In vitro models are useful in answering this question by providing information over the spatiotemporal nature of such dynamics. In the present work, we used physical quantities such as growth rate, velocity, and acceleration for the cellular proliferation and identified the fractal structures in tumor cell proliferation dynamics. We provide evidence that the rate of cellular proliferation is of nonlinear nature and exhibits oscillatory behavior. We also calculated the fractal dimensions of our cellular system. Our results show that the temporal transitions from one state to the other also follow nonlinear dynamics. Furthermore, we calculated self-similarity in cellular proliferation, providing the basis for further investigation in this topic. Such systems biology approaches are very useful in understanding the nature of cellular proliferation and growth. From a clinical point of view, our results may be applicable not only to primary tumors but also to tumor metastases.Biological systems are characterized by their potential for dynamic adaptation. One of the challenges for systems biology approaches is their contribution towards the understanding of the dynamics of a growing cell population. Conceptualizing these dynamics in tumor models could help us understand the steps leading to the initiation of the disease and its progression.  Population dynamics and population genetics provide a well-developed mathematical theory of evolution , 2 and mGenes manifest several patterns of differential expression in cancer , 7. Gene in vitro systems provide an excellent opportunity to study effects that are impossible to be measured in vivo. Most importantly, in vitro systems can be studied in the long term. This is required in order to reach conclusions regarding nonlinearity and chaotic behavior of a cellular system. Since primary cell cultures are short-lived when untransformed (15–20 days), the only way to apply such measurements is to use already established cell lines. For this reason, we developed a modeling approach in order to simulate the in vivo conditions, as best as possible. The nature of proliferation dynamics can give insight into the way that not only cells proliferate, but they also differentiate.Data regarding the dynamic nature of a tumor can only be collected after it has been diagnosed. Usually, this is too late for the patient, since all the critical steps for the progression of the tumor have already taken place. Therefore, in vitro cellular systems, using three central nervous system (CNS) tumor and a T-cell acute lymphoblastic leukemia (T-ALL) cell lines. These cells provide an excellent substrate for modeling proliferation dynamics, as previously shown  appeared to manifest the most stable oscillatory pattern, among all measurements that we performed  and the Apollonian Gasket . Our results confirmed two interesting points: (1) cell growth factors follow oscillatory dynamics (of nonlinear nature) and (2) different cell types followed similar dynamics of growth, irrespective of whether they grow as adherent or suspension cells, hinting towards a common mechanism of cellular proliferation.We measured the proliferation of the three CNS tumor cells and the CCRF-CEM cells Figures . In orde Figures . As a re Figures , charact Figures  and acce Figures , respect Figures . Therefo 0.80888 . In ordeIn the present work we identified nonlinear factors of cellular proliferation, in three CNS tumor cell lines and one leukemic cell line. Since our results show that cell growth is of nonlinear nature, we propose an initial theoretical framework for the analysis of such phenomena and for future considerations. This knowledge could be useful in treating tumors, since by understanding the mechanisms of cellular proliferation we could interpret the factors that determine the progression of the disease and/or metastasis. Biological systems are extremely complicated and manifest non-linear/chaotic phenomena. Among others , 32, we  Lyapunov exponents (one of the criteria of chaos existence). Such an example is the understanding of cellular proliferation in which we attempted to contribute with our hints. Here, we show that that different cell types follow similar dynamics with respect to proliferation, however their dynamics follow different trajectories. This difference arises from the probable reason that it is possible that all trajectories can be described by the same function, yet with different constants. Another question arising is that when cellular populations are measured with respect to time, they would give natural numbers corresponding to the exact or approximate number of cells present in the system. At the same time, all other physical variables calculated return oscillations. The question posed, concerns the role of gene expression in controlling cellular proliferation. A possible explanation is that cells follow oscillatory dynamics exactly the same way gene expression does [We have previously described the chaotic patterns of the leukemic cell line that we used . These pion does .Another issue in tumor proliferation dynamics that remains unanswered is the conditions at the time of the disease onset. The only knowledge we have thus far, concerning tumors, originates from the time of clinical presentation, at diagnosis. Practically, we have complete lack of knowledge from the time of tumor initiation to the time of tumor presentation. In that sense, the understanding of the proliferation dynamics of tumor cells is critical since it could provide insight into the understanding of tumor initiation. Supposing we could describe the dynamics of cellular proliferation in a formal, mathematical form, we would be able not only to predict the time zero (the starting point) but also to understand the mechanics of this progression. Additionally, if we suppose that such a formal description could be applied to several tumor types, we could conclude to a general rule of tumor proliferation. To the best of our knowledge, there are no previous works dealing with this subject and posing these questions.The implications of the understanding of proliferation dynamics are immense. To date, we do not have a sufficient theory that could allow us understand and predict cellular growth. This is easy to prove if we ask a simple question: given the population of cells today can we predict the population after 24 hours? The answer is no, since the only way we can do it is by approximation and this can be done only statistically. In other words, if we could predict the future cellular population based on the past population, this could lead us to the starting point of cellular proliferation, information that would be of extreme importance in cancer biology.Concluding, in the present work we aimed to set a framework for the detection of global patterns in cellular proliferation. Considering the fact that our knowledge in tumor biology comes only from the clinical presentation of the disease, the discovery of global models of tumor progression and proliferation could provide more insight in tumor biology and be used for therapeutic or prognostic purposes. Future work should focus on the investigation of the rates of cellular death in the same proliferation models and, most importantly, expand this model to gene expression for the same proliferation models, thus moving from phenotype to the genotype.


A striking puzzle about language use in everyday conversation is that turn-taking latencies are usually very short, whereas planning language production takes much longer. This implies overlap between language comprehension and production processes, but the nature and extent of such overlap has never been studied directly. Combining an interactive quiz paradigm with EEG measurements in an innovative way, we show that production planning processes start as soon as possible, that is, within half a second after the answer to a question can be retrieved . Localization of ERP data shows early activation even of brain areas related to late stages of production planning . Finally, oscillation results suggest an attention switch from comprehension to production around the same time frame. This perspective from interactive language use throws new light on the performance characteristics that language competence involves. There is a striking but largely unexplored puzzle at the heart of the language sciences. The core ecological niche for language use is conversational interaction, where speakers take rapid turns at talking. The puzzle is that the changeover between speakers is very quick, but the latencies involved in planning language production are three to ten times longer (depending on the nature of the turn under construction). This implies that to achieve this timing, participants must start production planning already during the incoming turn. Thus, there must be significant overlap between language comprehension processes and production ones – such dual-tasking forms an interesting challenge for the system since both processes draw on largely the same cognitive and neural resources.Earlier work has established the tight timing of turn-taking in conversational interaction278Given the predictive comprehension and early production implied by turn-taking, a crucial locus of investigation is what cognitive processes are going on in the recipient during an incoming turn to which he or she must respond. Using electroencephalography (EEG) as a non-invasive way of tapping into these processes with an excellent time resolution, we designed an innovative interactive turn-taking paradigm in the form of a quiz game (see Methods). This preserved the essential turn-taking structure, and is a familiar genre of interactive language use. Sitting in a shielded chamber, participants were asked to respond quickly (within 5 seconds) to Dutch quiz questions, and received live feedback from the experimenter. Unbeknownst to the participant the quiz questions themselves were pre-recorded by the same experimenter, providing a high degree of experimental control across participants.007, appears in the famous movies?EARLY: Which character, also called movies, is also called 007?LATE: Which character from the famous To vary when participants could start planning their answers, we created two different conditions.007, in bold) was available early on, whereas in ‘LATE’ questions (see example 2), this information was not available at the equivalent position , but only became available at the very end of the question. The position in the middle of the sentence  is hereafter referred to as the first time-locking point (or TL1) and the onset of the last word of the sentence is referred to as the second time-locking point (or TL2). Answers constituted one word or a small phrase. We were interested in how soon participants would start planning the production of their answer after the critical information became available, especially in the EARLY condition. Crucially, to distinguish production vs. comprehension processes, a control experiment was conducted, in which participants listened to the same quiz questions but instead of answering them, were asked to remember the questions and were tested using probes.In ‘EARLY’ questions (see example 1) the critical information essential to answer the questions  as the main predictor, and random intercepts for participant and item, showed that responses relative to question offset in the EARLY condition  were faster than in the LATE condition  based on the EEG data from 64 electrodes, time-locked to TL1 and TL2. Second, and most importantly, we found a large positive effect, starting around 500 ms after the onset of the critical information in both TL1  and TL2 . In the control experiment, in which participants did not answer the questions, positivities were present as well , but they were reduced in size, as confirmed by statistically comparing the two experiments . Note that the smaller positivities in the control experiments are not caused by the larger N400 effects found there, as shown by two additional analyses  and TL2  were localized at distributed sources in the brain, mostly in the left hemisphere . Local m007). In contrast, in the control experiment where no overt production was required, no frequency effects were found at TL1. At TL2 marginally significant reduced alpha power was found for LATE vs. EARLY questions between about 800 and 1100 ms after onset of the last word (p = .06). An analysis comparing the two experiments confirmed that the reduced alpha effect at TL1 (EARLY vs. LATE) was stronger in the main experiment than in the control experiment , but we found no differences between the experiments at TL2. Since the reduced alpha power was not present or smaller in the control (comprehension) experiment than in the main (production) experiment, this effect also appears to be related to production preparation.In a further analysis, we computed time-frequency representations of the oscillations of the EARLY and LATE questions, using the same time-locking as for the ERPs. We found that power was modulated around the alpha band (about 9–14 Hz) differentially across the two experiments . In the These oscillatory effects might be interpreted in two different ways. First, reduced alpha (and beta) power has been associated with motor preparation , as suggested by source localization of the positivity. The second effect was reduced power in the alpha band that we interpret as a switch of attentional resources from comprehension to answer retrieval and production processes. Importantly, both effects started already around 500 ms after the onset of the information that enables participants to retrieve the answer. This indicates that interlocutors started production planning within half a second of hearing the critical information necessary to start answer preparation – in the EARLY questions on average around 2.4 seconds before the end of the question. Additional analyses time-locked to the response . The 24 remaining participants  had a mean age of 21.3 years old (range 18 to 24). For the control experiment, another 32 participants were recruited from the same pool. Data from 2 participants was excluded from the analysis because of too many artifacts . The 30 remaining participants  had a mean age of 21.1 years old (range 18 to 25). All participants were native speakers of Dutch without hearing impairments. They gave informed consent before participating and received 8 euros per hour for their participation.007, komt voor in de bekende films? “Which character, also called 007, appears in the famous movies?”EARLY: Welk karakter, ook wel films heet ook wel 007? “Which character from the famous movies is also called 007?”LATE: Welk karakter uit de bekende We used 94 experimental Dutch question pairs in two different conditions. In EARLY questions (see example 1), the ‘answer-recognition’ point (the point at which the answer was clear) occurred early in the question, followed by 8.1 syllables on average (range 5–21) or 4.4 words (range 2–10), whereas in LATE questions (see example 2), this point occurred during the last word (or two words). The questions of each pair differed in the order of the constituents, but furthermore contained the same words as much as possible and fitted with exactly the same answer.A pretest (20 participants) ensured that the EARLY questions could be reliably answered correctly immediately after the answer recognition point  and the LATE questions could not reliably be answered correctly before hearing the last word . The same pretest showed that the average cloze probability of the last word was .07 for the LATE condition and .59 for the EARLY condition (t(93) = 13.89, p < .001). Thirty pairs (EARLY and LATE) of relatively difficult quiz questions served as filler questions, to create a more realistic quiz and to create more of a competition for the prizes (see Procedure). A pretest (10 participants) showed that they were answered correctly 31% of the time on average (range: 10–80%).All experimental and filler questions were recorded twice by a research assistant (RA) in the EEG room under the same circumstances as during the EEG experiment, and the best token of each question was chosen for the experiment. The experimental quiz situation was simulated by having a participant present who answered the questions. Some of the filler questions  were recorded with a hesitation or error to create the illusion of live speech. Recorded items were cut around 500 ms before question onset and exactly at question offset using PraatThe experimental questions could differ on the factor Answer . Two lists were created, which were both administered to half of the participants. List 1 contained half of the experimental and filler questions in the EARLY condition and the other half in the LATE condition, which was reversed for list 2. All items were presented in the same order for all participants. The items were divided into 4 blocks of 31 questions each with pauses in between. Question topics were spread over the 4 blocks and each block contained 7 or 8 difficult filler questions, but never twice in a row. The exact same design was used for the main and the control experiment.After EEG preparation, participants sat down in a sound proof booth in front of a computer screen. The same RA that recorded the materials instructed them from outside of the soundproof booth via a sound system. The RA used a microphone and participants heard her via loudspeakers. Participants were first informed that the two participants who answered most questions correctly would win a prize. Then, the RA instructed participants to answer quiz questions that varied from easy to difficult. They were urged to focus on giving the right answer, within a deadline of 5 seconds. They should look at a fixation cross on the screen (without moving or blinking) that was presented from between 1500 and 2000 ms before the question started until a button-press by the RA after the answer was given. Afterwards, the RA gave feedback while the word ‘knipperen’ (blink) was visible on the screen, indicating that participants could blink their eyes. They were told that the complete interaction was live, while in reality the questions were played by a button-press from the RA, while the feedback was given live. Participants started with a practice block of 16 questions, followed by the 4 experimental blocks. After the experiment, participants filled out a short questionnaire and received a debriefing sheet, informing them that the questions had been recorded.In the questionnaire after the experiment, in a first open question, none of the participants reported to have noticed anything strange during the experiment. When asked directly whether they thought (in retrospect) that any of the stimuli they heard could have been recorded, 6 participants (25%) reported that some of the questions could have been recorded. Also 3 other participants (12.5%) reported that the feedback after the questions could have been recorded . To keep enough power, we kept all these participants in the analyses. However, we performed additional ERP and time-frequency analyses for the main experiment without the 6 participants that reported that some of the questions might be recorded, to exclude the possibility that these participants could explain some of the differences we found see .The procedure was the same as in the main experiment except for the following. Participants were instructed that this was a control experiment for an earlier quiz experiment in which participants answered the questions. They were told that the RA read the questions to them live to keep as close to the quiz experiment as possible. Participants were asked not to answer but try to remember the questions. After each block of questions, they saw 10 probe questions on their screen, one by one, and had to indicate for each question whether they had heard it in the previous block or not. Participants received two practice blocks of 20 questions each, to give them an indication of the type of foils they could expect. The foils were very similar to the questions they had heard, differing either in answer or in content. For example, for the quiz question “Which element in coffee is the effective element?”, the foil was: “Which element in coffee wakes you up?”On average 92% of probe questions were remembered or discarded correctly, showing that participants paid attention to the questions. The questions from both conditions were remembered equally well (T < 1). In the questionnaire after the experiment, in a first open question, none of the participants reported to have noticed anything strange during the experiment. When asked directly, only 3 participants reported that some of the questions might have been recorded.EEG was recorded from 61 active Ag/AgCI electrodes using an actiCapFor behavioral data analysis of the quiz questions, we looked at the numbers of errors in the two conditions and we measured the response time from the end of the question to the start of the answer. Incorrect responses  and responses starting with hesitations (5%) were discarded before the analysis. We ran mixed-effects models on these two variables to assess the effect of Condition  in R.007 in example 1). As an equivalent point in the LATE condition, we took the word onset that was closest in time to the same point in the EARLY condition (as measured from sentence onset) for each item . The second time-locking point (TL2) was the onset of the last word in the LATE condition  and in the EARLY condition . See Preprocessing and statistical analysis of EEG data was conducted using FieldtripEpochs for analysis consisted of 200 ms before until maximally 2000 ms after TL1 and 200 before until maximally 1400 ms after TL2 since speech started earlier for TL2 . These preprocessed data entered the event-related potential (ERP) and time-frequency  analyses. For ERPs, epochs were filtered with a low-pass filter of 35 Hz and baselined with a baseline window of 200 ms immediately before the time-locking point. Then, trials of the same condition were averaged per participant. For time-frequency representations, no filtering or baselining was performed, but a linear trend was removed from the data before the analysis. The power of each frequency between 4 and 30 Hz (with steps of 1 Hz) was calculated on the extracted epochs of individual trials using a Hanning taperTo test for statistically significant differences between conditions and reduce the multiple-comparison problem, we used the cluster-based approachTo identify sources underlying the electrode-level effects, a BEM (boundary element headmodel)How to cite this article: Bögels, S. et al. Neural signatures of response planning occur midway through an incoming question in conversation. Sci. Rep.5, 12881; doi: 10.1038/srep12881 (2015).


Graphene exhibits unusual electronic properties, caused by a linear band structure near the Dirac point. This band structure is determined by the stacking sequence in graphene multilayers. Here we present a novel method of microscopically controlling the band structure. This is achieved by epitaxy of graphene on 3C-SiC(111) and 3C-SiC(100) thin films grown on a 3D microfabricated Si(100) substrate (3D-GOS (graphene on silicon)) by anisotropic etching, which produces Si(111) microfacets as well as major Si(100) microterraces. We show that tuning of the interface between the graphene and the 3C-SiC microfacets enables microscopic control of stacking and ultimately of the band structure of 3D-GOS, which is typified by the selective emergence of semiconducting and metallic behaviours on the (111) and (100) portions, respectively. The use of 3D-GOS is thus effective in microscopically unlocking various potentials of graphene depending on the application target, such as electronic or photonic devices. Carrier dynamics in monolayer graphene, a honeycomb lattice consisting of carbon atoms, are described in terms of quantum electrodynamics12368One of the major challenges to such device applications of graphene is that of establishing epitaxial growth of graphene on practical substrates using methods compatible with existing device technologies11131516171516171718191922In addition to these manufacturing advantages, GOS has another more profound advantage: the tunability of the band structure by altering the Si substrate. By controlling the crystallographic orientation of the Si substrate, we can select the surface orientation of the 3C-SiC thin film, which eventually makes it possible to modulate the stacking of graphene and its interface structure with SiC. The latter controls the presence or absence of the interfacial (6√3 × 6√3)-reconstructed buffer layer, which is a precursor for graphene in EG and GOS. On the Si(111) substrate, the buffer layer is present, and GOS is Bernal-stacked17171821In this work, we demonstrate the microscopic tuning of the band structure of epitaxial graphene on a 3D microfabricated Si substrate (3D-GOS) through stacking with the aid of microelectromechanical system (MEMS) technology, specifically anisotropic wet etching which produces (111) and (100) microfacets on a Si(100) substrate.2 film (90 nm) is grown on a Si(100) substrate  by the dry oxidation method. Electron-beam lithography is performed on the sacrificial SiO2 thin film, followed by fast atomic beam etching using SF6 as an etchant272 mask on the surface. Using the SiO2 mask, the surface is anisotropically etched in a 25% tetramethyl ammonium hydroxide (TMAH) aqueous solution at 353 K for 6 min. The etching by the TMAH aqueous solution is used to expose Si(111) microfacets on Si(100) substrates, which are frequently used as the standard fabrication procedure for MEMS devices2 mask with a treatment in a 5% HF aqueous solution. The 3C-SiC thin films (~100 nm in thickness) are grown on microfabricated Si(100) substrate by using gas-source molecular beam epitaxy with monomethylsilane as a gas source at a substrate temperature of 1353 Kin vacuo at 1523 K for 30 min to grow graphene epitaxially on the surface of the 3C-SiC(111) microfacet15161718The formation of 3D-GOS is schematically shown in The interface between graphene and the 3C-SiC microfacets is examined here because the graphene growth mode is determined by the presence or absence of the buffer layer because it works as the template for the Bernal stacking of graphene17183017171333The atomic structure of the interfaces between graphene and the 3C-SiC microfacets has been investigated by using cross-sectional transmission electron microscopy (X-TEM), as shown in To clarify the relation of the observed interface variation caused by microfacets with the graphene stacking, microscopic LEED (μ-LEED) analysis is performed for 3D-GOS, as shown in 1718As seen from the μ-LEED images of the two bevels, a hexagonal LEED pattern from graphene is clearly identified on the 3C-SiC(111) microfacets. The spots due to the specular reflection, labelled as (00) in the images, are located away from the center of the μ-LEED pattern because of the inclination of the 3C-SiC(111) microfacets. The μ-LEED pattern is almost the same as that of the graphene on the flat Si(111) substrate and graphene on 6H-SiC(0001)171836In contrast to epitaxial graphene on the 3C-SiC(111) microfacet, the LEED spots of graphene on both the top and bottom 3C-SiC(100) microterraces are rotated, as observed on the graphene on 3C-SiC(100) thin films on flat Si(100) substrates. The rotation angle between the adjacent spots indicated by the yellow arrows is about 15 degrees. One of the possible mechanisms for this rotated growth of graphene on the 3C-SiC(100) face is the {111} facet-induced mechanismechanism . There a−1), D (~1360 cm−1), and G′ (~2700 cm−1) bands, are visible in the spectra on the graphene on both 3C-SiC(100) microterraces and 3C-SiC(111) microfacets. The G band is the band originating from a single-resonant Raman process at the Γ point39The band structure of 3D-GOS is anticipated to vary with the microfacet and the microterrace orientation owing to a high susceptibility of the band structure to the stacking sequence, by analogy with the susceptibility of the band structure of GOS using flat Si substrates for stacking depending on the crystallographic orientation of the Si substrate17−1. This value is larger than that of epitaxial graphene on 6H-SiC(0001) (37 cm−1)−1) for graphene on a 3C-SiC(100) thin film on a Si(100) substrate172044Among these bands, the G′ band is analysed in detail to clarify the band dispersion41424344394244−1) than that on the 3C-SiC(100) microterrace (70 cm−1). These two features can only be accounted for by considering a set of multiple Lorentzian components, as shown in D/IG of the D band to the G band, which is a good measure of the defect density that broadens the G′ band, is substantially smaller in the graphene on 3C-SiC(111) microfacet (ID/IG = 0.64 ± 0.04) than that on the 3C-SiC(100) microterrace (0.75 ± 0.04). Thus, the G′ band on 3C-SiC(111) cannot be represented by a single Lorentzian. The broadening of the G′ band of the graphene on the 3C-SiC(111) microfacet is thus concluded to be due to presence of multiple components. The presence of multiple Lorentzian components in the G′ band suggests presence of multiple routes for this double-resonant Raman scattering process in the graphene on the 3C-SiC(111) microfacets. Several origins can be considered, which include the band splitting of graphene424347−1 is attributed to the so-called D′ band, induced by defects4742On the other hand, the G′ band of the graphene on the 3C-SiC(111) microfacet is asymmetrical and is much broader  substrate by anisotropic wet etching, which produces Si(111) microfacets as well as Si(100) microterraces. The realization of 3D-GOS is the first step to exploiting graphene-based multifunctional integrated circuits (combining electronics and photonics) compatible with existing Si-based electronics in the next generation of devices.E/ΔE > 104). The photon energy of the SR beam for the measurement is 1000 eV. Using 3D nano-ESCA enables us to obtain a high lateral resolution (70 nm) by focusing X-rays using a Fresnel zone plate. The energy resolution of the spectrometer was set to 300 meV, and the accuracy of the angle resolution is 0.9°. The binding energy scale was calibrated by the photoelectron peaks of a gold foil (Au 4f 7/2). Details of the experimental setup are described in the previous reports3017The spatially-resolved and angle-resolved C1s core-level photoelectron spectra are taken by using the 3D nano-ESCA system installed at the BL07LSU at SPring-83031H.F. performed the 3D-GOS fabrication and all of the microscopic characterizations, designed the study, and participated in the writing of the manuscript. T.I. performed the 3D-GOS fabrication and all of the microscopic characterizations. Y.K. participated in fabricating the 3D-GOS. T.S. participated in the microscopic characterization using 3D nano-ESCA. N.N., K.H. and H.K. performed the microscopic characterization using 3D nano-ESCA, and participated in discussions about the content of the manuscript. M.K., T.O. and T.K. participated in the microscopic characterization using μ-LEED, and participated in discussions about the content of the manuscript. M.O. participated in the microscopic characterization using 3D nano-ESCA, and participated in discussions about the content of the manuscript. M.S. participated in discussions about the content of the manuscript.Supplememtal Information


Our results show that this herbal medicine possesses strong activities including stimulation of angiogenesis, cell proliferation, and cell migration, which provide the scientific basis for its clinically observed curative effects on nonhealing diabetic wounds.“Jinchuang ointment” is a traditional Chinese herbal medicine complex for treatment of incised wounds. For more than ten years, it has been used at China Medical University Hospital  for the treatment of diabetic foot infections and decubitus ulcers. Three different cases are presented in this study. “Jinchuang” ointment is a mixture of natural product complexes from nine different components, making it difficult to analyze its exact chemical compositions. To further characterize the herbal ingredients used in this study, the contents of reference standards present in a subset of the ointment ingredients  were determined by HPLC. Two Medicine Comprehended, published in 1732. Clinical applications of this herbal medicine for diabetic foot infections and decubitus ulcers have been a successful course of treatment in the Division of Chinese Medicine, China Medical University Hospital, Taichung, Taiwan, for more than ten years. Despite its track record of curative effects, there is no literature published in the English language describing the clinical efficacy of “Jinchuang ointment” [ It is well-known that diabetic foot ulcers are extremely difficult to be treated and are the dominant complication leading to amputations . “Jinchu“Jinchuang ointment” is composed of lard, wax, starch, synthetic borneol, camphor, frankincense, dragon's blood, myrrh, and catechu. To further characterize the chemical content of each component in this complex, the ratio of stereoisomers in chemically synthesized borneol used in this study was analyzed by chiral gas chromatography (GC). Meanwhile, the content of reference standards in the herbal components, like frankincense, myrrh, dragon's blood, and catechin, was determined by high performance liquid chromatography (HPLC). Lard is the major component in this complex, and its weight percentage is as high as 67%. It is of great interest to determine the role of lard in this complex. Lard was therefore substituted for synthetic triacylglycerol, coconut oil, Vaseline®, and sesame oil. The activity of these reconstituted complexes was examined in this study. in vitro tube formation assay, a wound healing assay, and a cell proliferation test were carried out to examine the activity of “Jinchuang ointment.” Here, we report the outcomes of treating patients with “Jinchuang ointment,” the results of cell based activity assays, and characterization of herbal components by HPLC.“Jinchuang ointment” is directly applied to the wound surface during treatment. The components in this complex are neither digested nor absorbed in the gastrointestinal tract. It is therefore reasonable to evaluate its bioactivity by direct addition of this complex into media containing cultured human skin or endothelial cells. Wound healing is a very complicated process. In this study, anβ-boswellic acid, catechin, and epicatechin were purchased from the National Institute for the Control of Pharmaceutical and Biological Products . (E)-Guggulsterone and (+)-borneol were obtained from Sigma-Aldrich . (−)-Borneol and (±)-isoborneol were purchased from Alfa Assar . DMEM high glucose with sodium pyruvate media, Catalog number: I-26F55-I, was bought from Amimed® BioConcept . Chemically synthesized borneol and camphor were bought from Cheng Yi Chemical Co., Ltd. . Herbal medicine, dragon's blood, catechu, frankincense, and myrrh were bought from Healthy Beautiful Biotech. Co. Ltd. . Vascular endothelial growth factor (VEGF) was bought from B&D Systems . Antibodies for western blot analysis were bought from Santa Cruz Biotechnology, Inc. . Food-grade lard was obtained from I-Mei Food Company . Synthetic triacylglycerol  was obtained from InterMed Manufacturing Sdn. Bhd. . Food-grade sesame oil was from Fwusow Industry Co. Ltd. . Coconut oil was bought from First Chemical Co., Ltd. . Vaseline was bought from Unilever .The reference standards of dracorhodin perchlorate, acetyl-11-keto-The composition of “Jinchuang ointment” (100 g) is as follows: lard 67.3 g, dragon's blood 2.1 g, catechu 2.1 g, frankincense 2.1 g, myrrh 2.1 g, camphor 6.3 g, borneol 0.1 g, corn starch 8.4 g, and wax 9.5 g. For wound healing and tube formation assays, the DMSO stock solution of “Jinchuang ointment” is prepared as follows: two grams of “Jinchuang ointment” is dissolved in 10 mL DMSO and homogenized by ultrasonication just before use.μm, 250 × 4.6 mm, analytic column  was used for analysis.All experiments were carried out on a Hitachi L-7000 HPLC system, equipped with L-7100 quaternary gradient pump and a L-7450 photo diode array detector. Hitachi HSM software was used for machine controlling, data collecting, and processing. A Mightysil, RP-18, 5 μm syringe filter. For myrrh, 95% ethanol was used for extraction rather than methanol. Other preparation steps were identical to those of dragon's blood, catechu, frankincense, and myrrh.For samples of dragon's blood, catechu, and frankincense, 0.1 g grounded solids were weighed and dissolved in 10 mL of methanol. After ultrasonicating for 30 minutes at room temperature, methanol extracts were transferred to a new glass vial by using disposable glass Pasteur pipettes. 4 mL of methanol was then added and ultrasonicated for another 30 minutes at room temperature. The final volume of the extract was adjusted to 25 mL by adding methanol. Undissolved particles were removed by centrifugation at 2500 ×g for 10 minutes at room temperature and filtrated through a 0.22 3CN containing 0.1% formic acid) and solvent B (90% CH3CN containing 0.1% formic acid) with a flow rate of 1 mL/min [The methanol extract of dragon's blood was separated using a gradient elution of solvent A (10% CH1 mL/min . The elu2O containing 0.1% formic acid), B (10% CH3CN containing 0.1% formic acid), and C (90% CH3CN containing 0.1% formic acid) with a flow rate of 1 mL/min [The catechu methanol extract was separated using a gradient elution of A  and solvent B  with a flow rate of 1 mL/min [The frankincense methanol extract was separated using a gradient elution of solvent A  with a flow rate of 1 mL/min for 25 minutes [To determine the content of (E)-guggulsterones in myrrh, an ethanol extract was separated using an isocratic elution of 0.1% H minutes . The UV μm) was obtained from SGE Analytical Science . Chromatographic conditions were as follows: helium used as a gas carrier; a constant flow of 1.0 mL/min; 2 μL injection volume (splitless model), and a 280°C injector temperature. The GC temperature program was as follows: 90°C (1 min), 90°C to 130°C (41 min), 130°C (10 min), and 200°C (3 min). An Agilent 5973N quadrupole mass spectrometer was operated in selective ion monitoring (SIM) mode, with ionization source by electron impact at 70 eV, transfer line at 280°C, ion source at 280°C, and quadrupole at 150°C.Chiral GC was used to determine the ratio of stereoisomers in synthetic borneol. Analysis was performed using an Agilent GC system model HP 6890N  equipped with a split/splitless injector, liner of silanized quartz with a 4 mm i.d. (effective volume 0.49 mL), and an Agilent 6890 autosampler for 100 vials. A Cydex-B chiral GC column .Confluent HaCaT cells in 12-well plates were starved overnight in DMEM medium. The surface of the plate was scraped with a 200  in vitro wound healing assay by “Jinchuang ointment” were also examined using human microvascular endothelial cells (HMEC-1). Ibidi Culture-Inserts  were placed on the chamber of 24-well cell culture plates. About 70 μL of HMEC-1 (5 × 105 cells/mL) was seeded per well and plates were incubated at 37°C and 5% CO2. After 24-hour incubation, Ibidi Culture-Inserts were removed, and 1 mL of MCDB131 media containing 1 μL DMSO solution of “Jinchuang ointment” was then added into individual wells. The migration of cells was observed by microscopy over a period of 6–24 hours. The gap size was measured by using software Image J.Stimulation effects on4/well) were seeded in 96-well plates. The medium containing “Jinchuang ointment” at various concentrations was then added after cell adhesion. Cells were incubated in DMEM medium for the indicated time. The proliferation of HaCaT cells was subsequently determined by using Cell Proliferation Reagent WST-1 . The statistical method used is Student's t-test.HaCaT cells . Membranes were blocked for 1 h with 5% low-fat milk powder solubilized in phosphate-buffered saline (PBS) containing 0.05% Tween 20. Levels of Cdc25b, Cdc25c, CDK2, CDC D2, Cyclin B, Cyclin D3, and 5 cells/well) was placed into the wells of a 24-well flat bottomed plates precoated with 200 μL Matrigel . Cells were then mixed with 1 mL medium containing “Jinchuang ointment” . Cells were incubated at 37°C for a 24 h exposure [Human umbilical vein endothelial cells (HUVEC) were bought from Bioresource Collection and Research Center . 1 mL of HUVEC . A To the best of our knowledge, there are no English language case reports describing “Jinchuang ointment” treatment for nonhealing diabetic wounds. Three different cases are presented in this study. The first subject, Mrs. Wu, is a 75-year-old female patient with type II insulin-dependent diabetes accompanied by peripheral arterial occlusion disease (PAOD) which led to left lateral leg and ankle necrotizing fasciitis. She was treated with percutaneous transluminal angiography (PTA) to improve lower limb circulation on Feb 6, 2013. As a result of reperfusion injury, the ulcer had enlarged with erythema. After examination, a below-knee amputation was immediately scheduled two weeks later at the Surgery Division, the China Medical University Beigang Hospital. As suggested by a doctor from the Division of Chinese Medicine, she decided to use traditional Chinese medicine to treat her wound. She was referred to the Division of Chinese Medicine for wound management. Normal saline was first used to clean the wound. About 2-3 g “Jinchuang ointment” was applied directly to the wound once daily. Pictures depicting wound healing under treatment with “Jinchuang ointment” are shown in Mr. Tsai, a 71-year-old male, with past history of type II diabetes was diagnosed on September 8, 2013. He had a 3 × 0.8 cm and a 4 × 1.5 cm grade 3 pressure sore in the sacral region. 1 g of “Jinchuang ointment” was applied topically to the wound area once per day. Pictures documenting wound healing under treatment of “Jinchuang ointment” are shown in Mr. Wang, a 64-year-old male with a past history of hypertension, had a chronic wound measuring 6.2 × 5.3 cm which had not healed for more than six months. He received the topical application of “Jinchuang ointment” once per day beginning November 26, 2014. A great improvement was observed after two months of treatment as shown in One of the main problems associated with herbal medicine is the high level of batch to batch variation in the amounts of active components. The content of pure chemical reference standards in herbal products is therefore used as an indicator for the purposes of quality control and standardization. Accordingly, the content of reference standards in dragon's blood, catechu, frankincense, and myrrh used was measured by HPLC in this study. All the calibration curves of reference compounds were linear over the concentration range studied . A linea Daemonorops draco. It possesses antimicrobial, anticancer, and cytotoxic activity [Dracorhodin is a red anthocyanin pigment that is a major component in “dragon's blood” resin of the plantactivity , 10.  FiBoth catechin and epicatechin are phenol-type antioxidants in catechu, an extract of acacia trees. Catechu is a common component of herbal medicine. In addition to their ability to scavenge free radicals in plasma, the health benefits of catechin and epicatechin also include stimulation of fat oxidation, expansion of the brachial artery, and resistance of LDLs to oxidation . At the  Boswellia. In Africa and Asia, it is widely used in incense, perfume, and traditional medicine. Boswellic acids, a series of pentacyclic triterpene molecules, are one of the major components of frankincense. Theiranti-inflammatory properties and ability to induce cancer cell apoptosis have been reported in vitro [β-boswellic acid derivatives and acetyl-boswellic acid [β-boswellic acid in frankincense. The mass percentage of acetyl-11-keto-β-boswellic acid in the frankincense used in this study is 1.62%.Frankincense is a resin from plants in the genusin vitro –17. The lic acid , 18.  Fi Commiphora. The usage of myrrh is similar to that of frankincense. In fact, both myrrh and frankincense are frequently used in concert in many traditional Chinese medicine recipes. In western medicine, myrrh is also used in liniment and healing salves for minor skin ailments. The chemical composition of myrrh is rather complicated [Myrrh is also a resin from plants in the genusplicated . Notablyplicated . Both isplicated . The mas Blumea balsamifera have enjoyed more widespread use. The price of synthetic borneol is the lowest among the sources discussed above. Previous results show that borneol stereoisomers can interact with GABA receptors [Borneol is a bicyclic monoterpene plant secondary metabolite. (+)-Borneol, mainly isolated from the plant family of Dipterocarpaceae, is the major form used for analgesia and anesthesia in traditional Chinese medicine. More recently, chemically synthesized borneol and (−)-borneol isolated from the plant specieseceptors , 23 and eceptors . Chiral μg/mL, 20 μg/mL, and 2 μg/mL “Jinchuang ointment” treatment was 78.2 ± 5.3%, 78.2 ± 4.3%, and 63.8 ± 1.7%, respectively. In contrast, the negative control HaCaT cells without “Jinchuang ointment” treatment only showed 64.4 ± 4.0% wound closure. The positive control treated with 100 ng/mL EGF treatment showed 71.8 ± 1.0% wound closure. It is obvious that application of 200 μg/mL or 20 μg/mL “Jinchuang ointment” is more potent than 100 ng/mL EGF in promoting in vitro wound closure, showing statistically significant differences when compared to both positive and negative controls /initial wound area × 100%. The wound closure percentage with 200 controls .μg/mL “Jinchuang ointment” 24 hours after treatment was 85.0 ± 12.3%, whereas the wound closure percentage observed with the DMSO control was only 43.3 ± 8.2%  by WST-1 assay. The increased percentages of cell proliferation by 200 ectively . These rTo further investigate the underlying mechanisms involved in “Jinchuang ointment”-induced effects on cell proliferation in HaCaT cells, the expression of several key cellular proteins involved in cell cycle progression was investigated by western blot analysis. As shown in  in vitro angiogenic effect of “Jinchuang ointment” on HUVEC cells. As shown in μg/mL “Jinchuang ointment” for 24 hours can efficiently induce endothelial cell capillary tubes and network formation.The process of wound healing has been divided into three different stages, inflammatory, proliferative, and remodeling phases . Angioge in vitro wound healing and tube formation, were used to examine activity. Our results show that this herbal medicine possesses potent activities stimulating cell proliferation, migration, and angiogenesis. This provides a scientific rationale to account for the observed clinical curative effects on wound healing by “Jinchuang ointment.”“Jinchuang ointment” is a Chinese herbal medicine complex. It has been clinically used in the treatment of diabetic foot infection and decubitus ulcers in China Medical University Hospital for more than ten years. Because of its complicated composition, its biological activities have never been investigated. To further characterize its herb ingredients, the content of reference standards present in dragon's blood, catechu, frankincense, and myrrh was determined by HPLC. Two cell based assay platforms,According to current pharmaceutical regulations in Taiwan, only traditional Chinese herbal medicine manufactured from cGMP pharmaceutical factories can be sold in drug stores or by hospital marketing channels. However, for homemade traditional Chinese herbal medicine, they can only be administered to patients by the doctors who made the respective medicine. For this reason, “Jinchuang ointment” cannot be widely used in the Taiwan area since many clinicians are not able or unwilling to prepare this remedy. To facilitate the manufacturing process of “Jinchuang ointment” by cGMP factories, it will be very important to find out the activity indicator markers for components, such as dragon's blood, catechu, frankincense, and myrrh.


At high temperature, Mg can reduce not only carbon in the oxidation state of +4 in CO32− to form graphene, but also sulfur in SO42− from its highest (+6) to lowest valence which was hybridized into the carbon sp2 framework. Various characterization results show that sulfur-doped graphene with only few layers has an appropriate sulfur content, hierarchically robust porous structure, large surface area/pore volume, and highly graphitized textures. The S-doped graphene samples exhibit not only a high activity for ORR with a four-electron pathway, but also superior durability and tolerance to MeOH crossover to 40% Pt/C. This is mainly ascribed to the combination of sulfur-related active sites and hierarchical porous textures, facilitating fast diffusion of oxygen molecules and electrolyte to catalytic sites and release of products from the sites.Efficient metal-free electrocatalysts for oxygen reduction reaction (ORR) are highly expected in future low-cost energy systems. We have successfully prepared crumpled, sheet-like, sulfur-doped graphene by magnesiothermic reduction of easily available, low-cost, nontoxic CO CO2 emitted from fuel-fired power plant is captured by a well-developed chemical absorption process using aqueous amine-ammonia solutions in industrye.g. zeolites45etc) and CO2 moleculesBecause of the predicted detrimental effects  of a cell with the conventional graphene via chemical exfoliation of graphite2 (in the form of Na2CO3) in the presence of Na2SO4 as an inorganic sulfur source to graphitized, wrinkled, sulfur-doped graphene with only few layers.Except for CO2-hybridized carbon)18212325e.g. nitrogen , phosphorus , sulfur , boron , etc) with distinct electronegativity with respect to carbon atoms (electronegativity: 2.55) into sp2-hybridized carbon frameworks can remarkably modify their electronic structures and chemical activities as well as the Fermi level of the host. This strategy is simple and efficient to improve the properties of graphene. At the same time, the robust framework and most of the intrinsic properties of graphene are maintained after the doping of the heteroatoms. Chemical vapor deposition (CVD) and the solvothermal pathway have been adopted to produce doped graphene using liquid precursors27e.g. ureaetc). Compared to N- (or B-) doped graphenes, little attention has been paid to the doping of S atoms into the graphene framework3334Since the seminal work of Geim and co-workers on the preparation of single layer graphene (sp2 carbon lattice by two procedures: (I) CVD technique using liquid organics and (II) the pyrolysis of GO and S-containing precursors. For example, Gao and co-workers reported the formation of S-doped graphene on Cu foil using liquid S-hexane mixture vapor at 950°C332S, SO2 or CS2 gas is an efficient procedure of doping graphene lattice with S atoms342S-treated GO at 500 ~ 900°C has the content of 1.2–1.7 at% S, which was employed as metal-free electrocatalysts for ORR40412S, SO2 and CS2) gas flow as the S source could result in the production of large amount of exhaust waste. It is, however, yet a big challenge to directly generate S-doped graphene from easily available, low-cost, nontoxic Na2CO3 and Na2SO4 inorganic oxide salts as the C and S sources, respectively.S-containing functionalities have been successfully incorporated into the sp2 (in the form of Na2CO3) in the presence of small amount of Na2SO4 as an inorganic S source to form S-doped graphene, as shown in 32− to form black graphene products. At the same time, the SO42− ions could supply S atoms to build C-S sp2 bonds integrated into the graphene lattice; S in its highest oxidation state of +6 in SO42− is reduced to the lowest valence by Mg metal. It is observed that S-doping has a great influence on the morphologies of graphene sheets. Thus, the present novel procedure can efficiently reduce the CO2 concentration in the atmosphere by chemically fixing carbon in CO2 to valuable doped, high-quality graphene. We also showed that the as-formed S-doped graphene materials demonstrate high electrocatalytic activity for ORR with a dominant four-electron reaction pathway, as well as excellent durability and MeOH crossover compared to commercial Pt/C catalyst. Thus they can be considered as ideal candidates as metal-free ORR electrocatalysts.Herein, we have employed a one-step magnesiothermic reduction strategy to transform greenhouse gas CO2CO3, the product has a dark black color, suggesting the formation of a graphitic structure. 2CO3 at 800°C (CG-800), observed by scanning electron microscopy (SEM). Low-magnification SEM image shows that CG-800 has a loose, porous structure . SG-800 exhibits a similar morphology to that of CG-800, confirmed by low-magnification SEM image  crystal plane (0.335 nm) of bulk graphite due to slight expansion and distortion47at%) as well as O (3.6 at%) element in the graphene layers. Also, small amount of Na and Cl elements were found, which are mainly due to the trapped Na compounds between the graphene layers during the magnesiothermic reaction and the following acid-washing stepFurther observation on the morphology difference between pure graphene and S-doped graphene was confirmed by transmission electron microscopy (TEM). As shown in 0 layers . In Figuat% (ca. 0.5 ~ 0.6 at%) prepared by the pyrolysis of GO and BDS2S at high temperatures342-bonded carbons, exhibiting that all of them possess a crystalline graphene structure. Indeed, two strong and broad characteristic peaks can be observed at 26.1 and 43.3 degrees arising from the (002) and (100) planes of graphene sheets, respectivelyd-spacing of (002) crystal plane is about 0.35 nm, slightly larger than the interlayer spacing in graphite (0.335 nm). These results match well with those from TEM analysis, implying the formation of high-quality graphene structures in SG-800. In contrast, magnesiothermic reduction of CaCO3 at 800°C led to the production of amorphous carbons rather than crystalline graphene2CO3 is better carbon source for the synthesis of graphene nanostructures.In accordance with EDS analysis, X-ray photoelectron spectroscopy (XPS) survey scans of S-doped graphenes prepared at different temperatures imply the varied S contents of 1.8 ~ 2.6 at% . The S cat% 16. After2 graphitic layer) bands at ~1560 cm−1, D (related to defect in sp2 lattice) bands at ~1350 cm−1, and 2D bands at ~2725 cm−1 in the spectrum (ID/IG) is used to evaluate the defect content of the graphene. As shown in ID/IG for the resulting S-doped graphenes lower. For example, the value of ID/IG is 0.75 for the sample prepared at 700°C, and it decreases to 0.47 for SG-800, and further to 0.41 for SG-900. This implies that a higher reaction temperature is favorable for defect repair to form graphene with more perfect layers. Furthermore, it can be found that the ID/IG values of the resulting graphene materials are evidently higher than those of most chemically reduced GO−1) of CG-800 is shifted down to 1570 cm−1 for SG-800, which is an important characteristics of n-type doping of graphene by the substitution. The similar red shift is also reported for N-doped graphene49−1) for S-doped graphenes are significantly lower than 2725 cm−1 for pure graphite, showing the successful preparation of few-layer graphene nanostructures in all samples. When increasing reaction temperatures, the 2D position (2685 cm−1) for SG-700 shifts down to 2678 cm−1 for SG-800, and 2676 cm−1 for SG-900, respectively. It indicates that SG-800 and SG-900 have similar average numbers of the graphene nanosheets, both of which are smaller than the average numbers for SG-700.Further structural information about S-doped graphenes was obtained from Raman spectroscopy technique through the determination of the G  surface area, total pore volume, and pore size distribution. The corresponding nitrogen adsorption-desorption isotherms and pore size distributions for S-doped graphenes and pure graphenes are shown in 2/g and 1.887–1.936 cm3/g, respectively. These data slightly decrease to 188–199 m2/g and 1.383–1.604 cm3/g, respectively, for S-doped samples compared with those of pure graphenes. Among these S-doped graphenes, the sample prepared at 800°C demonstrates the largest specific surface area and pore volume. The specific surface areas of these graphenes are significantly lower than the theoretical surface area of 2630 m2/g for individual isolated graphene sheets0) of 0.45–1.0, which might be a result of small mesopores and intersheet textural large pores. The pore size distribution curves of pure graphenes and S-doped graphenes calculated by the Barrett-Joyner-Halenda (BJH) model, show a single well-defined peak at 3.8 nm in all curves, implying that all graphene samples have narrow mesopores. It is noted that a weak peak is found at about 35 nm, possibly ascribed to the intersheet textural pores.To get insights in pore structures of the resulting graphenes prepared by magnesiothermic reduction, the N2 carbon in the samples. Furthermore, there are very minor shoulder contributions to the XPS signals at binding energies of 286.0–288.0 eV, which are assigned to sulfur- and/or oxygen-bound carbon atoms on the material surfacee.g. S dominated in the graphene framework via the formation of the sulfide bridges (-C-S-C)2SO4 could be reduced to its lowest valence, which is then hybridized into the graphene layers during the magensiothermic reduction.To further investigate the chemical environments of S and C atoms in the graphene layers, high-resolution X-ray photoelectron spectroscopy (XPS) spectra in the C1s and S 2p regions were recorded in in-situ reactive doping of S into the graphene framework could be accomplished by magnesiothermic reduction of Na2CO3 in the presence of Na2SO4 as an inorganic S source. It is believed that Mg could reduce not only CO32− to the graphitized carbons, but also S in the highest oxidation state of +6 in SO42− to the lowest valence which was further hybridized into the carbon sp2 framework. The addition of Na2SO4 during the magnesiothermic reduction evidently not only affects their structure and morphologies, but also improves the electrocatalytic performance of the resulting S-doped graphenes for ORR as discussed below. The present S-doped graphenes possess an appropriate S content, hierarchical porous textures, and highly graphitized structure. Such unique features will promote fast access of oxygen molecules to the catalytic sites and can favor the swift diffusion of electrons in the electrode during the ORR process.It has been reported that S-doped graphenes could act as metal-free electrocatalysts for ORR. In present work, it has been proved that the 2- and O2-saturated 0.1 M KOH aqueous electrolyte at a scan rate of 50 mV/s. It was found that ORR on a GCE requires large over potentials ) with very low oxygen reduction current density (−0.38 mA/cm2); this reduction current was reported to be the two-electron reduction process of O2 to peroxide which is electrochemically mediated by the oxygen-containing groups at the surface of bare GCE. The CVs for S-doped graphenes exhibit nearly rectangular shapes, showing high conductivity with superior capacitive current. A well-defined oxygen reduction peak at ~−0.32 V (vs. SCE) is observed for S-doped graphenes in O2-saturated 0.1 M KOH solution, while there is no such a peak in N2-saturated one, indicating that O2 is reduced on S-doped graphenes  significantly increases, corresponding to methanol electro-oxidation. In contrast, no noticeable change except for a slight decrease in the current density of ORR is found for SG-800 under the same conditions. These voltammograms show that S-doped graphenes have higher selectivity for ORR with a remarkable improved ability to avoid the cross effects than commercial Pt/C. It is mainly ascribed to the highly graphitized nanostructures, which increase both the electronic conductivity and corrosion resistance of SG-800. Thus, this kind of S-doped graphenes has great potentials as the electrocatalytic material in direct methanol and alkaline fuel cells.To examine the possible crossover effects, the electrocatalytic selectivity of SG-800 and 40% Pt/C against electro-oxidation of methanol  by CVs in an O3 M MeOH . The cur2-saturated 0.1 M KOH aqueous solution at different rotating speeds on a rotating disk electrode (RDE) (sweep rate: 5 mV/s). The voltammetric profiles show that the current density is increased with an increase in rotating speeds . The S doping into the graphene framework evidently results in the positively shift of the onset potential from −0.18 V (vs. SCE) for pure graphene to −0.15 V (vs. SCE) for S-doped graphene. It implies the important role of S atoms doped into the graphene layers in adjusting chemical/electronic properties of graphene, thus improving the ORR catalytic performance of S-doped graphene. All S-doped graphene samples show the onset potentials are just negatively shifted by 0.12 V (vs. SCE) related to that (−0.04 V vs. SCE) for commercial Pt/C (e. g. N-doped graphene29etc) for ORR (vs. SCE) are significantly higher than those for all pure graphenes, strongly indicating that S atoms have been doped into the layers of graphene sheets, and have similar impact to nitrogen atoms doped into the graphene layers on the electronic structure of doped graphenes. Among these graphene samples, SG-800 demonstrates the largest ORR current density (3.3 mA/cm2) at −0.32 V , which are evidently superior or compared to heteroatom-doped porous carbon-based electrocatalysts for ORR  in an Og speeds . The corlectable , indicatial Pt/C . This va for ORR . Notably for ORR . The higvs. SCE) exhibit good linearity, the slopes of which are approximately constant. It also indicates the first-order reaction kinetics with respect to the concentration of O2 dissolved. The exact electron transfer numbers of S-doped graphene and CG-800 were calculated according to the slopes of the linear fitted K-L plots on the base of the K-L equations as followingJ and Jk are the measured current density and kinetic-limiting current density, respectively, n is the electron transfer number, F is the Faraday constant (96485 F/g), v is the viscosity of the electrolyte (0.01 cm2/S), CO2 is the concentration of O2 (1.2 * 10−6 mol/cm3), and DO2 is the diffusion coefficient (1.9 * 10−5 cm2/s). The coefficient 0.2 is adopted when the rotating speed is expressed in rpm. As displayed in 2 molecules (n = 3.1 ~ 3.8) in ORR is higher than two in the potential range of −0.5 V to −0.6 V, and SG-800 has the highest electron transfer numbers (3.7–3.8), possibly because of its highest surface area and pore volume and appropriate S content. This result indicates that the desirable four-electron reduction process for SG-800 to form water as the main product other than peroxide is predominant. In contrast, CG-800 has n value of ca. 2.2, which suggests that the two-electron reduction process with peroxide as the main product is dominant. Note that four-electron process is very important for ORR in fuel cells, because peroxides can poison the cell. This finding matches well with previous reports indicating that pure reduced GO can cause a two-electron pathwayBy using the Koutecky-Levich (K-L) equation, we further analyzed the RDE data collected by LSVs. As shown in vs. SCE) in O2-saturated 0.1 M KOH solution at a scan rate of 200 mV/s, implying the excellent capacitance stability of SG-800. Moreover, we also conducted a 3.5 h stability test for SG-800 and 40% Pt/C toward ORR using the i-t chronoamperometric response at a constant voltage of −0.4 V (vs. SCE) in a O2-saturated 0.1 M KOH solution at a rotating speed of 1600 rpm. As shown in 2SO4 as the S source is compared to commercial Pt/C for ORR.Another major concern is the stability of the S-doped graphene used as high-performance electrocatalyst for ORR. Accordingly, the continuous potential cycling to investigate the stability of SG-800 was performed. As shown in 2 into the atmosphere. By magnesiothermic reduction of Na2CO3 and Na2SO4 as low-cost, nontoxic carbon and sulfur sources, respectively, S-doped graphenes with few layers have been successfully prepared. The melting Mg can reduce carbon in the oxidation state of +4 in carbonate to form black graphene products. And sulfur in sulfate also can be reduced from its highest oxidation state to lowest valence and hybridized into the carbon sp2 framework. The resulting graphenes possess crumpled, sheet-like nanostructures with hierarchically porous textures and the addition of sulfate during the magnesiothermic reaction results in the formation of S-doped graphenes with more distorted nanostructures. The sulfur doping of the graphene framework evidently decreases the ORR onset potentials and increases limited current densities compared with pure graphenes. The resulting S-doped graphenes demonstrate not only high electrocatalytic activity for ORR with a four-electron reaction pathway, but also much better stability and increased tolerance to MeOH crossover effects than commercial Pt/C. The high activity for ORR of S-doped graphenes is mainly ascribed to highly graphitized structures, S-related active sites, and hierarchically porous textures. The present magnesiothermic reduction procedure is being investigated in the preparation of other heteroatom-doped (e.g. P and B) graphene materials potentially utilized in the fields of electrocatalysis, batteries, supercapacitors, biosensors, etc.In summary, this work has shown an efficient strategy to chemically fix carbon to form valuable, high-quality, doped graphenes, which also can efficiently reduce the emission of CO2CO3 and Na2SO4 were used as the C and S sources, respectively, and magnesium (Mg) powder were utilized as the reducing reagent. A calculated amount of these powders were mixed together and then put onto an alumina boat which was inserted into a stainless steel tube. The reaction temperature was controlled to be 700, 800, or 900°C for 1 h at a ramp of 5°C/min under argon (Ar) atmosphere. After cooling to room temperature, the resulting black solids were dispersed in sufficient 10% HCl solution and vigorously stirred for 4 h to remove any inorganic impurities. The carbon was collected by filtration and purified by distilled water and ethanol for three times each. Finally, the filtered product was dried at 80°C overnight and further annealed in Ar atmosphere. And the resulting S-doped graphene was denoted as SG-X, where SG is the abbreviation of S-doped graphene and X stands for the magnesiothermic reaction temperature in °C. For comparison, pure carbon-based graphene (CG) samples were synthesized by magnesiothermic reduction of Na2CO3 without any Na2SO4.Anhydrous Nahυ = 1486.6 eV) radiation. The graphene powders were glued onto indium (In) metal particles by pressing for measurements. All spectra were calibrated using 285.0 eV as the line position of adventitious carbon. Nitrogen sorption isotherms were measured using a Micromeritics ASAP 2010 surface area and pore size analyzer at liquid nitrogen temperature (−196°C). Prior to measurement, the hierarchically porous materials were dehydrated under vacuum at 200°C overnight. The specific surface areas were calculated by the Brunauer-Emmett-Teller (BET) method. The total pore volume was calculated from the amount of nitrogen adsorbed at a relative pressure of 0.99. The pore size distribution curves were calculated from the analysis of the desorption branch of the isotherm using the Barrett-Joyner-Halenda (BJH) model.Transmission electron microscopy (TEM) images of the graphene were recorded using a JEOL 2010F electron microscope equipped with an energy dispersive spectrometry (EDS) attachment operating at 200 kV. Samples for analysis were deposited on a thin amorphous porous carbon film supported by copper grid from ultrasonically processed ethanol solutions. The morphology of the graphene was observed by scanning electron microscopy (SEM) using a field emission microscope . Samples were mounted using a conductive carbon double-sided sticky tape. Raman spectra were recorded on a DXR Raman Microscope  with 532 nm excitation length. X-ray powder diffraction (XRD) measurements were carried out on a Rigaku D/MAX-2250 V diffractometer with Cu Kα radiation. X-ray photoelectron spectroscopy (XPS) was recorded with an ESCALAB 250 X-ray photoelectron spectrometer with Al Kα (ca. 2 h. Then 5% Nafion solution (25 μL) was added into the above suspension, and the further ultrasonic treatment was performed until a homogeneous ink was formed. 20 μL ink containing 62.5 μg catalyst was transferred onto the glassy rotating disk electrode with 5 mm diameter, yielding a catalyst level of 0.32 mg/cm2. For 40% Pt/C commercial catalyst (JM company), 31.2 μg catalyst was deposited on the glassy rotating disk electrode. The electrode with the catalyst was dried at 50°C which was used as the working electrode for further electrochemical measurements. Electrochemical activity of the working electrode was studied by cyclic voltammetry (CV) and rotating disk electrode (RDE) using a standard three-electrode cell with a Pt plate as the counter electrode and a saturated calomel electrode (SCE) as the reference electrode. An aqueous solution of 0.1 M KOH was used as the electrolyte for the electrochemical studies. The electrochemical cell was controlled by a bipotentiostat (Pine Instrument Co.) equipped with a RDE. Prior to the measurements, the electrolyte was saturated by bubbling O2 or N2 for 15 min. The working electrode was cycled at least thirty times before the CV data were recorded at a scan rate of 50 mV/s. The RDE measurements were performed at different rotating rates varying from 400 to 2025 rpm with the scan rate of 5 mV/s.The graphene sample (5 mg) was dispersed in the mixture of 0.5 mL water and 0.5 mL ethanol under ultrasonic irradiation for J.W. performed the experiments, and wrote the manuscript; R.M., Z.Z. and G.L. analyzed the data; Q.L. designed the experiments; all authors reviewed the manuscript.supporting information


With the support of SiO2, the stacked interlamination of graphene was held open to expose the powerful adsorption sites in the interlayers. The adsorption of phenanthrene, a model aromatic pollutant, onto the loaded graphene nanosheets increased up to 100 fold compared with pristine graphene at the same level. The adsorption of GCMs increased with the loading amount of the graphene nanosheets and dramatically decreased with the introduction of oxygen-containing groups in the graphene nanosheets. The highly hydrophobic effect and the strong π-π stacking interactions of the exposed graphene nanosheets contributed to their superior adsorption of GCMs. An unusual GCM peak adsorption coefficient (Kd) was observed with the increase in sorbate concentration. The sorbate concentration at peak Kd shifted to lower values for the reduced graphene oxide and graphene relative to the graphene oxide. Therefore, the replacement of water nanodroplets attached to the graphene nanosheets through weak non-hydrogen bonding with phenanthrene molecules via strong π-π stacking interactions is hypothesized to be an additional adsorption mechanism for GCMs.The substantial aggregation of pristine graphene nanosheets decreases its powerful adsorption capacity and diminishes its practical applications. To overcome this shortcoming, graphene-coated materials (GCMs) were prepared by loading graphene onto silica nanoparticles (SiO This type of composition has been used in solid-phase extraction material2 and the effects of graphene loading percentage on their adsorption performance need to be investigated for practical fabrication and environmental applications; and the true potential of the adsorption mechanisms of the loaded graphene nanosheets need further elucidation to consider the effects of OCGs on the graphene-water interface. Silica (SiO2) is an ideal framework material for mitigating graphene aggregation because it is an inexpensive, nontoxic and widespread material that can be manufactured in megatons per year302 in the environment, interactions between SiO2 and graphene are unavoidable when graphene is discharged into the environment. Thus, the adsorption capabilities and mechanisms of graphene-coated silica composites with different loadings and various OCG contents are worth further investigation.One promising strategy is to develop graphene-based materials by loading graphene nanosheets onto low-cost substrates192 particles with different loading percentages to probe the effects of graphene loading percentages and the OCG contents. The structures of SiO2, NH2-SiO2, pristine graphene and GCMs were characterized by elemental analysis, surface area, surface charge, FTIR, SEM and TEM to reveal the materials’ microscopic properties. Phenanthrene was selected as a model aromatic pollutant to determine the materials’ adsorption abilities and as a probe molecule to understand the interaction mechanisms at the graphene-water interface. In addition, the roles of attached water molecules on different graphene-water interfaces are discussed.The main objective of this study is to fabricate a highly efficient graphene-based adsorbent for organic pollutant management. To optimize practical performance of graphene materials in pollutant management, a series of GCMs were prepared by loading graphene oxide (GO), reduced graphene oxide (rGO) and graphene (G) nanosheets onto SiO2-SiO2 (surface area = 109 m2/g) can support 82.9 mg of monolayer graphene (surface area = 2630 m2/g) at maximum degree. Four different SiO2-graphene ratios  were designed to investigate the maximum loading percentage considering the surface area reduction of NH2-SiO2 caused by aggregation. As presented on 2-SiO2 (positive) and graphene oxide (negative) were oppositely charged at pH 1 to 8; thus, the self-assembly of graphene oxide on NH2-SiO2 was easily performed via electrostatic interactions. The synthesis pH was controlled at 7 to maintain the maximum surface potential difference between graphene oxide and NH2-SiO2, according to surface potential analysis. The digital images presented in 2, as well as the fact that not all of the graphene oxide completely combined with the SiO2. Once the loading percentage surpassed 2%, large quantities of graphene oxide were suspended in the supernatant  and with various degrees of reduction . The macroscopic morphologies of the obtained GCM powders are shown in 2 was added. In this case, no graphene oxide was dissolved, even after ultra-sonication  of graphene sheets. The surface areas of GCMs largely depended on SiO2, when graphene was wrapped around SiO2 particles. There were no changes in the surface area of GCMs with different reduction processes and loading percentages. After wrapping, the loaded graphene was unfolded after being attached to the surface of the SiO2, which was demonstrated by the SEM and TEM investigations /(mg/L)N] is the Freundlich affinity coefficient, N is the exponential coefficient, Qe (mg/g) is the equilibrium adsorption concentration of the sorbent, and Ce (mg/L) is the equilibrium solution phase concentration of the sorbate. According to the regression coefficient (R2), the isotherms of the nine different graphene-based materials were well fitted by the Freundlich model. The Freundlich N index was 0.698−0.612 for ASGO at 100:0.5, 100:1 and 100:2, and decreased to 0.387−0.324 for ASrGO and ASG, suggesting that the surfaces of the ASrGO and ASG were more heterogeneous than those of ASGO. The adsorption of GCMs increased as the number of OCGs on the graphene nanosheets decreased (where ecreased  and increcreased . These o2-SiO2. Based on Qe) at Ce/Cs = 0.01 was 3.62 × 10−2 mg/g for ASGO (100:2) which was approximately 660 times higher than that of SiO2 (5.44 × 10−5 mg/g)Qe at Ce/Cs = 0.01 for ASrGO (100:2) further increased to 1.08 mg/g and up to 1.74 mg/g for ASG (100:2) after deeper chemical reduction. The adsorption capacity of ASG (100:2) was approximately 32,000 times greater than that of bare SiO2. Meanwhile, the exact graphene content in ASG (100:2) was only 2.35% according to the elemental analysis. Considering the widespread existence of SiO2 in the environment and the mass production of graphene, the combination of graphene and SiO2 is unavoidable. Companied with graphene’s commercial process, this dramatic improvement in the adsorption of SiO2 should increase our focus on controlling graphene as an environmental risk. Similarly, the adsorption capacity of GCMs with loading percentages of 100:1 and 100:0.5 (Qe of pristine graphene (2.45 mg/g at Ce/Cs = 0.01)2 with phenanthrene, the adsorption capacity of graphene in ASG (100:2) was 73.73 mg/g, which was 30 times greater than that of pristine graphene (2.45 mg/g) (based on the same graphene content at Ce/Cs = 0.01). This surprising increase indicated that the potential adsorption capacity of the well-dispersed graphene was successfully exposed in higher graphene loading percentage and that the results were higher than the relevant data presented in previous reports1723According to the loading percentage test, the maximum graphene loading degree is 2% under which graphene oxide can mostly be loaded onto the surface of NH 100:0.5  increase2 produced a synergistic effect in aromatic pollutant removal, and the large-scale improvements in the adsorption of graphene suggested that wrapping graphene around SiO2 particles is a facile method for using graphene to manage environmental pollution. In addition to the degree of reduction, the influence of the graphene loading percentage on the adsorption performance was also investigated , ASG (100:1), and ASG (100:2) were 0.424, 0.775, and 1.74 mg/g, respectively, at Ce/Cs = 0.01. When normalized by the graphene contents of the GCMs, the adsorption of phenanthrene onto wrapped graphene in ASG (100:0.5), ASG (100:1) and ASG (100:2) were 71.86, 52.01 and 73.73 mg/g, respectively, at Ce/Cs = 0.01. This excellent adsorption performance illustrates that the potential adsorption ability of the graphene nanosheets was readily available after the graphene was applied to silica particles under the loading ratio below 2%. Because the adsorption performance of the GCMs was influenced by the synthesis conditions, the ratio of graphene to SiO2, and the oxygen content in the graphene surfaces, this study provides a reference for fabrication parameters in designing optimal graphene-coated materials for potential environmental applications.The combination of graphene and SiOstigated . The Qe Kd = Qe/Ce) was introduced. To better exhibit the mutual interaction mechanism beyond the hydrophobic effect, the normalized adsorption coefficient (Kd) relative to the hexadecane-water partitioning coefficient of phenanthrene (KHW = 35481 L/kg) was also applied17Kd/KHW values of phenanthrene adsorption on the GCMs are presented in Kd/KHW values of phenanthrene adsorption on ASG (200:0.5) was cited from our previous study for comparisonKd/KHW ratio increased with the sorbate equilibrium concentration and formed a sharp peak at relatively low concentration ranges. This uncommon phenomenon for the adsorption of GCMs occurred by varying the concentrations of the sorbate over 3 orders of magnitude rather than over a narrow concentration range. The increase in Kd/KHW with increasing sorbate concentration suggests that greater surface energy adsorption sites were created on the graphene nanosheets during the initial adsorption stage. With more phenanthrene attached to the graphene surface, the original and subsequently created sites were gradually occupied, resulting in a decline in the Kd/KHW. A sharp peak in Kd/KHW appeared for all ASGO, ASrGO, and ASG samples. The turning point clearly shifted to lower equilibrium concentrations from ASGO, to ASrGO, and further to ASG, with enhanced reduction degree Kd/KHW values based on the graphene contents of different GCMs are compared in Kd/KHW values of phenanthrene on ASrGO (100:2) and ASG (100:2) were approximately 75 and 205 times greater, respectively, than that of ASGO (100:2). The maximum Kd/KHW ratio of graphene in ASG (100:2) was 1280, which was approximately 100 times greater than that of pristine graphene. However, the enrichment capacity of loaded graphene with regard to aromatic molecules was almost 1000 times higher than biological interfaces, which can be determined because KHW is widely used as a surrogate to simulate organic molecule partitioning on biological interfacesKd/KHW (1280) for phenanthrene on ASG was much higher than for other popular adsorbents, such as single-walled carbon nanotubes (maximum Kd/KHW = 280 for phenanthrene)Kd/KHW = 0.493)Kd/KHW  = 1.13 × 10−3)Kd/KHW = 1.83 × 10−4)Kd/KHW = 1.13 × 10−3)Kd/KHW = 0.564 and 0.324–1.27)402 nanoparticles acted as a superior sorbent for aromatic pollutant removal.Based on 2 nanoparticles are known to possess a hydrophilic surface with a large quantity of –OH322 nanoparticles are naturally covered by a hydration shell30312 surface, the adsorption of phenanthrene onto SiO2 in the aqueous solution was minimal. The Freundlich N index of SiO2 (1.06)2 and phenanthrene primarily resulted from the distribution effect2 involved its transfer from the bulk liquid phase to the vicinal water phase2 showed little change because the bound –NH2 was a monolayer and multiple –OH groups were preserved2 particles, the adsorption capacity of the GCMs significantly improved. This improvement was mainly attributed to the existence of graphene nanosheets acting as a hydrophobic cloth. Once wrapped, the hydrophilic surface of the SiO2 particles became hydrophobic and possessed an additional π-electron conjugated structure. The strong π-π interaction between the phenanthrene and graphene nanosheets was accompanied by a hydrophobic interaction, replacing the weak distribution behavior in the vicinal water phase. Consequently, the GCMs displayed an excellent affinity for phenanthrene.As a well-studied nanomaterial, SiO2 particles, the adsorption capacities of the coated graphene on the SiO2 significantly improved relative to pristine graphene at the same concentration. After wrapping around the surface of the SiO2 particles, the microscopic morphology of graphene was loose and porous 4845472 substrate, both highly oriented water clusters and water adsorbates can lead to graphene dopingIn aqueous solutions, water molecules are unavoidable adsorbates on hydrophobic surfaces of graphene nanosheets4546et al. also suggested that aromatic molecules may be better aligned on graphene sheetsKd/KHW values at low concentration ranges and may provide new insights into the phenanthrene adsorption mechanisms at the graphene-water interface.The hydrated shell functions as a hydrophilic barrier to weaken the hydrophobic character of the graphene and further prevents π-π interactions between the phenanthrene and the graphene oxide surface . TherefoKd/KHW values shifted to lower values from ASGO to ASrGO and ASG.The distributions of water clusters and water microdroplets/nanodroplets are regulated by oxygen-containing groups, which are influenced by the degree of reduction of the graphene-coated materials. During the reduction process, the π electron structure and hydrophobic surface of the graphene are recovered17502 particles, which then act as a framework. The potential adsorption sites concealed in stacked graphene were exposed in the GCMs, strongly enhancing the adsorption of the graphene nanosheets. Through surface potential manipulation, the potential adsorption capacity can be well expressed at its maximum loading degree (2%). Additionally, the separation of hydrophobic and hydrophilic graphene materials from water improved when SiO2 particles were used. The water molecules (microdroplets) attached to the exposed graphene nanosheets via non-H bonds are easily replaced by phenanthrene molecules, and the displacement efficiency can be regulated by oxygen-containing groups of graphene nanosheets. The graphene nanosheets and silica particles produce a synergetic effect in pollutant-removing GCMs, presenting a facile method for large-scale production of graphene-coated materials. Graphene-coated materials using silica particles as a framework are cost-effective and highly efficient adsorbents for removing aromatic pollutants from water and will result in enormous opportunities for using novel 2D nanomaterials (graphene) for environmental applications.The substantial aggregation of graphene nanosheets can be easily overcome via the coating of SiO2 nanoparticles were first modified using a silane coupling agent (APTES) to transform the surface potential by binding monolayer –NH22 particles were dispersed in deionized water (50 mL) by stirring. A selected concentration of graphene oxide solution (50 mL) was prepared using ultrasonication. Subsequently, the graphene oxide solution was gradually added to the mixed NH2-SiO2 colloids using continuous mechanical stirring. The whole process was kept at 35 °C for 1 h, and the final pH was controlled at 7. After completion of the reaction, the mixture was undisturbed for 2 h to observe the settling situation. The final aminosilica-supported graphene oxide (ASGO) was collected through filtration, washed with water and dried under vacuum. The loading percentage of graphene was controlled by modulating the initial concentration of the graphene oxide solution and was determined by elemental analysis. ASGO (100:0.5) was synthesized by adjusting the initial concentration of the GO solution to 0.1 mg/mL to obtain the SiO2-to-GO ratio of 100:0.5. GO solutions of 0.2 mg/mL, 0.4 mg/mL and 0.8 mg/mL were used for ASGO (100:1), ASGO (100:2) and ASGO (100:4), respectively.Graphene oxide (GO) was prepared from natural graphite flakes using a modified version of the Hummers method2, respectively. Compared with solvothermal reduction, the degree of deoxygenation via chemical reduction was much higherSolvothermal reduction and chemical reduction were used to deoxygenate of graphene oxide in the ASGO to prepare the reduced graphene oxide (rGO) and graphene (G) loading on SiO2 adsorption-desorption at –196 °C with a NOVA-2000E surface area analyzer and were calculated using the multipoint Brunauer-Emmett-Teller (BET) method. The exact concentrations of C, H, and N were determined using an EA112 CHN elemental analyzer (Thermo Finnigan). The surface charge properties were evaluated at different equilibrium pH values using a Malven Zetasizer analyzer. The surface functional groups were monitored with a Bruker Vector 22 FTIR spectrometer at a resolution of 1 cm−1 using a mixture of 1 mg graphene-coated materials and 200 mg KBr. The surface morphologies and structures were characterized using an S-4800 field-emission scanning electron microscope (FE-SEM)  and a high-resolution transmission electron microscope (HR-TEM) . For the FE-SEM observations, samples were placed on the conductive base without any extra treatment to detect the actual morphologies and gold nano-film was sprayed for better conductivity. For the HR-TEM observations, the samples were dispersed in a water solution to yield individual particles and dropped onto the micro grid network.The surface areas of the GCMs were measured using N2 and 200 mg/L NaN3 to control the ionic strength and inhibit microbial activity). For any given GCM, the adsorption capacities obtained after 36 h and 72 h were the same, suggesting that equilibration was achieved before 36 h. Isothermal experiments with different materials were conducted at selected water-to-solid ratios to maintain removal percentages of 20%–80%. Different volumes of phenanthrene in acetonitrile were dissolved in the background solution to achieve the desired concentration. The volume of acetonitrile in the background solution was maintained at less than 0.1% to avoid cosolvent effectsPhenanthrene was selected as the model aromatic pollutant to explore the adsorption capacity of GCMs with different loadings and various oxygen contents. Adsorption experiments were conducted at room temperature (25 ± 1 °C) in a background solution .


The progression of amyotrophic lateral sclerosis (ALS) through the brain has recently been staged using independent neuropathological and neuroimaging modalities. The two schemes tie into the concept of pathological spread through corticofugal axonal transmission that stems from observation of oligodendrocyte pTDP-43 aggregates along with neuronal inclusions. Here, we aimed to assess evidence of transmission along axonal pathways by looking for pTDP-43 oligodendrocyte pathology in involved white matter tracts, and to present a first validation of the neuropathological staging scheme.N = 34). Double-labelling immunofluorescence was performed to confirm co-localisation of pTDP-43 immunoreactivity to oligodendrocytes.pTDP-43 immunohistochemistry was performed in select white matter tracts and grey matter regions from the staging scheme in postmortem-confirmed ALS cases  and disease duration (shorter duration than Stage 3 and similar to Stage 2).Four stages of ALS neuropathology can be consistently identified, although evidence of sequential clinical progression requires further assessment. As limited pTDP-43 oligodendrocyte pathology in deep corticospinal and other white matter tracts from the motor cortex was observed, the propagation of pathology between neurons may not involve oligodendrocytes and the interpretation of the changes observed on neuroimaging should be modified accordingly.The online version of this article (doi:10.1186/s40478-015-0226-y) contains supplementary material, which is available to authorized users. Motor neuron disease covers a group of neurodegenerative disorders that affect different parts of the motor system, be it the upper motor neurons in the primary motor cortex and brainstem or the lower motor neurons in the spinal cord. Amyotrophic lateral sclerosis (ALS) with both upper and lower motor neuron involvement is the most common presentation, accounting for more than 75 % of cases with motor neuron disease , 2. The Recently, Brettschneider and colleagues defined four neuropathological stages of ALS , proposiThe idea of disease propagation from one brain region to the next has also been reinforced by cell models  and neurWhile white matter involvement is a prominent feature in neuroimaging investigations of the ALS brain and substantiates the idea of spread along these tracts, to our knowledge no study has tried to verify this concept by looking for the presence of pathology in the core white matter regions implicated. The primary objective of this investigation was to identify pathological evidence of transmission along axonal pathways by looking for pTDP-43 oligodendrocyte pathology in regions implicated by neuroimaging, namely the corticospinal tract in the posterior limb of the internal capsule, the corpus callosum and the cingulum bundle . We alson = 21 males, n = 13 females, age range = 47-81 years) and were matched for age and sex to controls with no evidence of a neurological or neuropathological disease . ALS cases had variable deficits at disease onset, including upper limb onset (n = 11), lower limb onset (n = 14) and bulbar onset (n = 9), but these subsets did not differ in age at onset, age at death or disease duration  was taken from the hemisphere contralateral to the side first affected clinically, in 4 cases (12 %) the sampled region was ipsilateral to the first affected side and in 11 cases (32 %) laterality of onset was bilateral or unknown. In all but three cases, validation of grey matter disease staging was performed using the following recommended blocks [ % was taThe following regions implicated in the neuroimaging literature –10 were Validation of the ALS neuropathological staging scheme was performed in all but three cases by assessing the presence (+) or absence (−) of pTDP-43 pathology in the regions listed above. Based on the regional involvement outlined in Table The severity of Betz cell loss and pTDP-43 pathology  was also evaluated. For Betz cell loss, the motor cortex from 32 the cases was assessed in sections taken from 3–5 consecutive blocks by an experienced neuropathologist, and the number of healthy-looking, normal-sized Betz cells in the lower half of the motor strip in each section was graded from 0 (no Betz cells) to 3 . The scores from each section were averaged to produce a final grade for each case.For pTDP-43 severity, the densest region of pTDP-43 pathology in the grey and white matter of 31 cases was independently graded under a 20x objective using a semi-quantitative scoring system: 0- pathology absent; 1- sparse pathology with no more than 5 inclusions in the entire region; 2- mild pathology with on average 1–5 inclusions in the densest region; 3- moderate pathology with an average of 6–10 inclusions in the densest region; 4- severe pathology with an average of 11 or more inclusions in the densest region  revealed <8 % difference between two investigators.Double-labelling immunofluorescence was performed using antibodies for ALS neuropathology  and oligodendroglia   to confiχ2 analyses performed to determine relationships between clinical and pathological variables. p <0.05 was considered statistically significant.All statistical analysis was performed using SPSS software . Means and standard deviations are shown. Kruskal-Wallis and Mann–Whitney U statistics were used to determine differences between groups and Spearman rank correlations and As expected, all ALS cases were immunopositive for pTDP-43 pathology in one or more of the grey matter regions examined: i.e. the motor cortex, brainstem, sensory cortex, striatum and hippocampus see Fig. . As expeIn the motor cortex, pTDP-43 immunopositive neuronal cytoplasmic inclusions Fig. , glial cThe distribution of ALS cases across the neuropathological stages is outlined in Table The severity of Betz cell loss was variable across all stages and did not influence the severity of pTDP-43 pathology in the motor cortex. Correlations revealed no relationship between the loss of Betz cells and disease stage or the severity of pTDP-43 pathology  with the grey matter having more severe pTDP-43 pathology on average . While the severity of pTDP-43 pathology in the motor cortex and the underlying white matter did appear to increase with stage, the stages themselves did not appear to reflect the same clinical course of disease for all groups (see below).The severity of pathology in the motor cortex Fig.  and its age Fig. . A signin = 6), double-labelling immunofluorescence was performed to confirm that the pTDP-43-immunoreactive aggregates observed in the motor cortex and underlying white matter were indeed mainly in neurons and oligodendrocytes. Double labelling with the oligodendrocyte specific p25α antibody showed pTDP-43-immunoreactive inclusions either surrounding oligodendroglial nuclei . However, the ALS cases with the greatest spread of pathology (Stage 4) were significantly older at onset and had short disease duration, suggesting a difference between this group and other disease stages. Further exploration of these findings is warranted, especially in ALS cases with co-existing dementia as such cases were excluded from this study.The present study validates the neuropathological staging scheme and confirms that regional assessment of pTDP-43 pathology allows for the identification of four specific stages as described previously . Our dat