There have been relatively few studies on sign language interaction carried out within the framework of conversation analysis (CA). Therefore, questions remain open about how the basic building blocks of social interaction such as turn, turn construction unit (TCU) and turn transition relevance place (TRP) can be understood and analyzed in sign language interaction. Recent studies have shown that signers regularly fine-tune their turn-beginnings to potential completion points of turns Groeber, . Moreove Subsequent research in Conversation Analysis (henceforth: CA) has further demonstrated the robustness of Sacks et al.'s  signers actively accomplish smooth transitions between the current and the next signer, collaborating thereby in a situated and collaborative construction of turns. The findings add to recent research in spoken and in signed interaction that proposes to conceive turn boundaries as flexible and interactionally achieved.With this paper I intend to add to this topic by a detailed investigation of those instances in signed interaction where participants actually overlap each other's turns, by focusing specifically on the sequential environment of these overlaps . This type of investigation sheds light on two issues. First, the analysis of the sequential moment of overlaps aims at revealing whether they present an orderly organization, as it has been shown for overlap in spoken language interaction (cf. Section Overlaps in Spoken and Signed Interaction). Second, the analysis of overlapping signing provides insight into how four participants of a signed language conversation accomplish the actual transitions from one signer to the other. On the basis of a 33-min video-recording of a multi-party interaction with 4 female signers in Swiss German Sign Language (DSGS), the paper shows that (1) signers overlap each other's turns regularly I start with providing some details with regard to turn-taking and overlap in signed languages (Section Research on Turn-Taking and Overlap in Sign Language), presenting my conception of turn and further detailing the issue of this study. Then I present the methodology and procedure I followed for the current study (Section Method), specifying the annotation practice and the established categories for analysis. In Section Sequential Environments of Overlapping Signing, I present the results on different types of overlaps before I discuss these findings in Section Discussion.sign, which consists of a distinct combination of four sublexical manual parameters, namely handshape, location (in the signing space), orientation  and movement , the stroke or independent hold  and the retraction  . Put simply, a participant can design a complete syntactic construction but prosodic resources may indicate that there is more to come . Within such a perspective, the delimitation of TCUs becomes less important an issue, because the focus of analysis is not on linguistic constructions but on the practices for the organization of activities  are traditionally conceived of as linguistic units, several researchers put into question a logocentric definition of turns-at-talk, where the turn and the TCU have to be conceived of in signed interaction are still scarce . This would be an argument to consider such non-lexical resources as proper parts of signed turns.A first fundamental issue in research on signed interaction is how to define the beginning and the end of a turn. Lackner  considerturn includes the preparation phase of the turn-initial sign. The preparation phase of a turn-initial sign is comparable to a hearable inbreath in spoken interaction, described as a pre-beginning element  and post-completions  as not being part of the core TCU, but as a part of the possible turn. Against the background of this, I maintain a differentiation between turns as participant's overall contributions including resources beyond grammar, and the grammatical units that such turns (can) host.The beginning of the stroke of a potentially last sign builds a first TRP, constituting also the end of the core grammatical unit . Consequently, holds after the stroke of the potentially last sign as well as additional gestural elements such as palm-up gestures are considered as being part of the turn. These additional elements extend the turn and finally propose a new TRP , providing a first visual index that the participant launches a turn. In case of absence of mutual gaze between participants, the incipient signer uses an attention-getting device  in order to establish recipiency. Baker . This recalls the difference that is made in spoken interaction between overlapping conducts that are troublesome and others that are not troublesome. For example, in spoken interaction an overlap between the end of a lexical unit by speaker A with the inbreath of the incipient speaker B is of a different quality than the overlap between two lexical units . An issue on this behalf is that all types of overlaps are indicated with the same transcription symbol —as mentioned in Groeber and Pochon-Berger  between four young deaf women: Denja, Nathalie, Isaline and Melinda. All participants have deaf and signing parents and learned DSGS as their L1. The data comes from a larger corpus of DSGS narratives and interactions that has been gathered within the project “Gaze and Productive Signing in a Corpus of Interactions of Deaf and Hard-of-Hearing Signers of Swiss German Sign Language (DSGS),” conducted at the University of Applied Sciences of Special Needs Education, and funded by Swiss National Science Foundation. Within this project, participants were invited to attend to a whole day of recording at a film studio producing programs in sign language. While there are some semi-experimental data, the film under study here comes from the lunch break that was recorded with the aim of having a maximally natural interaction. All data was filmed with three cameras. Two cameras were positioned to record two participants frontally, and the third camera captured all participants together.The current analysis is based on a 33-min four-party interaction in Swiss German Sign Language  that roughly describe the meaning of a sign. These glosses function as labels for a sign and are not a precise translation. In the transcript they are always written in capitals. As the project team is working with iLex, the glosses in the transcripts are automatically linked to the sign databank for Swiss German Sign Language. When a sign is not available in the lexicon, the annotators create a new gloss, which is automatically added to the lexicon.In the current state, the basic transcript consists of a content translation (done by two interpreters), an annotation of manual signs and participants' gaze conduct. Manual signs have been annotated with is the beginning of sign 2, and that there are no gaps between signs  Each overlap was tagged in ELAN on a separate analysis tier. On the basis of participants' gaze conduct I then chose to distinguish between two types of overlaps. Most frequently, overlaps occur between participants who are gazing at each other, either at the beginning, the end or over all their turns. This implies that at some point there is mutual orientation and participants can perceive their simultaneous production. Another type of overlap consists of simultaneous signing between two participants while they are addressing a third person through their action and gaze conduct. This type of overlap can go totally unnoticed by the participants  Description of the sequential environment of the overlapping turn's onset with respect to the overlapped turn. The analysis of all relevant overlaps resulted in a categorization of the overlaps in three main sequential environments:The overlap occurs at a first possible completion place reached by participant A (cf. cases A and B in the table below).The overlap occurs near the end of a unit or the potential end of a unit (but not exactly at the transition place) (cf. cases C and D in the table below).The overlap occurs in the midst of a unit (cf. case E).(3)turn-level, i.e., whether the overlap occurs at the beginning, in the midst or the end of a possible turn. The overlap onset at the lexical level  is visible also in the transcripts, but it is not the focus of analysis in this paper , another participant B , or it can be syntactically dependent (Section Overlapping a Current Signer's Dependent Unit at Places of Possible Completion).The first excerpt illustrates an overlap occurring at a place of possible completion between a current signer, Nathalie, and a new signer, Melinda. The segment stems from a repair sequence initiated by Melinda concerning an exchange that she missed between Nathalie and Isaline. Isaline said that she really dislikes her eyes without make-up because they appear so big. Nathalie responds to this by expressing her surprise. During this exchange, Melinda averts her gaze from the participants while she is drinking . After Nathalie's display of surprise, Melinda initiates repair by asking Nathalie what Isaline dislikes. When Nathalie reaches a potential completion of her repair, Melinda starts a turn  while Nathalie is continuing with a “new” unit, resulting in simultaneous signing.The excerpt is presented as follows: a rough gloss annotation of the larger sequence is provided first, followed by a content translation. The part of these transcripts that are in bold are then represented again in the format of the ELAN annotation, so as to provide some more details on the temporal unfolding of the overlap under discussion Figure . In the IX(isa) on the tier LH_Gloss_1_Nat; l.07) which is doubling the same pointing at the beginning of the unit. This type of reduplication of a pronoun is frequent in several signed languages and has been described as a prosodic marker , reporting her response to Isaline (highlighted in blue).Nathalie's repair turn (l.07-08) consists of two parts. She starts with reporting that Isaline told her that without make-up Isaline's eyes look so big (red rectangle). This part finishes with the referential pointing toward Isaline . Interestingly, Melinda's response to Nathalie's repair is finely tuned to the moment when the element she addressed as repairable has been mentioned, namely what does Isaline dislike (“her big eyes”). At this point, she therefore orients to pragmatic completion, as her repair initiation did not ask for anything more than “what does she/Isaline hate.” Note also that Melinda launches a response to the repair after the reduplication of the sign IX(isa), where the first part is markedly finished on a syntactic and prosodic level. What we see here is thus precisely what Jefferson  the current speaker, perfectly within his rights, keeps going.”  that is overlapped with a stroke by Melinda (RH_phas_Mel). The other overlaps concern strokes and preparation phases. Interestingly, the preparation phase of Melinda's I (RH_phas_Mel) is rather long (350 ms)I in response to the fact that Nathalie is extending her turn . This second part of the turn is overlapped with Nathalie's acknowledging response that can be translated as “you see” with a PALM-UP gesture directed toward Melinda (l.06).Excerpt 2 presents a simple case of a short overlap between Melinda and Nathalie within a discussion on the different possible origins of pimples Figure . MelindaPALM-UP gesture directed to Melinda , in precise overlap with the last item of Melinda's turn, the gesture PALM-DOWN . Considering the movement phases of the overlapping gestures, it becomes clear that Nathalie launches her responsive PALM-UP near the end of the stroke of Melinda's sign WORK, the final sign of her first unit. Thus, Nathalie orients to this moment as reaching potential completion where she can acknowledge Melinda's telling and display her understanding. Melinda's turn however continues with a sign PALM-DOWN, which she adds without any manual prosodic disruption . Despite the absence of a manual prosodic break after WORK, Melinda deploys a resource that indicates a possible completion after WORK—she averts her gaze from her co-participant (to the right side) during the deployment of WORK (cf. DD on the tier Gaze_3_Mel). Baker . In fact, the excerpt represents a recurrent way of how signers in the present data end their turns. We will take this issue up again with excerpts 3 and 5.Nathalie launches her acknowledging response to Melinda's telling, a ). Baker  describe Rossano  describeExcerpts 1 and 2 have shown the fine coordination of incipient signers to possible completion, which implies that the overlapping signing is not the result of mistiming. Signers therefore precisely time their turn beginnings to such places within the temporal unfolding of turns, where syntactic boundaries occur. The overlaps are the result of the fact that the current speaker is not ending her turn at this point, but extends her turn.A further environment where simultaneous signing occurs is just after a possible completion of a signer's unit, i.e., when some pause has occurred after the first possible completion place . This pause can consist of a full or partial retraction, or it can be filled with a holdWhen participant A continues with his turn after some pause after a possible completion, he can do this either by starting a new unit or by adding elements that are somehow dependent on the first part of the turn (as in Section Simultaneous Signing at Places of Possible Completion). In this section I present an example of the first case in excerpt 3 Figure . Denja fBUT LEGISLATION D-HAND BAD STOP -H, on the tiers RH_Gloss_Den/BH_Gloss_Den; l.01-02, with headshake that starts in parallel to the sign BAD). At the end of the sign BAD, Denja's turn reaches a first possible syntactic and pragmatic completion. She continues however with the sign STOP, after which again speaker change may occur. This sign STOP is then held for 0.6 s, and she expands her headshake that she began in parallel to the sign BAD). Nathalie provides an acknowledging response by means of a head nod that is precisely timed to the end of the sign BAD; this head nod extends simultaneously to the sign STOP and the further -H (hold). With this non-manual acknowledgment the course of action has reached some completeness and there is no constraint regarding who of the participants takes a turn. Nathalie might take a full turn during Denja's -H, i.e., she could launch a turn going beyond her non-manual acknowledgment, but her hands remain in rest position. By contrast to Nathalie's turn-ending in excerpt 2, where her gaze aversion from the co-participant was interpreted as a possible sequence closing resource, Denja is gazing at Nathalie during her -H. I suggest that by means of these resources she invites Nathalie to take a turn and elaborate on the topic . In overlap with the preparation phase of I, also Nathalie finally takes a turn by launching the preparation phase of her turn-initial sign SIMILAR , orienting thereby to Denja's previously deployed resources (-H and gaze) as yielding the floor to her. This simultaneous beginning after the completion point results in overlap (highlighted in blue).The segment starts with Denja affirming that Switzerland is a very safe country, but that there is poor legislation resulting in too mild sentences, as for example for the punishment of rapists or murderers  where participants deal with the determination of a next signer in a subtle and situated manner. This is a most relevant interactional task especially in moments where no projection is pending, which means that there is neither constraint with respect to the next action to be accomplished, nor with respect to whom of the participants will get the floor .I suggest that from the end of SCHOOL I, l.04) as soon as the simultaneous signing quits.It is interesting to note that when Nathalie has finally taken over the turn, Denja does not drop out of the overlap immediately, but she brings her new unit to an end (l.03). However, Nathalie clearly orients to the simultaneous signing as a potentially troublesome overlap, as she restarts the overlapped turn-beginning  ALREADY prod-MAN-needle TRY YOU, tier RH_Gloss_3_Mel; l.03; red rectangle]. Denja launches an answer  to this question while Melinda is still producing the stroke of her last sign of her question, the sign YOU . Such examples show that signers can project incipient turn completions and launch into the transition even before the current signer has actually finished. Surprisingly, in the data under study here at least, cases where a signer provides minimal responses or launches a new turn in overlap with a sign “under way” (during the stroke deployment) that will actually be the last sign of the turn are very rare . In the next section I will focus on those instances in more detail and propose a discussion on the possible reasons for the frequent accomplishment of this type of turn transition.Denja is reporting that she was not able to squeeze the pimple despite the fact that it was all yellow. In overlap with this , Melinda is addressing a question to Denja, namely whether she has already tried to pick it with a needle [Current signers are recurrently overlapped during a potentially last item of a unit, after which they continue their turn . The sign HOW-LONG (produced on the right hand) is accompanied with a head tilt back and raised brows  she deploys an acknowledging response that consists of a lifting of a PALM-DOWN gesture with a prolonged mouthing of the letter—e-PALM-DOWN, also Nathalie lifts her left hand (while on the right hand she still signs HOW-LONG) and produces a PALM-UP , which she holds for a moment.Nathalie says that she wonders for how long a rapist who is freed from prison will go before relapse (HOW-LONG). However, Nathalie still prepares and holds her PALM-UP on the left hand until Isaline has again retracted her hands to rest position. Different hypotheses can be drawn up for explaining Nathalie's -H: first, Nathalie may thereby indicate that she does not want to relinquish the turn, i.e., that she is holding the floor. However, -H as a floor-holding device has been described as being accompanied with gaze aversion from the participants . A second possibility is that, by means of her -H, Nathalie embodies her expectation of Isaline's response  during which Nathalie averts her gaze from the co-participant (cf. excerpt 2 for a similar case). It is only after the simultaneous and sequential combination of all these resources that Nathalie retracts her hands to rest position. However, even after having retracted her hands to rest position (on the table), she then further extends the turn with a short PALM-UP accompanied with a shrug of the shoulders . This is again accompanied with a glance toward Isaline, who still does not take a turn.Interestingly, when Nathalie sees Isaline's preparation phase of her acknowledgment (raising the hands out of rest position), Nathalie could relinquish the floor by retracting her hands into rest position .within units. Jefferson . While the sequential moment of these overlaps does not manifest any coordination between the participants with respect to turn management, an interesting question to ask is what actions participants accomplish by means of these overlapping turns. In what follows I provide a brief description of the principal actional environments in which these overlaps occurred. Note however that each case is very specific with respect to the involvement of the participants and the coordination processes .A first environment where participants overlap each other with interjacent onset is in courses of actions involving repair (7/35 cases). The overlapping party is either self-initiating other-repair, providing a self-initiated other-repair, or displaying his understanding after having launched a repair sequence . In order to understand how members of a community accomplish courses of action, and establish and negotiate meaning in a situated and dynamic way, we have to first understand how turns and turn-taking are organized. There is still little research on social interaction in signed languages that adopts a conversation-analytic perspective. To date, we have been provided with some descriptions of how signers indicate their incipient speakership, or their willingness to keep a turn or assign a turn to a next participant. These descriptions do not necessarily follow the analytical mentality of conversation analysis. As a consequence of this, the notions of turn, TCU and TRP are still rarely discussed in detail in this field. Moreover, a most basic issue that has given rise to contradictory assumptions among researchers is the question of whether Sacks et al.'s  turn-taksequential environments of overlaps in signed interaction. Using the conversation analytic tools for studying the organization of signed interaction data allowed us to see how participants themselves treat ongoing turns (as being complete or not) and overlaps (as being troublesome or not). The preceding analyses have shown that the vast majority of overlaps produced among four young female signers frequently occur within the sequential environment of possible completion , i.e., they rarely occur in the midst of syntactic units . This shows that participants finely tune their turn beginnings to those places in the dynamics of interaction, where turns are possibly complete. Hence, the majority of overlaps results from the fact that incipient signers anticipate a turn-end and overlap it, and/or that current signers continue beyond a first possible completion. The findings therefore underpin the observed orderliness of overlapping talk in spoken language interaction , or by accomplishing actions that can reasonably overlap ongoing turns because of their urgency (repair initiations) or because of the interactional effects it thereby creates . It seems therefore that the observations made on overlaps in spoken interaction are also applicable to the data under study in this article. Consequently, prolonged simultaneous signing and the existence of one-at-a-time principle are not necessarily mutually exclusive.The data showed that signed overlap is not messy but organized. Admittedly, however, this does not disprove the assumption that signed interaction presents n-Spence  or Lacknn-Spence . It mighPALM-UP, PALM-DOWN, STOP, -H). Moreover, after these add-ons, signers often finish their turns. This provides evidence, from a participants' perspective, that there is in fact no attempt to hold the floor. By contrast, on the basis of a fine-grained investigation of the collaborative work accomplished by the signer and his recipient's during possible turn transition, the present analyses suggest that participants deploy such short add-ons as an interactional resource for the management of turn transitions. Concretely, participants step out of their turns in a gradual and smooth manner.An important finding of the present study is that ‘last item overlaps’ are scarce (cf. Section Overlapping a Current Signer's Last Item of a Unit). By contrast, participant B often overlaps a potentially last item of participant A who however continues past a possible completion . This may raise the question of whether participants erroneously predict turn-endings. Another hypothesis would be that current signers who are confronted with overlapping signing by their co-participants continue their turns as a means of holding the floor. While the methodological tools of CA do not allow answering the first question, the sequential analyses of overlapping signing provide some evidence for answering the second one. The analyses have shown that at first places of possible completion, current signers' continuations regularly consist of one or more items, short add-ons. These added items often consist of non-lexical elements or signs that do not contribute substantially new information (These add-ons can occur after a pause (Section Simultaneous Signing after Places of Possible Completion) or latched to a first possible completion . In the first case, the turn continuation can serve as a resource for treating a problem of recipiency, similarly to turn extensions in spoken language  and a limited range of courses of action . Whether the same type of turn transition can be found with other participants  is an open question. Moreover, as the excerpts in Sections Simultaneous Signing after Places of Possible Completion and Simultaneous Signing before Places of Possible Completion suggest, a systematic analysis of turn-endings that set a strong actional projection as compared to those without strong projection  will be necessary for a more detailed understanding of signers' management of turn-taking.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The intestinal microbiome represents a complex network of microbes that are important for human health and preventing pathogen invasion. Studies that examine differences in intestinal microbial communities across individuals with and without enteric infections are useful for identifying microbes that support or impede intestinal health.n = 200) and 75 healthy family members to identify differences in intestinal community composition. Stools from 13 patients were also examined post-infection to better understand how intestinal communities recover. Patient communities had lower species richness, evenness, and diversity versus uninfected communities, while principle coordinate analysis demonstrated close clustering of uninfected communities, but not the patient communities, irrespective of age, gender, and race. Differences in community composition between patients and family members were mostly due to variation in the abundance of phyla Proteobacteria, Bacteroidetes, and Firmicutes. Patient communities had significantly more Proteobacteria representing genus Escherichia relative to uninfected communities, which were dominated by Bacteroides. Intestinal communities from patients with bloody diarrhea clustered together in the neighbor-joining phylogeny, while communities from 13 patients’ post-infection had a significant increase in Bacteroidetes and Firmicutes and clustered together with uninfected communities.16S rRNA gene sequencing was conducted on stool DNA from patients with enteric infections  contains supplementary material, which is available to authorized users. Salmonella spp. predominated (11 %) followed by Clostridium perfringens (10 %) and Campylobacter spp. (9 %). Shiga toxin-producing Escherichia coli (STEC) O157 and non-O157 also caused 0.7 and 0.6 % of infections, respectively, while Shigella spp. caused 1.4 % during this same time frame [C. jejuni can cause neuropathies  and paralysis [In the US, diarrheal diseases are mostly caused by 31 different foodborne pathogens and were reported to contribute to 9.4 million infections, 55,961 hospitalizations, and 1351 deaths each year [me frame , though 12 microbes/ml [Bacteroidetes and Firmicutes, have been found to dominate in the intestines of healthy individuals, though considerable variation has been observed [Bacteroidetes and Lachnospiraceae has been observed [Bacteroidetes and Firmicutes [Bacteroidetes and Firmicutes was suggested to result in enhanced susceptibility to enteric infections as it shifts the microbial balance away from a healthy state. A prior study in mice demonstrated that intestinal communities with a high abundance of species closely related to pathogens were more likely to be affected by those pathogens [E. coli, for example, were more susceptible to inflammation induced by Salmonella enterica infections, whereas diverse intestinal communities were more resistant to infection. Commensal microbes were also suggested to alter pathogen virulence gene expression profiles by modifying environmental conditions [Human intestinal communities were previously suggested to comprise >10robes/ml  with keyrobes/ml , 7. Membobserved . Prior sobserved  and thatrmicutes . Decreasathogens . Communinditions . These dCitrobacter rodentium, a model for human enterohemorrhagic and enteropathogenic E. coli infections, alterations in the composition and abundance of the intestinal microbiota could be restored to a pre-infection state upon elimination of the pathogen [Several studies have also demonstrated that intestinal communities are capable of recovering following perturbations. In mice infected with pathogen . Similarpathogen , 18.Despite the recent interest and research on the intestinal microbiome, few population-based human studies have been conducted to better understand how the microbiota is impacted by enteric pathogens with different lifestyles. Here, we have compared the composition and abundance of intestinal microbes between patients with acute enteric infections and their uninfected family members by 16S rRNA sequencing. We have also examined how the intestinal microbial communities from a subset of patients change up to 14 weeks post-recovery, and have identified specific microbial populations that are correlated with clinical symptoms.n = 615) identified between January 1, 2011 and May 7, 2014 to all cases (n = 7435) identified throughout the State of Michigan during the same time period. For each pathogen, the frequency of cases was similar even after stratifying by gender, though a difference was observed after stratifying by age group (data not shown). Because the ERIN network targeted hospitals with the largest pediatric populations, we have captured more pediatric cases, particularly those in the youngest age group.As part of the Enteric Research Investigative Network (ERIN) Cooperative Research Center (CRC) at Michigan State University, we developed an active surveillance system to identify patients with enteric infections at four participating hospitals and the Michigan Department of Health and Human Services (MDHHS). To ensure that the ERIN surveillance network is representative of statewide enteric infection frequencies and that our results are generalizable, we compared the frequency of all ERIN cases (Campylobacter (n = 71), Salmonella (n = 66), Shigella (n = 34), and STEC (n = 28); the pathogen was not known for one patient. A total of 75 stools from otherwise healthy family members were also included for comparison, with 64 (85 %) representing 22 families ranging in size between two and six participants.A total of 310 stool samples were included for 16S rRNA sequencing analysis. These stools originated from 200 patients infected with P < 0.0001) and Bray-Curtis dissimilarity index (P < 0.0001). Moreover, principal coordinates analysis demonstrated tighter clustering of intestinal communities from uninfected family members relative to patient communities based on both the number and abundance of OTUs  and abundance , respectively. Significant differences were also observed using UniFrac weighted and unweighted distances calculated by the analysis of similarities (ANOSIM) and multivariate ANOVA based on dissimilarities (ADONIS) methods (data not shown).Rarefaction analysis revealed that communities were sampled completely with between 60 to 120 OTUs per sample and at 1000 sequences per sample  cases; two patients infected with Salmonella and STEC had communities comprising 99 % Proteobacteria. A total of 28 OTUs representing phylum Proteobacteria were classified in the uninfected communities, though variation in OTUs was observed in the communities infected with the four different pathogens . Variation in the abundance of Proteobacteria was also observed as 37 % of patients with Campylobacter, 29 % with Salmonella, 18 % with STEC, and 38 % with Shigella infections had over 50 % Proteobacteria. Among these infected communities, Gammaproteobacteria represented more than 92 % of the Proteobacteria detected  across the infected communities relative to the uninfected communities. The mean abundance of Escherichia in communities infected with Campylobacter, Salmonella, Shigella and STEC was 0.21, 0.14, 0.24, and 0.21, respectively, relative to uninfected communities (0.01). More specifically, the population of Gammaproteobacteria from Campylobacter cases represented 29 genera with Escherichia comprising 60 % followed by Trabulsiella (7 %) and unclassified Pseudomonadaceae (7 %). Genus Campylobacter was only detected on average in 0.01 % of 41 of the 71 Campylobacter cases, which represented 3.7 % of the total Proteobacteria levels. Salmonella cases had 27 genera represented from Gammaproteobacteria with Escherichia and Trabulsiella predominating at 45 and 21 %, respectively, followed by Citrobacter (12 %) and unclassified Enterobacteriaceae (10 %). Only 22 of the 66 Salmonella cases had detectable levels (average 0.002 %) of genus Salmonella, which represented 0.52 % of the total Proteobacteria levels. For STEC cases, 21 genera were identified with Escherichia comprising 77 % of the Gammaproteobacteria population identified, which represented the total level of Proteobacteria detected in these cases. A total of 23 genera were detected in Shigella cases with most belonging to Escherichia (69 %), unclassified Pseudomonadaceae (8 %), and Klebsiella (6 %). Genus Shigella was not detected in any of the cases, though unclassified Enterobacteriaceae was found at an average of 0.007 %. None of the cases had levels of unclassified Enterobacteriaceae exceeding 10 %. Together, these data demonstrate that the distribution of genera differed among the intestinal communities from patients, which was also indicated in the NPMANOVA analysis; however, Escherichia predominated in most infected communities regardless of the pathogen causing the infection.To further demonstrate that the differences observed across patients infected with the different pathogens were not attributable solely to an increase in the abundance of the actual pathogen, we also examined genera abundance and composition across patients. Notably, genus Enterobacteriaceae followed by Pateurellaceae, Lactobacillales, Streptococcaceae, and Lachnospiraceae. At the genus level, Bacteroides dominated in 86.7 % of the samples from healthy family members with a range between 6 and 75 %, while Prevotella were found in 13.3 % with a range of 16 to 65 %. Among the intestinal communities from healthy individuals, a total of 38 microbial features were differentially abundant. At the family level, most of these features were classified as Rikenellaceae, Bacteroidaceae, Ruminococcaceae, Porphyromonadaceae, Bifidobacteriaceae, Alcaligenaceae, Odoribacteraceae, Barnesiellaceae, and Lachnospiraceae. The genera Roseburia, Lachnospira, and Blautia of the Lachnospiraceae family were most abundant in healthy family members compared to patients. Overall, no microbial features were differentially abundant in the communities of patients infected with different pathogens.Similar findings were observed utilizing the linear discriminant analysis (LDA) effect size (LEfSe) method , which in = 153) of the infected communities belonged to clusters I or II, whereas 91 % (n = 68) of the uninfected communities belonged to clusters III and IV. Based on the placement within the dendrogram, communities of cluster V, which originated from one uninfected individual and five patients, were grouped together with communities belonging to clusters III and IV for subsequent analyses. Overall, intestinal communities from patients were significantly more likely to group within clusters I or II, while uninfected communities grouped within clusters III–V (likelihood ratio chi-square (χ2) = 109.2; P = <0.0001).Microbial community composition was examined based on the Bray-Curtis dissimilarity index to compare microbiota profiles between patients (infected communities) and healthy family members (uninfected communities). Five distinct clusters were identified with the infected and uninfected communities separating into different sections of the neighbor-joining tree Fig. . SpecifiP < 0.0001) when compared to clusters III–V. A significantly higher average percentage of phylum Proteobacteria (42 %) was observed in clusters I/II relative to clusters III–V (6 %), while Bacteroidetes (38 %) and Firmicutes (17 %) were more common in the latter three clusters. Among the uninfected communities belonging to clusters III–V, the abundance of Bacteroidetes (59 %) and Firmicutes (33 %) was even higher. Significant differences were also observed at the genus level. Intestinal communities representing clusters I and II (n = 160) were dominated by Escherichia, Trabulsiella, Citrobacter, unclassified Pseudomonadaceae, or Bacteroides, whereas communities of clusters III–V (n = 115) were dominated by Bacteroides or Prevotella. Notably, Escherichia was significantly more abundant in communities belonging to clusters I and II compared to communities of clusters III, IV, and V as were Trabulsiella and unclassified Enterobacteriaceae (P < 0.0001). Bacteroides, Parabacteroides, Faecalibacterium, Roseburia, Ruminococcus, Suturella, and unclassified Lachnospiraceae, however, were significantly more abundant in clusters III–V . Indeed, only 14 of the 85 patients (17 %) with bloody diarrhea had intestinal communities belonging to clusters III–V. No association was observed between specific intestinal microbiota profiles and length of hospitalization, patient age group, or history of nausea, headache, fatigue, and chills.To determine whether differences in the intestinal microbiota profiles were associated with disease severity and other patient characteristics, we evaluated epidemiological data by the cluster groups defined in the Neighbor joining phylogeny Fig. . No signSalmonella, Shigella, and Campylobacter . Nonetheless, these data suggest that individuals with more severe disease, as measured by the presence of bloody diarrhea, were more likely to have intestinal communities with similar profiles regardless of age and infection by three of the four pathogens.While adjusting for age, gender, and intestinal microbiota profile, logistic regression analyses demonstrated that patients with intestinal communities belonging to clusters I/II were significantly more likely to have bloody diarrhea (odds ratio (OR), 2.3; 95 % CI, 1.12, 4.82). Male gender, however, resulted in a protective effect , and no association was observed for length of hospitalization. Addition of pathogen type to the logistic regression model resulted in similar associations for gender and cluster, but also showed that patients with STEC infections were significantly more likely to have bloody diarrhea when compared to patients with Campylobacter (n = 3), Salmonella (n = 6), and Shigella (n = 4) infections were examined to assess how intestinal communities changed following an enteric infection. In each case, a shift in the intestinal community was observed following recovery. Collectively, the recovered intestinal communities had greater richness, evenness, and diversity relative to the communities that were present during the acute infection , while the level of Proteobacteria was significantly greater (P < 0.0001) . Such differences are likely attributable to variation in the time that each recovered patient was evaluated as patients submitted a follow-up sample between 1 and 14 weeks post-infection. Indeed, Proteobacteria levels decreased over time across the 14 week follow-up period in all patients regardless of the pathogen causing each infection. At the genus level, this decrease in Proteobacteria was mostly attributable to a decrease in Escherichia, which coincided with an increase in Bacteroides, Prevotella, Parabacteroides, and Rikenellaceae in phylum Bacteroidetes and Faecalibacterium belonging to phylum Firmicutes  intestinal communities when compared to the uninfected communities of healthy family members. Both Bacteroidetes and Firmicutes have previously been associated with intestinal health [Proteobacteria compared to an average of only 4.3 % of the healthy individuals (n = 75). Although the total number and relative abundance of OTUs uncovered in the intestinal communities from individuals were lower than in prior studies, all stool samples were preserved in the same conditions with Cary-Blair media, which had little effect on the presence and abundance of specific community members following experimental inoculation of cattle feces  that are characterized by an increase in Proteobacteria and not Bacteroidetes and Firmicutes, which represent dominant commensals and the most common phyla in human intestinal communities. Patients with similar microbiota profiles were more likely to have severe disease outcomes, suggesting that increases in specific microbe populations, which were primarily attributable to Escherichia, occur during an infection. This finding is supported by the result that patient intestinal communities recovered over time to mimic those communities of healthy family members, which were classified by an increase in phyla Bacteroidetes and Firmicutes and decrease in Proteobacteria belonging to genus Escherichia. Future enteric disease prevention strategies could therefore aim to decrease intestinal Escherichia levels at the time of an acute infection while simultaneously boosting microbes including Bacteroidetes that are most important for restoring intestinal health.Enhancing our understanding of how intestinal microbial communities change during an enteric infection and identifying factors that influence these changes are important for the development of novel prevention and treatment strategies. In our analysis of 200 intestinal communities from patients and 75 healthy family members, we found that specific intestinal microbiota are more common among patients during an acute enteric infection regardless of the type of enteric pathogen. Specifically, the intestinal microbial communities from patients were more frequently dominated by Campylobacter, Salmonella, Shigella, and STEC at four participating Michigan hospitals. Surveillance began on January 1, 2011. Stools from each patient were added to Cary-Blair transport media and submitted to the MDHHS for de-identification, culture, and transport to MSU for community DNA isolation and 16S rRNA sequencing. Family members of patients (n = 75) were enrolled by phone and asked to submit a stool specimen preserved in Cary-Blair transport media and to complete a questionnaire containing demographic information, exposures , and signs of illness including duration, type, and severity of clinical symptoms. Such information was also available for each patient through the Michigan Disease Surveillance Network (MDSS) maintained by the MDHHS Bureau of Epidemiology. A subset of 13 patients was also re-contacted post-infection for stool submission and questionnaire completion. Each participant was required to provide informed consent prior to enrollment and was given a monetary incentive after samples were received at MDHHS. All protocols were approved by the Institutional Review Boards at MSU (IRB# 10-736SM), the MDHHS (842-PHALAB), and each of the four participating hospitals .An active surveillance system was created through the MDHHS Bureau of Laboratories to identify 200 patients infected with n = 741) received via the ERIN surveillance system through May 7, 2011. Stool DNA recovery (260/280 nm) and yield (ng/μl) were initially compared using the QIAamp DNA Stool Mini Kit  and the MoBio Fecal Isolation Kit  and standardized using the NanoDrop (Thermo Fischer Scientific). Four stools were compared using the manufacturer’s instructions, except for the addition of a denaturation step (95 °C) followed by bead beating for the QIAamp kit to enhance DNA isolation of gram-positive bacteria as described [Stool samples were processed soon after receipt by both MDHHS and MSU and time period from collection to processing was noted. Following homogenization and centrifugation, aliquots of stool were stored in triplicate at −80 °C until further use. A total of 310 stools were examined in this analysis, which represent 41.8 % of the total number of stools  in triplicate with initial denaturation at 95 °C for 2 min followed by 30 cycles of 95 °C for 20 s, 50 °C for 30 s, and 72 °C for 5 min. An amplicon library of 48 communities were prepared for each sequencing run on 454 GS Junior Titanium chemistry, by individually purifying the product using the Agencourt AMPure XP  beads, quanitification by pico green assays  followed by normalization and pooling.Isolated community DNA was amplified for the 16S rRNA gene spanning the V5–V3 regions using universal primers 357F and 926R tagged with the 454 universal primers A and B at either end with barcodes for multiplexing samples. PCR amplification was performed using AccuPrimeOver 1 million reads were processed in the Quantitative Insights Into Microbial Ecology (QIIME) software  and aligThe phylogenetic data from QIIME was further analyzed using Paleontological Statistics Software Package For Education and Data Analysis (PAST) based on the NPMANOVA and to calculate genera important for dissimilarities between groups using SIMPER. Additional 2D principal coordinate analysis plots based on both the Bray-Curtis and Jaccard similarity coefficients were also constructed for a weighted and unweighted analysis (28). Box plots were constructed using SigmaPlot 10.0.http://tree.bio.ed.ac.uk/software/figtree/). We tested for differences in the frequency of clinical characteristics including HUS, diarrhea, bloody diarrhea as well as for various factors including age and gender, across the different microbial community clusters using the likelihood chi-square test or Mantel-Haenszel chi-square (for variables with more than two outcomes) distributions using odds ratios with 95 % confidence intervals performed with Statistical Analysis Software (SAS) (version 9.3).Links to epidemiological data were made after clustering relationships were identified between the stool communities based on abundance profile calculated by Bray-Curtis dissimilarity index in PAST for generation of neighbor-joining tree followed by tree construction in FigTree (Raw sequence data is available at (http://dx.doi.org/10.6084/m9.figshare.1447256), while the metadata and taxonomic classifications have been included as Additional files


The crosslinking mechanism under different temperatures indicated two crosslinking pathways: (a) crosslinking on the sulfonic acid groups; and (b) crosslinking on the backbone. It was observed that membranes crosslinked at a temperature of 150 °C lead to low proton conductive membranes, whereas an increase in crosslinking temperature and time would lead to high proton conductive membranes. High temperature crosslinking also resulted in an increase in anisotropy and water diffusion. Furthermore, the membranes were investigated for a Vanadium/Air Redox Flow Battery application. Membranes crosslinked at 200 °C for 30 min with a molar ratio between 2:1 (mol repeat unit:mol benzenedimethanol) showed a proton conductivity of 27.9 mS/cm and a 100 times lower VO2+ crossover compared to Nafion.Highly conductive and low vanadium permeable crosslinked sulfonated poly(ether ether ketone) (cSPEEK) membranes were prepared by electrophilic aromatic substitution for a Vanadium/Air Redox Flow Battery (Vanadium/Air-RFB) application. Membranes were synthesized from ethanol solution and crosslinked under different temperatures with 1,4-benzenedimethanol and ZnCl These applications require highly conductive and stable membranes. The vanadium/air redox flow battery (Vanadium/Air-RFB) is one of the most promising energy storage technologies based on such membranes.2+/V3+, where the second half-cell contains either water vapor or air, depending on the charge or discharge modes of the system. The Vanadium/Air-RFB has been chosen as a special type of redox flow system, since it employs only vanadium in its 2+/3+ oxidation state, but not in the 4+/5+ oxidation state. Due to that, stability issues caused by the 4+/5+ oxidation state  on the square of the wave vector (q2) is shown in q2 range, where a “fast” diffusion occurs and from which apparent diffusion coefficients, namely Dfast, can be extracted.The authors of a recent paper  measuredmposites . The watη = (D⊥ − D∥)/D⊥, where D⊥ corresponds to the through-plane diffusion coefficient and D∥ the in-plane diffusion coefficient, respectively. The anisotropy of water diffusion, as shown in slowD, is more affected by the experimental errors and, therefore, is not shown in this work.The “fast” diffusion is characterized by a higher diffusion coefficient for the perpendicular orientation (through-plane) of THE gradient relative to the membrane plane than for the parallel one (in-plane). The SPEEK membrane crosslinked at 200 °C for 60 min shows the highest in-plane and through-plane diffusion among the studied membranes. The anisotropy of water diffusion in the cSPEEK membranes is defined as fastD in the crosslinked SPEEK membranes at elevated temperatures (200 °C) would indicate an improvement of proton conductivity through the membrane, which is beneficial for applications, such a vanadium redox flow batteries or fuel cells.The increase of 3.3.i.e., From eat unit . To provi.e., after exposition at 200 °C for 60 min. The resulting conductivities support the previous hypothesis of the sulfonate ester appearance at lower crosslinking temperature and the change of the crosslinking position of the 1,4-benzenedimethanol at higher temperatures and longer crosslinking duration.As illustrated in i.e., IEC for the non-crosslinked SPEEK  data. Indeed, IEC is directly related to the number of the ed SPEEK . Therefo3.4.4 compartments over time, where 2+ through these membranes.The vanadium permeability of the synthesized and commercial membranes were studied. This was done in a diffusion cell, which consists of two half-cells separated by the membrane under investigation. i.e., increasing the crosslinking). However, the vanadium permeability remains still higher than through a Nafion 117 membrane. This may be a result of the highly sulfonated SPEEK, which has a high water uptake. To overcome this issue, the ratios of crosslinked It can be seen that the vanadium permeability decreased with decreasing the amount of SPEEK (fion 117 . HoweverThe reason for the low vanadium cross over in the case of the crosslinked SPEEK lies in its glassy properties and rigid backbone. Furthermore, the dead end hydrophilic channels of SPEEK also decrease the vanadium permeability, since these are not as well interconnected as in the case of Nafion.4.Highly sulfonated crosslinked SPEEK membranes for a Vanadium/Air-RFB application were prepared as described elsewhere ,23. For


Functionalized graphene sheets were customised as a matrix for covalent immobilization of Fenugreek β-amylase using glutaraldehyde as a cross-linker. The factors affecting the process were optimized using Response Surface Methodology based Box-Behnken design of experiment which resulted in 84% immobilization efficiency. Scanning and Transmission Electron Microscopy  and Fourier Tansform Infrared (FTIR) spectroscopy were employed for the purpose of characterization of attachment of enzyme on the graphene. The enzyme kinetic studies were carried out for obtaining best catalytic performance and enhanced reusability. Optimum temperature remained unchanged, whereas optimum pH showed shift towards acidic range for immobilized enzyme. Increase in thermal stability of immobilized enzyme and non-toxic nature of functionalized graphene can be exploited for production of maltose in food and pharmaceutical industries. It is essential for the generation of maltose from cereal grain starch, thus making its role important in mashing and brewing process. The saccharifying activity is also exploited in bread making and in the use of malt as an additive in food stuffs. Maltose has properties such as mild sweetness, good thermal stability, low viscosity in solution and lack of colour formation, which accounts for its applicability in pharmaceutical dispensing and in the production of non-digestible sweetner . Major use of β-amylase is in being the exclusive source of carbon in the production of diphtheria pertussis tetanus vaccine. The characteristic property of enzyme of yielding a single product maltose, is utilized in the structural studies of starch and glycogen Enzymes being highly selective catalyze efficient reactions with few by products, thus they are environmentally favourable alternative to conventional chemical synthesis, particularly in the food and pharmaceutical industries where high reaction selectivity is essential. But, lack of long term operational stability, difficult recovery and reuse of the enzyme, limit their industrial applications. These limitations can often be overcome by immobilizing them on suitable matrices. The interaction between the enzyme and carrier material governs the properties of immobilized enzyme, thus, influencing the chemical, biochemical, mechanical and kinetic properties. The property of immobilized enzyme is also affected by particle mobility which is governed by particle size and solution viscosity Nano-materials and nano structures generally provide large surface area, low mass transfer resistance and high enzyme loading capability, which enables better interaction with the enzyme, resulting in increase in immobilization efficiency and enhancement in long term storage and recycling ability of the enzyme. Nanomaterials can also be engineered to present multiple surface functional groups for interacting with biomolecules. A wide range of nano materials have been studied and their positive effect on enzyme was shown through immobilization process Various graphene based nano materials have been used to fabricate functionalized biosystems integrated with nucleic acids, peptides, proteins and even cells. Graphene is a free standing 2D crystal with one atom thickness. This allotrope of carbon comprises layers of six atom rings in a honey combed network and can be conceptually viewed as a true planar atomic macromolecule. Graphene also gets benefitted by biological modification which improves its biocompatibility, solubility and selectivity. Proteins can exfoliate and modify graphene due to presence of various functional groups, through physical adsorption or chemical bonding Response surface methodology (RSM) is a widely practiced approach for the production and optimization of different industrially important biotechnological and biochemical products. It is a collection of statistical and mathematical techniques useful for developing and optimizing processes. RSM is a method for constructing global approximations to system behaviour based on results calculated at various points in the design space. It also determines effect on specific response resulting from the changes in the level of the factors.The choice of experimental design is affected by the shape of the experimental region, which is determined by the ranges of the variables. The Box-Behnken design is an independent rotatable or nearly rotatable quadratic design. It excludes the corners, where all variables are simultaneously at the maximum levels. The use of the Box-Behnken design is popular because it is an economical design and requires only three levels for each factor. Therefore, it requires few experimental combinations of the variables β-amylase on functionalized graphene using RSM based Box-Behnken design of experiment and to study the effect of immobilization on kinetic parameters. Immobilized enzyme was characterized using SEM and TEM. The chemical interactions and bonding involved during immobilization were characterized by FTIR spectral analysis.The basic objective of the present study include immobilization of Fenugreek Trigonella foenum-graecum) were purchased from local market. The chemicals for buffers preparation were of Analytical or Electrophoresis grade from Merck Eurolab GmbH Damstadt, Germany. All other chemicals and reagents were purchased from Sigma Chem. Co. Milli Q  water with a resistance of higher than 18 MΩ cm was used all throughout the experiment.Dry seeds of fenugreek (β-Amylase was purified from dry seeds of fenugreek (Trigonella foenum-graecum) and had a specific activity of 732.59 units/mg. Homogeneity of the purified preparation was checked by SDS-PAGE Protein concentration was determined by using Bradford's method (1976) Enzyme activity was measured by following Bernfeld's method (1955) β-amylase were incubated with 0.5 mL of starch solution (1%) prepared in 50 mM sodium acetate buffer, pH 4.5 for 3 min at 37°C, followed by centrifugation at 5000 rpm for 2 min at 4°C. Supernatant was pipetted out in test tube and 1 mL of 3,5 dinitrosalicylic acid was added, followed by placing it in boiling water bath for 5 min. It was diluted with 10 mL of Milli Q water before recording absorbance at 540 nm.Graphene sheets cross-linked with Fenugreek β-amylase is defined as the amount required for release of 1 µM of β-maltose per unit at 30°C and pH 5.0, under the specified condition.One unit of The immobilization efficiency was calculated using following formula:Enzyme units were calculated using starch as substrate, under standard assay conditions as described earlier. The immobilized protein was determined by subtracting the protein estimated in supernatant after immobilization from the total amount of protein used for immobilization.2SO4, HNO3 and KClO3 at room temperature under constant stirring condition. The graphite oxide solution obtained was washed with distilled water and 10% HCl solution for removal of sulphate and other ionic impurities. It was then dried under vaccum at 80°C. The graphite oxide powder was placed in an alumina boat and inserted into a 1.5 m long quartz tube with outer diameter of 25 mm. The sample was flushed with Ar gas for 15 min followed by insertion of quartz tube into a tube furnace pre heated at 1050°C and was kept in it for 30 sec. The functionalized graphene sample was allowed to cool down to room temperature under Ar gas flow. Thermally exfoliated functionalized graphene thus prepared, was light weighted shiny black powder which differed in appearance from brownish graphite oxide. The sample prepared was suspended in double distilled water and was sonicated at room temperature for 10 min. It was then left undisturbed for 30 min for the settlement of the larger non exfoliated flakes and the suspended functionalized graphene was collected.Functionalized graphene sheets were prepared by thermal exfoliation of grahite oxide, following the protocol described by Staudenmaier The enzyme was coupled to the functionalized graphene with the help of glutaraldehyde as a cross linker. A preparation of 1 mg/mL of graphene was made by dissolving functionalized graphene in 50 mM sodium phosphate buffer (pH 7.0), which was divided into 17 aliquots according to the design of the experiment . These a−1 wave number. A good signal to noise ratio was achieved with 100 scans of each sample.The characterization studies of functionalized graphene sheets (both native and coupled) wereperformed by Transmission Electron Microscope , Scanning Electron Microscope  and Fourier Transform Infrared Spectroscopy . TEM studies were done by placing a drop of sample on electron microscope 200 mesh copper grid and the water was allowed to get evaporated for complete dryness of the sample, followed by its loading into machine. SEM studies were carried out by sprinkling the samples on the stub having layer of silver glue for striking the particles. Secondary electron imaging mode was used for obtaining fine structural details. FTIR was performed in the range of 650 to 4000 cmβ-amylase onto functionalized graphene were: amount of graphene ; concentration of glutaraldehyde  and amount of enzyme . The quadratic polynomial equation relating the variables to the responses was used for calculation of the mathematical relationship:The experiment was designed using Box-Behnken design for analysis and optimization of the factors  which influence the process. The initial values of the factors affecting the process of immobilization were determined by carrying out some preliminary experiments (data not shown). ‘Design Expert’ software  was used for experimental design and analysis. The Box-Behnken design used for fitting the quadratic model led to the experiment consisting of 17 trials and all the factors studied were varied at three different levels. The variables and their levels selected for carrying out immobilization of Fenugreek iY is the predicted response, iXjX are input variables which influence the response variable Y; oβ is the offset term; iβ is the ith linear coefficient; iiβ the ith quadratic coefficient and ijβ is the ijth interaction coefficient. The co-efficients were estimated by performing the 17 trials and the generated mathematical model was validated by carrying out experiment at given optimal condition. The model was statistically analysed for evaluation of the anlaysis of variance (ANOVA), which determines the validity of the model based on Fischer's F-test, associated probability, correlation coefficient R and lack of fit test.Where, In all cases, control experiments using soluble enzyme were carried out.β-amylase was studied in the pH range of 3.0–9.0 . 50 mM buffers were used in each case and assay procedure was performed under standard conditions. The optimum temperature was studied by carrying out assay procedures at temperatures between 25–75°C. The thermal stability was determined by incubating the enzyme at 55°C for different time intervals, followed by residual activity assay under standard condition. Starch concentration was varied in the range of 0.5–10 mg/mL for observing effect of substrate concentration on immobilized enzyme. Data thus obtained was used for calculation of mK and maxV from Lineweaver-Burk plot. For the assessment of reusability, the immobilized enzyme was used for carrying out assay procedure repeatedly. After each assay, the immobilized enzyme was washed with 50 mM sodium phosphate buffer, pH 7.0. The aliquots of functionalized graphene coupled with enzyme were stored at 4°C and the residual activity was determined during 4 months storage.The effect of pH on immobilized Fenugreek +0.5).For obtaining optimal conditions for enzyme immobilization, RSM was employed. Central Composite design and Box-Behnken design are the two designs of experiment commonly utilised in RSM. A comparative study between the two designs of experiment was done by Ferreira et al (2007) µg) and C, enzyme (µg).These experimental results formed the base for building of model for obtaining maximum immobilization response as presented in For establishing the adequacy and significance of predicted quadratic model, ANOVA was performed . The ModSystematical characterization of the enzyme immobilized on functionalized graphene was done by carrying out SEM and TEM, followed by FTIR spectroscopy. Bright field TEM images showed typical transparent sheets of functionalized graphene at 50 nm resolution, the characteristic Selected Area electron Diffraction (SAD) pattern obtained is shown in inset of −1 (C = C band), which has been assigned to skeletal vibrations of graphitic domains. Functional groups present in graphite oxide  are utilized in its further functionalization. The peak at 1740 cm−1 is characteristic of −COOH (−C = O in carboxylic acid) which is known to appear after execution of oxidation process. The O-H stretching mode of −COOH groups and −OH groups attached on the surface of functionalized graphene leads to appearance of peaks of O-H at ∼1400 cm−1 together with the band at 3450 cm−1. The vibration of C-O in carboxylic acid groups or epoxy groups results in appearance of the peak at ∼1054 cm−1. The cross linking of functionalized graphene has also been characterized by FTIR. Peaks corresponding to the C-H stretch modes of the cross linked molecule was located at 2927 and 2858 cm−1. An increase in the relative intensity between peaks at 1054 cm−1 and 1630 cm−1 signifies the increase in the intensity of C-O stretch mode and suggests that cross linking between functionalized graphene and glutaraldehyde has taken place. The hydroxyl groups can react with aldehyde through formation of hemiacetal structure. The aldehyde functional groups present in the molecule of glutaraldehyde, react with the sheets of functionalized graphene as a cross-linker. Glutaraldehyde binds to the −OH group of the graphite oxide through its one arm containing −CHO group and attaches to the enzyme via lysine amino group through its another −CHO group. Glutaraldehyde is also known to play important role in the self-assembly of graphite oxide nanosheets in aqueous solution, as the self-assembly of nano sheets occur in more ordered fashion in presence of little amount of glutaraldehyde −1, representing carbonyl amide I bond whereas bands at 1481 and 1567 cm−1 were observed due to amide II bonds suggesting immobilization of enzyme on functionalized graphene.The natural graphite shows a characteristic peak located at 1625 cmAn alteration in kinetic parameters is often brought about by immobilization process due to changes in the microenvironment of immobilized enzyme β-amylase with respect to activity and stability. The optimum pH of the immobilized Fenugreek β-amylase was found to be 4.5, while the soluble enzyme had pH optima at 5.0. The extent of displacement of optimum pH is influenced by interaction between the enzyme and the matrix and also by the properties of matrix. Hydrolytic activity was observed in a wider pH range by the immobilized enzyme. Compared to soluble enzyme, immobilized one showed better activity in the alkaline range (pH 7.0–9.0). β-Amylase from sweet potato immobilized on chitosan and α-cyclodextrin also showed similar results −1 and 0.0213 min−1, respectively. The immobilization support generally provides protecting effect against enzyme deactivation occurring at high temperature. The immobilization process affects the conformational flexibility and results in increase in enzyme rigidity, which is observed in form of stability towards denaturation resulting from rise in temperature β-amylase from soybean showed a small increase in thermostability on being immobilized on chitosan beads β-amylase immobilized on functionalized graphene showed better activity at higher temperatures as compared to β-amylase immobilized on phenyl boronate agarose mK value showed slight change from 1.56 to 1.82 mg/mL on being immobilized on functionalized graphene. These differences can be related to the change in microenvironment resulting from the enzymatic hydrolysis of substrate.Industrial applicability of any enzyme is judged by the knowledge of its storage stability. Stabilization of enzyme by immobilizing it on suitable matrix has been the subject of considerable research, as the intra and inter molecular cross-linking leads to a more rigid molecule that can resist conformational changes. The immobilized enzyme was found to be quite stable when stored at 4°C for 120 days with retention of 60% residual activity, whereas soluble enzyme showed residual activity of 20% during storage for same duration (β-amylase immobilized on functionalized grapheme nanosheets (Another important parameter for industrial application is reusability. Reusability in terms of retention of more than 76% enzymatic activity after 10 repeated uses was shown by Fenugreek nosheets . Repeateβ-Amylase covalently attached onto the functionalized graphene showed wider range of pH operability, better thermal stability and greater storage ability as compared to soluble enzyme, making it suitable for production of maltose at industrial scale.Optimization of immobilization using RSM based Box-Behnken design of experiment was proved to be efficient and economical, due to requirement of less number of experiments to be performed. Fenugreek


A predictive model is proposed that quantitatively describes the synergistic behavior of the electrical conductivities of CNTs and graphene in CNT:graphene hybrids. The number of CNT-to-CNT, graphene-to-graphene, and graphene-to-CNT contacts is calculated assuming a random distribution of CNTs and graphene particles in the hybrids and using an orientation density function. Calculations reveal that the total number of contacts reaches a maximum at a specific composition and depends on the particle sizes of the graphene and CNTs. The hybrids, prepared using inkjet printing, are distinguished by higher electrical conductivities than that of 100% CNT or graphene at certain composition ratios. These experimental results provide strong evidence that this approach involving constituent element contacts is suitable for investigating the properties of particulate hybrid materials. Carbon nanotubes (CNTs) and graphene have been intensively studied for various applications123467912151619et al. suggested that the synergism of the conductivities is derived from the graphene particles filling the space between the CNTs and thereby providing a lower junction resistance between the graphene and the CNTs. These two explanations for the synergistically improved electrical conductivity of CNT:graphene hybrids, i.e., CNTs acting as conducting bridges between the graphene flakes and graphene particles filling the space between CNTs, have the common feature of an increased number of contacts between the graphene particles and the CNTs. This study aimed to establish theoretical and experimental explanations for the synergistic effect. CNT:graphene hybrids prepared by simple dispersion and a film processing method were used in the experimental work.CNT:graphene hybrids have been studied in many researches due to its enhanced properties than that of graphene and CNT itself. In some work, it was found that the thermal conductivity increases by hybridizing CNT/graphene1222232425262728302730323323242528Various processing methods have been developed to make CNT:graphene hybrid films. Vacuum filtration was the first solution approach24122023151738This paper presents a predictive model for quantitatively explaining the synergistic behavior of CNT:graphene hybrids. A quantitative analysis carried out using the model demonstrated that the hybrids can exhibit higher electrical conductivities than pure CNT or graphene films at certain composition ratios. CNT:graphene hybrids prepared by inkjet printing were used to validate the predictive model.A predictive model was developed to explain the synergistic behavior of graphene and CNT electrical conductivities in CNT:graphene hybrids. The conductivity of these hybrids is governed by inter-particle contacts. A random stick network model can be used to estimate the electrical conductivity of a CNT filmns is the number of conductive sticks and nj is the number of their junctions, i.e., the number of stick-to-stick contacts. Here, b is a constant, Gs is the stick conductance, and Gj is the junction conductance. Because the conductivities of individual CNTs and graphene are much higher than the conductivity of their junctions (contacts)42where et al. defined the orientation of fibers in space using two angles , with the assumed geometries and sizes of graphene particles and CNTs, then the total number of CNTs and graphene particles in a specific volume is k. Equation Equation α and β are positive constants. In this system, 56.7 wt% is the CNT fraction that can bring about the maximum number of contacts in the modeled CNT:graphene hybrids. At this concentration, the proportion of graphene-to-CNT contact is the largest (75.0%), followed by CNT-to-CNT (19.2%), and graphene-to-graphene (5.8%) contacts. A synergistic behavior of CNT:graphene hybrids can be observed at this CNT content.where Next, the effect of graphene particle size on the number of contacts was investigated. For simplification, the size of the graphene particles was assumed to be where x μm, the number of CNTs can be expressed as The effect of CNT size on the number of contacts can be investigated by changing either its diameter or length. For simplicity, the mass of a CNT is assumed to be independent of its diameter and proportional to its length. By changing the length from 5 to 5The number of contacts that graphene and CNT particles can make in their hybrids can thus be calculated using a simplified geometry for the CNT and graphene. The model demonstrates that the condition of maximum contact occurs at a specific concentration of both materials. We can deduce that properties sensitive to such contact, e.g., electrical conductivity see Eq. , will beoC)There are several requirements for inks to be printed acceptably by an inkjet printer. The inks should have relatively low viscosity  and low volatility . The graphene particles were randomly deposited on the paper . The PAN–1, respectively. Although pure graphene has a higher electrical conductivity than pure CNTs4239The electrical conductivity of printed ink was measured using the four-point probe method. The electrical properties of the CNT:graphene hybrid inks are shown in The number of CNT-to-CNT, graphene-to-graphene, and graphene-to-CNT interparticle contacts was calculated using an orientation density function of graphene and CNT in a hybrid assembly. CNTs and graphene particles were assumed to be straight cylinders and disks, respectively. The probability of each type of contact was then calculated, from which the number of contacts was obtained. The total number of contacts followed a parabolic equation as a function of CNT concentration, suggesting the existence of a maximum at a specific composition ratio. This indicates a synergistic effect of the properties of the CNT:graphene hybrids, in particular those properties that are contact-dependent. This synergistic effect was experimentally investigated by using inkjet printing to prepare CNT:graphene hybrids. Hybrid inks, prepared at a certain composition ratio, had higher electrical conductivities than those of pure CNT or graphene, i.e., a synergistic effect. These findings support the use of our statistical approach to investigate the properties of particulate hybrid materials involving contacts of the constituent elements.Our model was aimed to estimate the electrical conductivity of CNT:graphene hybrids. However, we have found out that the percolation threshold of CNT composites can be estimated using the equations derived in our work. In previous research, the critical volume fraction ensuring the percolation was estimated by calculating the probability of one cylinder intersecting at least two other cylinders. In a work by Munson-McGee, the critical volume fraction was chosen when the probability that one cylinder intersecting at least two other cylinder is 0.5The size distribution of graphene particles and CNTs can be considered in the calculation of the number of contacts through size distribution function. The number of CNT-to-CNT, graphene-to-graphene, and graphene-to-CNT contacts can be derived as follows  was then added to each PAN solution. The graphene solutions were stirred and then ultrasonicated. Then, the solutions were centrifuged at 5000 rpm for 15 min and the supernatant was vacuum-filtered through filter paper having a pore size of 1.2 μm . CNT and graphene inks were prepared in the same manner. The separate graphene and CNT inks were then mixed together in various proportions to make different CNT:graphene hybrid inks.Graphene particles , multi-walled carbon nanotubes , poly(acrylonitrile) , and N,N-dimethylformamide  were used to prepare CNT and graphene hybrid inks using the following process. First, PAN solutions were prepared at 0.25, 0.5, 1, and 2 wt% concentrations in DMF (70 g). The solutions were stirred on a hot plate at 90 °−1 at 25 °C to adjust the printing parameters. The average size of the particles in an ink was measured using a dynamic light scattering spectrophotometer . The zeta potential was also measured using an electrophoretic light scattering spectrophotometer  to evaluate the degree of dispersion of the particles. TGA was carried out to calculate the amount of graphene, CNT, and PAN in the inks.The viscosity of each solution was measured using a rheometer  at shear rates of 4.642, 10, 21.55, 46.42, 100, 215.5, and 464.2 s2 with a drop spacing of 25 μm, varying the number of printings from 1–25. The thicknesses and morphologies of the printed inks were investigated using AFM and SEM, respectively. The electrical properties of the printed inks were measured using the four-point probe method.An inkjet printer  was used to print the prepared inks on a substrate. The droplet formation of the inks was controlled by changing the cartridge settings, including the jetting voltage, waveform, meniscus control, and temperature. Photo paper was selected as the substrate. The inks were printed in a square pattern 7 × 7 mmHow to cite this article: Shim, W. et al. Optimally conductive networks in randomly dispersed CNT:graphene hybrids. Sci. Rep.5, 16568; doi: 10.1038/srep16568 (2015).


Angiogenesis is an important process involved in the pathogenesis of diffuse parenchymal lung diseases. The aim of the study was to compare the angiogenic profile of patients with sarcoidosis and idiopathic pulmonary fibrosis (IPF) based on analysis of circulating factors.Serum concentrations of angiopoietin-2 (Ang-2), follistatin, granulocyte-macrophage-colony stimulating factor (GM-CSF), interleukin-8 (IL-8), platelet derived growth factor-BB (PDGF-BB), platelet endothelial cellular adhesion molecule-1 (PECAM-1) and vascular endothelial growth factors (VEGF) were measured in the patients and the healthy subjects.Serum concentrations of G-CSF, follistatin, PECAM-1 and IL-8 were significantly higher in the IPF patients in comparison with the control group and the sarcoid patients. PDGF-BB concentrations were also significantly higher in serum of IPF patients than in sarcoid patients, but not than in the controls. In contrast, Ang-2 and VEGF concentrations did not differ significantly between the three groups. In the sarcoid patients, irrespective of the disease activity or the radiological stage, serum concentrations of these cytokines were similar to the control group.These results indicate that differences may exist in angiogenic activity between patients with parenchymal lung diseases. In contrast to sarcoidosis, IPF is characterized by a higher serum concentration of different molecules involved in the angiogenic processes . The interstitial lung diseases are a heterogeneous group of diffuse parenchymal lung diseases, including sarcoidosis and idiopathic pulmonary fibrosis (IPF). IPF is characterized by tissue damage and exuberant repair with an aberrant wound-healing response leading to a severe disruption of the pulmonary architecture . On the Angiogenesis, defined as the process of growth of new blood vessels, plays a pivotal role in wound healing and may contribute to the fibroproliferation and extracellular matrix deposition observed in IPF and in advanced stages of sarcoidosis –5.Among various markers of angiogenesis, angiopoietin-2 (Ang-2), follistatin, granulocyte-macrophage-colony stimulating factor (GM-CSF), interleukin 8 (IL-8), platelet derived growth factor-BB (PDGF-BB), platelet endothelial cellular adhesion molecule-1 (PECAM-1) and vascular endothelial growth factor (VEGF), seem to be involved in different steps of physiological and pathological angiogenic processes, such as proliferation, maturation and survival of new blood vessels .Ang-2, which is part of a family of vascular growth factors that play a role in embryonic and postnatal angiogenesis and is involved in controlling microvascular permeability, vasodilation, and vasoconstriction by signaling the smooth muscle cells surrounding vessels , 7. In aGM-CSF is a cytokine secreted by macrophages, T cells, mast cells, NK cells, endothelial cells and fibroblasts and it functions as a white blood cell growth factor. Additionally, GM-CSF plays a vital role in angiogenesis through the regulation of VEGF and sVEGFR-1 . PDGF-BBThe aim of this study was to analysis serum concentrations of these markers associated with angiogenic processes, rarely investigated simultaneously in patients with IPF and sarcoidosis; looking for any possible differences and correlations with the main clinical parameters.Forty three non-smoking patients with sarcoidosis and 17 non-smoking patients with IPF characterized in our previous report  were enrSeventeen patients with IPF were diagnosed according to the ATS criteria  .Serum concentrations of Ang-2,follistatin, GM-CSF, IL-8, PDGF-BB, PECAM-1 and VEGF were measured in duplicate  using commercially available multiplex bead-based sandwich immunoassay kits . All assays were performed according to the manufacturer’s instructions. The system allowed simultaneous identification of 9 different molecules in a 96-well filter plate. We used 9 different sets of fluorescent dyed beads with capture monoclonal antibodies specific for each molecule to be analyzed. The appropriate molecule standards (50 μl/well) and samples diluted (50 μl/well) in plasma diluents were added to a 96-well filter plate and incubated for 30 min. at room temperature. After 3 washes, premixed streptavidin/phycoerythrin was added to each well and incubated for 10 min. followed by 3 additional washes. The beads were resuspended with 125 μl of assay buffer, and the molecule reaction mixture was quantified via the Bio-Plex protein array reader.The data analysis was performed using the Bio-Plex Manager software version 4.1.1. (Bio-Rad Laboratories). Values with a coefficient of variation over 12 % were discarded before the final data analysis. The concentration (pg/mL) of different analytes in the plasma samples was identified using standard curves from the multiplex assays. The minimum level of detection for VEGF was 3.27, 11.32 for Ang-2, 5.15 for PDGF-BB, 33.29 for PECAM-1, 8.41 for follistatin, 6.66 for G-CSF and 1.6 for IL-8.p < 0.05.The statistical analysis was conducted using Statistica 6.0 software . The normal data distribution was assessed using Shapiro-Wilk test. Homogeneity of variance was evaluated using Levene’s test. Comparisons between the study groups were performed using the ANOVA, Mann–Whitney or Kruskal-Wallis test, if the distribution of data was abnormal. Correlations were analyzed by Spearman’s test. All results were statistically significant if r = 0.62, P < 0.01), follistatin and PECAM-1  and PDGF-BB and VEGF .In the IPF patients in comparison with the control group and the sarcoid patients we observed significantly higher serum concentrations of GM-CSF, follistatin, PECAM-1 and IL-8. Serum concentrations of PDGF-BB were also significantly higher in the IPF patients than in the sarcoid patients but not than in the controls. In the IPF patients, serum Ang-2 and VEGF concentrations did not differ significantly from these obtained in the sarcoid group and the controls Table , and 7. r = 0.62, P < 0.0001), follistatin and PECAM-1  as well as PDGF-BB and PECAM-1 .In the sarcoid patients concentrations of serum Ang-2, follistatin, GM-CSF, IL-8, PDGF-BB, PECAM-1 and VEGF were similar to the values noticed in the control group. In patients with progressive and non-active  sarcoidosis the serum concentrations of all molecules were similar. No significant differences were observed between stage I, II and III-only serum Ang-2 concentrations in stage I patients was significantly higher than in stage II and III Table . In the Both in the IPF group and the sarcoid group no significant correlations between concentrations of the examined molecules and the pulmonary function parameters  Table .In our study elevated serum concentrations of IL-8, GM-CSF, follistatin, PDGF-BB and PECAM-1 in the IPF patients, but not in the sarcoidosis patients even in active progressive disease have been observed. Surprisingly, VEGF and Ang-2 concentrations  both in the sarcoid and the IPF patients were similar to the concentrations observed in the controls. We did not find any differences in serum concentrations of the examined cytokines obtained from patients with progressive i.e., active sarcoidosis and stable non-active sarcoidosis. To the best of our knowledge we were the first to demonstrate the serum concentrations of Ang-2, PECAM-1 and follistatin in IPF and sarcoidosis.Keane et al. , 19 demoNevertheless, Sekiya et al.  demonstrIn patients with sarcoidosis we previously observed significantly higher concentrations of GM-CSF in the BAL fluid from patients with active sarcoidosis in comparison with the control group . RecentlWe noticed significant elevation of PDGF in serum of IPF patients. Cao et al.  demonstrWe observed an increased serum concentration of follistatin in IPF, but not in sarcoidosis. Follistatin is effective in treating bleomycin -induced fibrosis by blocking the actions of activin and TGF-beta . RecentlIn our study serum concentrations of ANG-2 were also similar in IPF, sarcoidosis and the controls. Serum Ang-2 concentrations in stage I was higher than in stage II and III patients with saroidosis. ANG-2 is an essential factor for the vascular formation and is normally expressed in the endothelial remodeling tissues, while it is higher expressed during hypoxic processes . RecentlThe low angiogenetic serum activity in our patients with sarcoidosis may be, at least in part, explained by the findings of Kambouchner et al. . These aWe are not able to replicate some previously published results of circulating cytokine alterations. Methodological differences may account for our different, yet not contradictory findings: whereas many studies have employed single-cytokine ELISA tests, used plasma or culture supernatant samples, or had cohorts of newly diagnosed disease, we employed a multiplex assay, used serum samples, and had a cohort of chronic disease.Our study has limitations. A relatively small number of patients, especially with IPF, was included into the study and cytokines examinations were performed only in serum, but not in BAL. The results of Diffusing Capacity of the Lungs for Carbon Monoxide (DLCO) were available only in few patients as no correlations were estimated between DLCO and cytokines concentrations. No relationships were checked between the examined angiogenic molecules and other potential markers of sarcoidosis or IPF activity, for example: ACE (angiotensin converting enzyme) or LDH (lactate dehydrogenase), as no such data were available. It would be interesting to compare results obtained from patients with IPF with those at stage IV of sarcoidosis i.e., with fibrotic lung changes, it was very difficult however to collect a sufficient number of previously untreated sarcoid patients in IV stage.These results indicate that differences may exist in angiogenic activity between patients with parenchymal lung diseases. In contrast to sarcoidosis, IPF is characterized by a higher serum concentration of different molecules involved in the angiogenic processes.


Atomic level engineering of graphene-based materials is in high demand to enable customize structures and properties for different applications. Unzipping of the graphene plane is a potential means to this end, but uncontrollable damage of the two-dimensional crystalline framework during harsh unzipping reaction has remained a key challenge. Here we present heteroatom dopant-specific unzipping of carbon nanotubes as a reliable and controllable route to customized intact crystalline graphene-based nanostructures. Substitutional pyridinic nitrogen dopant sites at carbon nanotubes can selectively initiate the unzipping of graphene side walls at a relatively low electrochemical potential (0.6 V). The resultant nanostructures consisting of unzipped graphene nanoribbons wrapping around carbon nanotube cores maintain the intact two-dimensional crystallinity with well-defined atomic configuration at the unzipped edges. Large surface area and robust electrical connectivity of the synergistic nanostructure demonstrate ultrahigh-power supercapacitor performance, which can serve for AC filtering with the record high rate capability of −85° of phase angle at 120 Hz. Atomic level engineering of graphene-based materials is highly demanded for the customized structures and properties. Here, the authors show heteroatom dopant-specific unzipping of carbon nanotubes as a reliable and controllable route to customized 'intact crystalline' graphene-based nanostructures. A better controllability over unzipping mechanism is highly demanded for the minimal damage to the genuine graphene-based carbon structures16242526Unzipping of graphene basal plane is a valuable route to tailor carbon nanostructures123456781011121322 hybridized carbon framework, enable atomic-scale site-selective unzipping reaction. Although pristine CNTs are stable up to the electrochemical potential above 0.8 V, nitrogen-doped CNTs (NCNTs) are readily unzipped below 0.6 V. Detailed investigation on the reaction mechanism reveals that substitutional pyridinic nitrogen (Np)-dopant can specifically initiate CNT wall unzipping. Such a dopant-specific unzipping at moderate potential enables fine controllability of unzipping level and intact crystallinity of the unzipped structures with well-defined edge configuration. Taking advantage of these unique features, we are able to synthesize intact crystalline graphene-based nanostructures, where unzipped graphene nanoribbons are seamlessly connected to the CNT strands. This structural feature offers synergistic properties comprising large surface area and robust electrical conductivity, which are highly desirable for electrochemical applications, such as energy storage. As a representative application, we demonstrate ultrahigh-power double-layer capacitors (DLCs) for alternating current (AC) line-filtering performance.In this work, we present dopant-specific electrochemical unzipping of CNTs as a controllable method for intact crystalline unzipping. Heteroatom dopants, such as nitrogen substitutionally incorporated into sp2SO4 aqueous solution at a positive potential of 0.6–0.8 V, electrochemically developed high-energy oxygen species react with the carbon atoms nearby the Np-dopant sites and initiate unzipping242SO4 solution and subsequently unzipped (unzipped . Eventuaunzipped  are formunzipped .29ID/IG) from 1.06 to 1.52 and up-shift of G band position from 1,581 to 1,588 cm−1 result from the decrease of in-plane sp2 domain size upon C=C bond breaking reactions263132Chemical structure of typical unzipped materials was characterized by X-ray photoelectron spectroscopy (XPS) and Raman spectroscopy. Deconvoluted C1s XPS spectrum  shows thp peak is specifically reduced during unzipping, whereas quaternary nitrogen (NQ) is not influenced. Concurrently, Fe2p XPS results (p atoms to generate FeN4 moieties in the graphene plane+) can replace the Fe2+ ion to form H2N4 moiety  is easily tunable by adjusting NH3 gas partial pressure during the synthesis of NCNTs36p-dopant-specific unzipping is triggered at the significantly reduced potential of 0.6 V  enables the fine controllability of unzipping propagation. Ic) is another measure to monitor the unzipping progress, particularly in terms of surface area enhancementIc rapidly increases in the early stage because of the enlargement of surface area by gradual exposure of the inner surface of NCNTs, and the rate enhancement of Ic slowdowns after 4 h  were measured at 1–400 V s−1 . Reproducan rate . This facan rate Electrochemical impedance spectroscopy further confirms the outstanding power performance of the DLCs with unzipped structures. 4142434445In a DLC, series resistance, which hampers the power capability of the devices, can be simply considered as the sum of electronic and ionic resistance. The vertically aligned unzipped morphology of our electrode materials can effectively minimize the resistive elements. The vertical alignment offers idealized straight pathway for facile ion transport and homogeneous distribution of stored charges at electrode surfaces4644CA, F cm−2) is also calculated  of DLCs fabricated with 5-μm-NCNT forest, 5- and 1-μm-unzipped structures are 616, 468 and 212 μs with 2.68, 1.24 and 1.15 Ω of total resistance at 120 Hz, respectively. These values are sufficiently smaller than the standard 8.3 ms period, which is crucial for the effective AC line-filtering at 120 Hz. The unzipped nanostructures also demonstrate a very small relaxation time constant  at 120 Hz, significantly higher than previous carbon based-electrodes. This CV value is approximately three orders of magnitude higher than those of typical AECs (∼10−4 F cm−3)4344In this work, we report the ultrahigh-power performance of unzipped structure-based DLCs. In contrast to the intensive research efforts for high-energy-density supercapacitors155253542-hybridized carbon plane allows the transformation of structural dimension among zero-, one-, two- and three-dimensions. Overall, customized dopant-specific unzipping of graphene-based materials possibly guides us to rarely explored opportunity for multi-dimensional material design with atomic level precision as well as physicochemical property tuning, which are generally desired for energy storage, environmental remediation, nanomedicines and other versatile applications. Moreover, this precisely controllable unzipping mechanism grants a significant step forward to idealized intact crystalline graphene nanostructures in conjunction with the potential advances in the precise control of dopant position, dopant density and the chirality of CNTs.As demonstrated from the record-high power DLCs, delicate manipulation of graphene-based nanostructures may offer straightforward routes to high-performance applications. The highly specific reaction features of our unzipping mechanism can serve as a generic principle to diversify graphene-based nanostructures, whose physicochemical properties are strongly dependent on their structural dimension and defect formation123456783 gas partial pressure and plasma power (2/Si was floated on diluted hydrofluoric acid (HF) solution to separate NCNT forests from SiO2/Si substrate by wet-etching of SiO2 layer. The floated NCNT forests were carefully transferred onto a glassy carbon electrode. To enhance the mechanical and electrical contact between NCNT forests and glassy carbon electrode, the recovered electrode was thermally annealed at 65 °C for 12 h. The NCNT electrode was used as working electrode in typical three electrode configuration with mercury/mercury (I) sulfate reference electrode and platinum wire (counter electrode). The unzipping was carried out at room temperature in 1 M H2SO4 solution with positive electrical potential from 0.5 to 1.0 V for various unzipping period. Before each unzipping reaction, the electrolyte was purged with high purity N2 gas over 10 min.NCNTs were grown by plasma-enhanced chemical vapour deposition with Fe catalyst. The density of N dopants was easily controllable by adjusting NHma power . As-grow2/Si substrate. Raman and XPS spectra were recorded with ARAMIS (Horiba Jobin Yvon) and Sigma probe (Thermo VG Scienfitic), respectively.HR-TEM images were collected on a double-aberration corrected HR-TEM  located at KAIST Analysis Center for Research Advancement, operating with a monochromator (excitation 0.06) at 80 kV accelerating voltage. The structural changes were also measured by field-emission scanning electron microscope . Samples were prepared by critical point drying to maintain the as-synthesized vertically aligned structure. AFM images were obtained with scanning probe microscope . Samples were prepared by drop-casting from ethanol suspensions onto SiO−2) on Pt foil were used as an electrode for ultrahigh-power DLCs. Before DLC assembly, the unzipped structures were electrochemically reduced at −0.6 V for 30 min to remove dispensable oxygen functional groups. The prototype DLCs were composed of two identical unzipped nanostructure electrodes and separator. All electrochemical characterization was carried out with a potentiostat  using 1 M KOH solution as an electrolyte under ambient environment. All CVs in CA, F cm-2)Unzipped nanostructures ; Z"(f) is the imaginary part of the impedance.where CV, F cm−3) was calculated with equation (2)The frequency specific volumetric capacitances  and C"(f),The specific capacitance of DLCs prepared with unzipped nanostructures could be calculated by using C′(f) is the real part of specific capacitance; C"(f) is the imaginary part of specific capacitance; Z′(f) and Z"(f) are real and imaginary parts of the impedance; |Z(f)| is the absolute value of the impedance.where Ab-initio Simulation Package codes. The Perdew–Burke–Emzerhof exchange-correlation energy functional and projected-augmented wave potentials were employed. A plane-wave kinetic energy cutoff of 400 eV and 1 × 1 × 1 gamma k-point were used for 12 unit cells of  single-walled CNT with 240 atoms. The supercell size is 20 × 29.514 × 20 Å3 with the tube axis along the y direction, and the diameter of model is about 6.726 Å. Atomic forces were relaxed <0.025 eV Å−1.To understand the mechanism for electrochemical unzipping of CNTs, first-principles DFT calculation was performed using Vienna How to cite this article: Lim, J. et al. Dopant-specific unzipping of carbon nanotubes for intact crystalline graphene nanostructures. Nat. Commun. 7:10364 doi: 10.1038/ncomms10364 (2016).Supplementary Figures 1-17, Supplementary Table 1, Supplementary Note 1 and Supplementary References.