Four patients suffering from advanced amyotrophic lateral sclerosis (ALS)—two of them in permanent CLIS and two entering the CLIS without reliable means of communication—learned to answer personal questions with known answers and open questions all requiring a “yes” or “no” thought using frontocentral oxygenation changes measured with fNIRS. Three patients completed more than 46 sessions spread over several weeks, and one patient (patient W) completed 20 sessions. Online fNIRS classification of personal questions with known answers and open questions using linear support vector machine (SVM) resulted in an above-chance-level correct response rate over 70%. Electroencephalographic oscillations and electrooculographic signals did not exceed the chance-level threshold for correct communication despite occasional differences between the physiological signals representing a “yes” or “no” response. However, electroencephalogram (EEG) changes in the theta-frequency band correlated with inferior communication performance, probably because of decreased vigilance and attention. If replicated with ALS patients in CLIS, these positive results could indicate the first step towards abolition of complete locked-in states, at least for ALS.Despite partial success, communication has remained impossible for persons suffering from complete motor paralysis but intact cognitive and emotional processing, a state called complete locked-in state (CLIS). Based on a motor learning theoretical context and on the failure of neuroelectric brain–computer interface (BCI) communication attempts in CLIS, we here report BCI communication using functional near-infrared spectroscopy —which measures brain hemodynamic responses associated with neuronal activity—could overcome this barrier. Four patients suffering from advanced amyotrophic lateral sclerosis (ALS), two of them in permanent completely locked-in state and two entering the completely locked-in state without reliable means of communication, learned to answer personal questions with known answers and open questions requiring a “yes” or “no” by using frontocentral oxygenation changes measured with fNIRS. These results are, potentially, the first step towards abolition of completely locked-in states, at least for patients with ALS.Despite scientific and technological advances, communication has remained impossible for persons suffering from complete motor paralysis but intact cognitive and emotional processing, a condition that is called completely locked-in state. Brain–computer interfaces based on neuroelectrical technology  have failed at providing patients in a completely locked-in state with means to communicate. Therefore, here we explored if a brain–computer interface based on functional near infrared spectroscopy ( Communication is the process of expressing and sharing feelings, thoughts, and intentions with one another by verbal and various nonverbal means. Communication skills appear automatic but can pose severe challenges to individuals suffering from motor neuron disorders. The most devastating of motor neuron diseases is amyotrophic lateral sclerosis (ALS) , which ifMRI) [fNIRS) [Brain–computer interface (BCI) represents a promising strategy to establish communication with paralyzed ALS patients –7, as itfMRI) , and fun [fNIRS) , to reco [fNIRS) . The fir [fNIRS) . With at [fNIRS) . Since t [fNIRS) ,16,17, s [fNIRS) –20, and  [fNIRS) –24, have [fNIRS) –27 for c [fNIRS) ,27–30. I [fNIRS) ,23, and In a meta-analysis of the scientific literature of all ALS patients in CLIS , it was Because none of the BCI techniques outlined above are able to provide viable means of communication , the patfNIRS to measure and classify cortical oxygenation and deoxygenation following the “yes” or “no” thinking of the CLIS patient in response to true or false questions, respectively. The report described a CLIS patient with ALS achieving BCI control and “yes” and “no” communication to simple questions with known positive answers or negative answers and some open questions over an extensive time period. Although it was not spontaneous and voluntary, controlled communication, it at least enabled the individual without any means of communication to transmit “yes” and “no” to questions framed by family members and/or caregivers. The result opened a venue to provide at least some means of communication to individuals in CLIS who are otherwise left communicationless. Hence, an extensive study was performed on four ALS patients in CLIS to train them to communicate “yes” and “no.” In the present study, which is the first of its kind, fNIRS-based BCI was used for binary communication in four ALS patients in CLIS. The fNIRS-based BCI was employed successfully to train patients to regulate their frontocentral brain regions in response to auditorily presented questions. After training a classifier separating “yes” from “no” answers for several days, the patients were given feedback of their affirmative or negative response to questions with known answers and open questions over weeks.A single case report by Gallegos-Ayala et al. (2014)  used fNI2Hb) during the “true/yes” and “false/no” sentences’ interstimuli interval (ISI)—which corresponds to patients’ response interval over the frontocentral brain region of patients F, G, B, and W—are shown in The relative change in oxygenated hemoglobin , shown in p > 0.05) between the true and false sentences’ ISI across each patient, as shown in t-test , as shown in 2Hb), EOG, and EEG power spectrum in response to true and false questions, obtained from the frontocentral region of the brain, across all the sessions from each patient was used to determine the SVM classification accuracy of "true/yes" and "false/no" answers. Successively, the daywise CA  of each patient was compared to the adjusted chance-level threshold (described in BCI effectiveness metric section), as shown in Figs 2Hb, EEG, and EOG signals for training sessions, while online CA is reported using only O2Hb for feedback and open question sessions because feedback was provided online only using the O2Hb signal. The answering concordance between semantically paired questions , expressed as the percentage of concordant answers over pairs' repetition, was as follows: F, 68%; G, 67%; B, 67%; W, 70%. Thus, the semantic concordance rate (SCR) ranges from 67 to 78% (see p < 0.0001), in which 50% is the SCR expectation of a random classifier.The support vector machine (SVM) classifier’s classification of true sentences as true, false sentences as false, true sentences as false, and false sentences as true was used to calculate the false positive rate (FPR) and true positive rate (TPR) for each training and feedback session and also for all the training sessions and feedback sessions separately for each patient. FPR and TPR was plotted to obtain the receiver operating characteristic (ROC) curve of the binary SVM classifier during training and feedback sessions for each patient, as shown in  78% see . Median t-test  suffering from juvenile ALS with an extremely rapid disease progression (2 y from diagnosis to CLIS) was not asked open questions because of the supposedly difficult emotional state at that early stage of BCI communication but continues to train the BCI at present.The results of ANOVA and post hoc p > 0.05). The middle-frequency bands’  spectral features comparison between sentence presentation interval and sentences’ ISI revealed some main effects of the intervals factor, as reported in p < 0.05). In patient W, a higher high-theta, low-alpha, and high-alpha bands’ mean power in the sentences’ ISI compared to the sentence presentation interval was found (ISI was synchronized compared to sentence presentation). Patient F did not show any significant difference in the middle-frequency bands’ mean power and “power variability” . See Results section of The patients showed the following stable dominant frequencies: F, 6.75 Hz; G, 6.25 Hz; B, 7 Hz; and W, 8 Hz. Power spectrum density of electroencephalographic signals corresponding to "true/yes" and "false/no" sentences’ ISI acquired from channel FC6 is shown in fNIRS classification accuracy and low-frequency bands’ (those more related to vigilance) mean power revealed some interesting results . However, in patient F, who had the longest time period in CLIS, the median of the positive averaged correlation between delta and high-theta band mean power and fNIRS classification accuracy was significantly different from zero . The low-frequency bands’ mean power distribution medians of successful and unsuccessful days  was further investigated for each patient to ascertain the difference, if any. In patient G, the low-theta band mean power of successful days was significantly smaller than that of unsuccessful days (p < 0.05). In patient B, the high-theta band mean power of successful days was significantly smaller than that of unsuccessful days (p < 0.05). This strengthens the above results of lower-frequency bands’ dominance for more unsuccessful performance. Additional details are provided in the Results section of The correlation analysis between fNIRS exceeded classification of EEG oscillations from 0–30 Hz and vertical and horizontal EOG classification. However, despite the absence of reliable eye communication in all patients as the inclusion criteria in the study and by the definition of CLIS condition, EOG classification was at some sessions, albeit rarely, above chance, mainly in patient W. Nonetheless, the inability of the social environment to perceive them and their instability and the eye tracker’s failure to use them for communication [Four patients in CLIS communicated with frontocentral cortical oxygenation-based BCI with an above-chance-level correct response rate over 70% during a period of several weeks. The performance of the binary SVM classifier across all the patients, except a few training sessions of patient B, was above chance level. None of the sessions were eliminated in the analysis, and only very few sessions had to be interrupted because of life-saving measures such as sucking saliva; thus, no bias for selecting “successful” sessions incriminates the results. Correct response rate for feedback and open questions sessions, as judged by the criteria mentioned in the Material and Methods section , exceeded 75% in three out of four patients . Patients F, G, and B answered open questions containing quality of life estimation repeatedly with a “yes” response, indicating a positive attitude towards the present situation and towards life in general, as reported in larger samples of ALS patients ,44. Repenication  preventsfNIRS responses to “yes” and “no” are different, as they are in three patients showing oxygenation increase during the answering interval (ISI) for “yes” responses with negligible topographical differences and oxygenation decrease during negative answers, again without topographical differences throughout the frontal cortical area. Only the very young patient W, with an extremely rapid course of the disease and a strong genetic variant, shows a variable fNIRS pattern with different slow oscillatory oxygenation changes between “yes” and “no” answering periods. In an animal study with nonhuman primates [The unreliable discrimination between "true/yes" and "false/no" sentences’ ISI by means of EEG signals  showed a negative averaged correlation between low-theta band mean power and accuracy , meaningFor a robust validation of the BCI binary communication system in CLIS, two main unsolved questions remain: (i) the physiological identification of the cognitive processes underlying the listening to “yes–no” questions and the answerer’s mental state and (ii) the online identification of decreased vigilance states that are detrimental (lowering performance) for BCI binary communication purposes, such as decreased alertness, drowsiness, and sleeping.fNIRS system and quantitative source analysis of the different frequency bands at different sites are necessary to clarify these questions. For the study reported here, only portable devices and a few EEG channels could be used in the interest of the bedside, home-based strategy selected. Thus, our interpretation of the EEG frequency bands’ variations remains speculative.Multielectrode EEG recordings used simultaneously with the fNIRS–BCI–system in a hybrid EEG–fNIRS–BCI, with the EEG allowing online corrections of excessive reduction of vigilance indicated by appearance of delta and low-theta periods. This new hybrid system should allow further improvement of communication in CLIS.In patients completely motionless over a period of years with restricted vision because of eye muscle paralysis and compromised vision because of drying and reduced or absent afferent input from the sensorimotor system, reduced vigilance measured with EEG and an irregular sleep–wake cycle was documented by Ramos et al. (2011)  and SoekThe results on four CLIS patients reported here allow the following conclusions:fNIRS–BCI [Even after extended CLIS in ALS spanning months and years, reliable, meaningful communication using questions requiring a mental affirmative (“yes”) or negative, rejecting (“no”) answer is possible with NIRS–BCI ,56. ThisNIRS–BCI  calculatNIRS–BCI . BecausefNIRS seems to provide better classification of patients’ answers compared to oscillatory EEG responses. The above statement of a superiority of fNIRS over EEG–BCI needs a word of caution. The EEG oscillations were analyzed using average power over the 15 s answering period, neglecting the temporal dynamics of the EEG across the answering period. Thus, any difference in the beta and/or gamma frequency range (less so in the low frequencies) that may occur at different time points of the answering period is ignored with this type of averaging. Replications of the comparisons reported here between fNIRS–BCI and EEG–BCI in CLIS should employ a more complex time-frequency analysis of the EEG signals during the course of the answering period. This kind of EEG temporal dynamics analysis should investigate, at the single-trial level, relevant frequency changes at different time delays of the mental and neurophysiological signatures of the answer. With the limited number of EEG channels and the employed bandpass filter because of the home-based BCI limitations, the covert nature of the dynamics of the mental answer, and the obvious pathology of the EEG in these patients, we had to abstain from further complex analysis. In the present study, only a preliminary EEG temporal dynamics analysis based on averaged data of each session for each patient was viable . However, because the time-frequency analysis was performed at session level , caution is necessary in the interpretation of the results. Similar difficulties encountered using different techniques are described in a previous study of patient F and two LIS ALS patients , albeit a slightly different semantic conditioning paradigm employing electrical stimulation was used. Compared to fNIRS, the EEG time-frequency analysis may identify specific neuroelectric frequencies’ oscillations at different time points with fine resolutions and with potential further insights in neurophysiological mechanisms underlying the engaged mental processes. Conversely, fNIRS signals may be more easily detected for binary classification. We have argued previously [fMRI neurofeedback experiments because of an existing feedback pathway between the vascular system in the central nervous system. While neuroelectric changes lack receptors systems of their own activity, the vascular bed provides accurate information of flow and diameter changes to the neuronal assemblies in its neighborhood. This allows adjustment to metabolic and cognitive needs and probably superior access to voluntary control [fMRI and fNIRS provides feedback to the brain about its state changes, while the neuronal neuroelectric changes may not be “perceived” by the brain and thus provide no feedback of its present state or state changes; thus, a physiological system should be superior for instrumental learning. To put it more colloquially: physiological feedback of our thoughts (“perceiving thoughts”) is encoded in the neuronal control structures of the brain through the vascular systems and not through neuromagnetic changes such as cellular membrane polarization changes and changes in neuroplastic synapses. While thoughts probably consist of neuroelectric changes and their underlying cellular polarization across the cell membranes, their physiological consequences or correlates may appear as metabolic. Thus, coregistration of fNIRS and EEG and other neuroelectric changes and oscillations from multiple sites maybe important in CLIS in order to monitor and eventually modify nonfavorable vigilance changes reflected in the EEG, with arousal stimulating activities to improve NIRS classification.eviously  the spec control , as requThe Internal Review Board of the medical faculty of the University of Tubingen approved the experiment reported in this study, and the patients’ legal representative gave informed consent for the study with permission to publish the results and show the face of patients in the publication. The study was in full compliance with the ethical practice of medical faculty of the University of Tubingen. The clinical trial registration number is ClinicalTrials.gov Identifier: NCT02980380. At the time of this study, prospective clinical trial registration was not mandatory for nonpharmacological studies; it was therefore registered retrospectively.fNIRS system, NIRSPORT (NIRX), which performs dual-wavelength (760 nm and 850 nm) CW near-infrared spectroscopic measurement at a sampling rate of 6.25 Hz, as shown in in A continuous wave (CW)-based Patient F  was diagnosed with bulbar sporadic ALS in May 2007, was diagnosed as locked-in in 2009, and was diagnosed as completely locked-in May 2010, based on the diagnoses of experienced neurologists. She has been artificially ventilated since September 2007, fed through a percutaneous endoscopic gastrostomy tube since October 2007, and is in home care. No communication with eye movements, other muscles, or assistive communication devices was possible since 2010. Further details of this patient are described in Gallegos-Ayala et al. (2014) .Patient G  was diagnosed with bulbar ALS in 2010. She lost speech and capability to walk by 2011. She has been fed through a percutaneous endoscopic gastrostomy tube since September 2011, artificially ventilated since March 2012, and is in home care. She started using assistive communication devices employing one finger for communication in February 2013. Later, she was diagnosed with degeneration of vision because of cornea defects in September 2013. After the failure of the finger-communication device, an attempt was made to communicate using eye tracking in early 2014. She stopped communicating with the eye in August 2014, before the BCI was introduced, and an attempt was made to communicate with the subtle twitch of an eye lid, which was not reliable. The husband and caretakers declared no communication with her since August 2014.Patient B  was diagnosed with nonbulbar ALS in May 2011. He has been artificially ventilated since August 2011, fed through a percutaneous endoscopic gastrostomy tube since October 2011, and is in home care. He started communicating with a speech device in his throat from December 2011, which ultimately failed, and he started using the MyTobii eye-tracking device in April 2012. He was able to communicate with MyTobii until December 2013, after which the family members attempted to communicate by training him to move his eyes to the right to answer “yes” and to the left to answer “no,” but the response was variable. No communication was possible since August 2014.Patient W  was diagnosed with juvenile ALS in December 2012. She was completely paralyzed within half a year after diagnosis and has been artificially ventilated since March 2013, fed through a percutaneous endoscopic gastrostomy tube since April 2013, and is in home care. She was able to communicate with eye tracking from early 2013 to August 2014 but was unable to use the eye-tracking device after the loss of eye control in August 2014. After August 2014, family members were able to communicate with her by training her to move her eyes to the right to answer “yes” and to the left to answer “no” questions until December 2014. In January 2015, eye control was completely lost, she tried to answer yes by twitching the right corner of her mouth, that too varied considerably, and parents lost reliable communication contact.All four patients reported in this manuscript were enrolled consecutively. Patients’ family approached us to get enrolled in the study because of the past work and public appearance of the corresponding author. Patients were never screened and excluded for this study. The only criterion for the inclusion in this study was that the patient should be in completely locked-in state (CLIS) or on the verge of CLIS, and family members could not communicate with eye movements or any other response with the patient. The CLIS state was then verified with confirmation of the attending neurologist, EOG recordings, and video recordings of the families’ failures to achieve contact with the patient.fNIRS and EEG data during BCI sessions is shown in The schematic depicting the experimental procedure, acquisition, and analysis of An auditory paradigm was employed to (a) train patients on questions with known answers, termed as training sessions; (b) give feedback on questions with known answers, termed as feedback sessions ; and (c) answer open questions, termed as open question sessions (“You have back pain”). Known questions are personal questions based on patient’s biography. For every known question with a clear “yes” answer, a semantically related question with a clear “no” answer was constructed and vice versa; for example, “You were born in Berlin” and “You were born in Paris.” Patients were asked to think “yes” or “no” answers and, if possible, also to use their previously successful eye movements. They were explicitly instructed not to imagine the answer or visually or auditorily imagine the word  “yes” or “no”. Open questions are general questions related to quality of life and questions of caretakers whose answers can only be known by the patient. A total of at least 200 known questions and 40 open questions were constructed for each patient with family members before the initiation of the BCI study. Each patient was visited for 4 to 5 d in a month, except patient W. Three to four sessions were performed each day depending upon the health condition reported by the caretakers of the patient. Every session lasted for 9 min, and a session in progress was terminated extremely rarely . In such a rare event, the session was started again. Since each session lasted for 9 min, the caretaker or the family member was always instructed to take care of the needs of the patient before the start of the session, and the session was always started with the permission of the caretaker or the family member. A session, once in progress, was never terminated for patients F, G, and W. For patient B, a session was terminated while in progress three times because of removal of saliva, and the data were not included in any kind of analysis.fNIRS feature necessary to differentiate between “yes” and “no” answers during ISI was extracted and classified. Only training sessions were performed during the first few days, and upon several successful training sessions (as described below in BCI effectiveness metric section), the online feedback session was performed. During training sessions, both the patient and the algorithm were trained. Patients learned to mentally answer the question, and the algorithm learned to classify the “yes” and “no” fNIRS pattern of a particular patient. This kind of “mutual learning” seems important to optimize the “yes” versus “no” classification outcome and to customize and/or adapt the BCI system to each individual patient. At the end of each training session with 20 sentences (questions), patients were told the average classification accuracy of the session  to motivate and help patients in learning. In the course of an online feedback session, patients were presented the known questions as described above, but now at the end of the 15 s ISI they were given auditory feedback of accuracy, during which the computer said, “Your answer was recognized as yes” or “Your answer was recognized as no” depending upon the question . Feedback to strengthen the conditioned response was provided only if the classification accuracy was greater than the chance-level upper limit to guide the conditioned learning toward meaningful answers and to avoid frustration by negative feedback already at the beginning of a daily session. Feedback was driven by the fNIRS classifier, calculated using the data acquired during the training sessions. After successful training and feedback sessions, the patients were presented with open questions, during which they were always given the auditory feedback of their answer.Acoustically presented instructions about the procedure were given repetitively before each training, feedback, and open questions sessions, allowing patients to recall and consolidate the required task to listen and answer mentally. Each BCI session started with training sessions, during which the patients were instructed to listen to 20 personal questions (with known answers) consisting of 10 true and 10 semantically equivalent false sentences. The sentences were presented randomly in such a way that two semantically related questions never played one after another. Family members were always present throughout the BCI session, and they never prompted the patient to answer the question. Complete pin-drop silence was maintained during the session, and only the recorded sentences were presented via audio presentation software connected to sound box with the voice of a family member or caregiver. Patients were asked to think “ja, ja…” (German for “yes”) and “nein, nein…” (German for “no”) for 15 s during the ISI until they heard the next sentence after an interval of 5 s of rest, as shown in The validity of answers to open questions can only be estimated by (a) face validity ; (b) stability over time; (c) external validity, estimated by family members and caretakers; and (d) internal validity between questions . The binary BCI system effectiveness and robustness depends on its capability of correctly classifying the neurophysiological correlates of “yes” and “no” answers to true and false questions. The proposed true and false questions have two possible outcomes only, which are equally distributed with a probability of 0.5. To ensure that the classification of “yes” and “no” answers is not at chance-level, a reliable metric has to be used. Based on binomial distribution theoretical background, Müller-Putz et al.  defined fNIRS data was acquired online throughout all the sessions, namely training, online feedback, and open question sessions. The fNIRS data acquired online was normalized, filtered using different bandpass filters (0.0016–0.3), (0.01–0.3) and (0.02–0.3) and processed using modified Beer–Lambert law [2Hb) and deoxyhemoglobin (RHb). The choice of bandpass filter had no effect on the waveforms of signal. The relative change in O2Hb computed online during each session was used to train a SVM classifier model. The mean of relative change in O2Hb across each channel was used as a feature to train the SVM model through a 5-fold cross-validation procedure. In this study, only the relative change in O2Hb was used, as after the end of sessions with known answers it was observed that O2Hb provided stable and higher cross-validation classification accuracy than RHb. In an invasive animal study with nonhuman primates, we have also measured a superior covariation of oxygenation changes compared to deoxygenation, with intracortically recorded neural activity [2Hb, the SVM model generated using O2Hb was used to provide online feedback for known as well as open questions sessions. If the classification accuracies for at least three consecutive “training” sessions with questions with known answers were greater than the chance-level threshold, a new model was generated using the relative change in O2Hb across three training sessions to give online feedback. During an online feedback session, fNIRS data acquired online corresponding to each ISI was processed to obtain the relative change in O2Hb, as described above, across all the channels. The mean of the relative change in O2Hb across all the channels was used as test feature to map onto model space. Upon mapping of this test feature onto the model space, the SVM predicted  the side of the hyperplane the test feature fell on. Depending on the value of the predict label, appropriate feedback was provided to the patient: if the predict label was 0, the patient was given feedback that his or her answer was recognized as “no,” and if the predict label was 1, the patient was given feedback that his or her answer was recognized as “yes.”The bert law ,61 to caactivity  supportifNIRS provides three different signals: oxyhemoglobin (O2Hb), deoxyhemoglobin (RHb) and total hemoglobin (THb) [2Hb, only the results from the offline processing of O2Hb data will be shown along with the EEG and EOG data. The relative change in O2Hb, EEG, and EOG data were processed offline to determine:in (THb) ,61. As ma)2Hb, EEG, and EOG) during the ISI of true (yes) and false (no) sentences (in the time domain).The statistical difference in the particular physiological signal  across different channels in a session.To ascertain the difference between the averaged ISI of true and false sentences, b)2Hb, the EOG signal, and the EEG signal power spectrum during ISI corresponding to true and false sentences.The statistical difference in the offline classification accuracy of the relative change in Ot-test were used.For the EEG, frequencies between 0 and 30 Hz, estimated by Welch’s method , were usc)fNIRS SVM classification accuracy.The statistical difference of frequency bands’  features averaged daily-wise from EEG signals and their relationships with fNIRS classification accuracy was computed to find relevant relationships of low EEG rhythms with the BCI experimental procedure outcome. Details are provided in the Methods section of Frequency bands’ mean power and their “variability” were estimated using Welch’s method ,58. For The performance of the binary SVM classifier was ascertained by plotting the ROC curve. The ROC curve was created by plotting the TPR against the FPR (obtained from the contingency table created for each session) and the average of all the sessions, separately for each patient, using the four possible outcomes of a binary SVM classifier. The formation of contingency table for training and feedback sessions for each participant is described in the Receiver operating characteristic curve section of Semantic concordance rate (SCR) was calculated to ascertain the consistency and/or concordance of the answers between semantically equivalent but contrasting true and false sentences requiring “yes” and “no” answers, respectively. SCR  was calculated for all semantically related sentences presented to each patient. The method employed to calculate the semantic concordance rate is described in the Semantic concordance rate (SCR) section of S1 TREND Checklist(PDF)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.S2 Text(DOCX)Click here for additional data file.S1 TableSection A. Lists the daily-wise EEG frequency domain results of each patient. The number of days for each patient were: F, 14; G, 17; B, 12; and W, 6. Middle-frequency bands’ mean power and their “variability” were compared between sentence presentation interval and sentence’s ISI. See p-value. Section B. Enlist the averaged correlation between daily-wise EEG frequency bands’ mean power and fNIRS CA for each patient. The averaged correlation was computed across selected intervals  and across all electrodes. Column 4 lists the mean and standard deviation of each averaged correlation across intervals and electrodes with CA. Column 5 lists the p-values of the tested null hypothesis of whether the median of averaged correlation was zero. Further, the number of days of each patient was split in successful and unsuccessful days using the chance-level threshold. Then, the hypothesis of whether the particular band mean power distribution medians of successful and unsuccessful days differed was tested . The effect of this comparison  is reported in column 7. “Rest” stands for resting interval before sessions. “ISI” stands for interstimuli interval. “SP” stands for sentence presentation interval. “Succ.” and “Unsucc.” stand for successful and unsuccessful days, respectively. The symbol ≈ means that the band mean power medians of successful and unsuccessful days were similar. The symbol < means that the band mean power medians of successful and unsuccessful days differed significantly. The symbol * denotes a significant p-value. See fNIRS classification accuracy for details. S1 Table data is located at https://doi.org/10.5281/zenodo.191929.(XLSX)Click here for additional data file.S2 Tablehttps://doi.org/10.5281/zenodo.192128.S2 Table data is located at (XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Tablehttps://doi.org/10.5281/zenodo.192398.Contingency table formed using the average of all the training sessions. S4 Table data is located at (XLSX)Click here for additional data file.S5 Tablehttps://doi.org/10.5281/zenodo.192400.Contingency table formed using the average of all the feedback sessions. S5 Table data is located at (XLSX)Click here for additional data file.S6 Tablehttps://doi.org/10.5281/zenodo.192401.Contingency table formed using the average of all the training sessions. S6 Table data is located at (XLSX)Click here for additional data file.S7 Tablehttps://doi.org/10.5281/zenodo.192402.Contingency table formed using the average of all the feedback sessions. S7 Table data is located at (XLSX)Click here for additional data file.S8 Tablehttps://doi.org/10.5281/zenodo.192406.Contingency table formed using the average of all the training sessions. S8 Table data is located at (XLSX)Click here for additional data file.S9 Tablehttps://doi.org/10.5281/zenodo.192407.Contingency table formed using the average of all the feedback sessions. S9 Table data is located at (XLSX)Click here for additional data file.S10 Tablehttps://doi.org/10.5281/zenodo.192408.Contingency table formed using the average of all the training sessions. S10 Table data is located at (XLSX)Click here for additional data file.S11 Tablehttps://doi.org/10.5281/zenodo.192409.Contingency table formed using the average of all the feedback sessions. S11 Table data is located at (XLSX)Click here for additional data file.S12 Tablehttps://doi.org/10.5281/zenodo.191982.S12 Table data is located at (XLSX)Click here for additional data file.S1 FigA. Power spectrum density. Power spectrum density (PSD) of electroencephalographic (EEG) signal corresponding to YES (red solid trace) and NO (blue dashed trace) sentences’ ISI acquired from channel FC6 in patients F, G, B, and W. In each subplot, the x-axis is frequency in hertz and the y-axis is channel FC6 EEG in dB (μV2/Hz). S1A Fig data is located at https://doi.org/10.5281/zenodo.192386; https://doi.org/10.5281/zenodo.192388; https://doi.org/10.5281/zenodo.192390; https://doi.org/10.5281/zenodo.192391. B. Electrooculogram signal. The electrooculogram (EOG) signal corresponding to YES (red solid trace) and NO (blue dashed trace) sentences’ ISI in patients F, G, B, and W. In each subplot, the x-axis is time in seconds and the y-axis is EOG in micro volt (μV). S1B Fig data is located at https://doi.org/10.5281/zenodo.192386; https://doi.org/10.5281/zenodo.192388; https://doi.org/10.5281/zenodo.192390; https://doi.org/10.5281/zenodo.192391.(EPS)Click here for additional data file.S2 FigReceiver operating characteristic (ROC) curve of the binary support vector machine (SVM) classifier. (A) Training and (B) feedback sessions. Each circle in the ROC curve space represents false positive rate (FPR) versus true positive rate (TPR) for each session. Sessions with the same coordinate points in the ROC space are represented by concentric circles. The red star along with the coordinate points in the ROC space represent FPR versus TPR of all the sessions combined. In the figure panels A and B, the x-axis is the FPR and the y-axis is TPR. The thick diagonal line dividing the ROC space represents chance level. Points above the diagonal represent good classification results (better than random); points below the line represent poor classification results (worse than random). S2 Fig data is located at https://doi.org/10.5281/zenodo.192398; https://doi.org/10.5281/zenodo.192400.(EPS)Click here for additional data file.S3 FigReceiver operating characteristic (ROC) curve of the binary support vector machine (SVM) classifier. The description of this figure is the same as described in https://doi.org/10.5281/zenodo.192401; https://doi.org/10.5281/zenodo.192402.(EPS)Click here for additional data file.S4 FigReceiver operating characteristic (ROC) curve of the binary support vector machine (SVM) classifier. The description of this figure is the same as described in https://doi.org/10.5281/zenodo.192406; https://doi.org/10.5281/zenodo.192407.(EPS)Click here for additional data file.S5 FigReceiver operating characteristic (ROC) curve of the binary support vector machine (SVM) classifier. The description of this figure is the same as described in https://doi.org/10.5281/zenodo.192408; https://doi.org/10.5281/zenodo.192409.(EPS)Click here for additional data file.


Staphylococcus aureus (MSSA) isolates can indicate the emergence of novel sequence types (ST). The diffusion of MSSA ST can be tracked by combining established genotypic profiles with phenotypic surveillance data. ST398 emerged in New York City (NYC) and exhibits resistance to clindamycin and erythromycin but tetracycline susceptibility . Trends of potential ST398 were examined in a national cohort of all Veterans Health Administration patients with MSSA invasive infections during 2003–2014.Changing phenotypic profiles of methicillin-susceptible A retrospective cohort of all patients with MSSA invasive infections, defined as a positive clinical culture from a sterile site, during 2003–2014 was created. Only isolates tested against clindamycin, erythromycin and tetracycline were included. Annual hospital-level proportions of potential ST398 were compared according to facility distance from NYC and region.p < 0.01) reflect increases in potential ST398 over time in hospitals nearer to NYC.A total of 34,025 patient isolates from 136 VA medical centers met the inclusion criteria. Of those, 4582 (13.5%) met the definition of potential ST398. Potential ST398 increased over the 12-year cohort and diffused outwards from NYC. Incidence Rate Ratios of >1.0 (S. aureus infections.We observe an increase in the phenotypic profile of potential ST398 MSSA isolates in invasive infections in a national cohort of patients in the US. The increase is not evenly distributed across the US but appears to diffuse outwards from NYC. Novel MSSA strain emergence may have important clinical implications, particularly for the use of clindamycin for suspected  Staphylococcus aureus (SA), either methicillin-resistant (MRSA) or methicillin-sensitive (MSSA), has implications for the availability and efficacy of empiric therapy options. Detecting and tracking such an emergence is difficult, however, given the lack of nationwide genotyping databases. Isolates collected in routine clinical care are seldom genotyped and retained in an isolate bank for future analysis. As a result, analysis of phenotypic proxies for genotypes, based on susceptibility panels completed during routine clinical treatment, provide a method for hypothesis development regarding the emergence of novel strains. A change in SA susceptibility patterns would suggest changes in the underlying genotypic strains that are circulating and provide impetus for funding large-scale directed genotypic studies.The emergence of novel genotypes of When genotyping studies for SA have been completed, they have overwhelmingly focused on MRSA . Yet eveA newly emerging strain that has been detected both phenotypically and genotypically in the US is sequence type 398 (ST398). ST398 MRSA is of human origin but is more often reported in the literature as having an association with livestock exposure –18. ST39ST398 MSSA presents a case study for how phenotypic analysis can be used to document the potential diffusion over space and time of an emerging SA strain type. In systematic surveillance of NYC hospitals for MSSA ST398, Uhlemann et al. indicated that the majority of detected isolates shared a resistance profile: 100% (64/64) were tetracycline-susceptible and 97% (62/64) were clindamycin-resistant and erythromycin-resistant . In contpotential ST398, was present in and diffusing among veterans across the US. The emergence and diffusion of this novel MSSA phenotype, with resistance to frequently used antimicrobials, has important clinical implications for empiric therapy in US healthcare settings.The Veterans Health Administration (VHA) represents the only nationwide healthcare provider in the US, with standardized procedures for testing and reporting of antibiotic susceptibilities. This study makes use of this singular large national database to evaluate a possible emergent phenotypic profile over space and time. Using susceptibility profiles from patients with MSSA invasive clinical isolates, we sought to examine spatial and temporal trends in tetracycline-susceptible and clindamycin-resistant and erythromycin-resistant MSSA bacteria to determine whether this phenotype, which we designate as A retrospective cohort of all patients from VHA facilities in the contiguous US with MSSA invasive infections during 2003–2014 was created. An invasive infection was defined as a positive clinical culture from a sterile body site such as blood or synovial fluid; specimens from non-sterile sources such as urine, sputum and wounds were excluded. Only those MSSA isolates that were tested against all three classes of antibiotics  were included in the analysis. All microbiology laboratories within the VHA system are required to follow guidelines recommended by Clinical Laboratory Standards  . The numThis study used medical, microbiological, and demographic data extracted from the Veterans Affairs’ (VA) electronic health record for the VA’s Corporate Data Warehouse and accessed through the VA Informatics and Computing Infrastructure, as previously described . The insThe distance of each VAMC from NYC was calculated in kilometers using ArcMap 10.2  Fig. . FacilitA Poisson generalized estimating equation (GEE) regression model, with counts of potential ST398 in each VAMC in each two-year period as the outcome variable, explored the relationship between phenotypically potential ST398 and distance from NYC over time while accounting for repeat observations in hospitals over time. Because the majority of VAMCs had zero isolates matching the phenotypic profile in one or more years, an overdispersed model was fitted. Total invasive MSSA infections were included as an offset variable. Distance was included as a categorical variable, with hospitals grouped at <50 km from NYC and then in 1000 km increments. An interaction term between year and distance category was included in the model to detect regional variability in temporal trends, and incidence rate ratios (IRRs) for temporal trends for each distance category were calculated based on the model. Category-specific IRRs were also calculated for each distance category with hospitals >4000 km from NYC as the reference category to detect regional variability in baseline prevalence rates. Model diagnostics, including residual plots and dispersion measures, were examined to assess appropriateness of the model. Statistical analysis was completed in SAS version 9.4 .In 2003–2014, 47,513 patients from 136 VAMCs had sterile-site cultures positive for MSSA. Of these, 34,025 (72.2%) were tested against all three antibiotics. The number of sterile site MSSA isolates tested against all three antibiotics varied over time, rising from 62.2% in 2003 to 76.05% in 2014. Among the included sterile site MSSA isolates, 4582 (13.5%) exhibited clindamycin resistance and erythromycin resistance with tetracycline susceptibility and were potentially ST398. The proportion of isolates with this phenotypic profile increased over time, from a low of 5.7% in 2003 to a high of 16.1% in 2010, falling to 14.9% in 2014  in NYC VAMCs  and the area closely surrounding NYC (50-999 km)  when compared to those facilities furthest from NYC (Table p < 0.01).Results of the Poisson GEE regression indicated that, at the facility level, there was a significantly higher proportion of potential ST398 among MSSA isolates for hospitals in NYC  guidelines, such as for testing inducible clindamycin resistance, we do not have information on adherence in individual facilities. Finally, the VHA patient population, comprised of predominantly older white males, is not representative of the US population as a whole.We cannot definitively conclude the observed change in phenotypic profiles from 2003 to 2014 in the VHA MSSA cohort is due solely to ST398. However, there is circumstantial evidence that ST398 MSSA is emerging based on similarities to genotyped isolates in the literature and a similar association with distance from NYC, the putative epicenter of ST398 MSSA in the US. Within the VHA data, we document higher prevalence of isolates with a specific phenotypic profile in NYC hospitals in 2003–2004 with lower prevalence at greater distances from NYC. This pattern changed over the years of the study as overall potential ST398 increased in facilities further from NYC.In contrast to MRSA, seldom are nationally representative sets of MSSA isolates stored and genotyped. Until a nationwide data bank of isolates becomes available for genotypic evaluation, phenotypic surveillance of novel type emergence, followed by genotypic analysis in local healthcare settings, will have to suffice. Such phenotypically informed and directed genotypic analysis is more efficient and cost-effective than efforts to genotype all clinical isolates.Based upon a retrospective analysis of MSSA susceptibility data, our findings document the emergence and diffusion of a changing MSSA susceptibility pattern, suggestive of potential ST398, which occurred within the VHA system between 2003 and 2014. The diffusion of a novel MSSA genotype that is resistant to commonly-prescribed antimicrobials, and that is potentially more fit and transmissible than other genotypes, warrants attention as it can limit the options for empiric antimicrobial therapy in patients with suspected SA infections. Phenotypic profiles generated during routine clinical care can be used to evaluate hypotheses about changing genotypic patterns and to target isolates for detailed genotyping by standard methods such as spa typing, pulsed-field gel electrophoresis (PFGE) or even whole genome sequencing. Finally, these findings also suggest that establishing a nationwide surveillance system with banked isolates, regardless of their resistance to methicillin and other beta-lactams, could have significant epidemiologic and clinical utility.


In this study we used a counterfactual approach to disentangle the effect of two closed areas on fishing location from other competing effects on the behaviour of the Indian Ocean tuna purse seine fishery. Our results revealed an inconsistent effect of the one of the closed areas between years, after taking into account the influence of environmental conditions on fleet behaviour. This suggests that the policy of closing the area per se was not the main driver for the fleet allocating its effort elsewhere. We also showed a marked difference in effect between the two closed areas resulting from their different locations in the fishery area. These findings highlight the need to account for other key fleet behavioural drivers when predicting or evaluating the contribution of area closures to achieving conservation and fishery management objectives.Spatial closures are widely used in marine conservation and fisheries management and it is important to understand their contribution to achieving management objectives. Many previous evaluations of closed area effects have used before-after comparisons, which, without controlling for a full range of factors, cannot ascribe changes in fleet behaviour to area closures  The use of area closures is commonplace in addressing problems of sustainability in fisheries management –3. ClosuIn the management of industrial tuna fisheries, area closures are most commonly used to reduce fishing mortality, either overall or for a particular component of the stock such as small or juvenile fish ,5. Shortper se. Policy change is only one of many factors that can cause or contribute to an observed management impact. Environment, economic and social forces also influence how fishing fleets behave. In offshore tuna fisheries, the physical environment is particularly important in determining the movement of target tuna in space and time, and safety and cost considerations play an important role in determining a fisher’s decision to visit a particular area. These forces, which are usually independent of any management action, may disrupt the intended effect of management. Thus, without accounting for these different effects and isolating the effect of policy, managers risk misinterpreting the relationship between an action and an outcome.This approach is helpful in acknowledging change but does not reveal the full picture of how a management action has performed. For instance, while a before-after analysis can identify whether a change in total catch has occurred, it cannot ascribe changes in catch to the use of an area closure http://ec.europa.eu/smart-regulation/guidelines/toc_guide_en.htm; accessed 27th February 2017) it has not been widely adopted for evaluation of fishery management interventions. Here, a counterfactual approach can be achieved by developing a predictive model of fishing effort allocation that accounts for a range of influences on fleet movement. By comparing the observed response of a fleet to a closure to predictions of how the fleet would have behaved if the closure had not been implemented it is possible to isolate the effect of closures on fishery dynamics. An interesting use of the counterfactual approach in a manner broadly similar to this is demonstrated by [A promising approach for diagnosing the reasons behind an observed change in a management indicator is to build up a counterfactual scenario. Counterfactual thinking considers what outcomes would have looked like in the absence of an intervention, yet while it is a common feature in some areas of policy impact evaluation  one-month fishery management closure off the Somalia coast, and the British Indian Ocean Territory (BIOT) marine reserve. We aimed to understand whether the policy of closing these areas Understanding the response of the purse seine fleet to the two area closures selected in this study is of interest for different reasons. In the case of the fishery closure, which was intended to temporarily reduce the fishing capacity of the purse seine fleet, it has been suggested that vessels may have reallocated effort in such a way as to maintain catches and therefore subvert the management outcome . In the http://www.iotc.org/English/resolutions.php; accessed 1st June 2013).The two closures examined differed in their size, location and intended management objectives. In November 2011 and 2012 the Indian Ocean Tuna Commission (IOTC) designated a one-month closure that extended from the Somali coast to 60°E and covered a large part of the productive northwest fishing grounds typically fished by the fleet during August-November. This region is a key fishing area for the purse seine fleet and is characterised by consistently very high catches, mainly around floating objects . The objIn late 2010 the British government designated their entire British Indian Ocean Territory a marine reserve. The BIOT reserve is positioned at the eastern periphery of the purse seine fishery area in a region typically fished during November-February and characterised by catches of free-swimming schools. Prior to the closure of BIOT, the importance of the area in terms of purse seine fishery production varied considerably from year to year, with the average proportion of total monthly catch taken within the Territory ranging from 0 to 23% . The designation of the BIOT reserve was not linked to regional fisheries management policy and whilst no formal management plan has yet been developed, several objectives of the reserve can be inferred, including the provision of a scientific reference site and near shore and pelagic biodiversity conservation .For the IOTC closure we focussed on the behaviour of the fleet during November, the month of the closure, in 2011 and 2012. For the BIOT reserve we focussed on the period December-January when the provision of fishing licences was historically highest . Fishing data were available until December 2012, allowing the analysis of two full fishing seasons. In both instances we chose to focus on the short term response of the fleet to the closures. Firstly, in a highly dynamic environment like the Indian Ocean it is difficult to accurately predict fleet behaviour in the long term (e.g. over a period of years), and any longer term effect of the closures on fishing location may be masked by other influences on fleet behaviour. Secondly, an understanding of the short term response of resource users is important for management decision making, which tends to operate over relatively short timeframes .We used the generalised additive model (GAM) described in  to generIn the absence of the closure the fleet was expected to have fished within the IOTC closure area in both years of its implementation. In this counterfactual scenario the predicted distribution of effort was noticeably different for the Spanish and French components of the fleet. The Spanish fleet component was expected to have fished across a large area extending from the Seychelles plateau in the south to the Omani coast in the north, with core fishing grounds predicted within the area of the IOTC closure. In comparison, the French fleet component was expected to have fished across a comparatively small area in subequatorial waters, with core grounds predicted on the Seychelles plateau to the south of the closure boundary . This diIn the observed scenario the fleet complied with the closure in both years of its implementation and fishing effort was allocated to the south and east of the closed area boundaries . There wIn the first year of the designation of the BIOT reserve the fleet was expected to have fished across a large area of the western Indian Ocean, with core grounds predicted along a subequatorial band extending as far east as BIOT . These cIn the observed scenario there was a marked difference in the allocation of effort by the French and Spanish components of the fleet in the first year of closure of BIOT. In the case of the Spanish fleet component, effort was mainly allocated in the Seychelles region as predicted, although some effort was allocated in the northwest Somali basin region . This suIn the second year of closure of BIOT the fleet mainly allocated effort throughout the Seychelles region, with very little effort allocated in the proximity of the BIOT closure. This largely corresponded with predictions of the model and thus suggests little disruption of fishing behaviour caused by the reserve.Retrospective analyses of the response of fishing fleets to closed areas are scarce, although from the handful of published examples a varied set of fleet responses has been demonstrated. For instance,  observedIn these examples, the reasons for the observed reallocation of effort around closed areas have generally been inferred and few studies have been able to disentangle the policy effect of a closure from other competing influences . This isper se caused the fleet to reallocate fishing effort. The construction of a counterfactual scenario proved to be crucial in isolating the effect of the closures from confounding environmental influences on effort allocation and our results showed a mixed and inconsistent closure effect on fleet behaviour. Our results also demonstrated the flexibility of the fleet in adapting to the closures, particularly its ability to explore new areas, and furthermore we identified varied responses within the fleet depending on flag nationality, suggesting that the impact of closures may vary for different components of the fleet.In this study we have demonstrated a counterfactual approach as a useful diagnostic tool in evaluating the performance of area closures. To our knowledge, this is the first use in the fisheries literature of a counterfactual approach for evaluation using model-based predictions, and therefore marks new territory. We did not focus on whether the closures achieved their stated fisheries and conservation objectives. Rather we focused on whether the policy of closing areas Of particular interest in our results was the inconsistent effect of the BIOT reserve on fleet behaviour. Comparison between the observed behaviour of the fleet and counterfactual predictions of effort allocation showed a response to the closure in the first year of its designation but not the second, most likely resulting from inter-annual differences in environmental conditions and hence the distribution of tuna schools. This result highlights the importance of placement of fixed closures in fishery systems characterised by high environmental variability where the complex suite of influences on fleet behaviour can make it difficult to predict or evaluate the contribution of a closure to achieving conservation and management objectives.A second key result was the varying magnitude of closure effect on the two fleet components observed for both closures. In the case of the IOTC closure, the Spanish fleet component showed the most marked change in behaviour, with a number of vessels fishing to the east of the closure in typically un-fished areas, whereas the French fleet component allocated effort mainly in traditional grounds to the south of the closure. This varied response is largely explained by differences in the seasonal movement pattern of the fleet components linked to fishing strategy, with Spanish vessels typically remaining in the northwest FAD-fishing grounds for a longer period than French vessels, which move into subequatorial free schools regions earlier. A switch in the relative impact on two fleet components was observed for the BIOT closure in 2010/11, when the French component showed a more evident response to the closure with considerable exploratory behaviour around the closure boundaries. This variation in response is probably again linked to company-level strategy and differences in the targeting of free and associated schools, with Spanish skippers belonging to FAD-orientated companies less inclined to search for free schools in the vicinity of the BIOT closure and the opposite true for French skippers.In this study we have examined the short-term response of the fleet to the two closed areas. It remains unclear how these or other similar closures might affect fishing opportunities over a longer period, including how any cumulative impact on catches in the long term might influence decisions at a company level to participate in the Indian Ocean fishery. Furthermore, we did not look at the overall effect of the IOTC and BIOT closures on catches of tunas or bycatch species and as such we cannot offer a conclusion as to the conservation efficacy of large offshore closures. The effect of closures on fish stocks can take years or decades to be demonstrated  and, as S1 TableAll variables were aggregated at monthly intervals and at a spatial resolution of 1° latitude/longitude.(DOCX)Click here for additional data file.


Interestingly, microglia activation by IL4 has been described to promote neuroprotective and neurorestorative effects, which might be important to slow the progression of neurodegenerative diseases. In the present study, we addressed the role of endogenous and exogenous IL4 during MPP+-induced degeneration of mDA neurons in vitro and further addressed the impact of IL4-deficiency on neurodegeneration in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD in vivo. Our results clearly demonstrate that exogenous IL4 is important to protect mDA neurons in vitro, but endogenous IL4 seems to be dispensable for development and maintenance of the nigrostriatal system as well as MPTP-induced loss of TH+ neurons in vivo. These results underline the importance of IL4 in promoting a neuroprotective microglia activation state and strengthen the therapeutic potential of exogenous IL4 for protection of mDA neurons in PD models.Microglia are involved in physiological as well as neuropathological processes in the central nervous system (CNS). Their functional states are often referred to as M1-like and M2-like activation, and are believed to contribute to neuroinflammation-mediated neurodegeneration or neuroprotection, respectively. Parkinson’s disease (PD) is one the most common neurodegenerative disease and is characterized by the progressive loss of midbrain dopaminergic (mDA) neurons in the substantia nigra resulting in bradykinesia, tremor, and rigidity. Interleukin 4 (IL4)-mediated M2-like activation of microglia, which is characterized by upregulation of alternative markers Arginase 1 (Arg1) and Chitinase 3 like 3 (Ym1) has been well studied  Our results clearly demonstrate that IL4 is important to protect mDA neurons in vitro but seems to be dispensable for MPTP-induced loss of TH+ neurons in vivo.Microglia are the resident immune cells of the central nervous system (CNS), and thus are involved in a plethora of physiological as well as neuropathological conditions . Similarin vitro , the roland IgG1 . Moreoveand IgG1 . CNS-derand IgG1 . In the ad libitum access to food and water. C57BL/6-IL4tm1Nnt/J mice as described by C57BL/6 mice were housed at 22 ± 2°C under a 12 h light/dark cycle with n = 37, IL4 KO n = 27). All procedures involving MPTP were conducted in strict accordance with published safety and handling guidelines (Adult (10–12 weeks) male wild type (WT) and IL4 KO mice were intraperitoneally injected with 20 mg/kg MPTP hydrochloride (Sigma-Aldrich) dissolved in 0.2 ml phosphate-buffered saline (PBS) once a day for three consecutive days as previously described . Mice weidelines .2 culture flasks. Cultures were kept in a 5% CO2/95% humidified atmosphere at 37°C. After 10–14 days in culture, microglia were shaken off (250–300 rpm for 1 h) from adherent astrocytes and were plated according to the experimental purposes.Primary microglia cultures were generated as previously described . Brieflyg for 4 min. Cell pellets were resuspended in DMEM/F12 medium (Gibco) containing either 10% FCS, 10% horse serum (HS) and 1% penicillin/streptomycin (Invitrogen) to obtain mixed neuron-glia cultures, or N2 supplement (Invitrogen) and 1% penicillin/streptomycin (Invitrogen) to obtain neuron-enriched cultures. Both cultures were plated on poly-D-lysine-coated (Sigma-Aldrich) glass coverslips (12 mm diameter) at a density of 1–2 midbrains/coverslip. On day in vitro 1 (DIV1) medium was refreshed and mixed neuron-glia cultures were maintained for additional 7 days to yield mature cultures. Neuron-enriched cultures were treated from DIV1 according to the experimental design. Cultures were kept in a 5% CO2/95% humidified atmosphere at 37°C. MPP+ (Sigma-Aldrich) treatments were performed as described recently (Neuron-enriched and mixed neuron-glia cultures from embryonic day 14 (E14) ventral midbrains of NMRI mice (Charles River Laboratories Inc.) were generated as described previously . Dissectrecently . IL4  obtained from Ebiosciences . Functionality and optimal concentrations were confirmed in IL4-treated primary microglia cultures and the IL4 nAB was used in mixed neuron-glia cultures at a dilution of 1:50.Total RNA was isolated from primary microglia using TRIzol reagent  according to manufacturer’s instructions. RNAs from SN and caudate putamen (CPu) were isolated after dissection and transfer to RNA later (Ambion). Tissues were homogenized in peqGOLD TriFast (PeqLab) using a Precellys 24 homogenizer (PeqLab). RNA concentration and quality was determined using the NanoDrop 2000 . 1 μg RNA from each sample was reverse transcribed to cDNA using RevertAid  according to the manufacturer’s instructions.TM system  in combination with the Quantitect SYBR Green PCR Kit . 1 μl of cDNA template was used in 25 μl reaction mixture. Results were analyzed using the Bio-Rad iQ5 Optical System Software and the comparative CT method. All data are expressed as 2-ΔΔCT for the gene of interest normalized to the housekeeping gene Gapdh and presented as fold change relative to controls. The following primers have been used throughout this study: Arg1for 5′- AACACTCCCCTGACAACCAG-3′, Arg1rev 5′-CTGAAAGGAGCCCTGTCTTG-3′ [NM_007482.3], Igf1for 5′-CTGGACCAGAGACCCTTTGC-3′, Igf1rev 5′-GGACGGGGACTTCTGAGTCTT-3′ [NM_010512], IL4for 5′-ATTTTGAACGAGGTCACAGGAGAAG-3′, IL4rev 5′-ACCTTGGAAGCCCTACAGACGAG-3′ [NM_021283.2], Gapdhfor 5′-GGCATTGCTCTCAATGACAA-3′, Gapdhrev 5′-ATGTAGGCCATGAGGTCCAC-3′[NM_001289726].Semiquantitative RT-PCR  was performed using the PCR Red-Mastermix 2x . PCR products were separated using agarose gel electrophoresis and visualized after staining with GelRed (Genaxxon Bioscience). Images were captured using a Biometra  gel documentation station. Quantitative RT-PCR was performed using the MyiQ+-treated groups after 48 h as well as in medium from primary microglia cultures from control and MPP+-treated groups after 24 h using the murine IL4 ELISA Development Kit . IGF-1 was detected in serum-free culture medium from control IL4-treated primary microglia cultures using the murine IGF-1 ELISA Development Kit (Peprotech). ELISAs were performed according to the manufacturer’s instructions. Color reactions were performed using 2,2 azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) substrate  for 30 min in the dark. Finally, absorbance was detected using Multiskan FC plate reader (Thermo Fischer) at the absorption of 405 nm. Concentrations of IL4 and IGF-1 were calculated from standard curves using the GraphPad Prism5 software (GraphPad Software Inc.).Interleukin 4 was detected in serum-free culture medium from E14 mixed neuron-glia cultures from control and MPPNumbers of tyrosine hydroxylase (TH) positive neurons in E14 ventral midbrain neuron-enriched as well as mixed neuron-glia cultures were determined after immunocytochemistry. After incubation of neuron cultures for different time points, coverslips were washed once with PBS and fixed using 4% paraformaldehyde in PBS for 15 min at room temperature (RT). Cells were washed three times with PBS for 5 min each, and subsequently blocked and permeabilised for 1 h at RT in PBS containing 10% normal goat serum  and 0.1% Triton-X-100 (Roche Diagnostics). Primary antibodies rabbit anti-TH , anti-Iba1  and anti-Gfap  were incubated overnight at RT. Afterward, coverslips were washed three times with PBS, and cells were incubated with goat-anti-rabbit peroxidase-conjugated secondary antibodies  for 1 h at RT. The immunoreactivity was visualized using diaminobenzidine  as described by + mDA neurons, Iba1+ microglia and Gfap+ astrocytes were estimated according to the optical fractionator methods using the Stereo Investigator software . Cell counting was performed using an oil immersion 63 × objective , counting frame of 50 μm × 50 μm and a grid size of 120 μm × 100 μm. The optical fractionator was optimized to reach a coefficient of error ≤0.1. For each animal four sections with an evaluation interval of three were selected and labeled cells were counted. The nucleus of each immunoreactive cell was chosen as the counting event. Cell numbers are either presented as total estimated cell numbers or percentages (MPTP model) compared to PBS-treated control animals.Numbers of THg for 5 min at 4°C and filtered through 0.2 μm filters. DA concentrations were detected using a HPLC pump , an autosampler  and an amperometric detector . The isocratic mobile phase consisted of sodium acetate (90 mM), citric acid monohydrate (60 mM), ethylene- diaminetetraacetic acid (2 mM), 1-octanesulfonic acid (3 mM), and 8% methanol and was adjusted to a pH of 5.3. DA was separated on a Prontosil 120-3-C18 AQ column  by using a fixed flow rate of 300 μl/min. Data were registered and analysed by using Chromeleon 6 software (Dionex).Dopamine levels were quantified as recently described . Striatat-test. Normality of samples was assessed using the D’Agostino and Pearson omnibus normality test (as recommended by GraphPad Prism) and non-parametric tests have been used where appropriate. Multiple-group analysis was performed using one-way ANOVA followed by Bonferroni’s multiple comparison post-test. P-values ≤0.05 were considered as statistically significant. All statistical analyses were performed using the GraphPad Prism5 software (GraphPad Software Inc.).Data are given as means ± standard error of the mean (SEM). Statistical differences between two groups were determined using Student’s +-induced mDA neurodegeneration, E14 ventral midbrain cultures were treated for 48 h with MPP+ (0.2 μM) in the absence or presence of recombinant IL4 (10 ng/ml). Figures 1A,B demonstrates that neuron-enriched cultures responded to MPP+ application with a significant loss of mDA neurons (39.39% ± 7.821). IL4 co-treatment was not sufficient to protect mDA neurons in neuron-enriched cultures from MPP+-induced neurodegeneration (40.59% ± 7.417). Next, mDA neurodegeneration was induced by MPP+ in mixed neuron-glia cultures. As shown in Figures 1C,D, MPP+ treatment alone resulted in a significant decrease of mDA neurons in mixed neuron-glia cultures (54.93% ± 6.361). Interestingly, IL4 co-treatment significantly increased the survival of mDA neurons in mixed neuron-glia cultures (77.98% ± 4.655). These results indicate that IL4 only exerts its neuroprotective effects against MPP+-induced neurodegeneration in the presence of glia cells.In order to address the neuroprotective potential of IL4 against MPP+ astrocytes were negative for IL4, microglia labeled with FITC-coupled tomato lectin displayed a strong immunoreactivity for IL4 . Using an IL4 ELISA, we further analyzed the levels of IL4 in supernatants of control- and MPP+-treated cultures. Figure 2B demonstrates that 0.821 ng/ml (± 0.179) IL4 was detectable after 48 h in supernatants from control cultures and a slight but not significant increase of IL4 levels (1.123 ng/ml ± 0.5164) could be determined after treatment with MPP+. Moreover, we addressed whether primary microglia respond to MPP+ treatment with changes in IL4 secretion. 24 h after treatment with MPP+ no change in IL4 secretion from primary microglia cultures was detectable. Interestingly, the data demonstrate that microglia in mixed neuron-glia cultures release higher levels of IL4 than primary microglia. We next aimed to inhibit endogenous IL4 in mixed-neuron glia cultures in order to determine the role of microglia-derived IL4 during MPP+-induced neurodegeneration. Thus, the functionality of a neutralizing IL4 antibody (IL4 nAB) was validated. Therefore, primary microglia cultures were treated with recombinant IL4 (10 ng/ml) in the presence or absence of IL4 nAB at dilutions of 1:100 and 1:50, respectively. Using semiquantitative RT-PCR, expression of the IL4-induced M2 marker Arg1 was analyzed. As shown in Figure 2C, Arg1 expression was induced after treatment of primary microglia with IL4 for 24 h. Almost complete abrogation of IL4-triggered Arg1 expression was observed in the presence of the IL4 nAB at a dilution of 1:50, which was used for further IL4 neutralization experiments. Next, mixed-neuron glia cultures were treated with MPP+ and endogenous IL4 was blocked . Figures 2D,E show that inhibition of endogenous IL4 resulted in a significant increase in MPP+-induced neurodegeneration (38.75 % ± 6.116) as compared to MPP+ treatment alone (54.08% ± 4.72). These data clearly demonstrate that microglia-derived IL4 in mixed-neuron glia cultures is important to protect mDA neurons from MPP+ intoxication.Since IL4 has been recently described to be expressed by microglia , we analFigure 3A, results of the array using the proteome profiler kit for inflammatory cytokines revealed that IL4 did not influence the secretion of the vast majority of cytokines/chemokines from microglia. Notably, MCP-5 and MIP2 were two candidates with increased secretion after IL4 treatment (10 ng/ml) for 24 h. We further used microglia-conditioned medium to treat E14 neuron-enriched cultures in order to determine the survival promoting effects of microglia-derived factors on mDA neurons. Figures 3B,C show that microglia-conditioned medium after treatment with IL4 for 24 h (MCM IL4) significantly increased mDA neuron survival (157.4% ± 11.13) compared to microglia control medium (MCM). The observed neuroprotective effect of MCM IL4 was not promoted by IL4 itself, since direct IL4 treatment of E14 neuron-enriched cultures did not result in an increase of surviving TH+ neurons (108.5% ± 2.849). This observation demonstrates that IL4 treatment of microglia induces the secretion of neuroprotective factors, which increase the survival of TH+ neurons in E14 neuron-enriched cultures. IGF-1 has been identified as a survival-promoting factor secreted by macrophages after IL4 treatment . Moreover, Figure 3E clearly demonstrates that secretion of IGF-1 from primary microglia was significantly increased after 24 h (4.919 ng/ml ± 0.2868) compared to untreated control cells (2.061 ng/ml ± 0.3321). As IL4 was able to protect TH+ neurons from MPP+-induced degeneration in mixed neuron-glia cultures, we wanted to address whether microglia-derived IGF-1 was responsible for this protective effect. Thus, recombinant IGF-1 was used in order to determine whether TH+ neurons in E14 neuron-enriched cultures could be protected from MPP+-induced neurodegeneration. As shown in Figure 3F, MPP+ treatment significantly reduced the numbers of TH+ neurons (38.2% ± 9.848) and a slight non-significant increase of neuron numbers in the presence of IGF-1 could be observed. Although IGF-1 was able to increase the survival of mDA neurons in the presence of MPP+ (56.24% ± 10.39), the protective effect did not reach significance. Together, these results indicate that IL4 treatment of primary microglia results in the release of protective factors including IGF-1, which might at least partially mediate the neuroprotective properties of microglia-conditioned medium.The abovementioned results indicate that IL4 might shape the functions and properties of microglia in mixed neuron-glia cultures to promote survival of mDA neurons. Hence, primary microglia were treated with IL4 and secretion of cytokines and chemokines were analyzed. As shown in reatment . Therefoin vivo, IL4-deficient mice were used. IL4 KO mice are viable and fertile, develop normally and show no phenotypical abnormalities. However, these mice show impaired secretion of IL5 and IL10 and reduced IL4-dependent expression of IgE and IgG1  were not altered in IL4-deficient mice. Quantification of Iba1+ microglia in SNpc and SNpr revealed a slight and significant decrease in microglia numbers in SNpc  but no impairment of Iba1+ microglia numbers in SNpr . Analysis of Gfap+ astrocytes showed that the numbers of reactive astrocytes are neither changed in the SNpc, nor in the SNpr . Similar results were obtained to the basal ganglia (CPu). The numbers of Iba1+ microglia in the CPu of IL4 KO mice were comparable to WT mice . Numbers of Gfap+ astrocytes in the CPu were increased in IL4 KO mice without reaching statistical significance . These data indicate that loss of IL4 does not impair development and maintenance of the nigrostriatal system in vivo.In order to address the role of endogenous IL4 during MPTP-induced degeneration of mDA neurons and IgG1 . As a pr+-induced neurodegeneration in vitro, we used the MPTP mouse model to address the role of IL4 in this model for PD in vivo. Figure 5A displays the injection scheme and the time points used for RNA isolation, immunohistochemistry, and analysis of DA levels using HPLC. We first determined the expression of IL4 in the MPTP model. As shown in Figure 5B, a slight but not significant increase in IL4 expression was observed in the SN 1 day after MPTP injection. All other time points analyzed revealed no increases in IL4 expression in SN or CPu after intoxication with MPTP. Noteworthy, the relatively high CT values (between 32–36) indicate that in contrast to E14 mixed neuron-glia cultures, IL4 is hardly expressed in the nigrostriatal system in vivo. Two different time points for analysis of TH+ neuron numbers and striatal DA levels were chosen according to the acute neurodegeneration phase (7 days) and the regeneration phase (90 days) of this model  and IL4 KO mice (56.67% ± 14.39) without showing significant differences between both genotypes. Similar results were obtained after analysis of striatal DA levels. IL4 KO mice displayed a significant reduction of DA levels (46.4% ± 10.51) comparable to WT mice . Analysis of neuron numbers and DA levels 90 days after MPTP administration revealed that WT and IL4 KO mice showed a similar restoration of the MPTP-intoxicated nigrostriatal system. As shown in Figures 5F,H, numbers of mDA neurons in the SNpc recovered similarly in WT (84.82% ± 10.86) as well as in IL4 KO mice (93.29% ± 10.4). Moreover, striatal DA levels increased in both genotypes  compared to 7 days after intoxication, but were still significantly reduced at 90 days compared to PBS-injected control mice . Taken together, these data demonstrate that loss of endogenous IL4 does not increase the susceptibility toward MPTP-induced degeneration of mDA neurons in vivo. Moreover, the regeneration of the nigrostriatal system after MPTP administration is not impaired in IL4 KO mice, indicating that endogenous IL4 is dispensable in the MPTP mouse model for PD.Since endogenous IL4 was sufficient to protect mDA neurons from MPPis model . Figures+-induced degeneration in the presence of glia cells in vitro. Moreover, we provide evidence that endogenous microglia-derived IL4 in mixed neuron-glia cultures is important to reduce mDA neurodegeneration after MPP+ application and might facilitate neuroprotection by regulating microglia-mediated secretion of neuroprotective factors such as IGF-1. In vivo, the expression of IL4 was hardly detectable under basal conditions or after MPTP administration, and the loss of IL4 did not alter the susceptibility of mDA neurons toward MPTP-induced degeneration. Moreover, the regeneration of the nigrostriatal system after MPTP intoxication, as evident by restoration of TH+ neuron numbers as well as striatal DA levels, was not impaired in IL4-deficient mice.In the present study, we have demonstrated that exogenous IL4 protects mDA neurons from MPP+ neurons seem to be mediated by other microglia-derived factors. However, IL4 has been reported to induce IL6-producing macrophages that inhibit neuroinflammation in vitro and in vivo (+-driven degeneration (in vivo. IGF-1 increased survival of TH+ neurons and reduced neuroinflammation after intoxication with MPTP (+-induced degeneration, by adding recombinant IGF-1 to neuron-enriched cultures. Although the number of surviving neurons increased, the protection was not as effective as IL4 treatment of mixed neuron-glia cultures. It is high likely that microglia-derived neurotrophic factors and neuron–microglia interactions contribute to the neuroprotective effects observed after IL4 application in neuron-glia cultures. The fact that IL4 secretion from microglia is not a direct effect of MPP+ suggests that challenged neurons in mixed neuron-glia cultures trigger IL4 release from microglia. Damaged neurons have been shown not be passive targets of microglia-mediated neurotoxicity but are capable of regulating microglia functions. Release of CX3CL1 or IL34 from neurons and the subsequent binding to microglia-specific receptors CX3CR1 and CSF1R are well described mechanisms to shape microglia functions toward a neuroprotective phenotype (+-challenged neurons regulate microglia functions.Interleukin 4 has been described to promote a microglia activation state, which is characterized by functional features such as tissue repair and cellular protection . Here, w in vivo . Interesneration  and reduneration . Neverthneration . IL4 hasneration . Moreoveneration  indicatiith MPTP  as well ith MPTP . We furthenotype . Such neIn vitro, neutralization of endogenous IL4, expressed by microglia, worsens MPP+-induced degeneration of mDA neurons. This result suggests that IL4 acts in an autocrine manner on microglia in order to silence their activation. In vivo, loss of IL4 did not change the extent of neurodegeneration and neuroregeneration, indicating that IL4 is dispensable in the MPTP mouse model for PD. One of the reasons for these observations, might be the fact that IL4 expression in the nigrostriatal system is hardly detectable under basal conditions as well as after MPTP intoxication. The main cell type expressing IL4 are Th2 T-cells (+ T-cells (+ T-cells to protect mDA neurons from MPTP-induced neurodegeneration has been described after adoptive transfer of CD4+/CD25+ regulatory T-cells ( T-cells . However T-cells  and also T-cells  are able T-cells  but the  T-cells . This is T-cells . Lack of T-cells  and high T-cells  might be T-cells . Microgl T-cells  and coulThe therapeutic potential of exogenous IL4 has been well documented in several CNS pathology models including models for spinal cord injury , APP/PS1in vivo but the presence of IL4 in vitro is important to limit degeneration of TH+ neurons after MPP+ intoxication. Our results strengthen the hypothesis that IL4-treated microglia are capable of facilitating protection of mDA neurons and underline the therapeutic potential of IL4 administration in models of PD.Taken together, the results presented in this study demonstrate that IL4 is able to shape microglia functions to promote survival of mDA neurons. We provided evidence that endogenous IL4 is dispensable for MPTP-induced neurodegeneration BS conceived the project. LH, JR, XZ, VM, and BS performed experiments and analyzed the data. RG performed HPLC analyses. BS wrote the manuscript. All authors have read and approved the final manuscript and further agreed to be accountable for the content of the work.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 is currently at the forefront of cutting-edge science and technology due to exceptional electronic, optical, mechanical, and thermal properties. However, the absence of a sizeable band gap in graphene has been a major obstacle for application. To open and control a band gap in functionalized graphene, several gapping strategies have been developed. In particular, hydrogen plasma treatment has triggered a great scientific interest, because it has been known to be an efficient way to modify the surface of single-layered graphene and to apply for standard wafer-scale fabrication. Here we show a monolayer chemical-vapour-deposited graphene hydrogenated by indirect hydrogen plasma without structural defect and we demonstrate that a band gap can be tuned as wide as 3.9 eV by varying hydrogen coverage. We also show a hydrogenated graphene field-effect transistor, showing that on/off ratio changes over three orders of magnitude at room temperature. The absence of a band gap in graphene hinders its use in electronics. Here, the authors open a band gap as large as 3.9 electronvolts in graphene grown by chemical vapour deposition by treating it in hydrogen plasma, and then use this material to create a room temperature field- effect transistor. Graphene has opened a new arena in the field of two-dimensional condensed matter physics since its first isolation from graphite by means of micromechanical exfoliation124567912121591415161718Here, we show that monolayer graphene hydrogenated by indirect hydrogen plasma at room temperature acquires a band gap up to ∼4.0 eV that can be tuned by varying hydrogen coverage while preserving the structural integrity of the original graphene. We first demonstrate that the intrinsic band-gapped graphene we created fully functions as a field-effect transistor (FET), showing that conductivity at gate voltages changes over three orders of magnitude at room temperature.η on graphene increased. We found that the hydrogen coverage η saturated at ∼25% when we further increased the exposure time  graphene20time see . When thtime see  and 3.5 π and the π* orbitals, a fingerprint of graphene, disappeared and were significantly reduced, respectively, with the coverage. In other words, the sp2 orbitals rehybridized with the local sp3 orbitals as a result of hydrogenation. In addition, the C*=O orbital, located at ∼3.2 eV above the π* orbital, also disappeared as the hydrogenation continued, indicating that the hydrogen replaces oxygen chemisorbed on the CVD graphene. It is noteworthy that the pronounced π* band signifies topological ripples and corrugations, and that the intermediate states between π* and σ* contain residual adsorbates commonly observed in CVD graphene2021A close investigation of these spectra reveals that the band-gap opening indeed arises from the hydrogenation of graphene. The in-situ heating PES measurements of H-Gr directly prove that our indirect hydrogen plasma indeed inflicts minimal damage on graphene, that is, that H-Gr has no significant defects in the honeycomb basal plane after hydrogenation.  The indirect plasma technique is well known to significantly reduce damages to a surface, because the surface is not in direct contact with the plasma, which contains energetic particles. Both the band gaps of H-Gr created at an η of 12% and 25%, for example, returned to 0 (−10 Torr) at 550 K for 2 h. Besides, the recovery of the π orbital below the Fermi energy proves that hydrogen that had bonded with the pz-orbital of carbon was desorbed during heating and that H-Gr was reduced to metallic graphene. Such reversibility has been observed only when the graphene framework was not seriously damaged by surface treatments. Our observation of the full recovery of H-Gr to graphene bears a critical implication for the modulation of a band gap of graphene: we can control the band gap either by exposing the graphene to indirect hydrogen plasma or by thermal treatment of the resulting H-Gr.Our ned to 0  when H-Gπ plasmon located at ∼4.6 eV due to excitonic Fano resonanceπ plasmon peak is blue-shifted by up to ∼0.04 eV  band-to-band excitation. Therefore, the optical spectra often reveal the upper bound of the bandwidth in the case of indirect band-gap semiconductors. This is most likely the case in what we observed here—more of which later. Meanwhile, it is worth noting that the crucial reversible recovery from H-Gr to graphene was repeatedly confirmed through ex-situ optical absorption experiments. After we annealed H-Gr with an optical bandwidth of 4.6 eV at 550 K for 20 min under Ar atmosphere and carried out optical absorption experiments, the optical bandwidth decreased from 4.6 to 3.0 eV (Not only the transport band-gap measurements (PES and NEXAFS spectroscopy) but also optical absorption experiments support the wide tunability of a band gap of graphene induced by our plasma treatment. Initially, the absorption spectra of graphene acquired from near-infrared to ultraviolet at room temperature in air exhibited a general feature of a ∼0.04 eV . The opt04 eV  gradually increasedID/ID′) showed a similar saturation tendency and stayed at over 16, never dropping after saturation. The former ratio and its mapping imply that the defects are uniformly distributed and saturated at η of 25%, as the distance between defects no longer increases, and the latter ratio shows that the defect is not an atomic-vacancy type but an sp3 type in H-GrWe found that the hydrogenation or the formation of sp2 network can be ruled out by high-resolution transmission electron microscopy and by the evidence of the return of H-Gr to the pristine graphene state by the aforementioned thermal treatment. Neither structural damage nor disintegration of the innate symmetry of graphene was found in the micrographs  dependence of resistivity (ρ) of our H-Gr shows that the band gap stems from a long-range order at an η of 25%, whereas it can be opened by delocalized defect states at the lower hydrogen coverages (ρ−1/T of H-Gr (η=25%) showed three distinct behaviours: (1) intrinsic excitation, (2) variable-range hopping and (3) saturation towards the Mott minimum conductivity—all of which are essential features observed in crystal semiconductorsρ=ρ0 exp[Eg/(2kBT)]) in the intrinsic region was ∼3.8 eV, which is close to the band gap (∼3.9 eV) measured by transport band-gap measurements. On the other hand, when hydrogenation coverage was insufficient, the ρ−1/T showed the behaviour of variable-range hopping conductance, in which ρ is proportional to T−1/3 (not shown here) as others observedThe temperature  band-to-band excitation: optical absorption has been found to be very sensitive to the direct transitions at high-symmetry points, such as K, M and G, in the Brillouin zone of C4H-type hydrogenated graphene. The difference in the magnitude of the measured band gaps may also in part stem from the difference in the degree of hydrogenation or in the distribution of C–H bonds possibly caused by the different substrates used for transport measurements and optical absorption measurements—Si/SiO2 and sapphire, respectively (see Methods).Judging from the size of a band gap and the temperature-dependent resistivity of our H-Gr showing an C4H refs , 32, 33,2 gate dielectric showed a dramatic change in current of over 103 on/off ratio on the sweep of a back-gate voltage at room temperature as shown in 4. We believe such a limit can be ascribed to the high off-current due to leakage current through the thermally oxidized SiO2 of poor quality. I–V source-drain characteristics were initially nonlinear but became linear as the gate bias was increased over –20 V , indicating that the performance of FETs can be significantly improved by implementing a high-κ gate dielectric and an improved FET structure (20η∼12%), the on/off ratio was unexpectedly low. This is because the band gap was opened by random hydrogenationFinally, we demonstrate the functionality of a FET made from H-Gr . Our prof 3.9 eV . To showtructure . We noteThe very considerable advantage of our method is that a device can be built entirely of graphene: a conductor, that is, graphene, and a semiconductor and an insulator, that is, our hydrogenated graphene with small and large band gapsIn summary, we have demonstrated that the indirect exposure of CVD graphene to hydrogen plasma successfully modulated the band gap of graphene and, as a result, opened a band gap as wide as ∼4.0 eV without breaking the honeycomb structural symmetry of graphene; we have also shown that our hydrogenated graphene can fully function as a FET at room temperature. All of our hydrogenated graphene with a band gap retained very high structural integrity and was fully reduced to pristine graphene with a zero band gap simply by heating. Our study of monolayer graphene hydrogenated by indirect hydrogen plasma promises to provide answers to many controversial theoretical predictions and to trigger studies on new aspects of semiconducting graphene that have so far been hampered by experimental difficulties. Thus, this new form of graphene should play a significant role in enabling unique applications for long-envisioned carbon-based electronics and photonics.p-doped Si substrate with 300 nm-thick SiO2 for Raman, X-ray photoemission spectroscopy, PES and NEXAFS measurements. A sapphire substrate was used and CVD graphene showing ∼2.3% absorption in the visible range was chosen for hydrogenation and optical absorption measurements.Graphene was prepared under high vacuum by CVD on Cu foils and transferred onto a heavily Hydrogen plasma was generated in a separate gun with a microwave power of 180 W at 2.5 GHz. The flow rate of hydrogen was 25 s.c.c.m. and the working pressure was 10 mtorr. The as-prepared CVD graphene was downstream positioned 10 cm away from the end of the gun.p-doped Si substrate with 100 nm-thick SiO2. PMMA/HSQ bilayer resist on graphene was exposed by an electron beam and HSQ-uncovered PMMA/graphene layers were etched out by O2 plasma after developing. After removing them in acetone, FETs with graphene was obtained. They were annealed at 300 °C for 1 h in a gas mixture of Ar/H2 (10:1) to remove residual contaminations. Finally, FETs were treated by hydrogen plasma. We measured the electrical properties at various back-gate bias from 2 K to 300 K in vacuum. For a two-probe measurement, we applied constant source-drain voltage (Vsd) and FET mobility was extracted from the differential curve of the electric field effect. For a four-probe Hall measurement, constant source-drain current of 100 nA was applied and the Hall voltage (VH) was measured. The mobility and carrier density were estimated from VH measured by the four-probe Hall measurement.At first, to fabricate graphene-based FETs, electrodes were patterned by a lift-off process after Cr/Pt (5/25 nm) was sputter-deposited on the patterns prepared by photolithography. Then, CVD graphene was transferred onto a heavily The optical transmission and absorption coefficient of graphene and H-Gr were measured at room temperature via visible-ultraviolet spectrophotometry  over the wavelength range of 175–3,300 nm at normal incidence. Raman spectra were obtained with a Horiba Jobin-Yvon Lab Ram HR spectrometer with an Ar laser wavelength of 514.5 nm at room temperature. Diffraction patterns and atomic images were taken by Cs-corrected STEM  with 80 keV electron beam. PES and X-ray photoemission spectra were measured using the synchrotron source at the 4D beam-line and NEXAFS spectra were obtained at the 2 A beamline at Pohang Accelerator Laboratory. The NEXAFS spectra were obtained with partial electron yield at a 55° angle of incidence of the synchrotron photon beam with respect to the surface of the samples.The data that support the findings of this study are available within the article and its How to cite this article: Son, J. et al. Hydrogenated monolayer graphene with reversible and tunable wide band gap and its field-effect transistor. Nat. Commun.7, 13261 doi: 10.1038/ncomms13261 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures 1-4, Supplementary Table 1, Supplementary Notes 1-4 and Supplementary References.