In every-day conversations, the gap between turns of conversational partners is most frequently between 0 and 200 ms. We were interested how speakers achieve such fast transitions. We designed an experiment in which participants listened to pre-recorded questions about images presented on a screen and were asked to answer these questions. We tested whether speakers already prepare their answers while they listen to questions and whether they can prepare for the time of articulation by anticipating when questions end. In the experiment, it was possible to guess the answer at the beginning of the questions in half of the experimental trials. We also manipulated whether it was possible to predict the length of the last word of the questions. The results suggest when listeners know the answer early they start speech production already during the questions. Speakers can also time when to speak by predicting the duration of turns. These temporal predictions can be based on the length of anticipated words and on the overall probability of turn durations. Recently, there has been increasing interest in interactive language processing in psycholinguistics e.g., . For a lOne striking aspect of natural conversations is the give and take of turns between conversational partners. A systematic way of turn-taking appears early during human development and it precedes the development of linguistic competence. Moreover, it has also been suggested that turn-taking appeared prior to language in phylogeny . During Therefore, listeners are probably required to execute parallel cognitive tasks for an immediate response. They need to understand the message of the current turn and start to prepare an answer. The short transitions also suggest that next speakers time the production of the answer to the end of the current turn.In order to prepare an answer before the current turn ends, listeners must know what they want to say. Therefore, it is likely that they anticipate the message of the turn they are listening to. Studies using eye-tracking and electrophysiological (EEG) measurements have shown that people anticipate information at many different levels of language processing. Eye-tracking studies have demonstrated semantic predictions made by listeners e.g., . Event-rRegarding the timing of a response, the tight transition-times also suggest that next speakers wait for the right moment to deliver the answer . A timedOthers have argued that timed production of turns is not a result of anticipation. This view holds that turn endings are signaled to the listener through a variety of means, including eye-gaze  and chanHowever, it remained an open issue how syntactic and lexical information facilitates turn-end predictions. One possibility is that the syntactic-semantic content of turns informs the listener whether the currently heard linguistic element will terminate the turn or if it will be followed by further information. In this case, the terminal element serves as a cue for response initiation. Speakers do not have expectations of when the turn end will occur, but they will start speech production when they recognize the terminal element  of the turn. This account is also compatible with some studies which have investigated when speakers begin speech planning while listening to their conversational partner. For instance, The findings of these two studies  have beeEven if the initial stages of speech planning could start early, the question still remains how speakers time the articulation of their turn. We have already presented several studies which have suggested that speakers start articulation (or speech planning) as a reaction to cues in their partner’s turn see . These cIn another study, However, there is still little empirical evidence that listeners can make predictions about the duration of linguistic information and whether these temporal predictions influence turn-transitions. A related open issue is the span of such temporal predictions. Listeners might predict the durations of the final syllable of turns by entrainment to the speakers’ speech rate. They might also be able to make predictions about the duration of longer segments, e.g., several syllables or words. To address this issue, The “reaction on a cue” and the “temporal preparation” accounts are not necessarily incompatible with each other. When next speakers are already prepared for the motor response, they will respond faster when they encounter a turn-ending cue or the turn-end. They can also inhibit the execution of the articulatory movements by attending to cues which signal that the turn is not ending yet. Such “talk-projecting” cues have already been described in intonation and in non-pitch phonetic features of words .Most of the studies of turn-taking presented thus far have focused on the different features of the linguistic content of turns  which can help listeners, i.e., next speakers of a conversation in timing their turns. However, little attention has been paid to why estimation of turn durations could lead to better timed turn-transitions. Previous research on temporal preparation has shown that participants not only wait for a signal to react on but they prepare for the moment when the signal is most probably going to appear. These studies have usually employed non-verbal tasks where the time intervals between a warning signal and an imperative response signal have been manipulated. Several experiments have shown that reaction times to a response signal will decrease if participants can estimate the point in time when the response signal is delivered . Reaction times decrease when the occurrence of the response signal can be estimated, because participants can prepare for their response . MoreoveTo summarize, studies have shown inconsistent results whether speech planning starts already as soon as the answer is known to questions , or it sThus, our experiment was designed to manipulate preparation of what to respond independently of preparation of when to respond. The participants were tasked with answering recorded questions about objects presented on a display. Regarding the first experimental manipulation (preparation of the content of a response), participants could guess the correct answer either (1) early, i.e., toward the beginning of the question or (2) late, i.e., only when they recognized the last word of the question. In the first case, the correct answer to the question could be guessed only by looking at the picture. With respect to the other manipulation (anticipation of the number of syllables of last words of questions), the two possible last words of the questions were either (1) similar in the number of syllables (either both 3–4 syllables long or both monosyllabic) or (2) different . In the first case, participants could certainly anticipate the length of final word based on the number of syllables of the possible candidates. In the other case, it was uncertain before the recognition of the last word how many syllables of the last word contained. In none of the conditions, participant could guess in advance which word finished the questions.We reasoned that decreased response times when the answer was known in advance, would suggest that response preparation starts prior to recognition of the last word of the questions. However, if next speakers start to prepare their answer when they recognize the last word, there would be no difference in the response times. If response times increased when it was more uncertain how many syllables the final word of a question had, it would show that word-durations are anticipated and taken into account in response-timing. If next speakers do not predict the duration of words, there would be no differences in response times following this manipulation.Forty university students between the ages of 18 and 25 years  participated in the experiment. They were registered in the subject pool of the Max Planck Institute for Psycholinguistics in Nijmegen and were paid for their participation. All were native Dutch speakers. At the time of commencement of the study, ethical approval was not required for this study design according to the local legislation.Figure 1). The drawings were from the picture database of the Max Planck Institute for Psycholinguistics or from free web sources.Participants looked at pictures on a computer screen and heard questions about the presented pictures. On each picture, black and white drawings of two animals, a tiger and a rabbit and two circles with drawings of colorful objects were presented . The first two questions were the same in each trial. These questions aimed to control if participants named objects as intended and required participants to observe the picture carefully. These control questions asked which objects each animal had. The tiger and the rabbit were said to “have” the objects in the circle behind them. The third question was the critical question which was different across experimental trials. The critical question always named two objects, and it asked to which animal these objects belonged. The participants’ response time was measured only after this last  question.An experimental trial consisted of presenting a picture with two scenes (one with a rabbit and one with a tiger above each other) and three questions. Participants were asked to answer the questions while looking at the picture . The left column of Figure 1). When the name of these two objects had the same length measured in the number of syllables, participants could more certainly predict the duration of the question before hearing the last word  (CONGRUENT conditions). When the number of syllables was different between the possible final words, the prediction was less certain  (INCONGRUENT conditions). The four experimental conditions were created from these two main experimental manipulations: EARLY-CONGRUENT , LATE-CONGRUENT , EARLY-INCONGRUENT , LATE-INCONGRUENT .The other experimental manipulation measured if participants could estimate with certainty how long the critical question would take to complete (CONGRUENCE: INCONGRUENT vs. CONGRUENT). There were always three different objects presented in each picture stimulus. Two of these objects were mentioned in the critical question. When the first object was mentioned , the final word of the question named one of the two remaining objects  and the other half in a long word (3–4 syllables long). Similarly, the correct answer was “het konijn” (the rabbit) and “de tijger” (the tiger) 10 times, respectively. Each participant was presented with these 20 critical questions after the two control questions. The order of the critical questions was randomly determined and was the same for each participant. Each of the participants was assigned to one of four experimental lists. While the questions, answers and their order were identical in each list, different versions of a picture were presented with the same question in the different experimental lists. So, the experimental condition of each question varied across lists. Such a set of pictures belonging to one critical question is shown in To summarize, participants were presented with the same 20 critical questions in the same order and 2 control questions before each of the critical questions. Each group of participants was assigned differing conditions for each critical question and each condition was assigned an equal number of times. Additional features were balanced across the critical questions: the length of the last words of the critical questions, the type of the answer (“het konijn” or “de tijger”) and the position of the object representing the final word in the visual scenes. A list of the critical questions and the competitors of the final words of these questions can be found in the Supplementary Table Participants were seated in a soundproof cabin in front of the screen of a computer presenting the experimental stimuli, wearing headphones, and with a microphone and button-box in front of them. Participants’ voices were recorded continuously throughout the experiment using Digital Audio Tape (DAT). Pictures and sound stimuli were presented using the software Presentation 12.1. Button-presses were recorded in log files and on DAT using a beep. The pre-recorded sound stimuli (words and questions) were also recorded when they were played. The sound stimuli and the beep of button presses were recorded on a different track than the participants’ voices.First, a warm-up session was conducted to familiarize participants with the object-images and their names. This session consisted of each object being displayed along with an audio stimulus of its name. This round was run twice. In a third round, the participants were asked to produce the name of the displayed object. Both written and verbal instructions were provided. The round started when the participant pressed a button. Then, each image was displayed for 3 s in a random order along with the audio of its name. In the third run, a trial started when participants pressed a button. First, a cross appeared for 500 ms. Then, the image appeared on screen, the participant produced the name of the object and pressed a button to initiate the next trial.After the warm-up session, written and verbal instructions were provided for the second part of the experiment. This part started with five practice trials. These trials were similar to the experimental trials but used different stimulus material. Participants were asked to look at the pictures displayed in front of them and answer the three questions about each picture as quickly as possible after the end of the question. With this instruction, we aimed to avoid a quiz-like situation in which participants would answer as soon as they knew the answer. The instruction also encouraged participants to respond quick after the end of the questions, as one might do in real conversation. A trial began when the participant pressed a button, and the picture stimulus appeared on the screen. After 250 ms, the first question was played. Once the participants answered, they had to press a button to hear the next question. When all three questions for the picture were answered, a new picture appeared on the screen after a button-press.The recordings of the audio stimuli and the participants’ voice were converted to WAV files using Audacity 1.2.6. The files were analyzed with Praat 5.1. Response times were measured as the duration between the end of the critical questions and the beginning of the participant’s answer. Negative response times indicate an overlap between question and answer. The ending of a critical question and the beginning of an answer were manually coded using the intensity wave, the spectrogram, and by ear. The beginning of the first speech-sound  was marked as the beginning of an answer.P-values were derived from a likelihood-ratio test with a 0.4 alpha-level for model-selection  mixed-effects model with maximum likelihood estimation. The lme4 package  of R, anelection . At the We analyzed the effect of the control and the experimental variables on the response times. Control variables were variables which could affect response times but were not consistently manipulated in the experiment. These variables were (1) age of the participants (AGE), (2) gender of the participants (GENDER), (3) the order of stimulus presentation (ORDER), (4) the experimental list (LIST), (5) whether the answer contained a determiner , (6) whether the answer was “het konijn”/“konijn” or “de tijger”/“tijger” (ANSWER_TYPE). Experimental variables were variables which were manipulated consistently in the experimental design. ANSWER was a factor with two levels which showed whether participants could know the answer for the critical questions early or late, CONGRUENCE was a factor with two levels which coded whether the candidate final words of the critical question were the same or different in length and FINAL_WORD was a continuous variable. FINAL_WORD coded the duration of the last words in milliseconds. It was included because the questions differed in the length of the last words as a result of the experimental design. All continuous variables were centered.SD = 258, min = -427 ms, max = 1442 ms; 22,5% of all data was excluded) .One participant’s data was excluded from the statistical analysis because of remarkably different responses compared to all other subjects . Answers that were wrong, contained hesitations, false starts, laughter were excluded. Answers were also excluded if participants already pressed the button for the next trial while they were answering the critical question. The analyzed data had 620 data-points , whether the competitor of the final word was the similar or different in length (CONGRUENCE), the duration of the final word of the critical question as a continuous variable (FINAL_WORD) and the control variables . The experimental variables participated in the initial model with all of their interactions; the control variables were included as single main effects. Subjects and items were random effects. After model simplification and the expansion of the random effects, the final model contained the main effect of all variables and the interaction of CONGRUENCE and FINAL_WORD. Subjects, items and the slope of ANSWER under subjects were the random effects in the final model. Figure 4). In the EARLY ANSWER condition the mean of response times was 320 ms and the median was 269 ms, in the LATE ANSWER conditions the mean of the response time was 361 ms and the median was 314 ms.ANSWER, FINAL_WORD and the interaction of FINAL_WORD and CONGRUENCE showed significant effect on the response times. When the answer was known earlier, participant answered earlier .The durations of the final words also affected response times significantly. When the words were shorter, participants answered later  leads to less preparation which results in longer response times. This has been not confirmed, because CONGRUENCE did not have a significant main effect. However, Figure 6 shows that response time increased in the incongruent condition when the final words were shorter.The interaction between the duration of the final words and CONGRUENCE shows that the duration of the competitor word affected the response differently for shorter and longer final words. Figure 7 shows the differences of the mean response times in the congruent and in the incongruent conditions for each critical question in the order of the durations of their final words.post hoc analysis, we computed Wilcoxon signed-rank test on the mean difference of response times between the congruent and incongruent conditions separately for the critical questions with the 10 shortest  and with the other 10 final words (light gray). We tested whether the difference between conditions was different from 0. For the shortest final words, there was a trend in the deviation from 0 mean , while there was no trend or significant difference for the other group . This might suggest that uncertainty of the final word duration increased response times only when the final words had a short duration.In a In an additional analysis, we checked whether participants’ reaction to phonetic or lexical cues could lead to the effect of final word duration. It was possible that participants started the production of their answers as a reaction to such cues. For example, if cues are closer to the end of questions with shorter final words, it could explain why the response times vary as a function of final word duration. However, if the effect of final word duration cannot only be explained by reaction to cues, other mechanisms for explanation can also be proposed. We checked whether cues cause the effect of final word duration on the response times by using two aspects of this process: First, if speakers start the production of their answer by encountering a turn-yielding cue, and this cue is closer to the turn-end in shorter final words, responses will be produced later to questions ending with short words. To examine this assumption, we checked whether there were correlations between the durations of the final words and the durations from possible cues to the end of the questions . Second, if speakers start the production of their answer as a reaction to a cue, response latencies relative to the given cue will be not affected by the rest of the question measured from the cue to the end of the last word. For this, we calculated the response latencies relative to the possible cues and used mixed-effect modeling to reveal their relationship with the duration of the rest of the question.One of the most obvious cue to which speakers could time-lock the speech preparation process or the articulation is the beginning of the last word. If the final word of a question is longer, there is more time for speech production, so speakers can answer to questions faster relative to the end of the question. However, there might be also other potential turn-yielding cues in the questions which could help in timing speech production. We examined the effect of the following features in the questions: beginning of the final word, beginning of the question, beginning of the vowel in the final stressed syllable, peak of the final rise in pitch and uniqueness point of the final word. The uniqueness point of a word is the moment during listening to a word when only one real word is consistent with all of the input received up to that point. We examined the effect of this point because word recognition can take place sooner if a word has an earlier lexical uniqueness point .r(18) = 0.851, p < 0.001] and the duration between the uniqueness point and the word-end  showed significant correlation with the final word duration. The correlation was not significant with the other two features .First, we tested whether there was a correlation between the durations of the final words and the durations from the different possible cues to the end of the last word. If there are cues which are closer to word-end in shorter final words, these cues are potential candidates for explaining the effect of final word durations on the response times in the main analysis. The duration of the questions .Separate mixed-effects models were run with the newly calculated response times, respectively, for the uniqueness point, the beginning of the final word and the beginning of the questions. In the first two models the duration of the questions before and after the cues were included as fixed effects. Before applying the regression model, it was checked if there were correlations between the durations before and after these cues. There were no significant correlations [uniqueness point: Table 2 summarizes the results.When the beginning of the question was tested as the potential cue, the duration of the question was included as the only fixed effect in the model. We followed the statistical modeling procedure described in Section “Statistical Analysis.” When response times were calculated from the uniqueness point of the final word of questions, the duration of questions before the uniqueness point and the duration after the uniqueness point have affected the response times. As the duration before the uniqueness point increased, the response time decreased. However, when the duration of the question after the uniqueness point was longer, response time increased. When the response time was calculated relative to the beginning of the last word, the duration of the question before the last word did not show a significant effect on the response time. But the duration of the question after the beginning of the last word, i.e., the duration of the final word positively correlated with the response time. When response times were calculated relative to the beginning of questions, response times significantly increased at longer questions. In sum, all analysis showed that all type of response time positively correlated with the duration between the potential cues at the end of the questions.These analyses showed that none of the examined features  served as the only cue for starting speech production. It is still possible that these features helped in recognizing when the question was going to end and when speech production could start. However, the position of cues relative to turn-ends cannot alone explain the decrease in response times when the final words were shorter in our earlier analysis . Hence, it is likely that the expected duration of the final words influenced response times.We were interested to know more about how interactants are able to achieve fast transitions from listener’s to speaker’s role during turn-taking. In our experiment, we examined whether speakers started the speech production process before the terminal element, i.e., before the last word of questions when they already had the possibility to guess a correct answer; and (2) whether they anticipated the length of final word of questions to predict durations.In the experiment, we measured participants’ response times when they answered pre-recorded questions about images presented on a screen. Response time was measured between the question end and the beginning of the answer. We varied in different experimental conditions whether people knew the correct answer early or late and whether it was possible to predict with certainty the number of syllables of the words which ended the questions. The level of certainty was manipulated by presenting questions that could end in the names of one of two objects in the presented image. The objects were chosen so their names either have the same (congruent condition) or different number of syllables (incongruent condition). The questions also differed in duration of their final words.The results confirmed one of our predictions. When participants could only prepare the answer late, their response times were longer. Our second prediction was that participants answer later when they are uncertain about the duration of the question. There was no significant main effect of the certainty-manipulation, but there was an interaction effect between congruence and the duration of the final words. When the final words of questions were shorter, response times were longer in the incongruent conditions in which participants could not predict the length of the final words. The duration of the final words also had a main effect. When the last words were longer, response times decreased.When participants already knew the answer at the beginning of the questions, they had responded in average 310 ms after the question end. This shows that participants did not answer as soon as they knew the answer but were waiting for the right moment to start to speak. This is not surprising because we explicitly instructed participants to respond quickly after the question ended. The difference in response latencies in the early and late answer conditions also shows that participants started speech production before the recognition of the last word of the questions in the early condition. The results confirm that next speakers in a conversation will prepare their turn earlier if they know what to respond. This finding is in line with a study of When the last words were longer, participants answered faster. One explanation of this effect could be that participants answered in a fixed time interval relative to a feature in the last words, for example, the beginning of the last word, the uniqueness point, the final pitch or the final stressed syllable. If any of these features occurs earlier relative to question end in longer final words, and participants time the start of the production of their answer to this point, it could explain the shorter gap after longer words. However, in our additional analysis we found that none of these cues alone could explain the difference in response times according to final word duration. Therefore, it is likely that the expected final word durations influenced response time based on the probability distribution of final word durations throughout the experiment. In interval-timing experiments when the momentary probability of the end of an interval is higher, reaction times decrease . In our post hoc analysis. Therefore, the interpretation of the results should be handled carefully. The interaction might suggest that there is a difference in the process of preparation when the time-interval until the turn-end is estimated to be shorter or longer. Such explanation corresponds to conclusion of studies where a symbolic cue predicts the duration of the interval between a warning and a response signal. When there is a mismatch between the symbolic cue and the actual interval duration, reaction times increase. However, when the duration between the warning and response signal is longer, reaction times are less affected by the mismatch. This is explained by re-orientation of temporal attention. When a response signal is expected to appear early but it appears late, participants still have time to focus their attention on the later time-point . This is longer than the average duration and mode of turn-transition times reported at answers to polar question in Dutch face-to-face conversations  and when the answer was already known at beginning of the questions or was known later (at the recognition of the last word of a question).Our results suggests that participants prepared the production of their answers before the questions ended and tried to time their production to the end of the questions, yet there was still a substantial gap between the end of the questions and the beginning of the answers. The average response latency was 336 ms with a mode between 200 and 400 ms (median = 299 ms)  of every-day conversations can be only produced when speakers are prepared for the turn-end already before the last syllable.To summarize, we showed that speakers will start to prepare for the production of their answer before the current turn ends and they also prepare for the time to speak. This preparation can be influenced by linguistic and non-linguistic temporal expectations. Non-linguistic temporal expectations can arise from the overall distribution of turn durations. With other words, addressees know that they will need to respond and as the turn continues, the probability of its end increases, so the level of preparation for speaking will also increase. This preparation can occur even before addressees would know what to respond. For example, the neuronal network of speech production can be already set up before the actual content of the to-be-produced speech is retrieved . FurtherThe study was conducted at the Max Planck Institute for Psycholinguistics. Before the experiment, the study was presented and approved by the research staff of the Department of Language and Cognition with attention to ethical considerations in compliance with the Declaration of Helsinki. At the time of commencement of the study, ethical approval was not required for this study design according to the local legislation.Participants were recruited from the subject pool of the Max Planck Institute for Psycholinguistics via email and were paid for their participation. The email has already contained a description of the experimental task. When participants arrived to the experiment, their task was verbally explained to them again, and they only started the experiment when they agreed to participate.Healthy participants between 18 and 25 years participated in the study, therefore, additional considerations are not applicable.LM, JD, and SL have designed the study, interpreted the results and written the article. LM has conducted the experiment and analysed the data.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Graphene has been actively investigated as an electronic material owing to many excellent physical properties, such as high charge mobility and quantum Hall effect, due to the characteristics of a linear band structure and an ideal two-dimensional electron system. However, the correlations between the transport characteristics and the spin states of charge carriers or atomic vacancies in graphene have not yet been fully elucidated. Here, we show the spin states of single-layer graphene to clarify the correlations using electron spin resonance (ESR) spectroscopy as a function of accumulated charge density using transistor structures. Two different electrically induced ESR signals were observed. One is originated from a Fermi-degenerate two-dimensional electron system, demonstrating the first observation of electrically induced Pauli paramagnetism from a microscopic viewpoint, showing a clear contrast to no ESR observation of Pauli paramagnetism in carbon nanotubes (CNTs) due to a one-dimensional electron system. The other is originated from the electrically induced ambipolar spin vanishments due to atomic vacancies in graphene, showing a universal phenomenon for carbon materials including CNTs. The degenerate electron system with the ambipolar spin vanishments would contribute to high charge mobility due to the decrease in spin scatterings in graphene. Graphene has attracted a great deal of attention because graphene shows excellent physical properties, such as quantum anomalous Hall effect24589To further understand the high mobility and to apply graphene to the high-speed transistors, the elucidation of the spin and charge-impurity states that cause charge carrier scatterings11121315161718Electron spin resonance (ESR) spectroscopy using device structures is the most effective method to study the spin states in electronic materials under device operations222324252223242527Here, we report the ESR study of electrically induced spin states in single-layer graphene that are carried out using driven transistors fabricated with large-area high-quality graphene. To carry out the ESR measurements sensitively, we use an ion gel which is capable of forming high charge-density states in graphene2930To attain high signal-to-noise (S/N) ratio of the ESR signal by increasing the active area of the device, we utilized a rectangular graphene-transistor structure 3 mm × 30 mm) in an ESR sample tube with an inner diameter of 3.5 mm. 29 mm in anVG) region to present the microscopic investigations of the spin states due to the Fermi-degenerate two-dimensional electron system and those due to the atomic vacancies in graphene. VG. In the negative VG region, the ESR signal intensity increased with increasing the absolute value of the VG. The parameters of the ESR signal at VG = −1.5 V were obtained as the g factor of g = 2.0036 and the peak-to-peak ESR linewidth (ΔHpp) of 1.45 mT. In contrast, the ESR spectrum and the VG dependence dramatically changed under the positive VG region, as shown in VG increased and showed a maximum at VG = 0.8 V, and then decreased when the VG increased from VG = 0.8 V. The ESR parameters at VG = 0.8 V were obtained as g = 2.0033 and ΔHpp = 0.74 mT. As discussed later in detail, the signals at VG = −1.5 and 0.8 V are ascribed to the charge carriers and the atomic vacancies in graphene, respectively.The spin states of single-layer graphene are clearly reflected in the ESR signals. We show the ESR signals of graphene transistors under a wide gate-voltage (VG dependence of the ESR intensity and the sheet conductivity (σ2D) of the graphene transistor. To present the ESR intensity, we evaluated the spin susceptibility (χ) from double integral value of the ESR spectrum, considering the active area of the graphene transistor. The σ2D value showed a minimum at VG = 0.8 V or at VG = 0.6 V when the source and drain electrodes of Ni/Au or Au were used, respectively. The observation of the σ2D minimum indicates an existence of the charge neutral point, namely, the Dirac point in the graphene transistors. Such behavior is consistent with that reported for single-layer graphene transistors32VG at the charge neutral point as VCNP. χ and the σ2D on VG − VCNP. The χ value shows a maximum at VG = VCNP where the σ2D shows the minimum. The increase in the χ has been observed for VG − VCNP < −1 V. It is worth noting that the maximum of the χ at VG = VCNP with the drain-current minimum is fully consistent with that observed for the CNT transistorsThe correlation between the transport characteristics and the observed ESR signals were examined by measuring the σ2D of the graphene transistor. We extracted the Lorentzian component from the ESR spectrum of the graphene transistor because mobile charge carries show an ESR signal with the Lorentzian lineshape. We define the spin susceptibility due to the charge carriers as χL, which was evaluated from double integral value of the ESR spectrum with the Lorentzian lineshape. The χL at different VG was evaluated by performing the ESR spectrum simulation using Lorentzian and Gaussian functions. VG = −1.5 V (or VG − VCNP = −2.3 V), where most of the signal component is found to be described by the Lorentzian function. In contrast, the fitting analysis for the ESR spectrum at VG = 0.8 V (or VG − VCNP = 0 V) exhibits a different result, where the signal component is found to be described practically by the Gaussian function, as shown in VG = 0.8 V because the χL is proportional to the square of the ΔHpp and the ΔHpp of the Lorentzian component is considerably broader than that of the Gaussian component. σ2D and the χL of the graphene transistor on VG − VCNP, respectively. It is worth noting that the χL value shows a minimum at VG = VCNP, which is consistent with the minimum of the σ2D. The increases in the χL for VG < VCNP and VG > VCNP have been observed, which are ascribed to the accumulation of holes and electrons, respectively. Therefore, the clear correlation between the χL and the σ2D demonstrates that the ESR signal with the Lorentzian lineshape is originated from the charge carriers in graphene. Schematics of the energy diagram and the spin states in the graphene transistor are shown in χL intensity reflects the density of states at the Fermi surface in graphene shown in The ESR signal due to the charge carriers should have a correlation with the transport characteristics. To demonstrate the correlation, we discuss the relationship between the spin susceptibility due to the charge carriers and the χ, which is called as Pauli paramagnetism. Thus, it is interesting to discuss the temperature dependence of the χ. The previous studies have reported the temperature dependence of the ESR intensity of the graphene samples27χ of the graphene samples followed the Curie law or the Curie-Weiss law27χ using the graphene transistor to observe the Pauli paramagnetism in graphene. χ of the graphene transistor. The χ was evaluated from the ESR signal at VG = −1.5 V (or VG − VCNP = −2.3 V) for the hole accumulation state by double integrating the ESR spectrum. As shown in χ hardly depends on the temperature, which means the Pauli paramagnetism, not the Curie paramagnetism shown by dashed line in The Fermi-degenerate electron system is known to show no temperature dependence for the spin susceptibility ΔH1/2), which was obtained by integrating the ESR spectrum. As shown in ΔH1/2 increases with increasing temperature. This result can be explained by the Elliott mechanism observed for Fermi-degenerate electron systems, where the spin-lattice relaxation time (T1) is shortened with increasing temperature and the ESR linewidth is proportional to 1/T1, that is, ΔH1/2 ∝ 1/T1  due to the atomic vacancies shows a maximum at a charge neutral point (VG = VCNP) with a drain-current minimumNspin due to the atomic vacancies in the graphene transistors with that of the CNT transistors to present the universality of the electrically induced ambipolar spin vanishment in carbon materials. We evaluated the Nspin in the graphene transistors from the double integral value of the ESR spectrum with the Gaussian lineshape. The Nspin is related to the χG as μB is the Bohr magneton, S is a spin quantum number due to an atomic vacancy (S = 1/2), kB is the Boltzmann constant, and T is the temperature. Nspin of the graphene transistor and the CNT transistors on VG − VCNP, respectively. The data for the CNT transistor are taken from the literatureNspin similarly has the maximum at VG = VCNP and decreases for VG < VCNP and VG > VCNP in the ideal two-dimensional material single-layer graphene and in the one-dimensional material CNTs, respectively. Therefore, the electrically induced ambipolar spin vanishment seems a universal phenomenon for carbon materials with honeycomb structures formed with carbon atoms regardless of the dimensionality of the electron systems.The electrically induced ambipolar spin vanishments have been reported in the ESR study of the CNT transistors, where the number of spins (VG = VCNP where the Nspin (or the χG) due to non-bonding orbitals (NBOs) at the atomic vacancies in graphene shows the maximum value  in the CNT thin film due to a thick film thickness of CNT (approximately 300 nm)VG, resulting in the almost linear change in the Nspin . In the case of graphene, such charge-density distribution is not basically expected due to a monolayer film thickness.The electrically induced ambipolar spin vanishments are schematically explained in alue see . In the spin see . This ree VG see . That isIn contrast to the similar observation of the electrically induced ambipolar spin vanishments in the graphene and CNT transistors, the ESR signal due to charge carries was unable to be observed in the CNT transistors26352635Our result demonstrates the observation of the ESR signal due to the charge carriers and the electrically induced Pauli paramagnetism in the Fermi-degenerate two-dimensional electron system in graphene. The result is interesting due to the first direct ESR observation of the electrically induced Pauli paramagnetism for organic, inorganic, and carbon materials. The observation also demonstrates the existence of the Fermi surface of the two-dimensional material graphene from a microscopic viewpoint. Moreover, the electrically induced ambipolar spin vanishments due to the atomic vacancies in graphene are observed, which indicates that the spin vanishment is a universal phenomenon in carbon materials including carbon nanotubes regardless of the dimensionality of the electron systems. The spins due to the atomic vacancies vanish owing to the antiferromagnetic interactions with electrically induced electrons or the discharges of the vacancies’ spins due to hole doping, which would decrease the carriers’ spin scatterings due to the atomic vacancies. Therefore, the Fermi-degenerate electron system with the electrically induced ambipolar spin vanishments would contribute to high charge mobility in graphene. Our approach using the electrically induced ESR technique is the most appropriate method for investigating the spin states and their correlations with the transport characteristics in electronic materials from a microscopic viewpoint because the method can directly observe the correlations between the spin states and the transport characteristic. Applying the method to other various electronic materials would give new understanding of the microscopic properties which are also useful for improving the device performance.c-plane sapphire [α-Al2O3(0001)] by magnetron sputtering at high temperature. The Cu/sapphire substrate was placed inside a quartz tube (inner diameter 26 mm) and then annealed at 1000°C in the flow of H2 (2.5% in Ar) gases for 40 min. After elevating the temperature to 1075 °C, CH4 gas (10 ppm) was introduced for 90 min to grow graphene. High purity gases were used for this experiment . Confocal Raman (Tokyo Instruments Nanofinder30 with 532 nm excitation) measurements were performed for the graphene transferred on a quartz substrate by a poly(methyl methacrylate) (PMMA) mediated transfer technique. The obtained Raman spectra are consistent with that previously reported for single-layer grapheneWe synthesized large-area single-layer graphene with a chemical vapor deposition (CVD) method37b-methylmethacylate-b-styrene) (PS-PMMA-PS) (3 wt%) , and a solvent ethyl acetate (61 wt%) ; the mixture was stirred for over one and half day, drop-casted on the gate electrode and then thermally annealed at 70 °C under vacuum for over one and half day. The ion-gel insulator shows large electric double layer (EDL) capacitance and high ionic conductivity. The EDL capacitance is generally very large (~10–100 μF cm−2), leading to significant charge accumulation with low voltage and high on/off current ratios. The source and drain electrodes of Ni (1 nm)/Au (30 nm) or Au (30 nm) which had a channel length of 1.0 mm and a channel width of approximately 25 mm were fabricated with a vapour-deposition method on the quartz substrate. Finally, the PET substrate was placed on the quartz substrate, completing the transistor fabrication. The fabricated transistor was sealed into an ESR sample tube under a nitrogen glove-box atmosphere  after the transistor was connected to wires by Ag paste.The transistors were fabricated using two types of nonmagnetic substrates; one was a polyethylene terephthalate (PET) film with dimensions of 30 mm × 3 mm × 100 μm , and the other was a quartz glass with dimensions of 30 mm × 3 mm × 1 mm . Gate electrodes of Ni (1 nm) and Au (30 nm) were vapour-deposited on the PET substrate using an ULVAC VPC-260F vacuum evaporation system. Ion-gel solutions consisted of an ionic liquid, 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([EMIM][TFSI]) (36 wt%) , a gelator ABA-type triblock copolymer poly of the first integrated ESR spectrum. The spin susceptibility  was evaluated by integrating the ESR spectrum twice and comparing it with that of the Mn2+ marker sample. The number of spins, Nspin, was evaluated by integrating the ESR spectrum twice and by comparing it with that of the Mn2+ marker sample. The absolute value of the number of spins of the Mn2+ marker sample was calculated using a solution (220 μL) of 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPOL) as a standard. The calibration of the g factor was performed using a software program from the JEOL RESONANCE ESR system considering high second-order correction to the effective resonance field. Its correctness was also confirmed using 2,2-diphenyl-1-picrylhydrazyl (DPPH) as another standard sample.The ESR measurements were performed with a JEOL RESONANCE JES-FA200 X-band ESR spectrometer and a Keithley 2612A source meter. The ESR signals were measured as a function of How to cite this article: Fujita, N. et al. Direct observation of electrically induced Pauli paramagnetism in single-layer graphene using ESR spectroscopy. Sci. Rep.6, 34966; doi: 10.1038/srep34966 (2016).


Supplemental Digital Content is available in the text Latest study showed that a novel translocation between programmed cell death ligand 1 (PD-L1) (cluster of differentiation 274) and TP63 (tumor protein 63) can be found in diffuse large B-cell lymphoma (DLBCL), resulting in their conjunct overexpression in tumor cells at RNA level. However, the expressed pattern of these 2 genes at protein level in DLBCL remains largely unknown, and the clinical relevance of PD-L1 and TP63 expression in DLBCL are also unclear.Tumor tissues from 76 Chinese DLBCL patients were immunostained for programmed cell death 1 (PD-1), PD-L1, and TP63 using the EnVision system. Clinical relevance of PD-1, PD-L1, and TP63 in 74 DLBCL were analyzed by chi-square test, the Kaplan–Meier curves with log rank test, and Cox's proportional hazards regression model.P = 0.032, P = 0.026). DLBCL with PD-L1 or TP63 expression in tumor cells showed low International Prognostic Index (IPI) score . PD-1+ TILs was related to prolonged overall survival rate (OS) of DLBCL patients (P = 0.02), whereas PD-L1 expression was associated with worse clinical outcome of patients (P = 0.049). Immunoreactivity of TP63 was not correlated with patients’ survival time. Besides, PD-1 expression, patients’ age, Ann Arbor stage, and IPI score were significant prognostic markers for OS, but PD-L1 and TP63 had no prognostic significance.PD-1 was mainly expressed in tumor-infiltrating lymphocytes (TILs) of 39.5% patients. PD-L1 was expressed in tumor cells of 26.3% patients, and TP63 was immunostained in nucleoli of tumor cells of 31.6% cases. PD-1 expression was significantly associated with the patients’ gender and B symptoms (PD-1, PD-L1, and TP63 are frequently expressed in DLBCL. PD-1/PD-L1/TP63 blockade may be a potential therapeutic strategy for some patients. Recently, PD-1/PD-L1 blockade enhance anti-tumor immune response in many solid tumors, such as non-small cell lung cancer (NSCLC), melanoma, and renal cell carcinoma.–9 Therefore, targeting the PD-1/PD-L1 axis may be a potential utility in hematologic malignancies.Programmed cell death ligand 1 , a member of the B7 receptor family, is an inhibitory receptor expressed on activated T cells, B cells, dendritic cells, macrophages, and natural killer T (NKT) cells.–13 The blockade PD-1/PD-L1 pathway demonstrated beneficial therapeutic effect in cHL and follicular lymphoma.,15 In diffuse large B-cell lymphoma (DLBCL), PD-1 was expressed on tumor-infiltrating lymphocytes (TILs), and the number of PD-1+ TILs was associated with the favorable overall survival (OS) in patients with DLBCL.,17 Besides, the higher expression of PD-L1 was also found in tumor cells and it maybe a potential biomarker for DLBCL. Thus, the PD-1/PD-L1 pathway may also contribute to the proliferation of tumor cells of DLBCL and may be a novel applicable strategy to treat patients with DLBCL.In previous clinical trials, PD-1 and PD-L1 were found to be aberrantly expressed in variety of lymphoma, including T-cell lymphoma, mediastinal large B-cell lymphoma, classical Hodgkin lymphoma (cHL), and anaplastic large cell lymphoma. Similarly, the PD-L1 and TP63 fusion translocation pattern was newly found in 5% (1/20) DLBCL, leading to jointly increasing the mRNA expression level of both genes. Based on this intriguing finding, in this study we investigated the expression of PD-L1 and TP63 in 76 DLBCL cases by immunohistochemistry, and studied the frequency of their conjunct aberrant expressions in DLBCL. The present study aims to identify whether PD-L1 upregulation accompanies with the abnormal expression of TP63 in DLBCL at protein level, and whether their expressions are significantly correlated with each other. Besides, our study also investigated the clinical implication of PD-L1/TP63 in patients with DLBCL. Importantly, our research wants to uncover that PD-L1/TP63 immunostaining may be a preliminary screening for further molecular detection of some DLBCL cases, and targeting PD-L1/TP63 may be an ideal treatment for some patients.Lately, some effort put on clarifying why PD-1 or PD-L1 is frequently expressed in some hematologic malignant cells. The structural variation frequently disrupting the 3′ region of PD-L1 was identified in 27% adult T-cell leukemia/lymphoma and in 8% DLBCL, which resulted in the arrangement of gene's open reading frame.22.1A total of 76 patients diagnosed with DLBCL at Shanghai Tongji Hospital affiliated with Tongji University School of Medicine were enrolled into this study. All cases were reviewed according to present World Health Organization criteria (WHO 2008). Among these cases, 43 were treated with chemotherapy, 22 received surgery treatment, and 3 cases received chemotherapy after surgery treatment. The clinical data and outcomes were obtained from the patients’ medical records, including patients’ age, sex, grade, International Prognostic Index (IPI) score, and so forth. The follow-up period ranged from 0.2 to 7.2 years. Response criteria were based on standard guidelines. A total of 74 cases had clinical information and were included in survival analysis. This research was approved by the ethic committee of Tongji Hospital, Tongji University School of Medicine. The informed consents were obtained from all patients.2.2 The information of antibodies was used for IHC detection as following: monoclonal mouse anti-human-CD10 , monoclonal mouse anti-human-Bcl6 , monoclonal mouse anti-human-MUM1 , monoclonal mouse anti-human-PD-1, monoclonal rabbit anti-human PD-L1 , and monoclonal mouse anti-human-TP63 .For immunohistochemistry (IHC) analysis, 3-mm-thick formalin-fixed paraffin-embedded sections were submitted. All histological and immunophenotypic data were reviewed by 2 pathologists under blind study. DLBCL was classified into the GCB and non-germinal center B-cell (non-GCB) phenotype according to the Hans algorithm based on IHC for CD10, Bcl-6, and MUM1.The immunohistochemical staining for detection of PD-1, PD-L1, and TP63 were performed using EnVision system, the detail process was described as the previous study. The proportion of the cells showing membranous and/or cytoplasmic staining was assessed as follows: no or any staining fewer than 10% of tumor cells are negative and staining of more than 10% of tumor cells are positive. The intensity of positive cells was scored as follows: 1, weak; 2, moderate; and 3, strong staining. The proportion of immunostained cells was evaluated among tumor cells and tumor-infiltrating immune cells, respectively. The number of PD-1+ cells in TILs was assessed semi-quantitatively and scored as the previous study:0, no positive cells/high-power field; 1, fewer than 10 positive cells/high-power field; 2, 10 to 30 positive cells/high-power field; 3, more than 30 positive cells/high-power field on average. We used a cut-off value of 30% immunoreactivity for TP63 based on the previous study.,24 The 10% cut-off value of PD-L1 expression and 30% cut-off value of TP63 expression were adopted to identify cases as positive or negative for these 2 proteins. Each case was randomly selected at least 5 fields and quantified.The expression of PD-L1 was evaluated as the previous study.2.3χ2 test (N >5) or Fisher's exact test (N). Two-sided P values were calculated and P <0.05 was considered significant. The Kaplan–Meier curves with log rank test were employed for the survival analysis. The correlations between PD-L1 and TP63 expression were evaluated using Pearson correlation and linear regression analysis. The Cox-regression analyses, both univariate and multivariate, were used to identify the independency of these genes expression status. Both hazards ratio and 95% confidence intervals were calculated for the univariate and multivariate analyses of OS, and P <0.05 was considered significant.The statistical analysis was performed using SPSS 16.0 . The clinical relevance of PD-1, PD-L1, and p63 in DLBCL was assessed using the 33.1The clinicopathological features of patients with DLBCL are shown in Table 3.2+, TP63+ in malignant cells, and PD-1+ in tumor-infiltrating cells was summarized in Supplemental Table S1. Overall, PD-1 was mainly stained in the TIL of 39.5% patients , although the relevancy is not quite obvious.The expression of PD-L13.3P = 0.032, P = 0.026, respectively, Table P = 0.007, P = 0.002, respectively, Table P = 0.031, Table The clinical implications of PD-1, PD-L1, and TP63 in patients with DLBCL are summarized in Table P = 0.002, P = 0.000, respectively, Supplemental Table S2). Similarly, patients with PD-L1 and TP63 expressions possess low IPI score and without B symptoms .Additionally, we investigated the clinical relevance of these genes in patients who only received the chemotherapy treatment. The results showed that PD-1 expression in TILs was significantly higher in male patients with low IPI score (P = 0.02). Whereas, PD-L1 expression in tumor cells possessed worse OS rate . Furthermore, TP63 positive cases were not associated with OS in DLBCL patients . Notably, patients with double-positive expression of PD-L1 and TP63 were significantly had worse clinical outcome compared with those with PD-L1/TP63 double negative . In chemotherapy-treated patients, Kaplan–Meier analysis revealed that PD-L1 expression in tumor cells was significantly related to patients’ poor survival rate , but patients with double positive of PD-L1 or TP63 expression were not associated with patient's survival rate.Kaplan–Meier analysis revealed that patients with PD-1 expression in TILs had prolonged survival time compared with PD-1-negative cases . Multivariate analysis was performed to identify whether they are independent prognostic marker for OS, and the result revealed that age, Ann Arbor stage, IPI score, and PD-1 score are independent prognostic factor for OS (Table P >0.05). In only chemotherapy cohort, univariate analysis revealed that PD-1, PD-L1, and TP63 expressions were not correlated with patients’ age, gender, B symptoms, Ann Arbor stage, and IPI score maybe because of the relatively small sample size.Univariate analysis showed that OS was associated with patients’ age, Ann Arbor stage, IPI score, and PD-1 expression  exhibited objective responses in cancer patients with high expression of PD-1/PD-L1 in contrast with PD-1/PD-L1-negative patients.,30 Evaluating PD-1 expression on immune cells or PD-L1 expression on tumor cells and immune cells may be valuable for predicting responsiveness to PD-1/PD-L1 immunotherapy in malignant tumors. In our cases, PD-1 was highly expressed on immune cells and PD-L1 was highly expressed in tumor cells and immune cells of some cases, indicating that the PD-1/PD-L1 axis may also be useful for some patients with DLBCL. To further analyze the clinical significance of PD-1 and PD-L1 in our cases, we found that DLBCL patients with PD-1+ TILs had prolonged survival time, which contradicts with the suppressed role of PD-1 in immune response. However, the increased PD-1+ cells might reflect previous active immune response as Dohee's described, thus PD-1 expression was associated with favorable survival rate of patients. Consistently, the multivariate analysis by the Cox hazard model showed that PD-1 is an independent prognostic marker for patients’ OS rate. Being consistent with Kiyasu and colleagues’ study, our data also revealed that PD-L1 expression was correlated with worse clinical outcome although it is not an independent prognostic marker for patients’ OS in our study, which suggested that treatments targeting PD-L1 might benefit DLBCL patients.Previous studies indicated that PD-1 and PD-L1 expressions in tumor cells or immune cells is meaningfully associated with the patients’ response to PD-1 /PD-L1 blockade.Additionally, only a chemotherapy-treated cohort can be used to investigate the clinical relevance of PD-1 and PD-L1. Consistently, PD-L1 is significantly associated with patients’ survival rate, but PD-1 is not related to patient's survival rate. The multivariate analysis by the Cox hazard model displayed that all of them are not independent prognostic marker for patients’ OS rate. Therefore, more samples may be needed for further studies to validate the clinical application of them in DLBCL. The structural variation frequently disrupting the 3′ region of PD-L1's open reading frame, which leads to its overexpression. Interestingly, Georgiou and colleagues discovered that a novel translocation juxtaposing between PD-L1 and TP63 in DLBC, and RNA-seq data also showed that the high expression of PD-L1 is accompanied with TP63 expression. It is well known that TP63 is critical for cell homeostasis and embryo and epidermal development.,34 Several studies showed the aberrant expression and structural abnormality of TP63 was found in multiple types of lymphoma.,36 Moreover, Fukushima et al uncovered that TP63 was expressed in the nucleoli of tumor cells in 34% DLBCL, and non-germinal center DLBCL with TP63 positive displayed worse prognosis. In the present study, TP63 expression was found in 32.9% patients. Additionally, since previous study showed that PD-L1 may accompanied with TP63 expression, thus we wonder whether their expression was associated with each other, and whether they are prognostic marker for DLBCL patients. Our results demonstrated that TP63 positive expression was associated with PD-L1 expression though the significance was not obvious perhaps because of small sample size. Besides, our results also showed that patients with double positive of PD-L1 and TP63 had worse survival. A recent study indicated that the AKT/mTOR signaling pathway could be directly activated by PD-1/PD-L1 during the malignant progression of DLBCL. Similarly, TP63 also could regulate mTOR signaling, which play a critical role in the progress of DNA damage. Our previous study showed that PD-L1 dysfunction could promote the progression of leukemia via regulating JNK/cyclin D2 signaling. Thus, we speculated that PD-L1/TP63's overexpression may activate some important intracellular signaling results in tumor development, which need more further studies to elucidate. However, in only chemotherapy-treated cohort, no significance was to be found in correlation between PD-L1 and TP63 expression as well as the correlation between double positive of PD-L1/TP63 and patients’ survival rate. Therefore, more sample size and further studies need to clarify whether PD-L1 and TP63 have synergistic effect and whether the traslocational fusion of them play a critical role in the progression of DLBCL.Recently, some studies indicate that the high expression of PD-L1 can be induced by many oncogene or genetic aberrations. Loss function of PTEN was reported to up-regulation PD-L1 expression in tumor cells.In summary, our study uncovers that PD-1, PD-L1, and TP63 are highly expressed in some DLBCL. PD-1 predicts favorite patients’ survival rate and is an independent prognostic marker of DLBCL. PD-L1 is associated with patients’ poor prognosis regardless of the whole cohort and only chemotherapy-treated cohort. However, PD-L1 and TP63 show weak correlation in all patients, and no significance was found in the only chemotherapy-treated cohort. Additionally, double positive of TP63 and PD-L1 is not related to patients’ OS rate in only chemotherapy-treated patients, but their expression is associated with the OS rate in all patients. Thus, further studies are needed to clarify the expression pattern of PD-L1/TP63 and the role of PD-L1/TP63 translocational fusion in DLBCL by a large sample size.


The near-field plasmon hybridizations between individual Ag nanodisks and the Ag substrate form four independent magnetic dipole modes, which result into multiband absorption enhancement of monolayer graphene at optical frequencies. When the resonance wavelengths of the magnetic dipole modes are tuned to approach one another by changing the diameters of the Ag nanodisks, a broadband absorption enhancement can be achieved. The position of the absorption band in monolayer graphene can be also controlled by varying the thickness of the SiO2 spacer or the distance between the Ag nanodisks. Our designed graphene light absorber may find some potential applications in optoelectronic devices, such as photodetectors.It is well known that a suspended monolayer graphene has a weak light absorption efficiency of about 2.3% at normal incidence, which is disadvantageous to some applications in optoelectronic devices. In this work, we will numerically study multiband and broadband absorption enhancement of monolayer graphene over the whole visible spectrum, due to multiple magnetic dipole resonances in metamaterials. The unit cell of the metamaterials is composed of a graphene monolayer sandwiched between four Ag nanodisks with different diameters and a SiO Graphene, a monolayer of carbon atoms tightly arranged in two-dimensional 2D) honeycomb lattice, was first separated from graphite experimentally in 2004 . Since tD honeycoTo overcome this problem, various physical mechanisms –43 to en2 spacer on an Ag substrate. The near-field plasmon hybridizations between individual Ag nanodisks and the Ag substrate form four independent magnetic dipole modes, which result into four-band absorption enhancement of monolayer graphene. When the magnetic dipole modes are tuned to be overlapped spectrally by changing the diameters of Ag nanodisks, a broadband absorption enhancement is achieved. The position of the absorption band in monolayer graphene can be also controlled by varying the thickness of the SiO2 spacer or the distance between the Ag nanodisks.In this work, by employing similar multi-resonator approach, we will numerically demonstrate multiband and broadband absorption enhancement of monolayer graphene in the whole visible wavelength range, which arise from a set of magnetic dipole resonances in metamaterials. The unit cell of metamaterials consists of a graphene monolayer sandwiched between four Ag nanodisks with different diameters and a SiO2 spacer on an Ag substrate. We calculate the reflection and absorption spectra, and the distributions of electromagnetic fields by the commercial software package “EastFDTD, version 5.0,” which is based on finite difference time domain (FDTD) method (www.eastfdtd.com). In our numerical calculations, the refractive index of SiO2 is 1.45, and the frequency-dependent relative permittivity of Ag is taken from experimental data [σ of graphene is the sum of the intraband term σintra and the interband term σinter [The designed metamaterials for multiband and broadband absorption enhancement of graphene at optical frequencies are schematically shown in Fig. tal data . Under tm σinter , 68, whiω is the frequency of incident light, e is electron charge, ħ is reduced Planck constant, Ef is Fermi energy , τ is the relaxation time of electron-phonon, kB is Boltzmann constant, T is temperature in K, and i is the imaginary unit. Graphene has an anisotropic relative permittivity tensor of εg expressed aswhere ε0 is the permittivity of the vacuum, and tg is the thickness of graphene sheet.where λ1 = 722.9 nm, λ2 = 655.7 nm, λ3 = 545.5 nm, and λ4 = 468.8 nm. At four absorption peaks, the light absorption in graphene can reach as high as 65.7, 61.2, 68.4, and 64.5%, respectively. Compared with a suspended monolayer graphene whose absorption efficiency is only 2.3% at optical frequencies [Figure quencies , 17, thequencies –75, whicquencies –81.Fig. λ1, λ2, λ3, and λ4. At the resonance wavelength of λ1, the electric fields are mainly concentrated near the left and right edges of the first Ag nanodisk with a diameter of d1  absorption efficiency more than 50% (73%). For the diameters of the Ag nanodisks to be increased gradually, this broad high-absorption band is red-shifted, as shown in Fig. In our designed metamaterials, the near-field plasmon hybridizations between individual Ag nanodisks and the Ag substrate form four independent magnetic dipole modes, which result into multiband absorption enhancement of monolayer graphene in the visible wavelength range from 450 to 800 nm, with an average absorption efficiency exceeding 50% , so the center distance l between the nearest-neighbor Ag nanodisks is 200 nm. By varying l, we can also tune the position of the absorption band in monolayer graphene. Figure l to be decreased from 220 to 160 nm. With the decreasing l, the absorption band in monolayer graphene is slightly blue-shifted, owing to the plasmon interactions among the Ag nanodisks.In the above calculations, the coordinate points of four Ag nanodisks are (±2 spacer on an Ag substrate. The near-field plasmon hybridizations between individual Ag nanodisks and the Ag substrate form four independent magnetic dipole modes, which result into multiband absorption enhancement of monolayer graphene in the visible wavelength range. When the magnetic dipole modes are tuned to be overlapped spectrally by changing the diameters of Ag nanodisks, a broadband absorption enhancement is achieved. The position of the absorption band in monolayer graphene can be also controlled, by varying the thickness of the SiO2 spacer or the distance between the Ag nanodisks. The numerical results may have some potential applications in optoelectronic devices, such as photodetectors.In this work, we have numerically investigated multiband and broadband absorption enhancement of monolayer graphene at optical frequencies from multiple magnetic dipole resonances in metamaterials. The unit cell of the metamaterials consists of a graphene monolayer sandwiched between four Ag nanodisks with different diameters and a SiO


Idiopathic pulmonary fibrosis (IPF) is an inexorably progressive lung disease of unknown origin. Prognosis is poor, with limited treatment options available, and the median survival remains just 3–5 years. Despite the use of pirfenidone and nintedanib for the treatment of IPF, curative therapies remain elusive and mortality remains high. Regenerative medicine and the use of cell-based therapies has recently emerged as a potential option for various diseases. Promising results of preclinical studies using mesenchymal stem cells (MSCs) suggest that they may represent a potential therapeutic option for the treatment of chronic lung diseases including IPF. Encouraging results of Phase 1 studies of MSCs various have reduced safety concerns. Nonetheless, there is still a pressing need for exploratory biomarkers and interval end-points in the context of MSCs investigation. This review intends to summarize the current state of knowledge for stem cells in the experimental and clinical setting of IPF, present important safety and efficacy issues, highlight future challenges and address the need for large, multicenter clinical trials coupled with realistic end-points, including biomarkers, to assess treatment efficacy. Idiopathic Pulmonary Fibrosis (IPF) is a progressive debilitating lung disease of unknown etiology –4. The dCurrent evidence suggests that the areas of fibrosis seen in lungs of patients with IPF share many features with normal aging lung, such as genomic instability, telomere attrition, mitochondrial dysfunction, cellular senescence, and immune dysregulation , 15, 16.There are two approved compounds for the treatment of IPF: pirfenidone and nintedanib. Pirfenidone is an antifibrotic compound with an unclear mechanism of action targeting several molecules including transforming growth factor-β (TGF-β), tumor necrosis factor-α (TNF-α), and interleukin 6 . NintedaMSCs are multipotent cells able to differentiate into a number of different cell lines and exert immunomodulatory, anti-proliferative, and anti-inflammatory effects. Their multipotency, migratory ability, and immunoprivileged state has led to extensive research efforts for therapeutic applications in several diseases including cardiac ischemia –39, ischin vitro with minimal modifications. MSCs represent the most extensively studied stem cell population  and may be expanded pulation . Researcpulation , 55, 56.pulation .Recent studies on the pathophysiology of IPF suggest that early alveolar injury activates abnormal alveolar epithelial cells and stimulates the release of mediators including matrix metalloproteinases and TGF beta-1 , 58–61. 6 and 4 × 106 cells, in pathways associated with lung injury and pulmonary fibrosis and several end-points had been set  is a debilitating lung disease characterized by a progressive decline in lung function ultimately resulting in death. The lack of curative treatments for this disease has created an urgency for other potential therapeutic options. Preclinical studies suggest that because MSCs have immunomodulatory, anti-inflammatory, and potentially anti-fibrotic properties, they may be efficacious in the treatment of IPF. Early clinical trials have shown that MSCs may be safely administered to patients with IPF, but large multicenter randomized trials still need to be performed.AT, RT and TK wrote the initial manuscript. The manuscript was supervised and significantly modified by MG, AT, DB. KM, IN, VA offered significant intellectual contribution. All authors approved the final form of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


K+) ions on air-cleaved mica is important in many interfacial phenomena such as crystal growth, self-assembly and charge transfer on mica. However, due to experimental limitations to nondestructively probe single ions and ionic domains, their exact lateral organization is yet unknown. We show, by the use of graphene as an ultra-thin protective coating and scanning probe microscopies, that single potassium ions form ordered structures that are covered by an ice layer. The K+ ions prefer to minimize the number of nearest neighbour K+ ions by forming row-like structures as well as small domains. This trend is a result of repulsive ionic forces between adjacent ions, weakened due to screening by the surrounding water molecules. Using high resolution conductive atomic force microscopy maps, the local conductance of the graphene is measured, revealing a direct correlation between the K+ distribution and the structure of the ice layer. Our results shed light on the local distribution of ions on the air-cleaved mica, solving a long-standing enigma. They also provide a detailed understanding of charge transfer from the ionic domains towards graphene.The distribution of potassium ( KAl2O10(OH)2. The structure of mica consists of an octahedrally coordinated Al3+ sheet which is sandwiched between two tetrahedrally coordinated sheets of Al3+ and Si4+, see Si4+ by Al3+ in the tetrahedral sheet results in a negative charge. Interlayer potassium cations hold together these 2:1 layers and balance the negative charge of the mica crystal. After cleavage of the mica, half of the potassium ions are expected to remain on each of the two created surfaces in order to preserve charge balance. The atomic lattice periodicity of mica has been obtained by Atomic Force microscopy (AFM) and Friction Force Microscopy (FFM)24K+ ions. In many cases the large forces exerted on the surface result in significant damage and loss of lateral resolution.Muscovite mica has been the substrate of choice for a large number of scientific studies and technological applications owing to its atomically flat surface, high surface energy, low thermal and electrical conductivity and its perfect cleavage plane along the 0001) direction directioKAl2Al,SiO10(OH)2.et al.Although scanning probe techniques have been extensively used to study the surface of mica immersed in aqueous ionic solutions679K+ ions is unlikely due to the randomness of the cleaving process. Other studies suggest the formation of K+ islands, leaving thus ion-free mica regionsUnderstanding the complexity of the lateral organization of ions on air-cleaved mica surface is key in crystal growth and self-assembly of molecules on mica and justifiably has received intense scientific interestK+ distribution and the conductance of graphene. Immediately above the K+ ions, graphene is significantly less conductive than over ion-free locations. This result indicates charge transfer from the underlying surface to graphene and is in good agreement with previous studies152526Here we use graphene as an ultra thin coating for enabling the visualization of single ions and ionic clusters on the mica surface. Graphene has been shown to be able to accurately stabilize and conform to water layers13141516171819212223Ih-ice. A cartoon of the experimental system is shown in μm2), we are unable to determine the exact number of graphene layers. Nevertheless from the size of the observed fractals we do not expect the thickness to be larger than 4 graphene layersFreshly cleaved mica is reactive and immediately a thin water film is adsorbed and covers its entire surface14pm and 35 pm, respectively. The difference of the RMS roughness between the two regions is a result of the extra water layer at the environment of the ice crystals, which can smoothen the interface by filling atomic scale defects. The obtained RMS values are larger than those reported in earlier AFM studies14Small scale STM images, nN in order to avoid any irreversible damage of the surface. Lateral force images, nm. The protrusions unambiguously indicate the presence of K+ ions at the graphene-mica interface. Surprisingly, the ions appear to preferentially form rows and small clusters. A reference mica lattice can be reconstructed by FFT and overlayed with the locations of the K+ ions, Si4+ or Al3+. In agreement with our observations, Loh et al.K+ ions are stable  graphene-mica interface under low humidity conditions. Note that LFM can resolve atomic lattice periodicities of solid surfaces4able see , demonstK+ ions a histogram of nearest neighbours is extracted and reveals a preference for having 2 and 3 neighbours  is used. In C-AFM a conductive tip is maintained in mechanical contact with the surface while it measures the current generated by the applied bias voltage. K+ ions is found inside the fractal. The simultaneously recorded conductance map is given in 5c. As expected, the regions above the double layer of water are substantially less conductive than the graphene regions just above the ice crystals. Since the conductance depends on the local electron density, i.e. the density of states around the Fermi level, the doped graphene shows a higher conduction because of a higher density of states15In order to confirm the existence of an ice-like layer covering a nonuniform distribution of K+ rows (vacancies) exhibit higher electrical conductance as compared to graphene directly above the ions. The correlation becomes better visible by overlaying the two images, as shown in K+ ions is negatively charged, whereas the mica surface with K+ ions is neutral. At the positions where the K+ ions are located the negatively charged surface is induced by the polarized ice layer. However, at locations where the K+ ions are missing, the negatively charged surface is induced by the ice layer as well as the mica surface. Therefore the local electron density is higher and a higher conductance is measured at the locations were K+ ions are missing. The small overlap  due to repulsive ion-ion interactions by forming specific arrangements. The repulsive ion-ion forces are screened by the presence of ordered water layers. The ordered water/ice-K+ interface is studied by measuring the electronic properties of the graphene cover using conductive AFM. At locations where the water molecules arrange in a fractal-like crystalline structure, graphene has a higher electrical conductance due to the higher local electron density induced by charge transfer from the ice layer. High resolution conductance images reveal single ions and and show distinct variations of the conductance exactly above the ice layer. These variations show a direct correlation with the nonuniform distribution of K+ ions. The current results are of importance in crystal growth and self-assembly on mica surfaces and give new valuable information on charge transfer phenomena in the graphene cover.Using scanning probe microscopies in combination with graphene as an ultra thin cover, we have managed to obtain information on the local organization of ionic domains on air-cleaved mica surfaces. Lateral force images show that the N/m and resonance frequency of 30 kHz. The AFM scanner was placed inside an environmental glove box, in which the relative humidity (RH) could be controlled. The RH was tuned by an adjustable 2N flow and was measured using a humidity sensor , with an accuracy of 1.8% between 10–90% RH. Scanning tunneling microscopy (STM) was performed in an RHK Technology UHV3000 variable temperature STM operating at a base pressure of 1 × 10−10 mbar, using chemically etched W tips. All the STM images are aquired at room temperature. For the STM and C-AFM measurements the graphene flake was mechanically connected with a bigger graphite flake acting as an electrode.Graphene was obtained from highly oriented pyrolytic graphite  through the microexfoliation process and immediately deposited on a freshly cleaved mica surface . Optical Microscopy  and tapping mode Atomic Force Microscopy, AFM  were used to determine the thickness of grapheneHow to cite this article: Bampoulis, P. et al. Graphene Visualizes the Ion Distribution on Air-Cleaved Mica. Sci. Rep.7, 43451; doi: 10.1038/srep43451 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Shigella mainly resides in the use of a Type 3 Secretion System (T3SS) to inject several proteins inside the host cell. Three categories of proteins are hierarchically secreted: (1) the needle components (MxiH and MxiI), (2) the translocator proteins which form a pore (translocon) inside the host cell membrane, and (3) the effectors interfering with the host cell signaling pathways. In the absence of host cell contact, the T3SS is maintained in an “off” state by the presence of a tip complex. We have previously identified a gatekeeper protein, MxiC, which sequesters effectors inside the bacteria probably by interacting with MxiI, the inner‐rod component. Upon cell contact and translocon insertion, a signal is most likely transmitted from the top of the needle to the base, passing through the needle and allowing effectors release. However, the molecular mechanism underlying the transmission of the activation signal through the needle is still poorly understood. In this work, we investigate the role of MxiI in the activation of the T3SS by performing a mutational study. Interestingly we have shown that mutations of a single residue in MxiI (T82) induce an mxiC‐like phenotype and prevent the interaction with MxiC. Moreover, we have shown that the L26A mutation significantly reduces T3 secretion. The L26A mutation impairs the interaction between MxiI and Spa40, a keystone component of the switch between needle assembly and translocators secretion. The L26A mutation also sequesters MxiC. All these results highlight the crucial role of MxiI in regulating the secretion and transmitting the activation signal of the T3SS.The virulence of  This disease is responsible for more than 1 million deaths per year globally, essentially among children under 5 years of age in developing countries  to inject virulence proteins into eukaryotic cells , Salmonella (PrgJ), Pseudomonas (PscI), or Burkhoderia (BsaK). Recently, MxiI homologous protein, PrgJ, has been shown to interact with the cytoplasmic part of proteins composing the export apparatus in Salmonella  is composed of more than 20 proteins assembled into four parts: (1) a cytoplasmic part called the C‐ring, (2) an export apparatus localized in the inner‐membrane ring, (3) a basal body spanning the inner (IM) and outer (OM) membranes, and (4) an extracellular needle , an inner‐membrane protein, undergoes a conformational change following its autocleavage into two fragments, called Spa40CC and Spa40CN  secretion of effectors  highlights the role of the needle subunits  leading to an “effector mutant” phenotype, which is also rescued by the mxiC‐like phenotype. We have demonstrated here that some mutations of MxiI (on T82 residue) affect its interaction with MxiC and lead to exactly the same phenotype than the mxiC mutant. Moreover, the charge of the residue seems to play a key role in the secretion control. We have shown that the C‐terminal part of MxiI (74–93 residues), probably located inside the secretion channel, is sufficient for MxiC binding.In this study, we have undertaken a novel series of point and random mutations within MxiI to analyze its role in signal transmission aiming to find MxiI mutations that can lead to an 22.1Shigella flexneri strains were derivatives of the wild‐type strain M90T (serotype 5a)  were transformed with pET30a. Shigella were phenotypically selected on Congo red (CR) agar plates and grown in tryptic soy broth (VWR) at 37°C with the appropriate antibiotics at the following concentrations: zeocin 50 μg/ml, kanamycin 50 μg/ml, streptomycin 100 μg/ml, ampicillin 100 μg/ml, and chloramphenicol 25 μg/ml for E. coli strains and 3 μg/ml for Shigella strains.2.2mxiI), used to complement the mxiI mutant, was constructed by inserting a BamHI/XhoI digested PCR fragment, carrying native mxiI gene, into the BamHI/XhoI sites of the low copy vector pSU18 . Directed mutagenesis was carried out according to the procedure of the Quick Change Mutagenesis kit (Stratagene). The use of each primer in PCR creates a restriction site  containing 200 μg/ml CR, and incubating for 20 min at 37°C. Bacteria were centrifuged at 13,000 g for 15 min at RT and proteins present in the supernatant were analyzed by SDS‐PAGE. Western blotting was performed on polyvinylidene fluoride (PVDF) membranes  and developed using chemiluminiscence . Immunodetection was carried out as described by Botteaux et al.  was transformed with pGEX4T1 or its derivatives expressing, respectively, GST alone or GST fusion proteins and cultured in 100 ml of lysogeny broth (LB) at 37°C. Protein expression was induced with 0.1 mmol/L isopropyl β‐D‐1‐thiogalactopyranoside (IPTG) for 3 hr at 30°C. Bacteria were harvested, suspended in PBS, and then lyses by sonication in presence of 1% Triton X‐100. The lysates were then clarified by centrifugation and the supernatants mixed with glutathione‐Sepharose 4B matrix beads  previously equilibrated with PBS buffer during 1 hr at room temperature on a rotor shaker and then washed three times with PBS. Then the beads were incubated 16 hr at 4°C in a rotor shaker with cleared extract of E. coli strains (Rosetta DE3) expressing His‐tagged recombinant proteins. Beads were washed eight times and proteins eluted by incubating beads for 10 min with elution buffer . The eluted proteins were resolved by SDS‐PAGE and analyzed by Coomassie blue staining or Western blotting.2.52. Briefly, cells were seeded at 1 × 105 cells/well in 24‐well plates 24 hr prior infection. Shigella strains were grown at 37°C to mid‐log phase, washed once with PBS, and suspended in DMEM. Bacteria were then centrifuged onto plates (MOI of 100) at 2,000 g for 10 min and further incubated 45 min at 37°C. Infected cells were washed three times and incubated 1 hr with gentamicin (50 μg/ml). Finally, cells were lysed with PBS‐Triton 0.1% and intracellular bacteria were diluted and plated on TSB agar Petri dishes for colony‐forming units (cfu) counting. Hela cells invasion was defined as 100% for the wild‐type strain (M90T).Bacteria ability to invade HeLa cells was tested with a gentamicin protection assay  and grown at 37°C to mid‐log phase, washed once with PBS, and suspended at a concentration of 1,010 bacteria/ml. Horse red blood cells (Oxoid) were centrifuged at 2,000 g for 10 min at 4°C and washed twice with cold PBS. Then 50 μl of each sample was mixed in 96‐well flat bottom and centrifuged at 2,000 g for 10 min. After 1 hr incubation at 37°C, the reaction was stopped by the addition of 100 μl of cold PBS. Cells were suspended and further centrifuged at 2,000 g for 10 min. Optical density of the supernatant was measured at 540 nm. Red blood cells lysis was defined as 100% for the wild‐type strain (M90T).The contact‐mediated hemolysis assay was performed as previously described  in the same buffer. After serial dehydration samples were dried at critical point and coated with platinum by standard procedures. Observations were made in a Tecnai FEG ESEM QUANTA 200 (FEI) and images were processed by SIS iTEM (Olympus) software.Whole bacterial cells were applied to glow discharged carbon‐coated Formvar copper grids. Bacterial cells were negatively stained with 4% ForMol. Observations were done on a Tecnai 10 (FEI) microscope coupled to a Veleta charge‐coupled device (CCD) camera (Olympus iTEM), and images were captured and analyzed using SIS Olympus iTEM software. Whole bacterial cells were applied to glow discharged carbon‐coated Formvar copper grids and negatively stained with 4% Uranyl acetate. Observations were done on a Tecnai 10 (FEI) transmission electron microscope coupled to a Veleta CCD camera (Olympus iTEM), and images were captured and analyzed using SIS Olympus iTEM software. For SEM, samples were fixed overnight at 4°C in glutaraldehyde 2.5%, 0.1 mol/L cacodylate buffer (pH 7.2), and postfixed in OsO3mxiI which could result in an mxiC‐like mutant phenotype. As MxiI shares 18% of sequence identity with MxiH, which is also implicated in signal transmission, we first generated 8‐point mutations of conserved residues between these two proteins , and to invade HeLa cells. As shown in Table mxiI mutants show a phenotype similar to the wild‐type strain for colony color on CR plate, proteins secretion, hemolysis, and cell invasion, one presents exactly the same phenotype as the mxiI mutant (mxiIL63A), and another presents a global reduction in proteins secretion (mxiIL26A). Nevertheless, none of all the mutations led to a hyper‐red‐colony phenotype on CR plate suggesting that none of them is able to abolish the interaction between MxiI and MxiC.We decided to perform site‐directed mutagenesis within 3.1mxiIL26A strain  and YscI (Yersinia), play a role in substrate specificity switching and functional needles formation . In contrast to unmodified GST‐MxiI, GST‐MxiIL26A did not co‐elute His‐Spa40CT nor His‐Spa40CN (205–258)  even if produced at a similar level  were subsequently analyzed for their constitutive secretion phenotype. Two of them, harboring a mutation of the T82 residue, into a lysine (T82K) or an arginine (T82R), presented the same phenotype than the mxiC mutant as they constitutively secrete early and late effectors  and a GST‐MxiC , we thought that charge of the residue could influence its capacity to bind MxiC and to control secretion. To answer this question, we have replaced the T82 residue by a negatively charged (glutamate) and a hydrophobic nonpolar  residue on the pSM6. The proteins secreted by the different mxiI mutants (expressing MxiIT82E and MxiIT82A) under both constitutive and induced conditions were analyzed. Unlike mxiIT82K and mxiIT82R strains, mxiIT82E and mxiIT82A strains allow proteins secretion under induced and noninduced conditions like the wild‐type strain  or a positively charged one (mxiIQ67K). We found that the mxiIQ67E, like the mxiIQ67A, presents an “effector mutant” phenotype while the mxiIQ67K allows proteins secretion like the wild‐type strain  leads to a mxiC‐mutant phenotype in terms of early effectors secretion . To do so, we have first mutated conserved residues between MxiI and MxiH as the latter is also implicated in signal transmission  harboring an mxiC‐like phenotype (hyper‐red colonies). As expected these mutants were no longer able to interact with MxiC, supporting our initial hypothesis. Moreover, the loss of MxiC–MxiI interaction leads to an earlier secretion of MxiC (before induction) and explains why MxiC is no longer able to ensure its role in promoting translocators secretion in these backgrounds. Indeed, unlike the MxiCF206S variant, the MxiIT82K and MxiIT82R show the same defect in translocators secretion after CR induction than the mxiC mutant. Interestingly, the T82 residue is conserved with PrgJ, the MxiI homologue from Salmonella, which can interact with the MxiC counterpart, InvE  and shown that they are effectively sufficient to bind MxiC, supporting the conservation of the regulatory mechanism. As the C‐terminal part of MxiI harboring the T82 residue is not well conserved with MxiH  or negative (E) residues. Moreover, the charge seems to impact directly MxiC binding as MxiIT82K and MxiIT82R totally abolish MxiC binding. Thus, we can postulate that this deregulated phenotype is due to the loss of interaction between the MxiI variants and MxiC which results in MxiC early secretion. Interestingly, the change in negative charges on the surface‐exposed residues of MxiC by positive ones also leads to a deregulated secretion phenotype by an unexplained mechanism  and is rescued by mxiC inactivation, but the mxiHK69A needles are shorter than the wild‐type strain  that decrease, even abolish, translocators and effectors secretion. As we failed to detect the MxiI variants in Shigella background by Western blot, we cannot exclude a lack of expression in the mxiIL63A strain explaining the absence of the needle, especially given that the MxiIL63A counterpart in Yersinia (YscIL80A) is not expressed . Indeed Spa40CT is known to control substrate specificity switch between needle components and translocators secretion. Interestingly MxiIL26A is no longer able to interact with Spa40CT and Spa32 seems weakly secreted. So, as previously shown for MxiI homologous proteins (Marlovits et al., Salmonella strain‐expressing PrgJL29A but InvJ, the Spa32 counterpart in Salmonella, is secreted and the needle complexes are similar to wild‐type ones in this strain (Lefebre & Galán, In the course of our study, we also found two mutations within MxiI (MxiImxiI can lead to the same phenotype than the mxiC mutant by impairing their mutual interaction. The domain responsible for this interaction was also identified and its localization in a highly conserved domain within MxiI homologous proteins suggests that this mechanism is probably conserved among others T3SSs. Nevertheless, further structural and electrostatic studies of the inner rod would allow a better understanding of the mechanism of signal transmission through the T3SS needle.The results presented here strengthened our previous model in which the MxiC–MxiI complex regulates the effectors secretion. In fact, we have shown that point mutation in None declared. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.