, is observed for the dark current, as shown in the inset of Fig. 58. However, PV effect should not be the dominant effect, since it usually leads to photocurrents in the same direction. While, as observed in Fig. + or nn−, as described previously62. Although the doping seems homogeneous across large area of doped graphene, the non-uniform doping still exists locally, especially near the p–n junction interface  and responsivity (1.4~4.7 AW−1). Recently, high detectivity (~1012 cmHz1/2 W−1) is also achieved in the broad spectral range (300–950 nm) from the graphene p–n vertical-type tunnelling diodes, but the responsivity (0.4~1.0 AW−1) is inferior to our lateral graphene p–n junction63. The performance of photodetector built on graphene p–n junction in further evaluated by the pulsed light illumination (1550 nm) with variable intensity, as summarized in Fig. 2, and decreases subsequently as the intensity attenuates back to 15 mW/cm2. It should be noted that the photocurrent barely varies under the light illuminations at a fixed intensity, which suggests the photodetector built on graphene p–n junction exhibit good repeatability. In addition, the good reproducibility of the photodetector device is also confirmed by the measurements from multiple devices, as summarized in Supplementary Figure tr) and fall time (tf) are evaluated to be 1.2 and 0.8 μs, respectively. The response speed is superior to that reported previously from graphene-based photodetectors65. We believe that such an ultrafast response should be attributed to the lateral structure of our graphene p–n junctions at which fast transport of photo-generated carriers is maintained along the lateral direction due to the high in-plane mobility, which agrees with TCAD simulation results  mix atmosphere. Finally, CH4 was shutoff and the sample was cooled down to room temperature in a mixture of Ar and H2 (200:10 sccm flow rate). The optimized thermal procedures are summarized in Supplementary Figures The Ni/Cu bilayer substrate was formed by the deposition of a 300 nm thick Ni layer on a high-purity Cu foil  by an electron beam evaporator. Then, 60 keV B and N ions with different fluences were implanted into the selected areas specified by photolithography. The Ni/Cu bilayer substrates were firstly cleaned by acetone, isopropyl alcohol, and deionized water several times and then annealed at 950 °C for 30 min in Ar, HThe doped-graphene films were transferred onto an oxidized silicon substrate or quartz slides for analysis and characterization by a PMMA assisted wet transfer method. Detailed experimental procedures are provided in and Supplementary Note Raman scattering (HORIBA Jobin Yvon HR800) was performed to evaluate the thickness, quality, and uniformity of the doped graphene films. Crystallographic information and the number of doped-graphene layers were determined by transmission electron microscopy (TEM) (FET-Tecnai G2F20 S-7WIN). The STM measurements were performed in the constant current mode by applying a bias voltage to the sample. The polarization dependence of the π- and σ-resonances of the graphene- and graphite-functionalized systems were investigated by NEXAFS at beamline 20A1 of National Synchrotron Radiation Research Center at Taiwan. XPS  and EELS (FEI Tecnai G2 F20) were employed to determine the chemical composition of the doped graphene. The photoelectric measurements were conducted under ambient conditions using an Agilent (B1500A) semiconductor parameter analyzer together with the Keithley 4200 semiconductor characterization system.Supplementary Information


In this paper, we propose a broadband absorption-controllable absorber based on nested nanostructure graphene and a narrowband frequency-tunable absorber utilizing gold-graphene hybrid structure in the terahertz regime. The numerical simulation results showed that the absorption of the broadband absorber can be changed from 27% to more than 90% over 0.75 to 1.7 THz by regulating the chemical potential of graphene. With the same regulation mechanism, the absorbing peak of the narrowband absorber can be moved from 2.29 to 2.48 THz continuously with absorption of 90%. Furthermore, via the cascade of the two types of absorbers, an independently tunable dual-band absorber is constituted. Its absorption spectrum is the superposition of absorption-controllable absorber and frequency-tunable absorber. The absorptivity and operating frequency of the two absorbing bands can be tuned independently without mutual inference. Moreover, it is insensitive to the polarization and it maintains high absorption over a wide range of incident angle. For the flexibility, tunability as well as the independence of polarization and angle, this design has wide prospects in various applications. In recent years, terahertz (THz) wave, lying between the microwaves and infrared light in the electromagnetic spectrum, has attracted great attention of researchers for its potential applications in communication, imaging, sensing, spectroscopy, etc. ,2,3,4. WHence, the research on development of tunable THz absorbers based on graphene has been expanding fast recently. To enhance the absorption and tunability, THz absorbers usually take advantage of patterned graphene, graphene-gold hybrid structure, and gold patches along with monolayer graphene ,15,16,17In this paper, we propose a dual-band THz graphene absorber with a broad absorption-controllable band and a narrow frequency-tunable band using the cascade method. Firstly, a broadband absorption-controllable absorber is designed. This absorber consists of periodic nanostructure graphene layer placed over dielectric layers backed on a reflector. The nested square ring and patch of graphene sheet achieves a high switching intensity of absorption over a wide band when adjusting the chemical potential via external gate voltage. Secondly, a narrowband frequency-tunable absorber is proposed. It is composed of a graphene-gold hybrid layer, a substrate spacer, and a gold grid, exhibiting a typical sandwich structure. The graphene strips deposited on the top layer dissipate the resonant power of gold structure, thus forming an absorption band. Besides, via controlling the external gate voltage, the resonant frequency can be regulated for the perturbation caused by the regulation of chemical potential in graphene strips. Finally, replacing the gold reflector of absorption-controllable absorber with the frequency-tunable absorber of the sandwiched structure, an independent tunable dual-band absorber is constructed.Bk is the Boltzmann’s constant, e is the charge of an electron, and h/2π is the reduced Planck’s constant, FE is the chemical potential (Fermi energy), τ is the relaxation time set as 0.1 ps, which is typical for experimentally studied graphene [H(Ω) is defined as:Graphene is considered as an infinitesimally thin material, which can be modeled as a two-dimensional sheet ,27. The graphene , T is rogZ = 1/σg. The real and imaginary parts of the surface impedance as a function of chemical potential is illustrated in FE, the surface impedance decreases gradually. Therefore, the surface impedance of graphene can be effectively regulated by changing the chemical potential.According to the Pauli exclusion principle, the inter-band contribution of graphene conductivity can be safely neglected in low THz region . Then, tFv is the Fermi velocity, 0ε and rε are the permittivity of free space and the substrate between electrodes, separately. gV is the gate voltage, e is the electron charge, and sd is the thickness of the spacer between electrodes. Therefore, by regulating the surface impedance of graphene through the gate voltage, the intensity of surface plasmon resonance can be tuned effectively, thus controlling the absorption of THz absorber based on graphene.To manipulate the chemical potential of graphene via electrostatic doping, an external gate voltage is applied between the graphene layer and underneath layer. The relation between chemical potential and gate voltage can be approximately expressed by the equation ,33:(5)EF2-doped Si-SiO2 sandwich structure, a gold ground. The nested square ring and patch of graphene can form resonance and excite localized surface plasmon resonances, which are hybridized with each other leading to a broadband absorption band [2 layers function as spacers to separate the graphene, doped Si, and gold. It is a normal substrate and doesn’t have effects on the absorption. Doped Si plays the role of another electrode gate to realize the dynamical control of chemical potential of graphene [εAu = 1−ωp2/ω(ω+iγ0), where bulk plasmon frequency of gold is ωAu = 1.37 × 1016 S−1 and the collision frequency is γ0 = 4.08 × 1013 S−1 [Firstly, a broadband absorption-controllable absorber is proposed. ion band . The diagraphene ,35,36. T1013 S−1 . The str2-gold sandwich structure. The front layer is a square gold patch array surrounded by the gold grid, and graphene strips are evenly deposited at the bottom of the slot. The patch-grid periodic metal structure generates a parallel capacitance and inductance (LC) resonance [2 acts as a spacer and also as a support layer. The back gold grid in staggered arrangement plays the role of electrode gate. As mentioned above, graphene sheet behaves like the series of inductance and resistance. By increasing the voltage between front and back layer, the inductance of graphene drops gradually, the total paralleled inductance of the resonance decreases as well, thus moving the absorbing frequency. The detailed structural parameters are shown in the caption of Secondly, a narrowband frequency-tunable absorber is designed. esonance ,40, whos1f is controlled by the voltage between nanostructure graphene and doped Si layer 1V and the frequency of the second operating band 2f is tuned via the voltage between the gold-graphene hybrid layer and back grid layer 2V. Since the nanostructure graphene is almost transparent to incident wave of 2f and the frequency-tunable absorber reflects all of the transmitted wave of 1f, two absorbing bands can be controlled individually without impacting each other. In theory, the doped Si can play electorate for both two different absorbers, but the distance between doped Si and gold-graphene hybrid structure is quite long, which may lead to an extremely high bias voltage. The introduction of cross feed network can solve this problem effectively. Besides, as shown in Finally, an independently tunable dual-band absorber is constituted by substituting the frequency-tunable absorber for the gold ground of the broadband absorption-controllable absorber. The absorption of the first operating band R = ǀS11ǀ2 and T = ǀS21ǀ2. Then, absorption can be obtained through A = 1−R−T. The influence of the chemical potential on the absorption spectrum of the broadband absorption-controllable absorber is given in FE = 0.7 eV. As FE decreases from 0.7 to 0 eV, the surface impedance of graphene drops rapidly, the absorption changes continuously from 90% to 27% corresponding the intense of resonances weakened. In the process of chemical potential decline, the rate of change in absorption is gradually accelerated, consistent with that of surface impedance shown in The full-wave simulation is conducted in the Computer Simulation Technology (CST) Microwave Studio (2015). It is a high-performance 3D EM analysis software package for designing, analyzing and optimizing electromagnetic (EM) components and systems . The refs1t) is shown in the s1t, the absorption curves appear a red shift. Besides, the absorptivity drops down gradually at the upper frequency while grows slowly at the lower frequency. In ground backed absorber, the thickness of substrate between patterned graphene and ground has a negative relation with the resonant frequency. Hence, the high-frequency resonance is weakened while the high-frequency one is enhanced. As a result, the operating band of the broadband absorber shifts to lower frequency with the increase of s1t.The absorption spectrum of the broadband absorption-controllable absorber with different thick spacer thickness  sustains a continuous growth from 30% to more than 90%. At the same time, the second absorbing peak located at 2f = 2.3 THz maintains the absorption over 90% without frequency shift due to the fixed F2E. However, as shown in F1E, which leads to the gradual expansion of effective operating bandwidth (over 90%) of the second absorbing peak. F2E with F1E fixed at 0.7 eV. The effective absorbing band from 0.72 to 1.6 THz remains unchanged while the operating frequency of the second peak 2f moves from 2.3 to 2.5 THz, as F2E grows from 0 to 0.3 eV. During the blue shift of 2f, the effective bandwidth of the absorbing peak becomes narrow slowly, which is attributed to the decreased absorption of nanostructure graphene from 2.3 to 2.5 THz. In brief, the dual-band absorber combines the characteristics of broadband absorption-controllable absorber and narrowband frequency-tunable absorber. The absorption at 1f and the position of 2f can be tuned independently and freely.F1E and F2E are fixed at 0.7 and 0 eV. ϕ of incident wave. As ϕ varies from 0° to 90°, the absorption spectrum has no change since the C4 symmetry of the structure ensures the robustness. For TE (Transverse electric) and TM (Transverse magnetic) polarized wave under oblique incident angle θ, the absorption spectrums are shown in 1f and 2f remains over 90% up to 50° for TE polarized wave. However, as θ continues to rise, there is a decline tendency for the absorption at narrow absorbing peak 2f and the low frequency portion of the broad absorbing band 1f. The absorptions at these regions is triggered by the magnetic resonance on the same layer. With the increase of incident angle for TE waves, the transverse magnetic field decreases gradually, so does the intensity of magnetic resonance, resulting in a drop in absorption. As for TM polarized wave, the magnetic resonance and localized surface plasmon resonance are not affected by the incident angle so that the absorption remains more than 90% over a wide range of incidence angles up to 60°. Besides, the broad absorbing band is expanded to higher frequency when θ = 50°, because some parasitic resonances occur and become stronger at large incident angle [The independence of absorption with respect to angle and polarization is essential in practical applications. Therefore, the absorption under different incident angles and polarized wave is investigated. Here, the chemical potentials nt angle ,18. The A dual-band tunable absorber has been designed and demonstrated theoretically, by cascading two different types of absorber. The first broad absorbing band is produced by the hybridization of different resonances in the nanostructure graphene. By changing the voltage between the graphene and doped Si to control the resonance intensity, the absorption can be continuously tuned from 30% to 90% over 0.72 to 1.6 THz. The second narrow absorbing band is formed by the magnetic resonance of gold-graphene hybrid structure. The absorbing peak with 90% absorptivity can be varied between 2.3 to 2.5 THz via controlling the voltage between hybrid layer and back grid layer. The two operating bands are controlled with different electrode gates and can be independently regulated without interference, thus greatly expanding the scope of its applications. This paper presents the general method of designing broadband absorption-controllable absorber and frequency-tunable absorber. In addition, the integration technique for combining the mentioned reconfiguration capabilities is elaborated, which is also available in other frequency for the scalability.


Sarcoidosis is a multisystem granulomatous disease in which pulmonary involvement is the most characteristic feature. Even though extrapulmonary manifestations occur infrequently in the area of the head and neck, an occasional patient will have oral involvement. Oral involvement generally appears in patients with chronic multisystem sarcoidosis and seldom occurs in the acute stage. The oral lesions may be solitary, multiple or part of a generalized disease. In some cases, oral involvement is the first, or only, manifestation of the disease and appears as a nontender well-circumscribed brownish red or violaceous swelling, as papules, or as submucosal nodules that can occasionally either show superficial ulceration or be symptomatic. Gingival involvement presents as red gingival enlargement. Although the etiology of sarcoidosis is unknown. The diagnosis of sarcoidosis is established when clinical features are supported by histopathological evidence of typical non-caseating epithelioid granulomas and other laboratory tests. The differential diagnosis of oral soft tissue lesions must consider other granulomatous conditions, such as infections , Crohn's disease, Melkersson-Rosenthal syndrome (including Mieschers cheilitis or cheilitis granulomatosa), Wegener's granulomatosis, foreign body reactions and hairy cell leukemia. Management of oral sarcoidosis has been variable, ranging from no treatment to radiation therapy. Surgical excision and curettage were found to be the most common therapies, followed by systemic therapy such as steroids, methotrexate, hydroxychloroquine and minocycline. We report a case of sarcoidosis limited to oral involvement in a 12-year-old girl, treated with corticosteroid.


Formal validation studies are of critical importance in determining whether or not household survey questions are providing accurate information on what they intend to measure. These studies supplement an array of methods used to evaluate survey questions.This paper summarizes the methods used by the two major international household survey programmes – The Demographic and Health Surveys Program (DHS) and the Multiple Indicator Cluster Surveys (MICS) – to decide on possible modifications to the survey questions, nomenclature, tables, and interpretation of findings over time as additional information on the validity of the questions becomes available.Validation studies are most useful if they are conducted in a variety of different settings in low- and middle-income countries, preferably using representative samples and procedures that replicate DHS and MICS field conditions. Pilot tests, pre-tests in each country, feedback from interviewers and survey staff, and cognitive interviewing provide additional information about how well survey questions are understood and provide accurate information. The paper provides specific examples of changes that have been made in response to findings from validation studies and changes in international recommendations. Quantitative data are key to monitoring national and global-level commitments, as well as for prioritizing, planning, and evaluating programmes at the country level. For the Millennium Development Goals (MDGs), comparable data on a range of indicators were made available, in large part through nationally-representative household surveys. The UNICEF-supported Multiple Indicator Cluster Surveys (MICS) and the USAID-supported Demographic and Health Surveys (DHS) provided a substantial proportion of the data required to monitor progress toward the MDGs. For example, at the endline assessment for the MDGs, DHS and MICS contributed about half of the data on contraceptive prevalence, 60 percent of the data on child malnutrition, and 80 percent of the data on children’s use of insecticide-treated mosquito nets . It is wInterest in international survey methodology has recently gained additional traction. The SDGs have a tiering system for indicators which reflects the number and quality of measures available at the global level. Tier 1 indicators, for instance, are conceptually clear, have an established methodology and standards available to use and for which countries produce data on a regular basis. Tier 3 indicators, on the other hand, have no established methodology or standards and they are only now being developed . AgainstThe work of the Improving Coverage Measurement Core Group is particularly relevant in the Maternal, Newborn and Child Health (MNCH) sector, which relies heavily on data on population coverage from household surveys. The papers in this supplement are timely because they reflect on some key indicators and concepts that are becoming increasingly important for the international community and for measurement in household surveys. They typically focus on validating coverage indicators for priority interventions, although some studies start with a very long list of interventions of varying priority. In this paper, we discuss the mechanisms used to develop questions for the DHS and MICS surveys and examine the evidence and circumstances that the programmes use to make decisions about changing global standards. We also provide some key examples of how MNCH-related household survey questions have been adapted over time to take into account new evidence. Finally, we discuss the potential implications of the validation studies in this supplement for household surveys going forward.DHS and MICS work in rounds, allowing the programmes to periodically update tools to reflect global demands and emerging needs. Questionnaire revision, which is a part of this process, entails considerable effort to develop and test questions that accurately measure the intent of indicators  and for which measures are replicable across settings and over time (reliability).Deciding what to measure is the first step in question development. Key concepts are clarified and conceptual boundaries are defined. Through this process, concepts are mapped onto suitable indicators or measures which accurately represent the underlying concepts. DHS and MICS have a strong preference for indicators that are applicable across a range of settings and that are meaningful to both national and global audiences. Ultimately, indicators are translated into questions using a multi-stage process.There are two broad approaches to translating indicators into questions. One is to review validated questions, test them in the context of household surveys and adapt them to the survey instruments. The MICS child discipline module is a product of this approach. Literature reviews showed good validation and extensive use of the Parent-Child Conflict Tactics Scale in the US  and acroWhen no suitable questions exist to measure an indicator, a second option is to develop them from scratch. The general process is to examine the literature on the topic, review suggestions received from outside experts, and, based on the relevant indicators, define a set of questions that can be adapted for use in MICS or DHS surveys. This process relies heavily on the extensive questionnaire design expertise and field experience within the survey programmes and on close and sustained collaboration between the DHS and MICS programmes in the development of harmonized questionnaires for topics that are of interest to both survey programmes. Following question design, it is the usual practice for MICS and DHS to field test new questions and modules in one or more settings using a number of questionnaire evaluation techniques before these are included country surveys. The field testing includes fieldworker debriefing, behavioural coding of interviews , formal It is widely recognized that survey results are affected not only by question wording but also by the placement of questions in a questionnaire. As such, after new questions or modules are designed and validated, they are integrated into existing questionnaires that are tested with other modules under standard field conditions, and the data and experiences from these tests are studied for evidence of their performance (which is discussed in the next section). MICS implements a global pilot survey using all of the new and updated questionnaires, protocols, and tools before the beginning of a new round of surveys to see how all previously validated questions work together in a single instrument. The most recent global MICS pilot took place in Costa Rica, where a new questionnaire for children age 5-17 years was tested, along with a range of new modules and questions . At the beginning of the first six rounds of DHS, a large-scale pilot survey of the questionnaires, manuals, and field procedures was conducted. In many cases alternative approaches were incorporated in the pilots. A full-scale pilot survey was not conducted at the beginning of DHS-7 since the questionnaire content was similar to that in DHS-6. During the life course of each round or phase of surveys, additional insights on question performance are gleaned and questions which perform poorly may have to be changed.The DHS Program developed five new optional questionnaire modules in 2015 . These modules, along with a substantially revised module on adult mortality, were extensively pilot tested in Ghana . The queEvidence on question performance generally comes from the development stage of the questions (previously outlined) or studies during/after wide-scale implementation. Given that each piece of evidence carries varying levels of rigour and significance, DHS and MICS must consider an entire body of evidence to make global changes to questionnaires, rather than a single source in isolation.One important source of evidence is experience from the field. DHS and MICS survey specialists provide technical assistance to surveys and observe how questions work across countries. Countries implementing MICS surveys also produce structured pre-test reports wherein they outline implementation issues, including those on question administration. Pre-test reports are also produced for DHS surveys, although some of the reports are abbreviated summaries of key points. These observations are a starting point to accumulate global experience on question quality.Examining patterns in the data across a large number of countries is another means to interpret question performance. These can include standard data quality checks , as well as reports on data quality which the DHS produces during each cycle of surveys -16. ThesDHS and MICS particularly take into account the findings from special validation studies that examine their approaches in general and the accuracy of key indicator measures. These studies should be from a variety of different settings, such as low- and middle-income countries across different regions, preferably using representative samples and resembling DHS and MICS field conditions. With reference to the Improving Coverage Measurement Core Group’s work, studies are well designed and use conditions similar to MICS or DHS. However, certain elements require further consideration. Question design should reflect norms in the survey methodology field. Two studies validated uterotonic use immediately after birth ,18 and cCountry ownership is a key tenet of international development programmes. Household survey programmes must therefore take into account the needs and requirements of national governments, key stakeholders, and funding agencies. While evidence from the above sources provides good inputs for countries to use in designing their questionnaires and tools and while the two survey programmes are able to influence certain outcomes, country-level decision makers ultimately decide the questionnaire content based on a variety of factors in addition to scientific evidence. Countries often want to repeat questions from previous surveys to measure changes over time even if there is evidence that the questions do not perform well. Despite this, DHS and MICS work with countries to ensure that these issues are minimized and that even with departures from the standard questionnaires, the standard indicators can still be calculated.Apart from country ownership, at the global level, subtle changes in indicators or their metadata can also influence the need to change global standards. These changes may impact indicator numerators and denominators and the algorithms used to calculate the indicators. For example, DHS and MICS have measured sanitation indicators for many years. However, a few years ago, the WHO/UNICEF Joint Monitoring Programme for Water Supply and Sanitation recommended that household surveys also measure whether or not sanitation facilities are shared with other households and use that additional information in deciding on what sanitation facilities are considered to be improved. To this end, an additional question on shared services was included in both DHS and MICS surveys.Model questionnaires for DHS and MICS surveys have always evolved over time to reflect the most recent scientific evidence, as well as emerging needs and changing priorities. One good example of the historical changes that have taken place is the questions on the prevalence and treatment of symptoms of acute respiratory infection (ARI). DHS and MICS questions on child health originally focused on the three major causes of death for children under five years of age . Since household surveys are not an ideal vehicle for actually diagnosing these diseases in young children, the survey questions asked about symptoms related to these diseases  during the two-week period preceding the survey.The earliest DHS surveys in the 1980s) asked about recent episodes of diarrhoea, treatment-seeking behaviour, and the administration of oral rehydration salts or a home-based sugar, salt, and water solution. In the early 1990s, questions on fever and cough were added to the model woman’s questionnaire. For all children born in the last five years, the mother was asked whether the child had been ill with a cough in the last two weeks and in the last 24 hours, how many days the cough lasted, and whether the child was breathing faster than usual with short, rapid breaths during the illness. Even at that time, it was recognized that those symptoms were not specific to pneumonia, and that they could reflect a common cold, bronchitis, allergies, smoke exposure, or other conditions. The main purpose of those questions was to see if advice or treatment was sought for the child. Additional questions were asked about the type of treatment, but since it was not known what disease the child had, the questions could not determine whether or not the treatment was appropriate. By the time of the fifth round of the DHS surveys 2003-08), it was becoming increasingly clear that additional information on the symptoms of children’s respiratory illness would be needed. At that time, two important changes were made to the questionnaire. First, the question on cough with short, rapid breaths was expanded to include difficulty breathing. Second, a follow-up question was added for children with a cough with rapid breathing to determine whether the mother thought that the rapid or difficult breathing was due to a problem in the chest or a blocked or runny nose. These two additions to DHS were present from MICS3 (roughly 2005-2009). Since that time, only a cough with rapid or difficult breathing that was at least in part chest-related was considered to indicate symptoms of acute respiratory infection. The more recent strong evidence from carefully conducted validation studies in Pakistan, Bangladesh, and Nigeria [80s asked03-08, itAlthough the specific survey questions that are asked are of paramount importance, it is also important to pay attention to the way in which the results are tabulated and interpreted. Even from the beginning, the ARI questions were largely meant to determine whether parents were dealing appropriately with the symptoms rather than whether or not antibiotics were being appropriately administered to sick children. DHS and MICS surveys report on various types of treatment for symptoms of ARI in the standard tables. The inclusion of antibiotics as one type of treatment is not meant to suggest that children with symptoms of ARI should be treated with antibiotics, and the table is not currently interpreted in that way, although some outside agencies or researchers have chosen to interpret the results in different ways. Nevertheless, particularly given increasing resistance to antibiotics, survey reports should take great care to ensure that the reader does not interpret the findings in that way. Out of an abundance of caution, consideration should be given to the possibility of removing antibiotic treatment from the table to avoid confusion. This would be consistent with the conclusions of the three studies in Pakistan, Bangladesh, and Nigeria mentioned above that it is important to continue asking survey questions about the symptoms of ARI and care-seeking behaviour, but that since most young children with those symptoms do not have pneumonia, appropriate treatment cannot be determined.Attention also needs to be paid to appropriate terminology. DHS has always used the term “symptoms of acute respiratory infection” to refer to the symptoms asked about, even as the questions have changed over time. MICS referred to this indicator as “suspected pneumonia” beginning in 2009, but harmonized terminology with DHS around 2012.A similar path was taken in the malaria field. In the absence of a blood test for malaria parasites, WHO used to promote presumptive treatment of symptoms of malaria in young children in malaria endemic areas with an appropriate antimalarial medication. However, as the prevalence of malaria has dropped and the availability of rapid diagnostic tests for malaria has risen, WHO changed to its current recommendation to indicate that ‘all cases of suspected malaria should be confirmed using parasite-based diagnostic testing (either microscopy or a rapid diagnostic test) before administering treatment . TreatmeIn addition to the examples given above, some studies have tried to determine whether alternative question wording would improve the measurement of maternal, newborn, and child health questions or perhaps reduce confidence intervals for a given sample size. Some of these studies have examined the effect of changing the length of reference periods for the questions on childhood illnesses and other questions -24. ThesThe importance of skin-to-skin contact (SSC) of the newborn with the mother immediately after birth has been well established. Such contact promotes greater respiratory, temperature, and glucose stability, and less stress for the newborn. Based on the growing literature on the utility of SSC, DHS added a question on that topic in recent years. The question was worded ‘Immediately after birth, was (NAME) put directly on the bare skin of your chest?’ However, it was subsequently shown that using just one standard question on SSC does not provide accurate information about what actually occurred around the time of birth ,25,26. BImmediately after birth, was (NAME) put on your chest?IF YES: Was (NAME)’s bare skin touching your bare skin?MICS began including questions on SSC in round six of the surveys (beginning in 2016), which corresponded to the needs of the Newborn Indicators Technical Working Group. In 2015, MICS reviewed the available questions on SSC and tested an amended set in a field test in Belize. The test also included a photograph of the recommended SSC position, to assist respondents in understanding the SSC concept. Results of cognitive interviews of women in the study indicated that they generally recalled events around the time of delivery, including drying of the newborn and SSC. However, while SSC appeared common, women also said that newborns were often clothed or wrapped, and that SSC lasted only for short periods of time. With these observations in mind, MICS kept the additional question on whether the newborn was wrapped, with the intention of removing these cases from the numerator of the SSC indicator.The MICS6 questions are as follows:name) put directly on the bare skin of your chest?Immediately after birth, was . The questions should be sensitive to cultural norms, to the extent possible. They should be translatable into numerous languages without losing any of their original meaning. Questions asked on the standard questionnaires have generally been pilot tested overall and then pre-tested in each country in all of the languages that are used for interviewing in that country and in both urban and rural areas. During interviewer training, which includes mock interviews and field testing, questions that are difficult to understand in any language are identified. At that point, changes may be made in question wording to improve comprehension, with care being taken not to change the original meaning of the questions.Feedback received during the course of the fieldwork from interviewers and those monitoring the fieldwork, as well as a full debriefing of interviewers at the end of some surveys, adds to the body of evidence about respondents’ understanding of survey questions. Moreover, even if questions are well understood, they may not produce accurate information either because the respondent does not know the answer, for example, in the case of what happens around the time of birth for institutional births , or becaCognitive interviewing is increasingly being used by household survey programmes to provide an in-depth look at how people understand survey questions. It is likely that cognitive interviewing will be expanded in the future when designing new questions and reconsidering the wording of some problematic existing questions. Despite the usefulness of cognitive interviewing to gain insights into the understanding of key survey questions, the limitations of cognitive interviewing  also need to be considered in determining whether or not questions are valid or could be improved.Formal validation studies supplement the array of methods outlined above, and some examples have been given about how changes in questions or the interpretation of findings have already been made based on the results of validation studies. Even if a particular question is not well validated in one study, it is of concern. However, one study in one location, particularly if it is based on purposive sampling with a small sample of respondents, may not be enough to trigger global changes in question wording or the possibility of deleting some questions. It is necessary to consider the balance of evidence for the validity of an indicator or particular questions from several studies, if available, and the strength of that evidence. It is also important to note that validation studies of survey questions usually compare responses to survey questions to a ’gold standard,’ but the gold standard may itself be imperfect.The particular recommendations from validation studies should be carefully considered. Should a survey question be retained, changed, or dropped? Can a question that is not well validated be kept if one or more follow-up questions are added to clarify the original response? Are there any other viable sources that could produce reliable data on the same topic or that could provide an accurate estimate of an indicator? If not, are some data (with warts) better than no data at all, as long as the nature of the problems with the questions is well understood and the results are cautiously interpreted? Is a question needed to measure a specific Sustainable Development Goal indicator or another standard indicator  when there are no other viable sources for such data? A final consideration relates to country ownership of household surveys, as indicated above. We often find that countries want to retain (older) questions in order to measure trends even if it is found that a question is not well validated or the international indicator that is being measured has changed. In such cases, a thoughtful negotiation process may have to take place to ensure that some trend measurement is still possible while changes to ensure accurate measurement in the new survey are accommodated.Finally, we have a few suggestions to help guide the design of future independent validation studies of survey questions to make their findings most useful to household survey programmes. First, it is important that the basic question wording and translation in a validation study be exactly the same as in DHS or MICS surveys if the study largely aims to validate questions used in those surveys. This is already being done in almost all validation studies. However, it is equally important to try to anchor the study in the context of DHS and MICS surveys in terms of characteristics of respondents, interviewer training, use of the DHS and MICS interviewers’ manuals, field practice, fieldwork procedures, field monitoring, and data quality checks. Otherwise, questions that may be valid in a short survey that focuses on one topic may not be equally valid when integrated into a full DHS/MICS survey. It would be ideal if the questions could be tested in the context of a full DHS or MICS survey, although this may not be practical in most circumstances. It would also be desirable for validation studies to test the inclusion of additional questions, for example, questions on additional symptoms or the use of pill boards or videos, as was done in the ARI validation studies mentioned above. Without this additional information, the results of validation studies leave the survey programmes with the options of retaining or dropping questions, without providing guidance on how the questions might be improved, unless alternative question wording has been included. It would also be desirable to involve DHS and MICS in the development of validation study protocols or alternative strategies for data collection, since some of the alternative methods that might be tested are non-starters for large-scale surveys.The DHS Program has recently developed a new questionnaire module on maternal health, and the results of all relevant validation studies published to date have been taken into account in decisions about questions to include in this module. A pilot test of the new DHS maternal health module, including cognitive interviewing, is currently underway. DHS and MICS welcome further validation studies of survey questions to provide important information about changes in questions that may be needed or questions that should be considered for deletion because they do not perform well. When questions are well validated in these studies, not only can they be retained, but users of the survey data can have greater confidence in the reliability of questions and the results.


Copper-based materials are common industrial products which have been broadly applied to the fields of powder metallurgy, electrical contact, and heat exchangers, etc. However, the ease of surface oxidation limits the durability and effectiveness of copper-based components. Here, we have developed a powder metallurgy process to fabricate graphene/copper composites using copper powders which were first deposited with graphene layers by thermal chemical vapor deposition (CVD). The graphene/copper composites embedded with an interconnected graphene network was then able to be obtained by vacuum hot-pressing. After thermal oxidation (up to 220 °C) in humid air for several hours, we found that the degree of surface oxidation of our samples was much less than that of their pure Cu counterpart and our samples produced a much smaller increase of interfacial contact resistance when used as electrical contact materials. As a result, our graphene/copper composites showed a significant enhancement of oxidation resistance ability (≈5.6 times) compared to their pure Cu counterpart, thus offering potential applications as novel electrical contact materials. Copper is one of the most widely used industrial materials in the fields of powder metallurgy , electri2-hybridized lattice, has triggered great research excitement [−8 Ω m) [2 g−1) [Graphene, a two-dimensional atomically-thin carbon layer composed of an spcitement  due to i[−8 Ω m) , high sp [2 g−1) , and rob [2 g−1) ,19. In p [2 g−1) ,21,22. A [2 g−1) . Hence,  [2 g−1) ,24. Amon [2 g−1) . Chen et [2 g−1) . In addi [2 g−1) .D/IG ratio (≈0.86) in the Raman spectra suggests the graphene layer converted from the polymers is defective. As a result, currently, the fabrication of functional copper composites embedded with low-defect and well-defined graphene frameworks is still a great challenge.The advantages of graphene for barrier applications to protect copper from oxidation and corrosion have been demonstrated ,23. HoweIn this paper, a large-scale synthesis process was developed for preparing graphene/copper (Gr/Cu) microparticles by thermal CVD, followed by vacuum hot-pressing to fabricate Gr/Cu composites. Based on the uniform dispersion of low-defect few-layer graphene within the composites, the oxidation resistance of Gr/Cu composites was found to be 5.6 times greater than its pure Cu counterpart.−3 MPa. The surface of the obtained Gr/Cu composites was sequentially polished using 240, 400, 800, 1000, and 2000 mesh alumina waterproof abrasive papers. A pure Cu counterpart was also prepared using the same process for comparison.The production of Gr/Cu powders synthesized by catalytic CVD has been reported in a previous study of ours . High-quThe graphene quality was analyzed by a Raman spectrometer  employing a laser wavelength of 532 nm. The crystal structure of the composites was identified by an X-ray diffractometer . The sample morphology and elemental compositions were observed and determined by a field emission scanning electron microscope  equipped with an energy dispersive spectral analyzer (EDS).−3) and copper (8.9 g cm−3) commonly leads to difficulty in obtaining uniform dispersion of graphene in the copper matrix, especially for large scale production. Therefore, in order to solve this problem, our approach was to directly grow graphene layers on the surface of each copper powder by CVD, followed by hot-pressing consolidation to prepare our composites to be 12.6 mm in diameter and 1.9 mm in thickness. Based on our proposed process, the particle size of copper powders (average size ≈50 μm) before and after graphene growth may not change, as presented in The fabrication process of the Gr/Cu composites embedded with an interconnected graphene network is schematically illustrated in −1), G- (≈1580 cm−1), and 2D bands (≈2691 cm−1) [2D/IG ratio (≈1.07) and the full-width at half-maximum of the 2D-band (≈50.2), we concluded that the few-layer graphene was synthesized with a layer number of less than 5 [The quality and layer numbers of graphene in the Gr/Cu composites were identified by Raman spectroscopy. 91 cm−1) , respect91 cm−1) . In addis than 5 . In Figu2O is red. In order to investigate the improvement of oxidation resistance of the Gr/Cu composites based on the good barrier properties of low-defect graphene, both the Gr/Cu composites and the Cu counterpart were heated in air at 220 °C for 6 h at an ambient humidity of 50 ± 5%. According to previous studies , the con2O in the XRD pattern in The degree of surface oxidation of the samples was quantitatively identified by EDS mapping at an acceleration voltage of 5 kV. At least three spots (about 300 μm × 300 μm) were analyzed to produce the EDS spectra. A typical EDS spectrum of Gr/Cu composites after treatment is shown in −2, the ICR of the as-prepared samples (Gr/Cu composites and Cu counterpart) was similar. However, after thermal oxidation at different temperatures for 6 h, we found that the ICR of the Cu counterpart increased monotonically with an increase in treatment temperature. This increase in ICR can be attributed to the formation of an insulating oxide layer on the sample surface at high temperatures in air. By contrast, there was only a slight increase of ICR in the case of the Gr/Cu composites. The ICR increase of the samples compared to the as-prepared ones (at 30 kgf cm−2 clamping pressure) is plotted in One of most industrial applications for copper and its alloys/composites is their use as electrical contact materials . TherefoIn this study, copper-based composites embedded with an interconnected graphene network were fabricated by CVD growth of graphene on the surface of copper powders, followed by vacuum hot-pressing. Due to this characteristic microstructure and the barrier nature of low-defect graphene, the oxidation resistance of the obtained Gr/Cu composites was able to be significantly improved relative to that of the pure Cu counterpart. The ICR measurements indicate that the surface of the Gr/Cu composites was slightly oxidized even after thermal oxidation at 220 °C in atmosphere for several hours, leading to a small ICR increase. Our finding provides a new strategy for the development of copper-based electrical contact materials by modulation of the graphene microstructure within the matrix.


We found these RES-dependent introns were short, rich in GC and flanked by GC depleted exons, all of which are features associated with intron definition. Using these features, we developed and validated a predictive model that classifies RES dependent introns. Altogether, our study uncovers the essential role of the RES complex during vertebrate development and provides new insights into its function during splicing.Pre-mRNA splicing is a critical step of gene expression in eukaryotes. Transcriptome-wide splicing patterns are complex and primarily regulated by a diverse set of recognition elements and associated RNA-binding proteins. The retention and splicing (RES) complex is formed by three different proteins  and is involved in splicing in yeast. However, the importance of the RES complex for vertebrate splicing, the intronic features associated with its activity, and its role in development are unknown. In this study, we have generated loss-of-function mutants for the three components of the RES complex in zebrafish and showed that they are required during early development. The mutants showed a marked neural phenotype with increased cell death in the brain and a decrease in differentiated neurons. Transcriptomic analysis of  bud13, rbmx2 and snip1 mutant embryos showed a marked brain phenotype with a RES-dependent introns enrichment in genes with neurodevelopmental function. Altogether, our study unveils the fundamental role of RES complex during zebrafish embryogenesis and provides new insights into its molecular function in splicing.RES complex is essential for splicing in yeast but its function and role during vertebrate development are unknown. Here, we combined genetic loss-of-function mutants with transcriptomic analysis and found that a subset of introns is particularly affected in RES complex knock-out background. Those introns display the major hallmarks of splicing through intron definition mechanisms . Moreover,  Splicing is critical step in eukaryotic gene expression and is an important source of transcriptomic complexity . SplicinREtention and Splicing (RES) complex is a spliceosomal complex conserved from yeast to human. It is organized around the U2 snRNP-associated protein Snu17p/Ist3p (RBMX2 in human), which binds to both the pre-mRNA-leakage protein 1  and bud site-selection protein 13  . We. Webud13function . Zygoticnd snip1  Figs. Thesoderm) , consistr snip1)  or humanto 6 dpf . Interest 48 hpf . These rp53, casp8 and puma)  and nervous system development   , consistnd puma) , and ii)nd puma) . In cont, sox1a) .bud13∆7/∆7 mutants showed normal neural induction, morphogenesis and regionalization. For example, key brain areas such as the zona limitans intrathalamica , the mid/hind-brain boundary (pax2a expression), and rhombomeres 3 and 5 (krox20 expression) were properly specified . Other types of splicing events were less impacted upon RES depletion . Conversely, genes that were differentially expressed in the three mutants (down- or up-regulated) show significantly higher retention (higher ΔPIR) in the mutants compared to their WT siblings . Finally, genes with increased expression significantly overlapped with those with at least one strongly retained intron (ΔPIR>15)  across the transcriptome. Briefly, for any given intron, the percent intron retention (PIR) is calculated as the average number of reads mapping to the 5’ and 3’ Exon-Intron (E-I) junctions over the average number of reads mapping E-I junctions plus any Exon-Exon (E-E) junction that supports removal of that given intron  22]. We. We22]. epletion , althougepletion  in the lepletion , consistepletion , suggesth mutant , includiisorders ,24. GeneΔPIR>15) . Taken tTo identify features that are primarily associated with RES complex function, we first defined a set of introns that were confidently dependent on RES for proper splicing , (ii) had elevated GC content (P<0.001 Mann-Whitney-U test), (iii) were flanked by exons with lower GC content (RES-dependent introns (i) were significantly shorter than the control set   . The fal introns  showed F introns . Thus, aserpinb1l3 and col1a2 (predicted as RES independent) and wdr26b (predicted as RES dependent with ∆PIR>15) transcripts. Nevertheless, we detected no intron retention in ptch2, suggesting that its retention upon RES depletion may depend on its genomic context.Finally, to test our logistic regression model in isolation from the genomic context, we assayed four introns, two RES dependent and two RES independent, using minigene constructs including the tested intron plus the two flanking exons . Minigenin vitro studies on the single intron of the actin gene in yeast showed that the RES complex binds at the 3’ of the intron, between the BP and the acceptor site [in vitro but for U1 and U4 snRNP dissociation before the first catalytic step [in vivo and therefore that the biochemical functions reported in a limited set of RNAs reflect limitations of in vitro splicing reactions. Alternatively, the RES complex may not be involved in early splice site recognition, but could be a limiting factor for splice site pairing or other steps in spliceosome assembly progression in vivo, particularly for introns defined by intron definition, highlighting differences in molecular pathways for intron- and exon-defined splicing. These concepts are in line with increasing evidence that splice site selection can be modulated at late stages of spliceosome assembly or even catalysis [Splicing regulatory information is encoded by multiple sequence features, from the core signals (splice donor and acceptor and branch point) to other, less understood, sequence elements –29. Our tor site . We obsetor site . RES-deptor site ,21. Thistor site , and thatic step . One posatalysis ,27,30–35atalysis .irx1a, smn1, enc1, smad4, tbx2a, and nkx6.1/6.2). Specifically, zygotic mutants in bud13, snip1 or rbmx2 show microcephaly and decreased populations of GABAergic and glutamatergic neurons, despite normal specification and regionalization of the CNS  has been associated with epilepsy and skull dysplasia [BUD13 can rescue loss of bud13 function in zebrafish, and future studies will be needed to determine whether Bud13 has a conserved function during brain development in humans .bud13Δ7/Δ7, rbmx2Δ16/Δ16, snip1Δ11/Δ11 were obtained through natural mating of heterozygous bud13+/ Δ7, rbmx2+/ Δ16, snip1+/ Δ11 mutants, respectively (see below gene editing using CRISPR-Cas9). Tg(dlx6a-1.4kbdlx5a/dlx6a:GFP) lines [vglut:DsRed) [Wild-type zebrafish embryos were obtained through natural mating of TU-AB and TLF strains of mixed ages (5–17 months). Selection of mating pairs was random from a pool of 48 males and 48 females allocated for a given day of the month. P) lines ,46 were t:DsRed)  from theEmbryos were analyzed using a Zeiss Axioimager M1 and Discovery microscopes and photographed with a Zeiss Axiocam digital camera. Images were processed with Zeiss AxioVision 3.0.6.bud13Δ7/Δ7 mutant and their wild-type siblings respectively, two blinded raters segmented the raw z-stack images using ImarisCell module (Bitplane) and computationally counted the segmented cells in each channel (GFP and DsRed). Identical thresholds and parameters were applied to all samples for segmentation processing. Since the quantification performed by both independent raters yield consistent fold change in the respective cell counts between the bud13Δ7/Δ7 mutant and their wild-type siblings. Only one set of the analyzed results was displayed. Statistical analyses were conducted using Prism 6 (Graphpad).Whole-mount embryos were imaged in vivo by confocal microscopy  Mutant embryos and their wild-type siblings were scored at 30–32 hours post fertilization stage. Embryos were anesthetized using Tricaine and mounted in 0.6% agarose at an orientation where the frontal view of the brain was imaged. Embryos were imaged at 40x (1.3 Oil) using Z-stacks ranging from 70.41–96.02 μm, with a z stepping size of 0.4 μm. Z-stacks started at the first appearance of the GABAergic cells (GFP-labeled) and ended where GABAergic cells (GFP-labeled) could no longer be visualized. Each xy plane spanned 227.94 μm with a pixel size of 0.075 μm. Maximum intensity projections were shown for all confocal images. Images were processed using Fiji , Imaris To visualize apoptotic cells, vital dye acridine orange (Sigma) was used in live and dechorionated embryos. Embryos were incubated 2 minutes in PBS pH 7.1 with 2 ug/ml of acridine orange in the dark. After 3 brief washes in PBS, the embryos were placed in plates with 1% agarose and viewed with fluorescence microscopy, using the FITC filter set 1 .bud13, rbmx2 and snip1 ORFs were PCR amplified (bud13) in pT3TS [rbmx2 and snip1). bud13 PCR product was cut using NotI and EcoRI and ligated into pSP64T. rbmx2 and snip1 PCR products were cut with NcoI and SacII and ligated into pT3TS. To optimize Kozak sequence, the forward oligonucleotide used for rbmx2 ORF introduced extra aminoacid in the 2nd position (GGC). Human bud13 ORF was cloned in pSCDest [XbaI (pT3TS), BamHI (pSP64T) or KpnI (pSCDest) and capped mRNA was synthetized using the mMessage mMachine T3 (pT3TS), or SP6 (pSP64T and pSCDest) kit (Ambion), respectively and in accordance with the manufacturer’s instructions. In vitro transcribed mRNAs were DNAse treated and purified using the RNeasy Mini Kit (Qiagen). All mRNA rescued the mutant phenotypes when 50–100 pg were injected in one cell stage embryo.Zebrafish mplified  using cDin pT3TS  , is a Tol2 transposon-based, bipartite construct consisting of heat-shock promoter (hsp), a multiple cloning site (MCS), to insert the desired validation cassette, flanked by Xenopus globin UTR and polyadenylation tail (polyA) as well as a cis-linked CMV promoter and SV40 poly(A)-regulated eGFP reporter. Briefly the plasmid was built as following: pT2 [Xenopus globin, MCS and 3’UTR Xenopus globin was obtained from Integrated DNA Technologies (IDT) and PCR amplified with specific primers (The plasmid (modified from ) for intMV:eGFP)  was cut  primers . In-Fusibud13 mRNA start codon was obtained from Gene Tools and re-suspended in nuclease-free water. 1 nl of morpholino solution (0.6mM) was injected into wild-type dechorionated embryos at the one-cell stage.A morpholino targeting serpinb1l3, col1a2) and 2 predicted as retained  upon RES loss-of-function was injected in 1 cell stage embryo together with Tol2 mRNA (Addgene plasmid #31831) (33 pg/embryo). Embryos were sorted in bud13∆7/∆7 and bud13+/? at the onset of the phenotype (~ 28–30 hpf). Minigene expression under HSP promoter was induced by heat shock during 4 hs and then GFP positive embryos were collected for RT-PCR. PCR was performed using the specific Fw primer an a universal Rv globin (A mix of 4 plasmid (15 pg/embryo) with the desired cassette (minigene), 2 predicted as no retained . Strand-specific TruSeq Illumina RNA sequencing libraries were constructed by the Yale Center for Genome Analysis. Samples were multiplexed and sequenced on Illumina HiSeq 2000/2500 machines to produce 76-nt paired-end reads.Total RNA from 32 hpf RNA used for intron retention validation experiments was treated with TURBO DNase (Ambion) for 30 min at 37°C and extracted using phenol chloroform. Then, Polyadenylated RNAs were purified using Oligo d(T)25 Magnetic Beads (Invitrogen) following manufacter recommendations. cDNA was generated by reverse transcription with random hexamers using SuperscriptIII (Invitrogen). RT–PCR reactions were carried out at an annealing temperature of 59°C for 35–40 cycles. PCR products were run in a 1.5% agarose gel. Primers are listed in the For the qPCR experiment, total RNA was extracted as described above. GFP and dsRED mRNAs were used as spike-in RNA controls and 1 μg of total RNA was used to generate cDNA. 5 μl from a 1/50 dilution of the cDNA reaction was used to determine the levels of p53 in a 20 μl reaction containing 1 μl of each oligo forward and reverse (10 μM) , using PZebrafish embryos or a small amount of tissue from the end of the tail were used to extract DNA . Brieflycorrected-for-mappability Reads Per Kilobasepair of uniquely mappable positions per Million mapped reads [Danio rerio using BioMart . Gene Ontology analysis was performed with the online tool DAVID (https://david.ncifcrf.gov/ Version 6.8) using as background all genes that passed the initial filters (minimum expression and read count).To identify differentially expressed genes, we first filtered out genes that did not have cRPKM > 2 in all sibling control or all mutant samples and genes whose quantification was not supported by at least 50 read counts in at least 1 sample. Next, differentially expressed genes were defined as those that showed a fold change in expression of at least 1.5 in all 3 mutants and a fold change of at least 2 in 2 out of the 3 control vs. mutant individual comparisons  for each intron in a given RNA-seq sample, we used our previously described pipeline  with thecorrected-for-mappability intronic Reads Per Kilobasepair of uniquely mappable positions per Million mapped reads) for each intron and condition by dividing this number of counts by the number of uniquely mappable positions in that intron.To define the confident set of highly affected introns, potential false positives were filter out by comparing the density of the mapped reads in the introns bodies in the mutant vs the control. For this purpose, we extracted all intronic sequences and calculated the number of uniquely mappable positions per intron following a similar strategy to that used to calculate cRPKMs   were defined as those introns with ∆PIR > 15 and at least a 1.5-fold net increase in read density in the intron body calculated as:To analyze the number of retained introns in the 3 mutants and the inter-mutant overlaps Euler APE-3.0.0 software  was utilTo investigate the impact of non-sense mediated decay (NMD) on global intron retention upon RES mutation, all introns were separated as last or non-last introns (of the reference transcript) and between those predicted to trigger and not to trigger NMD. An intron was predicted to trigger NMD if its retention generated an in-frame stop codon that is located further than 50 nts upstream of an exon-exon junction . By defiTo identify features discriminating introns highly retained upon RES depletion from un-retained/un-affected introns, we compared the sets of confidently introns  and non-differentially retained introns (control) upon RES mutations. We focused on the set of confidently retained introns  versus control introns with an absolute ∆PIR < 0.5 in the three mutants . The binary response variable of the logistic regression models indicates for each intron if it belongs to the retrained or control group. As predictors we used 30 quantitative and qualitative features , includiTo investigate the overall classification performance, we randomly partitioned the data set “RESdep” of retained introns into 90%/10%  training/test data, and randomly sampled the same amount of training/test data from the control “Ctr” data set. We used the training data to learn a logistic regression model with Lasso variable selection and tested it on the test data. Next, to evaluate how well this model classifies specific subsets of "RESdep" introns and retained introns specific for each mutation, we applied the model trained with “RESdep” vs “Ctr” data having held fixed its parameters to the classification of the 141 control test-introns vs. 141 retained introns subsampled from the following sets: (i) “RESdep_∆PIR10” introns from the “RESdep” set with ∆PIR > 10 in all three mutants (871 introns); (ii) “NMD”, introns from the “RESdep” set predicted to trigger NMD when retained (574 introns); (iii) “bud13”, introns with ∆PIR>15 upon bud13 mutation at 32 hpf ; (iv) “rbmx2”, introns with ∆PIR>15 upon rbmx2 mutation at 48 hpf ; and (v) “snip1”, introns with ∆PIR>15 upon snip1 mutation at 48 hpf . We repeated this procedure, including model training and classification of test data, 10,000 times and report average ROC curves  and averTo study the potential of each feature to contribute to the discrimination between the "RESdep" and "Ctr" intron sets, we randomly partitioned the dataset of retained introns into 90%/10%  training/test data, and randomly sampled the same amount of training/test data from Ctr. Using the training data, we learned logistic regression models without Lasso variable selection using only a single feature at a time neglecting all other features. The test data were used to determine the AUC. This experiment was repeated 10,000 times and we report average AUCs for each feature in S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(TXT)Click here for additional data file.S6 Table(TAB)Click here for additional data file.S1 Summary Statistics(XLSX)Click here for additional data file.S1 Fig(A) Scheme illustrating the Cas9-nanos 3′-UTR strategy [(B-C) Bright field microscopy of RES mutant embryos and their corresponding phenotypically wild type sibling (WT), treated with PTU to avoid melanocyte production, in lateral view (C) or magnification . Increased levels of apoptosis, predominantly in the head, are observed upon RES loss-of-function. . (D) UCSC genome tracks showing mRNA levels of RES complex members at 2 cell and 48 hpf stages. (E) RT-qPCR showing p53 mRNA levels. Error bars represent SD of the mean from two independent biological replicates . A p53 up-regulation in the mutants compared to phenotypically WT siblings correlates with the increased cell dead observed in the brain. (F) In situ hybridization showing RES components sense (top) and antisense (bottom) probes. Lack of expression in the sense probes show the specifity of the RES expression pattern in the zebrafish developing embryos.strategy . The nan(TIF)Click here for additional data file.S2 Fig(A) Lateral view of WT embryos injected with 0.6mM of morpholino antisense oligonucleotide against bud13 mRNA (MObud13) showing different levels of developmental defects (types I to III). Phenotypes are fully rescue with human hBUD13 mRNA. . WT: represent phenotypically wild type sibling from the same mutant fish line. Stronger phenotype is likely due to a depletion of the maternal contribution. (B)bud13 mutant embryos fully rescued by providing 75 pg of hBUD13 mRNA, suggesting that Bud13 function may be conserved across vertebrates . WT: represent phenotypically wild type sibling from the same mutant fish line. (C) 48 hpf, Δ7/Δ7bud13 embryos showing a similar phenotype to Δ16/Δ16rbmx2 and Δ11/Δ11snip1 when partly rescued by injection of 2.5 pg of hBUD13 mRNA . WT: represent phenotypically wild type sibling from the same mutant fish line.(TIF)Click here for additional data file.S3 FigIn situ hybridization showing expression pattern of shh (notochord and floor plate), krox20  and pax2a (anterior midbrain-hindbrain boundary and hindbrain neurons) in WT (top) and mutant (bottom) embryos.(TIF)Click here for additional data file.S4 Fig(A) Biplot comparing transcript expression levels in RES mutants and their corresponding phenotypically WT siblings. Genes up- or down-regulation were defined as having a fold change in expression of at least 1.5 in all three mutants and at least 2 for two out the three control vs. mutant individual comparisons  (log10 cRPKM). (B-C) DAVID cluster analysis of enriched GO annotations for down-regulated (B) or up-regulated genes (C) in RES mutants compared with wild-type siblings. (D) Barplots showing expression values (using the cRPKM metric) for up- regulated genes associated with cell death . The significant up-regulation in the three mutants correlates with the increased cell dead observed in the developing brain.(TIF)Click here for additional data file.S5 Figptch2(A) and wdr26b (B) loci (upper panels). RNA- seq signal increases strongly (∆PIR>15) in RES mutants only on specific introns (dark blue). RT-PCR assays validating the increased retention (dotted square box) in bud13, rbmx2 and snip1 mutants compared with the corresponding phenotypically WT siblings (lower panel).Sequencing read density across the (TIF)Click here for additional data file.S6 Fig(A) DAVID cluster analysis of enriched GO annotations for genes that contain introns with highly increased retention (∆PIR>15) in at least two of the RES mutants. (B) Boxplots showing fold change in expression (FC-expr) for genes containing increased retention (∆PIR>15) compared with those genes in which all transcripts are not affected (∆PIR<2) in the RES mutants. P-values were calculated using Wilcoxon rank-sum tests. Genes with at least one strongly retained intron (ΔPIR>15) had significantly decreased expression in the mutants compared to genes with no substantial change in intron retention (ΔPIR<2). (C) Boxplots illustrating differences in intron retention between genes that were up-regulated (Up), down-regulated (Down) or did not show significant expression changes (Unaffected). Number of introns in each category: Up = 1,603; Down = 1,623; Unaffected = 69,646. P-values were calculated using Wilcoxon rank-sum test. Genes that were differentially expressed in the three mutants (down- or up-regulated) show significantly higher retention (higher ΔPIR) in the mutants compared to their WT-looking siblings. (D) Overlap between differentially expressed genes (up and down-regulated) and genes harboring retained introns. P-values were calculated using Wilcoxon rank-sum tests: down vs IR P = 0.4033681; up vs IR P = 0.000117.(TIF)Click here for additional data file.S7 Fig(A) and introns predicted not to trigger NMD upon inclusion (B) among the RES-dependent (RES-dep) introns. On the contrary, introns that are not affected by RES depletion (Ctr) are depleted for these types of introns. P-values were calculated using Fisher exact test. (C) Stacked barplots showing the percentage of introns affected by bud13, rbmx2 or snip1 mutation using different ∆PIR cutoffs. (D) Euler diagram showing the overlap of skkiped exons (∆PIR>5 and ∆PIR>15) affected by bud13, rbmx2, and snip1 mutants.Stacked bar plot showing enrichment for last introns (TIF)Click here for additional data file.S8 Fig(A) Boxplots showing the distribution of the median nucleotide (nt) distance from the top 3 predicted branch points (BP) to the 3’ splice site for each intron category. (B) Boxplots of the highest score for the human SF1 position weight matrix (PWM) in the 3’ intronic region (see Methods for details). (C-D) Boxplots showing the GC content ratio between up-stream (C) or down-stream (D) exons vs the retained introns. The lower ratio of no- NMD introns, which are enriched in last introns, is caused by the generally low GC content of last exons overlapping the 3' UTR. .(TIF)Click here for additional data file.S9 Fig(A) Boxplots showing the degree of change in intron retention (∆PIR) according to intron length. Number of introns per nucleotide bin: ≤100 = 13,059; 101–300 = 13,332; 301–1,000 = 11,808; 1,001–2500 = 18,164; >2500 = 16,563. (B) Boxplots illustrating the degree of change in intron retention (∆PIR) according to intron GC content. Number of introns per bin: 0–0.3 = 14663; 0.3–0.35 = 27361; 0.35–0.4 = 21873; 0.4–0.45 = 6721; 0.45–1 = 2308. P-values were calculated using Wilcoxon rank-sum test.(TIF)Click here for additional data file.S10 Fig(A) Logistic regression models were learned to discriminate between RESdep and Ctr introns with each feature individually and the fraction of the null deviance that was reduced was recorded. Values were averaged over 10,000 repeated holdout experiments. Training data sets consisted of 1,268 RESdep and 1,268 Ctr introns. Error bars indicate 95% confidence interval of reported averages. (B) Features call used in (TIF)Click here for additional data file.S11 Fig(A) ∆PIR values for the top 100 introns based on their likelihood to be bud13-dependent and -independent, as predicted by the regression model. Only introns with no read coverage across the six RNA-seq samples were used for this analysis . False positive rate (FPR) values are defined for retained introns to be the fraction of introns with ∆PIR<2, and for unaffected introns to be those with ∆PIR>5. (B) RT-PCR assays validating the predicted RES dependent and RES independent introns in bud13, rbmx2 and snip1 mutants compared with the corresponding phenotypically WT siblings (lower panel). Arrows indicate the retained intron. In the case of arf6a we could detect intron retention by RT-PCR only in the rbmx2 mutant. Unspecific upper band in pde4ba, rpp38, znf644b and c8h17orf59 are likely DNA heteroduplexes. The numbers underneath the introns in the cartoons represent their lengths in bp.(TIF)Click here for additional data file.S12 Fig(A) Intron predicted as RES independent were spliced in bud13∆7/∆7 (col1a2 and serpinb1l3) while RES dependent intron wdr26b, but not ptch2, was retained as predicted for the logistic regression model. (B) Scheme of the vector used in the validation assay. PCR product and primers used are depicted as specific Fw primer (blue arrow) and universal Rv primer (magenta arrow). All experiments were done using two independent biological replicates (rep#1 and rep#2).(TIF)Click here for additional data file.


Wrinkles are observed commonly in CVD -grown graphene on Cu and hydrogen etching is of significant interest to understand the growth details, as well as a practical tool for fabricating functional graphene nanostructures. Here, we demonstrate a special hydrogen etching phenomenon of wrinkled graphene domains. We investigated the wrinkling of graphene domains under fast cooling conditions and the results indicated that wrinkles in the monolayer area formed more easily compared to the multilayer area (≥two layers), and the boundary of the multilayer area tended to be a high density wrinkle zone in those graphene domains, with a small portion of multilayer area in the center. Due to the site-selective adsorption of atomic hydrogen on wrinkled regions, the boundary of the multilayer area became a new initial point for the etching process, aside from the domain edge and random defect sites, as reported before, leading to the separation of the monolayer and multilayer area over time. A schematic model was drawn to illustrate how the etching of wrinkled graphene was generated and propagated. This work may provide valuable guidance for the design and growth of nanostructures based on wrinkled graphene. As the first discovered two-dimensional system, graphene has already captured global attention owing to its extraordinary physical and chemical properties, which hold great potential for electronic and optoelectronic applications ,2,3. ComWhile it is a ubiquitous phenomenon for CVD graphene grown on Cu, the occurrence of wrinkles originates because of different thermal-expansion coefficients of graphene and Cu during post cooling down process, and can modify the electronic structure, transport properties, and surface properties of graphene ,17,18,19In this study, graphene domains with high density wrinkles were obtained by applying a fast cooling down process on polished Cu foil via CVD and then were etched by exposure to hydrogen/Ar flows at high temperatures. Significant differences in the wrinkle distribution and hydrogen-induced etching behaviors are observed for monolayer and multilayer graphene domains . By carrying out a systematic study along the time axis, a special etching pattern on wrinkled multilayer graphene domains is revealed and the corresponding mechanism is proposed. The result shown here may provide valuable guidance for the design and the growth of unique graphene nanostructures.2 flow of 0.5 sccm (5 mTorr). Then, a CH4 flow of 5 sccm (160 mTorr) and a H2 flow of 5 sccm (55 mTorr) were introduced to the CVD system for graphene growth, and the growth time was varied from 10 min to 60 min. After growth, the system was cooled down to room temperature with different cooling rates. We annealed the graphene samples in Ar/H2 gas mixture at 900 °C after growth to explore the influence of wrinkle-induced H2 etching behavior. Graphene formed on the inside surface of the Cu pocket was transferred onto 300 nm SiO2/Si substrates using the PMMA (Polymethyl Methacrylate)-assisted method for further characterization [A 25 μm thick Cu sheet  was electrochemically polished for graphene synthesis. After removing the polishing solution residuals, we folded the Cu sheet into a pocket-like structure in order to keep a clean and smooth surface ,22. The rization .These prepared wrinkled graphene samples were analyzed by Optical microscopy, Raman, atomic force microscope (AFM), and scanning electron microscopy (SEM). Optical microscopy studies were carried out with the digital optical microscope  in reflectance mode. The surface morphology of wrinkled graphene samples was characterized by SEM  using an acceleration voltage of 10 kV. Finally, structure and bonding properties of the samples were characterized by Raman analysis using a Witec laser Raman spectrometer  with laser excitation energy of 488 nm, power of ~10 mW, and resolution of ~207 nm. Single spectra were acquired with 0.5 s integration time for 10 times accumulation. Mapping images were obtained for 10 s/line scan speed, 100 points per line, and 100 lines per image. The topography of wrinkled graphene samples was evaluated by AFM .The typical LPCVD parameter used for the wrinkled graphene synthesis is shown in More quantitative information about the formed wrinkle was obtained using AFM characterization. The AFM image of a representative wrinkled area that consists of a range of widths of graphene wrinkles from ~60 nm to ~100 nm is shown in 2 etching experiment was conducted at 950 °C under a H2/Ar mixture gas flow. Different etching features of wrinkled graphene domains are observed in the optical microscope image  in the local gas phase, they will be hydrogenated immediately in the highly reductive environment. The etching process is attributed to hydrogenation and volatilization of carbon atoms. Since dangling carbon atoms at graphene edges are more reactive, H2 molecules dissociate exothermally and form bonds with carbon atoms at edges first. The etching begins from the graphene edges by removing carbon atoms [After the morphological characterization of wrinkled graphene, an Hpe image a. Figureon atoms . Meanwhion atoms ,30. FiguIn the monolayer graphene domain, the etching initiated from the edge and the wrinkled sites b. Now, w2/Si wafer and conducted Raman studies to investigate the structure properties. −1) and 2D band (2650–2750 cm−1) with different brightness contrasts, indicating the occurrence of monolayer, bilayer, and trilayer graphene. In wrinkled regions, there are peak height variations in both the G and 2D bands [−1 full width at half maximum (FWHM) of the 2D peaks. The red curve corresponds to the Raman spectrum of the bilayer graphene area, as shown by the red arrow in G/I2D and a FWHM of 2D band of ~52 cm−1, suggesting the strong interlayer coupling of the bilayer graphene [To further confirm the etching feature, we transferred the etched multilayer graphene domain onto the 300 nm SiO2D bands , and thegraphene ,32.2/Si is shown in 2 and was reduced to ~48 μm2 and ~12 μm2 after etching for 10 min and 20 min, respectively, giving an etching rate of ~3 μm2/min of multilayer graphene. When the etching time increased to 30 min, the multilayer area became invisible. While the diameter of the hexagonal hole along the multilayer area in the center region was ~18 μm, ~39 μm, and ~63 μm after etching for 10 min, 20 min, and 30 min, respectively, indicating that the broadening of the hexagonal hole accelerated with a nearly linear behavior. We also observed a considerably faster etching rate of ~133 μm2/min for monolayer graphene  the removal of dangling carbon atoms from exterior edges of monolayer graphene; (2) etching from interior edges of monolayer graphene; (3) taking carbon atoms away from the edge of the multilayer graphene; (4) hydrogenation at other wrinkled sites. Based on the finding of our experiments and facts from previous reports ,8,10, a We have demonstrated layer-dependent wrinkle distributions of graphene grown on Cu by LPCVD and the special hydrogen-induced etching phenomenon of wrinkled multilayer graphene domains. Under a relatively low strain condition, wrinkles tend to form in the monolayer area and break on the boundary of the multilayer area, resulting in a high density wrinkle zone along the multilayer area in the multilayer graphene domains. Due to the preference absorption of hydrogen atoms on wrinkled sites, the etching initiated from the boundary of the multilayer area, creating a large hexagonal hole to separate the monolayer and multilayer area. Then, the etching of the wrinkled multilayer graphene domain is accelerated through four feasible paths, including the exterior and interior edge of the monolayer graphene, edges of the multilayer graphene, and wrinkled sites. These findings shed light on the formation of wrinkles in CVD growth graphene domains, as well as their selective etching process, to fabricate well-defined structures.


The Australian Government Aged Care Quality and Safety Commission  is responsible for accreditation of Australian aged care services which are audited against the Australian Accreditation Standards. Accreditation reports are publicly available. Prior to 2017, some clients were interviewed about their experiences, but the resulting information could not represent the client experience within a service due to low numbers, biased sampling, and an unsystematic approach to asking questions. La Trobe University was engaged to develop and pilot an interview tool to measure client experience for use in accreditation. Potential questions were identified through a literature review, mapped against the Accreditation Standards, and workshopped with an expert reference group. Twenty-four questions and a visual analogue were then piloted. Consumer groups and groups of Indigenous and culturally diverse clients in residential aged care homes were consulted. The perspective of Quality Agency surveyors was sought on the questions’ usability. Statistical analyses sought to identify questions that minimised missing data, were responded to similarly by residents and their representatives, and elicited stable responses on retest. Twelve questions were identified as optimal. The 10 quantitative questions proved to reflect a single underlying dimension (consumer experience) and, when summed and explored through regression analyses, differentiated services significantly. The consumer experience interview tool is now used in all accreditation audits in Australia. Results are then used to generate consumer experience reports, which are published online and can support consumer choice of a residential aged care home.


In a traditional ADMIXTOOLS workflow, the user must first generate a set of text configuration files tailored to each individual analysis, often using a combination of shell scripting and manual text editing. The non-tabular output files then need to be parsed to extract values of interest prior to further analyses. Our package simplifies this process by automating all low-level configuration and parsing steps, making analyses as simple as running a single R command. Furthermore, we provide a set of R functions for processing, filtering and manipulating datasets in the EIGENSTRAT format. By unifying all steps of the workflow under a single R framework, this package enables the automation of analytic pipelines, significantly improving the reproducibility of population genetic studies.We present a new R package https://bioinf.eva.mpg.de/admixr.The source code of the R package is available under the MIT license. Installation instructions, reference manual and a tutorial can be found on the package website at Bioinformatics online. D, qpWave and qpAdm) for estimating the amounts of genetic drift shared between populations, testing admixture hypotheses and estimating admixture proportions, implemented as command-line utilities in the ADMIXTOOLS software suite , and a set of complementary functions for filtering and processing datasets in the EIGENSTRAT file format required by ADMIXTOOLS > snpsEIGENSTRAT object=================components:ind file: ∼/path/to/eigenstrat/data.ind snp file: ∼/path/to/eigenstrat/data.snp geno file: ∼/path/to/eigenstrat/data.geno All other functions in the package accept this object as their first argument, and perform either a requested calculation on it , or return a new, modified EIGENSTRAT S3 object (in case of filtering and processing functions) which can be used in additional downstream steps or calculations.f3, d, f4, f4ratio, qpWave and qpAdm, each implemented as a wrapper around one of the command-line programs distributed as part of the ADMIXTOOLS package.The core functionality of the package consists of the following set of R functions: Performing even the most trivial analysis using ADMIXTOOLS presents a significant amount of overhead for the user. For example, to estimate the proportion of Neandertal ancestry in a set of individuals, 4f statistics, a parameter file specifying the paths to a trio of EIGENSTRAT component files, then manually run the qpF4ratio command-line program, and then capture and parse its output to obtain relevant values , A = “Altai”, B = “Vindija”, C = “Mbuti”, O =  “Chimp”, data = eigenstrat(“<path to EIGENSTRAT data>”) ) f4ratio function performs all configuration and parsing work, and returns an R data frame which can be immediately used for further statistical analysis and plotting:Internally, the > resultA B X C O alpha stderr ZscoreAltai Vindija French Mbuti Chimp 0.019696 0.003114  6.324Altai Vindija Han Mbuti Chimp 0.024379 0.003364 7.248Altai Vindija Papuan Mbuti Chimp 0.032167 0.003499 9.193admixr wrapper functions have a similar interface and are described in the tutorial vignette on the package website in more detail.All other Reading and writing of ind, snp and geno file components.Filtering of SNPs based on regions specified in a BED file.Restricting analyses to sites carrying transversion SNPs.Renaming samples or grouping them into larger population groups.Merging of EIGENSTRAT datasets.Counting the number of sites present or missing in each sample.The fact that ADMIXTOOLS requires the data to be in EIGENSTRAT format presents additional challenges for quality control, processing and filtering, as this format is not supported by standard bioinformatics tools. Our R package therefore provides additional functionality to simplify the processing and filtering of EIGENSTRAT genotype data. This includes:btz030_Supplementary_InformationClick here for additional data file.