The generation of defects is studied at first by considering the influence factors . Then uniaxial tensile test simulations are conducted to uncover the evolution of the mechanical performance of graphene/MoS2 heterostructure after being irradiated by ions. At last, the control rule of interlayer distance in graphene/MoS2 heterostructure by ion irradiation is illustrated for the actual applications. This study could provide important guidance for future application in tuning the performance of graphene/MoS2 heterostructure-based devices by ion beam irradiation.The interaction between ion irradiation and two-dimensional (2D) heterostructures is important for the performance modulation and application realization, while few studies have been reported. This paper investigates the influence of Ar ion irradiation on graphene/MoS Over the past two decades, more than ten thousand articles have been published every year around the topics of 2D materials. Graphene, as a single layer of graphite, is the most important 2D material. The special atomic and electronic structure has endowed graphene unparalleled properties3 and therefore, great application potentials are expected5. Besides graphene, there are also other types of 2D materials such as hexagonal boron nitride, black phosphorus, molybdenum disulphide (MoS2), and other transition metal dichalcogenides being discovered in the last a few years. On one hand, these 2D materials behave lots of outstanding properties similar to graphene. On the other hand, they also exhibit special performance to compensate the weakness of graphene6. Together with graphene, the big 2D material family opens up a new era of nanomaterials research.Two-dimensional (2D) materials have become the focus of nanomaterials research since the experimental discovery of graphene in the lab in 20045 cm2 V −1 s−1 of graphene has indicate its significant potential application in electronic devices7, while the graphene-based field effect transistors (FETs) generally behave low on/off switching ratios due to the zero bandgap. In contrast, 2D MoS2 has the direct bandgap of 1.8 eV8, which enables the MoS2-based device on/off switching ratios of > 108, but the relatively low mobility limits its further applications9. To overcome the shortages of each material, the idea of combining different components together, i.e., generating 2D heterostructures, has emerged in recent years. 2D van der Waals (vdWs) heterostructures are one type of heterostructures which stack one layer of 2D crystal on top of another, analogous to building Lego blocks10. This vertical stacking feature provides the in-plane strong covalent bonds, whereas relatively weak interlayer vdW forces to the structure, which is able to integrate the merits of each 2D crystal, as well as generate some new features due to the synergistic effect, largely expanding the applications of 2D materials11. For example, by combining the high conductive graphene with semiconducting MoS2, we could fabricate high performance transparent and flexible memory devices12. Besides, integrating the high optical absorption and 1.8 eV direct bandgap features of MoS2 with high carrier mobility of graphene enables the scalable fabrication of phototransistors13.During the research of 2D materials, it is found that lots of the individual 2D material has the intrinsic drawbacks which impede the full realization of the applications. For example, the high mobility of 2 × 1015. However, it should be aware that the as-synthesized vdW heterostructures generally exhibit weak interlayer coupling and uncontrolled properties, which means the postprocessing and performance modulation should be applied to the heterostructures to adjust the performance before being used. Therefore, the discovery of suitable performance modulation methods for vdW heterostructures is important. The current proposed techniques, such as mechanical-based transfer-and-stack method18, external pressure field19, external electric field20 and thermal annealing method21, have been demonstrated to be able to be successfully applied in tuning the interlayer coupling of vdW heterostructures. However, these methods are either too complex , or unable to be precisely controlled , or relying on large external field . It is therefore desirable to explore new techniques to modulate the interlayer coupling in 2D vdW heterostructures for their further application.The outstanding properties of 2D vdW heterostructures have promised them wide application potential similar to 2D materials. To realize the applications, a lot of efforts have been made to synthesize vdW heterostructures24. After ion irradiation, the mechanical, optical and electrical properties of 2D materials can be adjusted efficiently, and this process is highly dependent on the irradiation conditions 26. The usage of ion irradiation in tuning the performance of vdW heterostructures is less reported28, especially for graphene/MoS2 heterostructures, which is the most typical 2D vdW heterostructure. To uncover the performance modulating mechanism of graphene/MoS2 heterostructures by ion irradiation, the first thing is to make clear the ion irradiation induced phenomena in the heterostructure. In this paper, we systematically studies the influence of Ar ion irradiation on graphene/MoS2 heterostructure by using molecular dynamics (MD) simulations. The irradiation induced structure evolution, and the influence factors are discussed, focusing on the generated defects and the sputtered atoms. The influence of irradiation on the uniaxial tensile properties of graphene/MoS2 heterostructure are then investigated for the actual application under loading conditions. At last, the possibility of controlling the interlayer distance by ion irradiation are discussed. The results in this paper could provide important supports for choosing the ion irradiation method in performance modulation of 2D vdW heterostructures.The ion beam irradiation technique has already been demonstrated in the application of tuning the properties of 2D materials, through generating defects, or forming doping, or thinning the materials29 is adopted due to its strong capability in nanomaterials simulation. Ar ions are chosen as the representative irradiation ions, and vertically irradiated onto MoS2/graphene heterostructures with various energy and dose, as shown in Fig. 30 to eliminate the side effect of the pressure waves generated due to the ion irradiation. The suspended graphene/MoS2 heterostructure has an in-plane size of 115 × 130 Å with 11,124 atoms. The initial interlayer distance between MoS2 and graphene is set as 3.5 Å owing to the minimized potential energy at around this value31. For the supported cases, SiO2 is chosen as the substrate with a thickness of about 58 Å. This thickness, which results in a total number of atoms of 74,988, is enough to withstand the irradiation effect in the studied energy range. The Ar ions are randomly irradiated from a cubic box 40 Å on top of the heterostructure plane, and only the nuclei collision is considered due to much weaker energy transfer from the electron collision32.For all the simulations, the large scale atomic/molecular massively parallel simulator (LAMMPS) package2 is described by a second-generation reactive empirical bond-order (REBO) potential developed by Liang et al.33, which was well demonstrated by different groups35. The interactions between the carbon atoms (C–C) in graphene is captured by adaptive intermolecular reactive bond order (AIREBO) potential36, which could consider the formation and dissociation of covalent chemical bonds, enabling it to model the bond breaking and formation during the irradiation process. A Tersoff potential37 is applied to consider the interactions in SiO2, and the Ziegler–Biersack–Littmark (ZBL) universal repulsive potential38 is adopted to model the energetic collisions between Ar and the target atoms due to its strong capability in describing the interactions at small separation. The MoS2 and graphene layers are coupled by vdW interactions, which are considered by the Lennard–Jones (LJ) potential39. Besides, the interactions between graphene/MoS2 with the SiO2 substrate are also described by the LJ potential. For the parameters used in the LJ potentials , please refer to Ref.41.To describe the multiple interatomic interactions between different atoms in the system, a hybrid potential strategy is applied. The interaction of the atoms in MoS42. Besides the simulation of irradiation process, the mechanical properties of the irradiated structures are also revealed by uniaxial tensile simulations. The irradiated configurations are extracted from the aforementioned results and then relaxed at room temperature for enough time. Afterwards, uniaxial stretching process is applied on the structure at room temperature condition with a strain rate of 0.003 ps-1. It was demonstrated that the tensile strength of single-layer 2D materials would increase with strain rate43, while the effect maybe limited for bilayered 2D structure44. The value of strain rate used in this study is comparable to the previous study, and the influence of strain rate would be studied in the future research. Thereafter, the information of stress/strain is derived based on the method in Ref.45, then the stress–strain curve is plotted and the values of fracture stress/strain are extracted. The configurations of the stretched heterostructures are visualized by the Visual Molecular Dynamics (VMD) package46.The MD simulation process for the ion irradiation is constituted by four stages. At the first stage, the system is relaxed at 300 K by an NVT ensemble for 5 ps to achieve an equilibrium state. At the second stage, consecutive ions with different parameters (energy and dose) are irradiated onto the heterostructure, for which the ions are emitted every 1000 timesteps and the system is kept at 300 K during the irradiation process. At the third stage, a high temperature (2000 K) annealing process is applied on the system for 30 ps to simulate the actual annealing effect in real experiment. At the last stage, the system is rescaled to room temperature (300 K) after the annealing process, and stayed at 300 K eventually. This four-stage scheme is successfully demonstrated in simulating the irradiation phenomena by different studies13 /cm2, the top-layer MoS2 receives most of the impact momentum, which results in the sputtering of Mo and S atoms and generates lots of vacancy defects , while the bottom-layer graphene is less influenced. Actually, under this irradiation condition, the MoS2 acts as a protective layer to avoid the graphene layer being damaged. The generation of vacancies in the 2D materials and 2D heterostructures can be used to modulate their properties. For example, H. K. Neupane et al.47 demonstrated that the vacancy defects at Mo sites could be used to efficiently tune the electronic and magnetic properties of 2D graphene/MoS2 heterostructure. Vacancies also affects the mechanical, electrical and magnetic properties of MoS248. The existence of vacancies can be beneficial or detrimental to the performance of the heterostructure, depending on the location and density of the defects. Therefore, to achieve the desired properties of the heterostructure, the ability to control the defects generation is significant. The next section of this paper focuses on the dependence of the generation of vacancies on ion irradiation energy, irradiation dose, substrate, stacking order, to offer important guidance for actual applications.Under ion irradiation, the structure of heterostructure would vibrate and the system becomes unstable. Defects would be induced into the system if the irradiation energy is high enough. As shown in Fig. 2 is limited, and the bottom-layer graphene is fully protected. With the increase of irradiation energy (200 eV), the generation of vacancies in both MoS2 and graphene would be intensified. Under this situation, the defects in top-layer MoS2 would be more severe than in bottom-layer graphene. If the irradiation energy is further increased (1000 eV), the defects in MoS2 is slightly changed, while the defects in graphene increase a lot, generating a more severe defect formation in graphene. For the low energy irradiation case, very little energy traverse through the top-layer plane, and the bottom layer is well protected. While for the high energy irradiation case, some energetic ions pass through top-layer, and collide with the bottom-layer, inducing the direct vacancy formation in the graphene. Besides, the sputtered Mo and S atoms from the top-layer MoS2 are also energetic at this condition, and they would fly to the graphene and lead to the second collision and indirect vacancy formation in the graphene layer. The generation of indirect defects in graphene become dominate if the ion energy is high enough, which could explain the reversed phenomenon observed in the 1000 eV case.For 2D materials under ion irradiation, the influence of irradiation energy and dose on the defect formation is important, so do 2D heterostructures. As shown in Fig. 2 and graphene layers, to obtain a graphene/MoS2 heterostructure, so that top-layer graphene acts as a protective layer to MoS2. Figure 2 layer for MoS2/graphene and graphene/MoS2 heterostructures. The case study of monolayer MoS2 is also provided as a reference. It shows that under the low ion irradiation energy (40 eV), the defects of MoS2 in the two types of heterostructures are always fewer than the pure monolayer MoS2 case, indicating a protective effect of the graphene to MoS2 layer, regardless of the stacking order of heterostructures. In the graphene/MoS2 heterostructure, the shielding effect of top-layer graphene could mostly relieve the irradiation damage onto bottom-layer MoS2, while in MoS2/graphene heterostructure, it is the interaction between bottom-layer graphene and top-layer MoS2 weakening the irradiation damage onto MoS2. If the irradiation energy is increased to 400 eV, it is found that the bottom graphene can still reduce the irradiation effect of MoS2. While for the graphene on top (graphene/MoS2 case), the irradiation damage is more severe, which is attribute to the indirect collision from the sputtered carbon atoms in top graphene layer. For the top-layer 2D crystal, the switch from protective effect to destructive effect at different energy levels corresponds to the results in Fig. The influence of the stacking order is investigated by switching the MoS2 layer under different irradiation energies for the aforementioned three cases. It clearly shows that the number of sputtered atoms in MoS2 for MoS2/graphene is always lower than monolayer MoS2 case, demonstrating a perpetual beneficial effect of the bottom-layer graphene regarding the irradiation damage on MoS2. For graphene/MoS2, there is no damage on MoS2 if the irradiation energy is smaller than 100 eV, then the number of sputtered atoms quickly climbs up to surpass the results in MoS2/graphene case. It even becomes the most severely damaged when the irradiation energy is above 400 eV, indicating that the indirect collision dominates under this energy level.Figure 2 and MoS2/graphene heterostructure, the number of sputtered atoms in MoS2 increases almost linearly with the increase of ion dose within the studied range. Due to the protective effect of the bottom graphene, the increase rate of MoS2/graphene is smaller. It is expected that at a higher dose beyond the studied range, the number of sputtered atoms would gradually achieve a steady value. In addition, due to the shielding effect of the top-layer graphene, the number of sputtered atoms in graphene/MoS2 is always zero under this irradiation energy.For the influence of the irradiation dose, we choose the irradiation energy of 100 eV and compare the data of three cases in Fig. 49. To make clear the influence of substrate on the ion irradiation effects of the heterostructures, we plot the relationship between the number of sputtered atoms and irradiation energy for the supported and suspended cases in Fig. The actual application of 2D heterostructures normally cannot be separated from the substrate. The existence of substrate has demonstrated to greatly affect the growth and properties of 2D heterostructures2 is more severe damaged under substrate supported case, the bottom-layer graphene remains undamaged, which means that the substrate influences the interaction between graphene and MoS2. It was described previously that the graphene-MoS2 interaction can bring beneficial effect to the irradiation resistance of MoS2, therefore, the existence of substrate weakens this beneficial effect. For the high ion energy irradiation (400 eV), the top-layer MoS2 is more severe damaged under supported case, in addition, the bottom-layer graphene is also a little bit more damaged under supported case. Especially, there exists some defects in the un-irradiated sites for the supported case. Under high energy irradiation, the impact of ions not only induced the direct collision with the heterostructure layers, but also it resulted in the sputtered of substrate atoms, which could generate second collision (indirect collision) between the substrate atoms and heterostructure atoms. The indirect collision from the substrate can intensify the damage effect in MoS2, and due to the random sputtering of the substrate atoms, there would be some defects randomly generated in graphene. Besides, the existence of substrate can also lead to the rebound of the carbon atoms in graphene, which would generate the indirect collision with the atoms in MoS2 as well. Therefore, there exists two different mechanisms for the ion irradiation phenomena under the supported case, i.e., the weakened MoS2-graphene interaction, and the generation of indirect collision due to the existence of substrate. Under low irradiation energy, only the weakened MoS2-graphene interaction mechanism exists, with the increase of irradiation energy, the generation of indirect collision mechanism dominates eventually.To uncover the mechanism underlying the phenomenon in Fig. 50. The ion irradiation process could provide tailored mechanical behavior to heterostructures if the generation of defects could be controlled. To understand the influence of the ion irradiation on the mechanical properties of the MoS2/graphene heterostructure, we conduct uniaxial tensile test simulations for the ion irradiated heterostructures with different irradiation parameters.2D materials and the heterostructures exhibit special mechanical behaviors such as high in-plane strength and stiffness, banding flexibility, which are highly valuable for their applications2/graphene and ion irradiated MoS2/graphene exhibit a two-stage fracture process under tensile loading. The phenomenon of a two-stage process is attributed to the different tensile behaviors of MoS2 and graphene layers. However, a quite different tensile fracture behavior could be observed. Firstly, for the original MoS2/graphene heterostructure, the initiation of cracks for the first and second stages happens at a larger strain when compared to the ion irradiation case , indicating a reduced fracture strain due to the ion irradiation process. Secondly, the cracks of the original MoS2/graphene heterostructure initiate at the bottom-layer graphene (first stage), then the cracks start to initiate at the top-layer MoS2 at a larger strain (second stage). While for irradiated MoS2/graphene heterostructure, the fracture sequence is reversed, i.e., the initiation of crack for the first stage happens at top-layer MoS2, and the initiation of crack for the second stage happens at bottom-layer graphene. The reduced fracture strain in irradiated MoS2/graphene is owing to the generated defects, which is commonly observed in the irradiated 2D materials. For the reversed fracture sequence, it is also related to the induced defects. The pristine perfect MoS2 layer has a larger fracture stress/strain compared to the perfect graphene layer, suggesting a preferential fracture process in graphene, as observed in Fig. 2/graphene under 100 eV ion irradiation, the defects are mainly generated in top-layer MoS2, resulting an inferior mechanical tensile strength compared to graphene. Therefore, the tensile stretching process leads to the cracks starting from the damaged MoS2.The typical configurations of the heterostructure under different tensile strains are provided in Fig. 2/graphene heterostructure at different irradiation energies. It clearly shows that all the structures behave a two-stage fracture process, owing to the different tensile behaviors of MoS2 and graphene layers, and the ion irradiation induced defects. The second fracture stage represents the continuous stretching process of top-layer MoS2 or bottom-layer graphene, depending on the ion irradiation parameters. The original MoS2/graphene has longest second fracture stage due to the defect-free feature, while the irradiated heterostructures have different lengths of second stage due to the distinct defects induced under the different situations. Besides, it is seen that the fracture stress (maximum stress) decreases with the increase of irradiation energy, which corresponds to the increased sputtered atoms with the increase of ion energy. The Young’s modulus also decreases with the increase of ion energy.Figure The experimentally synthesized 2D heterostructures usually exhibit weak interlayer coupling due to the loose interaction induced by the large interlayer distance. Ion irradiation could induce impact force to the 2D layers, bringing defects into the 2D crystal and meanwhile, induce the modulation of the interlayer distance.14/cm2) could induce a slight of the distance due to the limited impact momentum, while at a large ion dose (7.91 × 1014/cm2), the interlayer distance achieves a much smaller value. Figure 27. The precise modulation of the interlayer distance is significant to other properties as well, which would be the focus of our future studies.Figure However, the impact force would also bring defects to the 2D crystals if the irradiation energy is high. As shown in Fig. 2/graphene heterostructure. The main conclusions are as follows.Under ion irradiation, there would be some vacancies generated in the 2D crystals, while the 2D layers response differently to ion irradiation. For the low energy ion irradiation, the top-layer 2D crystal could shield the bottom-layer, while for the high energy ion irradiation, the indirect collision induced by the top-layer would intensify the irradiation-induced damage into the bottom layer. The bottom-layer 2D crystal would always relieve the defect generation in top layer, regardless of the ion irradiation energy. The irradiation induced vacancies would increase linearly with the increase of ion dose for different study cases. And the substrate would reinforce the irradiation effect because of two mechanisms, i.e., the weakened interlayer coupling in heterostructure at low ion energy and the indirect collision at high ion energy.2/graphene heterostructure due to the generated defects. The tensile behavior is highly dependent on the irradiation parameters.Ion irradiation would result in the reduction of tensile fracture stress and strain, and the reversed tensile fracture sequence for MoS2/graphene heterostructure could be well controlled at a certain ion irradiation dose and energy. While relatively high ion irradiation energy would lead to the coexistence of the defects with reduced interlayer distance.The interlayer distance in 2D MoSBased on classical molecular dynamics simulations, this paper studied the ion irradiation induced phenomena in 2D MoSThis study is meaningful for the application of ion irradiation on nanomanufacturing. The future research would focus on the electronic performance modulation and the actual application of ion beam irradiation on tuning the performance of 2D heterostructure-based devices.Supplementary Video 1.Supplementary Video 2.


In a conversation, recognising the speaker’s social action  early may help the potential following speakers understand the intended message quickly, and plan a timely response. Human language is multimodal, and several studies have demonstrated the contribution of the body to communication. However, comparatively few studies have investigated  conversational facial signals and very little is known about how they contribute to the communication of social actions. Therefore, we investigated how facial signals map onto the expressions of two fundamental social actions in conversations: asking questions and providing responses. We studied the distribution and timing of 12 facial signals across 6778 questions and 4553 responses, annotated holistically in a corpus of 34 dyadic face-to-face Dutch conversations. Moreover, we analysed facial signal clustering to find out whether there are specific combinations of facial signals within questions or responses. Results showed a high proportion of facial signals, with a qualitatively different distribution in questions versus responses. Additionally, clusters of facial signals were identified. Most facial signals occurred early in the utterance, and had earlier onsets in questions. Thus, facial signals may critically contribute to the communication of social actions in conversation by providing social action-specific visual information. A crucial prerequisite for having a successful conversation is to recognise the speaker’s social action, or what an utterance does in a conversation. For instance, this could be a request, an offer, or a suggestion   onot-face has been linked to negative messages during conversation and was argued to communicate negation or disagreement [facial shrug, which consists of an eyebrow raise and pulled down mouth corners. This facial expression signals indifference or lack of knowledge in a similar way to shoulder shrugs [thinking-face, consisting of a short gaze shift (away from the speaker) or closure of the eyes, can act as a signal that expresses effort while thinking of what to say, remembering something, or searching for a word or concept [Similarly, combinations of co-occurring facial signals have been associated with specific social actions. The facial expression  referred to as the greement . The notgreement . By usinr shrugs ,25,38, a concept . The thi concept . The expQuestions and responses are an important focus, for one, because they are foundational building blocks of conversation ,40. For Several facial signals have been linked to questions and responses in previous research. The eyes have been found to play a role in signalling questions and responses, albeit in different ways. Direct gaze has been linked to questions in both spoken and signed languages, where it is often held until a response is provided by the addressee ,44,45,46Like for gaze, many studies have found links between eyebrow movements, such as frowns, raises, and questions in spoken and signed languages ,54,55,56early recognition of the social action.Another critical component in addition to the form of the facial signals  is the timing of such signals  for fast social action recognition in a turn-taking context. Questions with manual gestures and/or head gestures have been found to result in faster responses than questions without such gestures ,59, sugg(1)Which facial signals occur with questions and responses, and what are their distributions across questions and responses?(2)combinations of co-occurring facial signals that map onto questions and responses?How do facial signals cluster, and are there specific (3)What are the timings of facial signals within questions and responses?To address the outstanding issues and questions highlighted above, the current study aimed to investigate a wide range of facial signals in multimodal face-to-face interaction, using a rich corpus of dyadic Dutch face-to-face conversations. We asked how the production of different facial signals mapped onto the communication of two fundamental social actions in conversation: asking questions and providing responses. To our knowledge, this is the first systematic investigation of conversational facial signals on such a large dataset and for these two specific social actions. The research questions we addressed were as follows:Due to the exploratory nature of the study and the relative paucity of research on facial signalling of social actions, we were only able to make predictions about a different distribution in questions versus responses for eyebrow movements (a domain with extant findings from at least a few studies), but did not make predictions about other facial signals. In line with studies showing eyebrow frowns and raises functioning as question markers, we hypothesised that they would occur more in questions versus responses ,54,55,56In agreement with the idea of early signalling as a facilitator of early action recognition in conversational interaction ,10,11,12M age = 23.10, SD = 8, 51 female, 17 male), without motoric or language problems, and with normal or corrected-to-normal vision, holding a dyadic casual conversation for one hour while being recorded.This study used 34 video dyads that form part of a multimodal Dutch face-to-face conversation corpus . The videos consisted of Dutch native speaker pairs of acquaintances  and clap to be able to synchronise audible and visible information .Informed consent was obtained before and after filming. Participants were asked to fill in a demographics questionnaire prior to the study, and four questionnaires at the end of the study. These contained questions about the relationship between the conversational partners and their conversation quality, the Empathy Quotient , the FeaThe conversations were recorded in a soundproof room at the Max Planck Institute for Psycholinguistics in Nijmegen, The Netherlands. Participants were seated facing each other at approximately 90 cm distance from the front edge of the seats .Two video cameras (Canon XE405) were used to record frontal views of each participant, two cameras recorded each participant’s body from a 45 degree angle (Canon XF205 Camcorder), two cameras (Canon XF205 Camcorder) recorded each participant from a birds-eye view while mounted on a tripod, and finally one camera (Canon Legria HF G10) recorded the scene view, displaying both participant at the same time. All cameras were recorded at 25 fps. Audio was recorded using two directional microphones (Sennheiser me-64) for each participant  and 4553 responses .The analysis focused on questions and responses. First, an automatic orthographic transcription of the speech signal was made using the Bavarian Archive for Speech Signals Webservices . Questioat 5.1; ,74). Thi. Thik = For the present analyses, facial signals were annotated in ELAN  based oFacial signals were all annotated except if they involved movements that obviously did not carry some sort of communicative meaning related to the questions or responses, as we were interested in the communicative aspect instead of the pure muscle movements. Like for questions and responses, the context of the conversational exchange was taken into account, to estimate the communicative meaning. Movements due to swallowing, inhaling, laughter, or articulation were not considered. Facial signals coded consisted of: eyebrow movements , eye widenings, squints, blinks, gaze shifts , nose wrinkles, and non-articulatory mouth movements   occurred. In those cases, the annotation lasted until the first evidence, or begun after the last evidence, of speech not related to the questions/responses or laughter.agr; [k; [r), standard error of measurement (SeM), and the mean absolute difference  of signal onsets, to access how precise these annotations were in terms of timing, if there was enough data to compare. One question and one response in one of the three parts were selected randomly for each participant in all dyads . This enabled us to compare all coders in a pairwise fashion on the same data. We excluded eyebrow lowering and mouth corners pulled back from all further analyses, since the paired comparisons including unmatched annotations showed low raw agreement and kappa scores. For all other facial signals, the paired comparisons showed an average raw agreement of 76%  and an average kappa of 0.96 , indicating almost perfect agreement.To avoid artefacts from potential timing discrepancies in ELAN between audio and image, any facial signal annotation that started or ended within 80 ms (two frames) of an unrelated speech boundary was excluded from the analysis. This prevented including facial signals that were related to any other speech from the speaker instead of the question or a response. Any facial signal that was in this 80 ms window generally continued throughout the unrelated speech, and was therefore potentially related to it. This resulted in the exclusion of 795 annotations. No annotations were made when there was insufficient facial signal data due to head movements preventing full visibility or due to occlusions. Similar to the questions and responses, interrater reliability between the coders was calculated with raw agreement agr; ,71) and  and agr;agr; [k; ) using aReliability for each individual facial signal was calculated to obtain a more detailed view on how reliable coders were for each specific facial signal. Non-reliable measurements because of insufficient data were excluded when calculating these averages . Results are shown in k > 0.81) for eyebrow frowns, raises, frown raises, unilateral raises, eye widenings, squints, blinks, gaze shifts, nose wrinkles, pressed lips, and smiles. There was a substantial agreement (k > 0.61) for mouth corners down. When there was enough data to perform a Pearson’s correlation, all signals showed r = 1 with a p < 0.0001, indicating a strong correlation. There was not enough data to perform a correlation for eyebrow frown raises, nose wrinkles, and mouth corners down.There was almost perfect agreement (SeM) as very low when SeM < 40, low when SeM < 80, moderate SeM < 160, and high SeM < 160. There was a very low variance for the coding of eyebrow unilateral raises, squints, blinks, gaze shifts, nose wrinkles, pressed lips, and mouth corners down. A low variance was found for and eyebrow raises and eye widenings. A moderate variance was found for eyebrow frowns, frown raises, and a high variance was found for smiles. The same rationale as the standard error of measurement was applied for the mean absolute difference: a very precise annotation timing was found for blinks and nose wrinkles (Mabs < 40), a precise annotation timing for eyebrow unilateral raises and squints (Mabs < 80), a moderate annotation timing for eyebrow raises, frown raises, eye widenings, gaze shifts, mouth corners down (Mabs < 160). A poor annotation timing was found for eyebrow frowns, pressed lips, and smiles (Mabs > 160). The complete list of all reliability pairwise comparisons per coder and per signal, as well as the reliability script can be found on the Open Science Framework project website https://osf.io/x89qj/ (last accessed on 28 July 2021).The measurement unit for the standard error of measurement was in milliseconds, and one video frame was equivalent to 40 ms. Thus, we considered the variance based on the reliability of the signals . With this analysis, we asked how likely a given question or response was to contain For the clusters, we aimed to identify specific combinations of co-occurring facial signals that map onto questions and responses. We did this by looking at combinations using three different approaches. First, we looked at the frequency with which pairs of signals co-occur in questions and responses. Then, we tested whether there were any particular facial signals that are statistically predictive (or strongly associated) with an utterance being a question or a response, and are therefore able to reliably differentiate between questions and responses based on their occurrence frequency. Finally, we assessed whether a particular set of signals is characteristic of questions and responses.For the first approach, we determined which pairs of facial signals frequently occurred together by analysing two data frames. One consisted of questions x facial signals, and the other of responses x facial signals. In these data frames, each row was either a question or response and each column the number of facial signals overlapping with that specific utterance. This provides a quantification of how frequently each pair of facial signals occurred together. This was performed as a test to see if there were any frequent co-occurrences of facial signals at all before examining potential clusters.p-value was obtained from calculating the percentage of cases where the random shuffle gave higher accuracies than the actual accuracy. We used the same data and holdout cross-validation as in previous classification analysis, and repeated the simulation a 1000 times.For the second approach, in order to find out if there are particular facial signals that differentiate questions from responses, we employed Decision Tree (DT) models . DT moden = 22) were created to produce stability plots. Stability plots tell us at which number the MCA clustering solution is optimal. The dendrogram was cut to the optimal clustering number to see in which clusters each variable should be allocated [For the third approach, after determining whether there were particular facial signals that are statistically predictive with an utterance being a question or a response, we asked whether there were specific combinations of signals that occurred within questions and responses using Multiple Correspondence Analysis . MCA isllocated .In order to study whether facial signals occur primarily early or late, and whether there were differences in questions and responses, we first looked at the difference in proportion of facial signals with an onset before the start of a question or response and after the start of a question or response by splitting the data in two data frames. The first consisted of facial signals with an onset before the start of a question or response, the second consisted of facial signals after the start of a question or response. The split in pre-onset and post-onset data frames was only used in this first analysis. As a second analysis, we plotted the onset of facial signals relative to the onset of questions and responses, to see where the facial signals started relative to the utterance onset. Finally, to get a better idea of how the facial signal onsets distributed within the utterances, utterance duration was standardised between 0 (onset utterance) and 1 (offset utterance), and facial signal onsets were plotted relative to that number.R .The analyses were conducted in   with RS.2.5019; ) using ach , FactoMeR , and Clar . Moreovyr , plyr , dplyr  , stringgr , reshap , purrr  , forcat , caret , and ca. For vi , factoectoextra ), gridExra , viridi , and sc . The ann = 895) co-occurred with questions and 39% (n = 374) with responses. Out of 2832 eyebrow raises, 53% (n = 1503) co-occurred with questions and 47% (n = 1329) with responses. Out of 233 frown raises, 59% (n = 138) co-occurred with questions and 41% (n = 95) with responses. Out of 344 unilateral raises, 59% (n = 204) co-occurred with questions and 41% (n = 140) with responses. Out of 446 eye widenings, 64% (n = 286) co-occurred with questions and 36% (n = 160) with responses. Out of 1172 squints, 66% (n = 771) co-occurred with questions and 34% (n = 401) with responses. Out of 9582 blinks, 53% (n = 5033) co-occurred with questions and 47% (n = 4549) with responses. Out of the 5193 gaze shifts away from the interlocutor that accompanied questions and responses, 51% (n = 2642) co-occurred with questions and 49% (n = 2551) with responses. Out of 138 nose wrinkles, 63% (n = 87) co-occurred with questions and 37% (n = 51) with responses. Out of 101 pressed lips, 45% (n = 45) co-occurred with questions and 55% (n = 56) with responses. Out of 91 mouth corners down, 53% (n = 48) co-occurred with questions and 47% (n = 43) with responses. Lastly, out of 2681 smiles, 58% (n = 1562) co-occurred with questions and 42% (n = 1119) with responses . With this analysis, we asked whether, when a signal occurs during a question or response, it is more likely to occur in one rather than the other. There were 13,214 facial signals that accompanied questions and 10,868 facial signals that accompanied responses. Specifically, we found that out of 1269 eyebrow frowns, 71%  were accompanied with eyebrow frowns, 20% (n = 1343) with raises, 2% (n = 136) with frown raises, and 3% (n = 189) with unilateral raises. Moreover, 4% (n = 276) were accompanied with eye widenings, 11% (n = 740) with squints, 51% (n = 3454) with blinks, and 35% (n = 2402) with gaze shifts. Furthermore, 1% (n = 82) were accompanied with nose wrinkles, 1% (n = 45) with pressed lips, 1% (n = 46) with mouth corners down, and 23% (n = 1528) with smiles. Out of all 4553 responses, 8% (n = 358) were accompanied with eyebrow frowns, 25% (n = 1145) with raises, 2% (n = 91) with frown raises, and 3% (n = 128) with unilateral raises. Moreover, 3% (n = 151) were accompanied with eye widenings, 8% (n = 375) with squints, 59% (n = 2679) with blinks, 49% (n = 2244) were accompanied with gaze shifts. Furthermore, 1% (n = 50) were accompanied with nose wrinkles, 1% (n = 54) with pressed lips, 1% (n = 43) with mouth corners down, and 24% (n = 1089) with smiles  a particular facial signal. With this analysis, we asked how likely a given question or response was to contain h smiles .combinations of co-occurring facial signals that map onto questions and responses. We first determined if groups of two facial signals frequently occurred together, to see if there were any groupings of facial signals at all before examining potential clusters. For both questions and responses, there was a high number of eyebrow frowns with squints, eyebrow raises with eye widenings, and eyebrow raises with smiles. Moreover, there was a high number of blinks with eyebrow frowns, raises, squints, gaze shifts, and smiles. Furthermore, there was a high number of gaze shifts with eyebrow raises, and with smiles. There was a higher number of co-occurrences in questions for eyebrow frowns with blinks, and squints with blinks. However, there was a higher number of co-occurrences in responses for blinks with eyebrow raises, gaze shifts, and smiles. Additionally, there were also more gaze shifts with eyebrow raises and with smiles. Overall, the largest number of co-occurrences between facial signals was found in responses  facial signals. We employed DT models, which constructed prediction models from specific configurations of facial signal combinations to statistically predict whether a verbal utterance was more likely to be a question or a response. With this analysis, we wanted to determine whether there is any evidence that the two social actions are distinguishable based on the frequency with which (a subset of) facial signals accompanied them. This analysis was performed on 11,331 observations. Results showed eight terminal nodes. A main pattern that can be gleaned from the tree is that eyebrow frowns appear to be amongst the most powerful visual question markers, since they are associated with the highest confidence values both when they occurred in the absence and in the presence of gaze shifts. Another pattern is that the verbal utterance was statistically predicted to be a question in all cases, except when there were gaze shifts with eyebrow raises. In that case, the verbal utterance was predicted to be a response. Although all other combinations of facial signals were predicted by the model to be questions, the confidence of this prediction changed depending on the combination. For instance, gaze shifts with blinks resulted in a 50% chance of being a question or a response . The perAfter determining whether questions and responses could be distinguished based on facial signals, we asked whether there were specific combinations of signals that occur within questions and responses by looking at the likelihood that particular facial signals occur with one another, irrespective of their frequency. The MCA analysis consisted of four steps. First, relationships were summarised between facial signals by using their (squared) distance, which was calculated based on how frequently they co-occurred in either questions or responses. The MCA analysis was performed on 7934 observations (4746 for questions + 3188 for responses), using 70% of the data for training. Like PCA and CA, we represented the cloud of variables by projecting it on orthogonal axes .n = 22) were created to produce stability plots. These plots suggest that the 12 variables of the MCA clustering can be combined into eight optimal groups of variables for both questions and responses, as the curve stops increasing around eight clusters , and gaze shifts with eyebrow raises were predicted by the model to be most likely in responses. Thus, questions and responses appear to be different in terms of individual facial signals. Finally, the MCA shows that, without taking into account relative frequency differences between questions and responses, the formal clusters between questions and responses are the same, and indicates that the signals that are likely to co-occur with one another are: (1) blinks and gaze shifts; (2) eyebrow frowns, squints, and nose wrinkles; and (3) eyebrow raises and eye widenings. These three clusters are stable combinations of signals within questions and responses, despite the frequency of these clusters occurring within each of these two social actions being different.n = 473), frown raises (Qn = 74), gaze shifts (Qn = 1456), and smiles (Qn = 903). Facial signals that had an onset mostly after or at the start of questions were eye widenings (Qn = 148), squints (Qn = 421), and blinks (Qn = 3788). Other facial signals occurring after or at the start of questions were eyebrow unilateral raises (Qn = 152), nose wrinkles (Qn = 51), pressed lips (Qn = 41), and mouth corners down (Qn = 34). Facial signals that mostly had an onset before the start of responses were eyebrow raises (Rn = 701), gaze shifts (Rn = 1505), and smiles (Rn = 709). Facial signals that had an onset mostly after or at the start of responses were eye widenings (Rn = 99), squints (Rn = 275), and blinks (Rn = 3522). Other facial signals occurring after or at the start of responses were eyebrow frowns (Rn = 236), frown raises (Rn = 50), unilateral raises (Rn = 99), nose wrinkles (Rn = 30), pressed lips (Rn = 46), and mouth corners down (Rn = 32)  for both questions and responses. Facial signals that mostly had an onset before the start of questions were eyebrow frowns (QRn = 32) .min = −18,599, Qmax = 12,960, Rmin = −12,418, Rmax = 17,694) , frown raises , eye widenings , squints , blinks , and nose wrinkles . Facial signals with a later onset in questions were eyebrow raises , unilateral raises , gaze shifts , pressed lips , mouth corners down , and smiles  , we looked at the onset of facial signals relative to the onset of questions and responses. Overall, facial signals had an earlier onset in questions compared to responses  . Facial  15,650) .Mdn < 0.50). Facial signals that had an onset early in the utterance were eyebrow frowns , eyebrow raises , frown raises , unilateral raises . Moreover, other early facial signals were eye widenings , squints , blinks  and gaze shifts , nose wrinkles , mouth corners down . Facial signals that had an onset later in the utterance (QRMdn > 0.50) were eyebrow frown raises , unilateral raises , pressed lips , mouth corners down , and smiles .To see how the facial signal onsets distributed within the whole verbal utterances, utterance duration was standardised between 0 (onset utterance) and 1 (offset utterance), excluding pre- and post-utterance onsets. Most facial signals had an onset early in the utterance  frequently co-occurred with all other facial signals, but generally co-occurred more with other facial signals in responses. There was a higher number of co-occurrences in questions for eyebrow frowns with blinks, and squints with blinks. However, there was a higher number of co-occurrences in responses for blinks with eyebrow raises, gaze shifts, and smiles. Additionally, there were also more gaze shifts with eyebrow raises and with smiles. In general, the largest number of co-occurrences between facial signals were in responses. Blinks and gaze shifts were the most frequent facial signals overall, so it is not surprising that they co-occurred the most. These results show that there are specific pairwise groupings of co-occurring facial signals that distribute differently in questions versus responses .To see whether it was possible to distinguish between a question or a response based on facial signals that accompanied them, we used DT models to construct prediction models. Results from the DT models showed that it is possible to statistically predict whether the utterance was a question or a response based on facial signals. Specifically, eyebrow frowns marked questions the most confidently (even if co-occurring with gaze shifts), and gaze shifts with eyebrow raises were predicted to often mark responses. This is in line with studies showing that eyebrow frowns are associated with questions in spoken and signed languages ,54,55,57After determining that questions and responses could be distinguished based on the co-occurring facial signals, we investigated combinations of facial signals characteristic for questions and responses by using MCA. This analysis indicated that the 12 facial signals could be clustered into eight stable clusters for both questions and responses. Both questions and responses consisted of the following clusters of facial signals: (1) blinks and gaze shifts; (2) eyebrow frowns, squints, and nose wrinkles; (3) eyebrow raises and eye widenings. The remaining facial signals consisted of eyebrow frown raises, eyebrow unilateral raises, pressed lips, mouth corners down, and smiles, each of which did not cluster with any other facial signals. The lack of clustering in the final five signals could be because they frequently associate with multiple facial signals from different clusters and therefore did not reliably fit with any one grouping. The second cluster  has some resemblance to the facial expression that was previously identified in the literature as the not-face , which tTurning now to timing, the majority of facial signals happened early . This confirms our hypothesis that facial signals may occur very early in the verbal utterance, or even prior to it, because this is how they may exert the greatest influence on early social action attribution: early visual signals may facilitate quick social action recognition for the following speakers and, thus, quick response planning, which is crucial for tight temporal coordination of conversational turn-taking . This esHowever, some signals occurred relatively late in the utterance too. This was the case for mouth movements, eyebrow frown raises and unilateral brow raises. It may be that the mouth movements were used for sarcastic or ironic intention, such as in studies on spoken and signed languages ,26,27,28The current study has some limitations. First, (extreme) laughter heavily affects the visibility of facial signals, since it involves many sub-movements, such as tilting back the head. Therefore, we coded the facial signals from the last evidence or until the first evidence of laughter. Although laughter occurred scarcely, it could be that the artificial cut-offs of facial signals in our data due to the occurrence of laughter still led to small artefacts in our calculation of timing. Second, multiple blinks sometimes occurred with gaps less than or equal to a single video frame (40 ms), which resulted in frames that showed only the white of the eyes, and made it difficult to make accurate annotations about blink onset or offset. Therefore, multiple blinks with gaps less than or equal to one frame were annotated as one blink. This could mean that more blinks were actually performed than we are reporting in this study. However, it could also be that multiple blinks following each other within one frame are perceived as a single communicative unit. Lastly, the present study focused on facial signals during questions and responses in dyadic conversations between acquaintances, but did not look at other categories of conversational social actions, sequential contexts other than question-response sequences, nor different intragroup and intergroup contexts.Future studies investigating participants’ facial signalling in other dyadic contexts and across different cultures would show whether our findings are representative of questions and response more generally, or if they only apply to dyadic conversations between acquaintances in a particular setting. Additionally, including other social actions or a more fine-grained categorization of the broader social actions of questions and responses investigated here may help to elucidate the extent to which facial signals encode specific social intentions in conversation. It is of course possible—and in fact likely—that the particular patterns identified here are characteristic for certain social actions that questions and responses perform, but not for all of them. Identifying commonalities and differences at these more fine-grained levels of social actions remains an important avenue for future research, as well as trying to disentangle social action from turn-taking signals by looking at different social actions within the same sequential context and vice versa. Moreover, investigating the temporal organization of facial signals within an utterance in relation to one another would help to determine whether there is a fixed order of facial signals that characterises different social actions, including cases where they appear to form social-action-specific clusters of visual signals. In addition, investigating how facial signals temporally synchronise with speech (including prosodic patterns) could show how closely they are aligned during different social actions. Another aspect requiring future research is consideration of the detailed interactional processes that underpin the communication of questions and responses, in particular with respect to the extent to which interlocutors may shape current speakers’ facial signalling during questions and responses. Finally, investigating whether the neural signatures differ when social actions are carried by a combination of facial signals and words rather than verbal utterances alone would provide us with important insights into the role of co-speech facial signals during social action recognition.In conclusion, this study showed that questions and responses are characterised by distinct distributions of facial signals, and consist of stable clusters of facial signals. Moreover, most facial signals occurred early in the turn. These findings suggest that specific facial signals, or combinations of facial signals, may be used to indicate different social actions, thus providing visual cues to speakers’ social intentions in conversation. The early timing of facial signals could provide a potential facilitative effect of facial signals for social action attribution, which in turn, may help potential following speakers to recognise the speaker’s intended message in a timely way during conversation; thus, facilitating fast responding. In sum, the results from this study highlight the potentially important role of the body in the pragmatics of human communication, and provide a foundation for investigating the cognitive and neural basis of social action recognition in face-to-face human communication.


Sarcoidosis is a granulomatous disease which can involve every organ, and can manifest with nonspecific clinical pictures that can be misdiagnosed with those observed with other disorders . DespiteIn the COVID-19 era, sarcoidosis is giving a great difficulty of differential diagnosis, especially when there is a predominant lung involvement or the two conditions exist ,3.The diagnosis of sarcoidosis can therefore be very challenging and a thorough evaluation, based on a multidisciplinary approach and holistic view, is often useful to achieve the correct diagnosis .A complete pictorial and literature essay aimed at showing the main diagnostic features of pulmonary sarcoidosis versus pneumonia from the SARS-CoV-2 infection is presented herein . AlthougBased on the necessity to reveal the typical presence of non-caseating and non-necrotizing granulomas, biopsy is often indicated although it can be invasive, especially in deep areas or those at risk of bleeding and or infection .Due to this often unbalanced cost–benefit ratio, every effort should be made to achieve the diagnosis in a way that is the least invasive, especially when sarcoidosis manifests with typical clinical pictures, such as the Löfgren syndrome (LS), characterized by fever, bilateral hilar lymphadenopathy, acute onset of erythema nodosum and migratory polyarthritis. An invasive approach can be temporary avoided in this situation, as LS is virtually diagnostic of sarcoidosis . Lungs are the organs that are predominantly affected, but other districts can be compromised such as skin, eyes, and the gastrointestinal and nervous systems. Laboratory exams in sarcoidosis are often nonspecific, and serum angiotensin converting enzyme (ACE) elevation is neither sensitive nor specific. In their study, Dr. Papasavvas et al. evaluated the usefulness of lysozyme versus ACE and of polyclonal antibody activation as laboratory tests supporting the diagnosis of ocular sarcoidosis. Their results are reported below .Other authors reported an extensive overview of the diagnostic workout of sarcoidosis and methods of follow-up. Some emblematic cases of potential misdiagnosis that can be major clinical problems in daily practice were moreover reported. Several conditions can indeed be misdiagnosed with sarcoidosis, such as infections, neoplasms, immunodeficiencies, and drug-induced diseases ,7,8. In We hope that readers will appreciate this issue, which is meant to be an attempt at reporting the last evidence on diagnosis and management of an old but always challenging clinical condition.


Physical exercise improves motor control and related cognitive abilities and reinforces neuroprotective mechanisms in the nervous system. As peripheral nerves interact with skeletal muscles at the neuromuscular junction, modifications of this bidirectional communication by physical activity are positive to preserve this synapse as it increases quantal content and resistance to fatigue, acetylcholine receptors expansion, and myocytes’ fast-to-slow functional transition. Here, we provide the intermediate step between physical activity and functional and morphological changes by analyzing the molecular adaptations in the skeletal muscle of the full BDNF/TrkB downstream signaling pathway, directly involved in acetylcholine release and synapse maintenance. After 45 days of training at different intensities, the BDNF/TrkB molecular phenotype of trained muscles from male B6SJLF1/J mice undergo a fast-to-slow transition without affecting motor neuron size. We provide further knowledge to understand how exercise induces muscle molecular adaptations towards a slower phenotype, resistant to prolonged trains of stimulation or activity that can be useful as therapeutic tools. Motor neurons (MNs) interact with skeletal muscles through the neuromuscular junction (NMJ) to regulate themselves in a feedback loop mode ,2,3. ThuExercise is the most common physiological stimulus in humans and animals and can modify tissue functionality. Through the years, the benefits of physical activity have been functionally and morphologically studied in both nerves and muscles . In partEndurance and resistance are the two major categories of exercise normally distinguished. While resistance, characterized by short periods of activity at maximal intensity, like weightlifting, is slightly studied in small animals like rodents, endurance is extensively examined. It is characterized by extended periods of continuous neuromuscular activity at moderate, submaximal intensity and is equivalent to running, swimming, and bicycling. Endurance refines the NMJ function ,16,17,18Fast-twitch muscles undergo more evident adaptations than slower muscles ,31. HoweNTR), whereas mature NTF forms, mBDNF, and neurotrophin-4 (NT4) selectively bind and activate the specific TrkB receptor [The BDNF/TrkB signaling is one of the most implicated in the NMJ stability and is essential for neurotransmission ,35,36,37receptor ,39,40,41receptor ,43. TrkBreceptor ,45,46, areceptor , that moreceptor ,49 by phreceptor ,51. Furtreceptor , which rreceptor . Thereforeceptor .Here we provide a relevant step between the external action of physical activity (whose effects are based in variations in protein transcription and translation), and the molecular reaction of the neuromuscular system by analyzing the full BDNF/TrkB signaling pathway, that works presinaptically to control synaptic vesicles exocytosis, including two classic and one novel downstream PKC isoforms , regulatory and catalytic PKA subunits and two components of the SNARE/SM exocytotic machinery proteins (Munc18-1 and SNAP-25). Thus, this study elucidates fast plantaris muscle’s ability to adapt to exercise, either running or swimming, resulting in a fast-to-slow molecular transition. The results show that molecular changes in the BDNF/TrkB pathway are essential for the plastic and synaptic-protective effect of exercise.NTR; three downstream PKCs  and its priming kinase (PDK1); the different PKA subunits; and two PKC and PKA targets related with neurotransmitter release (Munc18-1 and SNAP-25).We analyzed total and phosphorylated protein levels of the BDNF/TrkB downstream signaling pathway including BDNF and NT4 NTFs; their receptors TrkB and p75Results show that proBDNF is significantly increased by both training protocols compared with untrained animals. However, mBDNF protein levels are not affected by running but significantly decrease after swimming. Despite this difference in mBDNF, the mBDNF/proBDNF ratio decreases similarly and significantly after both protocols A. This mNTR and TrkB -). Running significantly increases all four receptors, but especially TrkB.T1, which results in TrkB.FL/TrkB.T1 and, consequently, pTrkB.FL/TrkB.FL ratios being significantly decreased  while swimming decreases total cPKCα. Therefore, the pcPKCα/cPKCα ratio increases but only significantly after swimming A. RegardFurthermore, because PKC maturation and priming include a first phosphorylation step mediated by PDK1, we also analyzed it. Results show that running increases total PDK1 levels while swimming decreases it. Consequently, the pPDK1/PDK1 (S241) ratio decreases with run and increases with swim D.Overall, these complex results show the increase of pcPKCα, nPKCε, and PDK1 protein levels after running and an important reduction of PDK1, cPKCα, and both cPKCβI and pcPKCβI after swimming. Interestingly, the exercise-induced values of cPKCβI after swimming are similar to the values observed in normal untrained slow soleus muscles rather than fast ones .We also analyzed the two catalytic PKA subunits  and the four regulatory subunits  as the T138 phosphorylation of SNAP-25 see . dependsWhen looking at two of the serine-threonine kinases targets, the results show that running increases Munc18-1 and reduces pMunc18-1 (S313) while swimming reduces both Munc18-1 and pMunc18-1. Consequently, after running the pMunc18-1/Munc18-1 ratio is decreased while after swimming it is maintained A. Regard2 are defined as alpha MNs (αMNs). They are responsible of muscular contractions and can be classified, from larger to smaller, into fast-twitch fatigable (FF), fast-twitch fatigue-resistant (FR) and slow-twitch fatigue-resistant (S) subtypes, in accordance with the myocytes they innervate [2, thinner axons, reduced conduction velocities [Since exercise influences the NMJ, we aimed to investigate whether running and swimming-based training protocols could influence MNs. To do so, we detected choline acetyl-transferase (ChAT) to identify MN in lumbar spinal cord sections and analyze the soma size. On one-hand, ChAT-positive MNs with soma size up to 300 µmnnervate . On the locities , and reglocities .Results show that exercise does not modify the total number of MNs in the lumbar sections of the spinal cord. Furthermore, the proportion of small and slow MNs in relation with big and fast ones is maintained, for all MNs categories , indicatNTR/PKC-PKA/SNARE-SM pathway, which is essential to modulate NMJ maintenance and promote neurotransmission [Neurotrophic changes at the NMJ influence its stability and functionality ,36,58,59smission ,51. Tablsmission .Our results show that after a prolonged period of physical training (45 days) there is an increase of proBDNF, similar to the one that was found in proBDNF and BDNF mRNA after 120 min of electrical stimulation in muscle cells . This suOn the other hand, acute 30 min) electrically-induced muscle contraction increases mBDNF without affecting mRNA in the mixed fiber type diaphragm 0 min ele,62,63. HNT4 is an activity-dependent neurotrophic signal for growth and remodeling NMJ. This is supported by the fact that exogenous NT4 induced sprouting of motor nerve terminals ,65,66. IIn addition, NT4 requires more time than mBDNF to activate TrkB endocytosis and stop downstream signaling . Thus, tNTR [NTR-mediated plasticity mechanisms to refine and optimize mature adult NMJ connections. However, despite proBDNF increases after both protocols, p75NTR does not behave symmetrically. This coincides with the fact that swimming muscles, where p75NTR is downregulated, attain a more complete fast-to-slow molecular adaptation than running muscles, which coincides with a faster morphological and physiological fast-to-slow transition [NTR accumulation may represent a signaling that is going through the molecular adaptation while in swimming p75NTR is less expressed as its adaptative mission may have been already achieved. These data add new evidence that p75NTR has a role on synapse refinement also in adult animals [During the development and after muscle stimulation, proBDNF promotes synaptic plasticity and refinement through p75NTR ,40,69. Aansition . Thus, i animals .NTR levels [Heterodimers of TrkB.FL with the truncated isoforms inhibit TrkB.FL trans-autophosphorylation, reducing BDNF signaling ,73,74,75R levels .Here, results show that after a long period of run or swim training, the TrkB isoforms change differently and so the ratios do. Thus, although TrkB.FL increases in both training protocols, pTrkB.FL only increases moderately after running and is reduced after swimming. The abundance of TrkB.T1 in running probably may downregulate the formation of pTrkB.FL, decreasing the downstream signaling. On the other hand, despite TrkB.T1 being unaltered after swimming, pTrkB.FL is strongly decreased. It could be that pTrkB.FL is being consumed due to utilization, but probably TrkB.FL phosphorylation is restricted as a result of the fast-to-slow transition, as the generalized decrease in the downstream pathway shows (see below). Overall, results suggest a downregulation of the TrkB pathway in accordance with the phenotypic fast-to-slow transition that the system undergoes, directly opposite to the adaptation to acute electrical stimulations ,76. FurtNTR interaction, we analyze the downstream proteins in the chain, PDK1, and PKC isoforms.Phosphorylated Y816 TrkB.FL residue activates the phospholipase Cγ (PLCγ) pathway, generating inositol-1,4,5-triphosphate IP3), and diacylglycerol (DAG). This results in calcium release from intracellular stores that activates PKCs . At the , and diaPDK1 directly regulates the priming of the presynaptic PKC isoforms nPKCε and cPKCβI and, consequently, their activity . FurtherConsequently, after running, cPKCβI maintains its levels and activity while pcPKCα and nPKCε increase (in these last cases as in slow muscles) . In accoOn the other hand, SNAP-25 synthesis and phosphorylation at S187 are independent of cPKCβI and exclusively depend on nPKCε . BecauseSwimming reduces Munc18-1 and SNAP-25 total levels either due to consumption after usage or lack of synthesis but always in the direction of a fast-to-slow transformation . InteresIt has been reported that in skeletal muscles, cPKCs are activated by fast, but not slow, patterns of activity  and do n2+ influx induces DAG production that does translocate and activate them. Thus, nPKCs could also work as a pattern-specific signal decoder of nerve-evoked activity in muscle. Indeed, Protein kinase D1 (PKD1), a downstream target of nPKCθ and nPKCε, is especially abundant in slow-type muscles [Additionally, although nPKCs lack the calcium binding domain, the Ca muscles ,88. Acco muscles .To investigate PKA function in the presynaptic component of the NMJ, the phosphorylation of its exclusively presynaptic target SNAP-25 has been studied, in accordance with its implication in acetylcholine release . This enFinally, PKA phosphorylation of SNAP-25 in the T138 residue is directly related with the control of the size of the releasable vesicle pools ,89. MEPP2 range the more abundant ones for the three groups. There is no difference in neither the number nor size of MN somas despite of the molecular adaptations reported at the NMJ. These results are in concordance with previous ones where no changes in soma volume due to exercise were found [The present results confirm that exercise protocols are not harmful for healthy mice MNs and that these cells do not change their soma surface despite of the molecular adaptations in the muscles they innervate. MN distribution follows an approximate Gaussian distribution, being the 500–600 μmre found ,98,99. Ore found  and funcre found .Physical exercise induces reversible changes that mediate tissue adaptability  to respoAccordingly, exercise modifies NMJs morphology and functionality patterns ,118,119 Neurotrophic factors as NT-4 and BDNF could mediate the effect of exercise over the NMJ  . In partThus, endurance protocols adapt the expression of genes in MNs and muscles towards a slower metabolic profile, indicating that both work differently after a training period despite that they still maintain their own identity. Similarly, under different training conditions, myocytes maintain the expression of myosin heavy chains but adapt their metabolism to the new requirements .Wild-type (WT) B6SJLF1/J male mice  until 115 days of age were kept in the animal facility under standard conditions: constant temperature (22 ± 2 °C), relative humidity (50 ± 10%), and a 12-h light/dark schedule and unrestricted access to food and water. Animal handling and experimentation were performed in line with approved Institutional Animal Care and Use Committee protocols at the University of Paris Descartes  and followed the national authority  guidelines for the detention, use and the ethical treatment of laboratory animals based on European Union Directive 2010/63/EU.−1) (Run) which is a low amplitude and frequency exercise that preferentially activates small MNs belonging to slow motor units and five mice were submitted to a high-frequency and amplitude swimming-based training in an adjustable-flow swimming pool (max. 5 L min−1) (Swim) which is a high amplitude and frequency exercise that also activates large MNs, belonging to fast motor units. These groups were compared with five untrained mice that only displayed an exploratory activity during the training time of the first and second groups. Sample size was calculated using previously stablished criteria [Mice were trained from 70 days of age (young adults), 30 min a day, five days a week for six weeks, until 115 days of age as previously described [criteria ,135 to oFour hours after the training was complete (P115), the animals were euthanized and plantaris muscles were dissected and immediately frozen in liquid nitrogen. Plantaris are fast-twitching extensors of the ankle which makes them a good model to study skeletal muscle adaptation to endurance training, either running or swimming. Plantaris muscles have been chosen because fast muscles are more susceptible to training effect ,31 and tWestern blotting procedure was performed as previously described . Musclesw/v) phosphoblocker or bovine serum albumin (BSA) for phosphorylated proteins and non-fat dry milk for non-phosphorylated proteins for an hour. Then, membranes were incubated in primary antibody (Protein samples of 30 µg were separated by 8% or 12% SDS-polyacrylamide electrophoresis and electro-transferred to a polyvinylidene difluoride (PVDF) membrane  using Trans-Blot Turbo Transfer System . For immunodetection, membranes were blocked with TBST containing 5% . The bands optical density was normalized in relation to (1) the background values and to (2) the total protein transferred on PVDF membranes, measured by total protein analysis . The re-1) and transcardially perfused with 20 mL of buffered saline then spinal cord were dissected and fixed in 4% paraformaldehyde overnight. The L1–L5 lumbar region of the spinal cord was sectioned with a vibrating blade microtome  at 50 μm thickness. One out of every six sections was processed for immunostaining on free-floating sections . The immunohistochemical analysis was based on detection of choline acetyltransferase (ChAT) to stain motoneurons. Primary and secondary antibodies conjugated to Alexa fluor used are specified in The spinal cord of P115 mice was also dissected after the animals were anaesthetized by intraperitoneal injection of 1% pentobarbital solution  within each group. Also, each dot represents the value of one animal to visualize their distribution. Statistical significance of the differences between the experimental groups was evaluated under a non-parametric Kruskal–Wallis test followed by Dunn’s post hoc test . The criterion for statistical significance was: * Physical exercise is a key modulator of the plasticity of muscles and nerves. Its results depend on the type, intensity, and duration of the induced contractions to trigger different intracellular signaling pathways. Activity dependent plastic modulation of the neurotransmission related structures in the normal adult occur in response to functional demands. Thus, the NMJ components change to adapt to new situations. Presynaptic nerve terminals are optimized regarding to the organelle machinery, branching complexity, and membrane densifications and active zones to sustain synaptic transmission. Moreover, postsynaptic membrane densification and fold crests adjust themselves and nAChR increases. Additionally, myocytes adapt their gene expression to a slower phenotype.The main result of the present study indicates that the change in the protein level of most BDNF/TrkB pathway and downstream neurotransmitter release related molecules in the trained fast plantaris resembles to levels in untrained slow muscles. Interestingly, slow muscles have lower quantal content and spontaneous quantal release frequency than fast muscles, due to their functional properties . HoweverOur results demonstrate that the BDNF/TrkB pathway, one of the most implicated in NMJ preservation, is precisely modulated by exercise, depending on the nature and intensity of the training, which permits to understand the benefits that physical exercise has over our health. Altogether, it seems that both training protocols studied optimize the BDNF/TrkB signaling pathway as, through the training, an amount of work is achieved to adapt the muscle to optimize its resources, which results in a transition to a slow way of working. This seems to have the objective to acquire a prolongated resistance and more stability to resist effectively prolonged trains of stimulation or activity. The molecular fast-to-slow transition induced by endurance exercise observed in the BDNF/TrkB signaling at the plantaris skeletal muscle occurs with some independence of the soma, where no changes in size occurs, thus, pointing to the autonomous capacity of the nerve terminal to respond to activity changes. However, further analysis of changes in MN soma gene transcription would be of interest, despite the morphology not reflecting them.


Tyrosine kinase activation plays an important role in the progression of pulmonary fibrosis. In this study, we analyzed the expression of 612 kinase-coding and cancer-related genes using next-generation sequencing to identify potential therapeutic targets for idiopathic pulmonary fibrosis (IPF).Thirteen samples from five patients with IPF (Cases 1–5) and eight samples from four patients without IPF (control) were included in this study. Six of the thirteen samples were obtained from different lung segments of a single patient who underwent bilateral pneumonectomy. Gene expression analysis of IPF lung tissue samples (n = 13) and control samples (n = 8) was performed using SureSelect RNA Human Kinome Kit. The expression of the selected genes was further confirmed at the protein level by immunohistochemistry (IHC).DCLK1 and STK33 were found to be upregulated in IPF lung samples compared to control lung samples. However, the top most upregulated genes were distinct in individual cases. DCLK1, PDK4, and ERBB4 were upregulated in IPF case 1, whereas STK33, PIM2, and SYK were upregulated in IPF case 2. IHC revealed that these proteins were expressed in the epithelial layer of the fibrotic lesions.Gene expression analysis revealed a correlation between the gene expression signatures and the degree of fibrosis, as assessed by Ashcroft score. In addition, the expression analysis indicated a stronger heterogeneity among the IPF lung samples than among the control lung samples. In the integrated analysis of the 21 samples, We performed a comprehensive kinase expression analysis to explore the potential therapeutic targets for IPF. We found that DCLK1 and STK33 may serve as potential candidate targets for molecular targeted therapy of IPF. In addition, PDK4, ERBB4, PIM2, and SYK might also serve as personalized therapeutic targets of IPF. Additional large-scale studies are warranted to develop personalized therapies for patients with IPF.The online version contains supplementary material available at 10.1186/s12931-022-01940-y. Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive lung disease that results in fibrotic scarring of the alveolar tissues. Globally, the incidence of IPF is increasing, with approximately 3–9 cases per 100,000 individuals being reported each year –5. HowevMultiple studies have shown that damage to the respiratory epithelium and impairment in its repair mechanism play a central role in the development of IPF . In partCurrently, there are multiple clinically approved kinase inhibitors for a wide range of diseases, including fibrosis and malignant diseases. Furthermore, there are a few similarities in the pathogenesis of IPF and non-small cell lung cancer (NSCLC), a chronic respiratory disease with abnormal cell proliferation. In addition, the activation of tyrosine kinases and overexpression of growth factors have been known to play important roles in the progression of both pulmonary fibrosis , 17 and In this study, we analyzed the expression of 612 kinase and cancer-related genes to identify the potential therapeutic targets of IPF. We used next-generation sequencing to perform gene expression analysis of 13 and 8 surgically resected lung tissues from patients with and without IPF, respectively. Further, we validated the expression of selected genes at the protein level in fibrotic lesions using immunostaining.This study (registered number: K1505-033) was approved by the Ethics Committee of Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences and Okayama University Hospital.Ⓡ Tissue System  or RNAlater™ . RNA was extracted from RNAlater™-fixed samples using the RNeasy Micro Kit according to the manufacturer’s protocol. The concentration and quality of RNA were measured using an Agilent 2100 Bioanalyzer .Patients with or without IPF were enrolled in this study between April 2015 and November 2016 after obtaining written informed consent. The tissue samples with or without IPF were obtained from the organs removed for lung transplantation or resected during the treatment of lung cancer at Okayama University Hospital. The diagnosis of IPF was based on the official ATS/ERS/JRS/ALAT statement . The colThe SureSelect RNA Human Kinome Kit (Agilent Technologies) that targets 612 genes, including 517 protein kinase-coding genes and 46 cancer-related genes, was utilized for library preparation using the RNA samples with an RNA integrity number > 7. Sequencing was performed on an Illumina MiSeq Sequencing System using the V2 Reagent Kit . The sequencing data were analyzed using the CLC Genomics Workbench . Gene expression data were normalized based on the reads per kilobase per million mapped reads (RPKM). Hierarchical clustering analysis was performed using Cluster 3.0 software with the following adjusting method: centralization with median value of each sample, and normalization by dividing centered values by the standard deviation of all samples. Gene expression diversity was calculated with the following adjusting method: the difference between the maximum and minimum RPKM values was calculated for each gene in each patient, and the obtained values were divided by the standard deviation of all samples.Ⓡ Tissue System were cut into 5-µm thick sections and stained with hematoxylin and eosin. The severity of fibrosis in these samples was evaluated using the Ashcroft score, as previously described [The paraffin-embedded tissue blocks fixed using PAXgeneescribed .Ⓡ Tissue System were cut into 5 µm thick sections, placed on glass slides, and deparaffinized in d-limonene and graded alcohol. The tissue sections were then incubated in 1 mM EDTA buffer (pH 8.0) for 10 min at 95 °C in a water bath and blocked for endogenous peroxidase activity with 3% hydrogen peroxide in methanol for 5 min. Following the incubation, the slides were rinsed with Tris-buffered saline containing 0.1% Tween 20 and blocked with normal goat serum or normal horse serum for 60 min. The sections were then incubated with primary antibodies overnight at 4 °C. The primary antibodies against doublecortin-like kinase 1 (DCLK1) (ab31704), pyruvate dehydrogenase kinase 4 , and spleen-associated tyrosine kinase  were obtained from Abcam . Anti-Pim-2 proto-oncogene, serine/threonine kinase  and anti-serine/threonine kinase 33  antibodies were purchased from Sigma-Aldrich , and anti-Erb-B2 receptor tyrosine kinase 4 (anti-ERBB4) antibody (19943-1-AP) was obtained from Proteintech Japan . Following overnight incubation, the sections were incubated with EnVision + Single Reagents HRP Rabbit  or ImmPRESS Reagent Anti-Mouse IgG  secondary antibodies for 20 min. Finally, the sections were stained with 3,3-diaminobenzidine and counterstained with hematoxylin.The paraffin-embedded tissue blocks fixed using PAXgenex) was calculated as described previously [x) is calculated as the weight for gene x as Sx = (μAshcroft≥6 − μAshcroft<6/σAshcroft≥6 + σAshcroft<6), where, for each gene, μAshcroft≥6 represents the mean value and σAshcroft≥6 represents the standard deviation for that gene in all samples of the Ashcroft score ≥ 6. In all, 26 genes were selected as “induced” genes showing (μAshcroft≥6 − μAshcroft<6) > 1, whereas 15 genes showing (μAshcroftt≥6 − μAshcroft<6) < -1 were selected as “suppressed” genes in Ashcroft score ≥ 6 samples  using weight Sx, boundaries bx = (μAshcroft≥6 + μAshcroft<6)/2, and weighted voting score Vx = Sx (gxγ − bx). The final voting scores were summed (∑xVx).Samples of IPF case 1 were used to select the genes for weighted-voting score calculation. Samples of IPF cases 2 and 3 were used as an independent validation set to evaluate the model versatility. The signal-to-noise statistic . Expression of targetable kinase genes between the individual case and the control lung samples were compared using two-tailed paired Student’s t-tests.The characteristics of five IPF and four control patients are shown in Table Gene expression analysis was performed using 13 IPF and 8 control (without IPF) lung tissue samples. Clustering analysis indicated that the IPF and control samples were clustered together . Expectedly, a stronger diversity was observed in IPF lung tissues than in control lung tissues, suggesting not only histological, but also genetic heterogeneity of IPF lungs  to be upregulated with the considered threshold of > twofold in 13 IPF samples compared to the 8 control samples  compared to control lung tissues  with a fold change of > 2. The integrated analysis indicated three genes  were excluded from further analysis because their RPKM values were lower than the overall median RPKM value. The findings from each of the IPF cases are shown below.Further, considering the heterogeneous nature of IPF, we independently compared the gene expression profiles between the IPF lung samples collected from each of the three cases and control lung. In addition, we evaluated the expression of the 46 selected genes encoding kinases having clinically available kinase inhibitors  and history of right upper lobectomy for squamous NSCLC. High-resolution computed tomography (HRCT) revealed bilateral honeycombing, indicating a usual interstitial pneumonia (UIP) pattern , but with no statically significant difference. Furthermore, IHC revealed that DCLK1 and PDK4 proteins were mainly expressed in the epithelial layer and smooth muscle cells of fibrotic lesions in IPF lungs, whereas they were expressed in the airway epithelium of control lungs  who was diagnosed with lung squamous cell carcinoma  along with IPF. HRCT revealed lung cancer of the right lower lobe and bilateral honeycombing, indicating a UIP pattern  who was diagnosed with IPF. HRCT revealed bilateral honeycombing, indicating a UIP pattern  with no statical significance. IHC indicated the expression of DCLK1 protein in the epithelial layer of fibrotic lesions and smooth muscle cells, similar to that observed in case 1 . Except for DCLK1 and STK33, these genes were neither identified by our integrated analysis nor found in the IPF Gene Explorer database, indicating that these genes are not universally expressed in IPF. However, these genes could serve as potential targets for personalized IPF therapy because they were uniquely upregulated in individual patients.Owing to the heterogeneous nature of IPF, its pathogenesis may vary in each patient. In some patients, IPF may be caused due to the dysfunction of AT2 cells, whereas in others it may be caused due to Among the selected genes having clinically available specific kinase inhibitors, ERBB4 was the most upregulated gene in IPF case 1. Dreymueller et al. reported that the release of inflammatory cytokines, such as CXCL8 and IL-6, from the smooth muscle cells was suppressed by inhibiting ERBB4 expression . In addiThis study had several limitations. First is the small sample size, potentially leading to skewed results because of selection bias. Second, of the 21 samples analyzed in this study, 13 were obtained from residual specimens of lung cancer surgery, which included a relatively high proportion of patients with lung cancer. Although we microscopically confirmed that the samples for this analysis did not contain lung cancer cells, we cannot completely rule out the possibility that the lung tissue that develops cancer may bias our results. Further, IPF case 3 was treated with pirfenidone prior to tissue collection, which may have affected gene expression . Third, We performed a comprehensive kinase expression analysis using RNA sequencing to explore potential therapeutic targets for IPF and found that DCLK1 and STK33 may serve as potential candidates for molecular targeted therapy of IPF. In addition, PDK4, ERBB4, PIM2, and SYK might also be attractive targets in individual cases. Additional large-scale studies are warranted to develop personalized therapies for patients with IPF.Additional file 1: Table S1. List of 41 genes selected based on the signal-to-noise statistic. Table S2. Genes expressed differentially by more than twofold, in moderate and severe fibrotic samples compared to control lung samples. Table S3. List of 46 kinases having clinically available inhibitors.Additional file 2: Fig. S1. a Clustering analysis of three samples from IPF case 2. b Clustering analysis of two samples from IPF case 3. c Signal-to-noise weighted-voting score based on 41 genes from IPF case 1. Fig. S2. Immunohistochemistry of ERBB4. Scale bar = 200 µm. Fig. S3. Expression of the 40 selected genes encoding kinases having clinically available kinase inhibitors in IPF cases 4 (a) and 5 (b).


The effects of a graphene bandgap and workfunction were studied. With an increase in the graphene workfunction and bandgap, the Schottky barrier height and series resistance increased and the saturation current decreased. This behavior was related to the tunneling rate variations in the graphene layer. Therefore, control of Schottky barrier diode output parameters was achieved by monitoring the tunneling rate in the graphene layer (through the control of the bandgap) and by controlling the Schottky barrier height according to the Schottky–Mott role (through the control of the workfunction). Furthermore, a zero-bandgap and low-workfunction graphene layer behaves as an ohmic contact, which is in agreement with published results.Controlling the Schottky barrier height ( In addirelation , the line dipole . Farzanae dipole  studied o et al.  used W, a et al.  studied a et al.  reportedIn this article, a new approach is proposed to improve and control the Schottky barrier height and other parameters of Ni/β-2O3 Schottky barrier diode (SBD) based on an epitaxial Si-doped (001) β-Ga2O3 drift layer was deposited on a Sn-doped (001) β-Ga2O3 substrate by halide vapor-phase epitaxy (HVPE). A nickel film was deposited on the drift layer using an electron-beam evaporation method followed by annealing at 400 °C. A schematic illustration of the SBD structure is shown in 2O3, and Sn-doped β-Ga2O3 was 0.3, 10, and A β-Galication .SILVACO ATLAS TCAD software  was used in this simulation to solve the basic drift-diffusion semiconductor equations, which are the Poisson and continuity equations.The Poisson equation is given by ,21:(1)diThe continuity equations for electrons and holes are defined in steady states by ,21:(2)0=ϵ, 2O3 [0 where m0 is the free electron mass [Traps are represented by their ionized density n levels ,23, has n levels :(6)JT=A*given by :(7)Γ(ϵ)=ron mass ), respecAs presented in 2) was observed after the insertion of this graphene layer. This decrease in The main goal of the present work was to study the effect of inserting a graphene monolayer between the Ni and the g et al. , who foun et al. , a similn et al.  also fouWith the graphene layer, the tunneling mechanism increased the density of the extracted free electrons from the Ni to the graphene and then to the β-We demonstrated that a graphene monolayer can enhance the SBD outputs by increasing the tunneling rate. Experimentally, the graphene bandgap can be controlled by several methods. Takahashi et al.  found th2 as shown in The This decrease in the tunneling rate led to a decrease in the saturation current as presented in The effect of the graphene workfunction on SBD parameters was investigated. In this study, a zero-bandgap graphene layer was considered. As the graphene workfunction increased from 4 to 4.8 eV, the output current was affected  and ϕB i(χGa2O3) :(8)ϕB=WGFurthermore, a decrease in the saturation current was obtained with the increase in the graphene workfunction.2. This is related to the decrease in the tunneling rate in the graphene layer. Generally, controlling the graphene workfunction means controlling As the graphene workfunction increased, ribed in ,32, the ribed in ,34. In an et al. .n increased. These effects were related to an increase in the tunneling rate. The graphene bandgap and workfunction were used to control the output parameters of the SBD. When the graphene bandgap increased, the Schottky barrier height and series resistance both increased. Similarly, In summary, this study investigated the effect of a graphene layer on characteristics of a Ni/β-