Graphene is the most recent superior material which could potentially initialize another new material Age. However, while being exploited to its full extent, conventional processing methods fail to provide a link to today’s personalization tide. New technology should be ushered in. Three-dimensional (3D) printing fills the missing linkage between graphene materials and the digital mainstream. Their alliance could generate additional stream to push the graphene revolution into a new phase. Here we demonstrate for the first time, a graphene composite, with a graphene loading up to 5.6 wt%, can be 3D printable into computer-designed models. The composite’s linear thermal coefficient is below 75 ppm·°C Tg) and extruded through the 3D printer’s nozzle, whose diameter defines the printing resolution. Computer-designed 3D objects are constructed through successively stacking thermoplastic layers. The printing process not only is easy and rapid, but also can realize exotic topological structures which challenges the traditional casting and welding techniques.Although 3D printing technology has been around since the 1980 s, it hasn’t gained enough momentum to be commercialized until recently12 carbon crystal, has never been used as a 3D printing feedstock. Its two dimensional honeycomb structure endows it with many unique properties9102/g)2 S/m for reduced graphene oxide monolayers and 2 to 3 orders of magnitude higher for pristine graphene)Driven by new applications, the printable category keeps expanding into many frontier scientific and engineering fields345673 C-C bonds242 bonds in graphitic domains. Chemical reduction of GO is a cost-effective strategy to prepare graphene sheets by utilization of reductant. Hydrazine has been commonly used to high yield production of reduced GO (rGO) owing to its ability to in situ reduce oxygenated functional groups. It was reported that rGO restores 80% sp2 in its structure, with the rest of sp3 bonds derived from residual oxygen (C:O atom ratio 12.5:1) after the reduction with hydrazineStarting with the graphene composites, graphene sheets have been successfully added into polymers to explore novel applications in mechanical reinforcement, flame retardant or electrical conductive composites16189202122in-situ formed without obvious phase separation. To fractionally precipitate G-ABS composites from NMP, the dispersion was added into water. The precipitated G-ABS composite is shown in Tg in the heated build chamber (HBC). Subsequently, based on layer-by-layer manufacturing technique1These graphene-polymer composites were further extruded into 1.75 mm diameter filaments to fit the commercialized 3D printer. As shown in σc) was investigated on hot pressed rectangular composite models using a four-probe method . Φc in previous graphene-based polymer composite was reported to be around 0.1 vol%303229The graphene dispersion’s homogeneity directly affects the composite’s printing ability and other physical properties. With scanning electron microscopy (SEM), graphene individuals can be captured from the polymer matrix with different contrast and detailed morphologies. SEM images were taken from both the surface and the cross section of the composite to give a spatial perspective of the graphene dispersion inside composites . Graphene method  and plote method  At 0.4 war model , its meailaments  may acco−1 and a G peak at ~1600 cm−1, related to the defects and sp2 structures in graphene~2240 cm−1 can be ascribed to the C≡N stretch in ABSRaman and ultraviolet-visible (UV-vis) analysis were used to further verify the presence of graphene in composites. A laser excitation with a wavelength of 785 nm was used to record Raman spectra from the samples . The repTg and then cool down from it to room temperature (RT). Therefore, before any 3D printing, Tg is the first and most vital parameter to be determinedTg values can be calculated and listed in Table S3Tg value close to ~105.8 °C. This value slightly shifted to ~110 °C with incremental graphene loadings , dynamic mechanical analysis (DMA), thermogravimetric analysis (TGA) and thermomechanical analysis (TMA). In FDM printing, printing materials need to be heated above Tg and thermal stability of the composites as compared to the pure ABS polymersIt was expected that polymer’s viscoelasticity would change after the incorporation of graphene sheets. The loss factor (tan δ), ratio of loss and storage modulus, is usually a characteristic indicator of how much energy dissipates in the polymer matrix322 atmosphere, the degradation of the samples occurred with a sharp weight loss around 400 to 480 °C (onset) is defined as the temperature when testing sample loses 5% of its total weightonset, the composite materials simply melt into viscous fluids rather than decomposing. The set temperature was also sufficiently higher than the G-ABS’s Tg, ensuring the filaments smoothly printed through the 3D printer.The HBC needs to get hot enough to soften the composites22o 480 °C , accompaTg. CTE values were extracted in the same temperature range, according to CTE = (dL/L0)·(1/dT), where dL/L0is the dimensional deformation along the vertical axis and dT is the temperature change. All G-ABS samples’ CTE values were below 75 ppm·°C−1, which exhibited no significant variation from pure ABS’s 78.25 ppm·°C−1, suggesting suitable for the 3D printing. With Tg values ranging from 104 to 110 °C, by calibrating the HBP temperature at ~80 °C, 20 to 30 °C below Tg and maintaining the nozzle and HBC temperature at 130 °C and 230 °C, separately, we can efficiently avoid thermal deformation during the printing process  with a printing area of 140 mm x 180 mm x 135 mm was used to print our graphene related materials. The nozzle can move back and forth at a rate of ∼20 mm/s with a horizontal resolution of 200 μm or 400 μm (depending on the inner diameter of the nozzles) and vertical resolution of 200–400 μm to print out 3D models. The temperatures of HBC, HBP and nozzle were set at 230, 80 and 130 °C, respectively, for G-ABS 3D printing. They were changed to 190, 60 and 130 °C, respectively, for G-PLA 3D printing.Full Methods and details of characterization for G-ABS and G-PLA composites are given in the How to cite this article: Wei, X. et al. 3D Printable Graphene Composite. Sci. Rep.5, 11181; doi: 10.1038/srep11181 (2015).


It requires the ability to process on-going turns at talk while planning the next, and to launch this next turn without considerable overlap or delay. Recent research has investigated the eye movements of observers of dialogs to gain insight into how we process turns at talk. More specifically, this research has focused on the extent to which we are able to anticipate the end of current and the beginning of next turns. At the same time, there has been a call for shifting experimental paradigms exploring social-cognitive processes away from passive observation toward on-line processing. Here, we present research that responds to this call by situating state-of-the-art technology for tracking interlocutors’ eye movements within spontaneous, face-to-face conversation. Each conversation involved three native speakers of English. The analysis focused on question–response sequences involving just two of those participants, thus rendering the third momentarily unaddressed. Temporal analyses of the unaddressed participants’ gaze shifts from current to next speaker revealed that unaddressed participants are able to anticipate next turns, and moreover, that they often shift their gaze toward the next speaker before the current turn ends. However, an analysis of the complex structure of turns at talk revealed that the planning of these gaze shifts virtually coincides with the points at which the turns first become recognizable as possibly complete. We argue that the timing of these eye movements is governed by an organizational principle whereby unaddressed participants shift their gaze at a point that appears interactionally most optimal: It provides unaddressed participants with access to much of the visual, bodily behavior that accompanies both the current speaker’s  The contrast formed by the white sclera surrounding a darker iris and pupil is unique to the human eye . This coIn social interaction, a system of turn-taking organizes opportunities to speak. According to Recently, a new experimental paradigm has been developed for the study of the cognitive processes that underpin turn-taking from a third-person perspective. The general procedure involves participants being presented with a pre-recorded dialog or conversation between two people on a computer screen while their eye movements are tracked and timed with respect to the turns at talk they hear. Experimental studies using this novel paradigm have shed light on the precise timing of eye movements and turns at talk by measuring where observers of dialogs look and when they do so.before he or she begins to speak  dialogs have not found evidence for anticipatory looks to the next speaker. While there is some discrepancy in findings, studies using the novel third-person perspective eye-tracking paradigm described above have provided us with valuable first insights into how adults may process turns at talk and transitions between them, as well as how children acquire this skill during development. However, two issues emerge from this work.The first issue has already been alluded to in the preceding section and concerns the third-person perspective as such. Recently, less cognitive resources available for projection in live conversation, meaning gaze shifts may be primarily responsive to the next speaker beginning to speak rather than anticipatory. However, it could also be that eye movements in face-to-face interaction do not reflect the projection of current or next turns at all, but that the social norms and dynamics of conversation determine where participants look and when. Thus, while third-person perspective eye-tracking paradigms allow for a high degree of experimental control and manipulation to investigate eye movements during turn-taking, and the role semantics and prosody play in this context  and ten dyadic (two of the three participants) conversations Figure 1 provides an overview of the laboratory set-up and equipment.The recordings took place in a sound proof room equipped with professional lighting suitable for high quality audio and video recording. Participants sat in standard height chairs with armrests, arranged in a triangle with the chairs equidistantly placed from one another. A ceiling microphone recorded the entire conversation. Each participant wore a head-mounted lightweight uni-directional microphone (Shure SM10A), which recorded only the respective participant’s voice, and a pair of eye-tracking glasses . In addition, three HD video cameras  recorded frontal views of each person . Due to the spatial arrangement of the chairs with respect to the cameras, each person was also visible from the right and left side in the recordings made by the respective other two video cameras. Figure 2). This resulted in a time resolution of approximately 41 ms, the duration of a single frame. The synchronization was based on audible and visible information from a clapperboard used at the beginning and end of each session.For each session, the recorded material resulted in three individual videos from the cameras, three individual videos from the eye-trackers , three individual audio files, and the audio file from the ceiling microphone. The audio tracks were recorded in sync using a four-channel audio recorder (Edirol/Roland R-44). The six video recordings and three individual audio recordings were combined and synchronized in Adobe Premier Pro CS4 and then exported as a single audio–video file for analysis (MP4) at 24 frames per second  and were handed study-packs, including information about the study and procedure of the session, forms asking about their language background, screening questionnaires ruling out motor and speech impairments, as well as consent forms and questionnaires about handedness and a variety of social dimensions. Once the study-packs had been completed by all participants  and any queries had been answered, participants were seated in their chairs in the recording room. All equipment was prepared beforehand, allowing immediate fitting of the microphones and the eye-trackers .Each recording session lasted approximately 40 min. in total, with the first 20 min. constituting a trialogue phase and the second 20 min. a dialog phase. Upon completion of the initial fitting procedure, the two investigators left the room and waited in an adjacent area until the first 20 min. had elapsed. At this point, they compared performance of the three eye-trackers and asked the person wearing the eye-tracker with the poorest calibration to leave the room. Once the remaining two participants had talked for another 20 min., all three were reunited in the recording room and asked to complete the social questionnaires contained in the study-packs. This was to ensure that questions about human communication and behavior  would not influence participants’ behavior during the conversations.  Participants were then asked one more time for their written informed consent relating to how their data should be handled, thanked, and financially compensated for their participation (26 euro per person). The entire test session lasted around 120 min. The study was approved by the Social Sciences Faculty Ethics Committee, Radboud University Nijmegen.N = 35). In a small number of cases (N = 2) the response was exclusively non-verbal ; in those cases the beginning of the response was timed to the first frame of visible movement. These annotations were then imported into ELAN 4.61  sequences in which the question was addressed to a single participant who then produced a response. All QR sequences were identified by an experienced conversation analyst (Kobin H. Kendrick), resulting in a total of 281 questions and their responses . Criteria for identifying QR sequences in our dataset were based on the coding scheme proposed by LAN 4.61 .All questions in the dataset were analyzed for the presence and location of points of possible completions before the end of the turn, drawing on conversation-analytic research on turn construction . For a pThe analysis of points of possible completion revealed a number of recurrent types. If a turn contained two complete questions, a point of possible completion – represented here by a vertical bar – was identified after the completion of the first, whether the two questions were produced one after the other  or with a short silence between the two  or is it just (0.2) any money is good?”). If a turn contained a possibly complete question together with an increment, a contingent addition to a turn that continues its grammatical structure , a pointK = 0.72  who independently identified the presence and precise location of points of possible completion in the dataset revealed strong reliability, The ELAN files containing the QR annotations were linked to the synchronized videos in order to annotate the unaddressed participants’ eye movements during the QR sequences. These annotations were done manually, on a frame-by-frame basis. At each frame during the QR sequence, the gaze fixation point generated by the SMI software for the unaddressed participant (henceforth referred to as the gaze cursor) was categorized as being (1) on speaker A, (2) on speaker B, (3) on self , (4) on the surroundings , or (5) as not identifiable from the eye-tracker data . Based on this coding scheme, 45 of the originally 281 QR sequences (16.0%) were discarded from further analysis of the unaddressed participants’ eye movements due to insufficient data. .Out of the remaining 235 QR sequences, unaddressed participants moved their gaze from speaker A to speaker B in 105 (45.5%) QR sequences. In order to be considered a valid gaze shift for our analysis, the trajectory had to be one that clearly moved from A to B, without the gaze pausing elsewhere in between (such as on self or background objects). In the remaining 131 sequences, unaddressed participants either did not shift their gaze at all and instead fixated speaker A, speaker B, themselves, or the surroundings throughout, or they did move their eyes but in the opposite direction, that is, from speaker B to speaker A. While these cases are interesting in themselves, they tap into a different phenomenon than the one under investigation here and require analysis and discussion elsewhere.Regarding those 105 QR sequences that did reveal a gaze shift from speaker A to speaker B , the average question duration was 2018 ms , and the average response duration was 1899 ms . Due to the highly dynamic nature of conversation brought about by, amongst other things, differences in personality, age, gender, closeness of friendship, and topic of discussion, the seven triads of course differed in the number of QR sequences they contributed to our analysis . Likewise, participants within the triads differed in the extent to which they contributed to the conversation by asking questions, but none of the conversations excluded participants . Basing analyses of QR sequences in conversation on samples that are determined by the participants’ spontaneous behavior, thus resulting in varying numbers of QR sequences across separate interactions, is the standard procedure for corpus studies and in line with existing research e.g., .when exactly this gaze shift occurred. The time window we took into consideration for identifying gaze shifts relevant for this analysis stretched from the beginning of A’s turn to the end of B’s turn. In all cases of gaze shifting from speaker A to speaker B within this time window, unaddressed participants looked at the face of speaker A and then moved their gaze from there to the face of speaker B. Using the frame-by-frame gaze annotations, we identified the first frame at which the gaze cursor left speaker A, defined as the frame at which the gaze cursor was no longer on, overlapping with, or directly adjacent to speaker A’s head or technical head-gear . At what time point before or during B’s turn the unaddressed participant’s gaze arrived at speaker B was not of relevance for the present analysis. Annotations were made in ELAN to measure the duration from the first gaze shift away from speaker A by the unaddressed participant to two points within the question turn: (1) the end of the turn and (2) the first point of possible completion of the question, for those questions that had a possible completion before the end of the turn. In addition to measuring the duration of these intervals (in ms), the values were set as either positive or negative. This was done to identify the temporal order of the respective events, with negative values indicating an anticipatory gaze shift before a point of possible completion or the end of a turn and positive values indicating the inverse.For these 105 QR sequences that did reveal a shift of the unaddressed participant’s gaze from speaker A to speaker B, we identified However, we need to consider that it takes time to plan and launch these eye movements before they are observable. This process is estimated to take on average around 200 ms . We therGaze coding was performed by two independent coders (LD and MvdG) blind to the study’s predictions and assumptions. In addition, their coding was checked by one of the two senior analysts (JH and KK), and any errors in coding  were discussed and corrected. Due to the considerably more objective coding criteria applied for our gaze analysis in comparison to the identification of points of possible completion, formal reliabilities were calculated for the latter only.estimate of the mode, all mode values given should be considered close approximations of the true value and decimal places are not stated for those values. Note also that these distributions are based on binned data brought about by our video frame rate providing a measure every 41.7 ms (24 fps).All statistical analyses were conducted in R 3.1.1 . The denN = 57) of the questions had at least one possible completion before the end of the turn. Here, we focus our analyses first on the end of the turn and then on its first possible completion (which corresponded to the end of the turn for 45.7% of questions).Out of the 105 QR sequences analyzed here, 54.3% . Because, as we have already noted, experimental research indicates that overt eye movements are planned about 200 ms in advance of them being observed, the covert initiation of unaddressed participants’ eye movements from speaker A to speaker B occurred most frequently around 250 ms prior to the end of questions .First, we measured the time point of each first observed gaze shift away from speaker A (and toward speaker B) with respect to the end of speaker A’s turn. This showed that the estimate of the mode of these data is located very close to the end of the question, namely just 50 ms prior to turn end (see N = 63) of QR sequences were associated with observable gaze shifts that anticipated the end of the question turn. When taking into account the time it takes to prepare these gaze shifts, the percentage of anticipatory gaze shifts increases to 73.3% (N = 77).On the whole, 60.0% . Taking into account the 200 ms required to plan and launch observed eye movements, the distribution yields a mode of 40 ms just prior to the first possible completion .Because many of the questions in our data had a point of possible completion prior to turn end (as seen above), we carried out a second analysis in which we timed unaddressed participants’ first gaze shift away from speaker A with respect to the first possible completion of each question; this corresponded to the end of the turn for those questions with only one possible completion. When plotting our data with respect to this reference point, the distribution yields a mode of about 160 ms just after the first possible completion (see N = 36) of cases, unaddressed participants shifted their gaze from current to next speaker before the first possible completion, and in 55.2% (N = 58) of cases unaddressed participants’ gaze shifts had at least been planned prior to this point.When timing the gaze shifts with respect to the first possible completion of the question, we still see that a considerable number of gaze shifts from current to next speaker happen prior to the first possible completion, but much less so than when timing these gaze shifts with respect to the end of the turn: in 34.3%  = 0.234, p < 0.05). This means that for responses that coincide with first possible completions of questions, gaze shifts could either be due to unaddressed participants recognizing the possible completion or reacting to the onset of the response. In order to tease these two factors apart, we carried out two further analyses by looking at two subsets of our data.Above we have shown that unaddressed participants are sensitive to first possible completions, as can be seen from the timing of their gaze shifts. However, considering that first possible completions mark points at which transition to the next speaker becomes relevant, addressed participants, too, are likely to be sensitive to these points and time their responses to them. This means that the first possible completion of speaker A’s question and the onset of speaker B’s response may often coincide. We therefore also measured the timing of the response 3 after the first possible completion (N = 54). If the timing of the unaddressed participants’ gaze shifts we observed based on the sample as a whole is explained by response onset rather than by first possible completions, then the mode for this subset of data should be at least 200 ms later than the mode for the distribution based on the entire sample. However, as can be gleaned from Figure 6, the mode for this subset is 105 ms (Range = –2337–2419), which differs only slightly from the mode of 160 ms for the entire sample. If anything, unaddressed participants’ observed gaze shifts occur slightly earlier when B’s response occurs 200 ms after the first possible completion, and certainly no later than when we consider the entire sample. Thus, unaddressed participants’ eye movements in our data do indeed appear to reflect sensitivity to the first possible completion of the question, rather than being a mere reaction to the onset of the response.For the first analysis, we considered only those QR sequences where speaker A’s first possible completion and speaker B’s response onset did not coincide but where the response comes after the possible completion. For this comparison, we selected those sequences where the response occurred more than 200 ms N = 15). If response onset alone also attracts unaddressed participants’ attention and, as a consequence, their gaze, then we should see that the mode of the distribution of gaze shifts for this subset is earlier than that for the distribution based on the sample as a whole. As can be seen from Figure 7, this was indeed the case, with the mode of observed gaze shifts for the subset of early responses being -35 ms, compared to an overall mode of 160 ms. This means that the eye movements within this subset must have been planned -235 ms before the first possible completion, which corresponds closely to the onset of these early responses at -200 ms or less.However, we of course do acknowledge that response onset may also play a role in the timing of unaddressed participants’ eye movements. In order to explore this further, we looked at another subset of our data, namely those cases in which speaker B’s response began at least 200 ms before speaker A’s first possible completion , they move their gaze from one speaker to the next in about half of all question–response sequences. This confirms that, even when participating in actual ‘on-line’ interaction, unaddressed participants show a tendency to track current speakers cf., , at leasWhen we examined the timing of gaze shifts with regard to turn ends, we found that the most frequent gaze shifts from current to next speaker were planned around 250 ms and observable around 50 ms prior to turn end. Thus, the results suggest that unaddressed participants’ gaze shifts are predominantly anticipatory in nature when timed with respect to the end of question turns. As such, it replicates the findings from third-person perspective eye-tracking studies that have found evidence for adults shifting their gaze to the next speaker prior to turn end e.g., . OverallHowever, despite the comparability, gaze shifts prior to turn end appear to be more common in actual conversation. In the present study, the majority of gaze shifts from current to next speaker occurred prior to turn end (60% of cases) or were planned and launched prior to this point (73% of cases). In contrast, in studies using the third-person perspective paradigm, either only a relatively small proportion of gaze shifts occurred prior to turn end e.g.,  or none Figure 5) would occur after the possible completion, not before.The present study went further than just measuring eye movements with respect to turn ends. Here, we have taken into account the intricate structure of questions, and, more specifically, the first possible completion of each question, which for half of our questions was not the actual end of the turn. These points of possible completion create opportunities for a next speaker to take the turn, and it has been argued that participants in conversation are sensitive to these transition-relevance places . Indeed,well before it reaches possible completion  At the same time, however, we have been able to show that early responses which precede the first possible completion also attract unaddressed participants’ gaze, and thus may certainly be a contributing factor in those instances where first possible completion and onset of the response coincide. Hence, taking overlap between current and next speakers into account appears crucial if we aim to understand unaddressed participants’ eye movements in natural conversation. Importantly, the effect of both factors – first possible completions and response onset – is based on a process of To date, all studies of the phenomenon under investigation here – the redirection of gaze by unaddressed participants from current to next speakers at turn transitions – have used it to gain insight into processes involved in turn-taking. But the conclusion that the eye movements of unaddressed participants do not anticipate the first possible completion of the current turn, and thus do not necessarily reflect a projection of it, leads us to reconsider the nature of the phenomenon and to look elsewhere for principles that can account for the fine temporal coordination that we observe.It has long been argued that among the many functions of gaze behavior in social interaction the use of gaze to display attention, engagement in the interaction, and recipiency to the current speaker is paramount . Gazing 4. Our findings that unaddressed participants’ gaze behavior during question–response sequences appears to be organized according to a principle that optimizes recipiency also fits well with the notion of an ‘intrinsic motivation for participation,’ as it were (Although the gaze behavior of unaddressed participants does not necessarily reflect projection of the current turn, optimizing recipiency between current and next speakers does make use of the turn-taking system in other ways. Our results provide new and quantitative evidence that the recognition of points of possible completions are indeed core to the turn-taking system in conversation, as argued in  it were . Both ScIn addition, it appears from our results that response onset can trump first possible completions, at least when these responses come prior to the first possible completion. In such cases, the timing of the response appears marked and may signal a marked social action see . That unThe present study looked at eye movements with respect to one particular type of turn, that is, questions. The present study has provided us with a first glimpse of the intricate connections between turns at talk and unaddressed participants’ eye movements in spontaneous, multi-person interaction. On the one hand, we have here reproduced the basic findings from studies using the third-person perspective eye-tracking paradigm in spontaneous, live conversation. On the other, our data have provided us with stronger evidence that gaze shifts by unaddressed participants toward next speakers precede the end of the current turn than previous studies have. As such, our findings corroborate the notion that interactive paradigms do, at least in part, provide different insights than paradigms involving passive observation . FurtherThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Antibodies targeting the checkpoint molecules programmed cell death 1 (PD-1) and its ligand PD-L1 are emerging cancer therapeutics. We systematically investigated PD-1 and PD-L1 expression patterns in the poor-prognosis tumor entity high-grade serous ovarian carcinoma.in silico validation of mRNA data was performed in The Cancer Genome Atlas (TCGA) dataset.PD-1 and PD-L1 protein expression was determined by immunohistochemistry on tissue microarrays from 215 primary cancers both in cancer cells and in tumor-infiltrating lymphocytes (TILs). mRNA expression was measured by quantitative reverse transcription PCR. An p < 0.035 each), and most being significant for OS. Most factors also had prognostic value that was independent from age, stage, and residual tumor. Moreover, high PD-1+ TILs as well as PD-L1+ TILs densities added prognostic value to CD3+TILs . The significant positive prognostic impact of PD-1 and PD-L1 mRNA expression could be reproduced in the TCGA gene expression datasets .PD-1 and PD-L1 expression in cancer cells, CD3+, PD-1+, and PD-L1+ TILs densities as well as PD-1 and PD-L1 mRNA levels were positive prognostic factors for progression-free (PFS) and overall survival (OS), with all factors being significant for PFS (Despite their reported immune-modulatory function, high PD-1 and PD-L1 levels are indicators of a favorable prognosis in ovarian cancer. Our data indicate that PD-1 and PD-L1 molecules are biologically relevant regulators of the immune response in high-grade serous ovarian carcinoma, which is an argument for the evaluation of immune checkpoint inhibiting drugs in this tumor entity. High-grade serous carcinoma (HGSC) is the major histological subtype (approximately 70%) of ovarian carcinomas. It is a poor-prognosis tumor , which is due to late diagnosis (75% in FIGO III/IV) as well as the development of resistance to standard platinum-based chemotherapy. However, high numbers of tumor-infiltrating lymphocytes (TILs) have repeatedly been shown to provide a significant survival advantage in ovarian carcinoma. Particularly the presence of T cells CD3+) and various T cell subpopulations  are indicators of a better prognosis, , 3 stron+ and varImmune checkpoint inhibitors constitute a novel class of cancer therapeutics that do not target the cancer cell itself but rather ligands and receptors on T cells that attenuate the anti-tumoral immune reaction and increase immune tolerance . Emevia AKT or STAT3, a mechanism termed intrinsic immune resistance, or by IFN-gamma produced by activated T cell or NK cells (adaptive resistance). [PD-L1 is induced on monocytes and epithelial cells, upon IFN-gamma stimulation , whereasstance). , 5 The mPD-1 inhibitors  have been approved in therapy-refractory malignant melanoma, and together with PD-L1 inhibitors  are investigated in clinical trials on various recurrent or metastatic malignancies to date. Early clinical trials in ovarian carcinoma are ongoing. , 10 ReliTo estimate if a tumor entity might constitute a candidate neoplasm for evaluation of a specific cancer therapeutic, the expression pattern of the drug target as well as its clinical relevance are of interest. We therefore systematically evaluated the expression of PD-1 and PD-L1 in a cohort of 215 primary ovarian high-grade serous carcinomas by determining protein expression in cancer cells and TILs as well as mRNA expression.n = 201 and n = 202 cases, respectively. If positive, both markers showed a membrane-accentuated expression, which was also often accompanied by a cytoplasmic expression . A significant number of cases did not show any membranous expression on cancer cells , and for further statistical analyses we decided to split the study group into cases with no PD-1/PD-L1 expression (IRS = 0) and cases with any expression. Membranous PD-1/PD-L1 expression were not correlated to each other . As no prognostic effect of cytoplasmic PD-1 or PD-L1 expression in cancer cells could be detected (not shown), only data on membranous expression of these markers will be given subsequently.Data on PD-1 and PD-L1 expression in cancer cells were available for n Figure . Membrann = 200 for each marker). All TILs markers showed a positively skewed distribution, which means that the median was lower than the mean: Intraepithelial CD3+ TILs numbers (per 5 HPF) ranged from 0 to 543, however most cases had rather low numbers of CD3+ TILs  for each marker. Statistical analyses are shown for these values subsequently. CD3+, CD4+, CD8+, PD-1+, and PD-L1+ TILs/mm2 highly correlated with each other . PD-1 expression in cancer cells showed a borderline positive correlation with CD3+ TILs/mm2 , but not with PD-1+, PD-L1+, CD4+, or CD8+ TILs, and PD-L1 expression in cancer was not significantly associated with TILs at all (p > 0.05 each).As stromal TILs had a weaker (CD3) or non-significant impact on prognosis  as compared to intratumoral TILs (data not shown), data for the latter only are are given subsequently. We separately counted CD3+ as well as PD-1+ and PD-L1+ TILs in identical tumor areas ). We furthermore investigated the potential dependence of CD4+ and CD8+ TILs density from combined cancer cell and TILs expression of PD-1 and PD-L1. As shown in PD-1 and PD-L1 cancer cell and TILs expressions were further investigated as to their relationship to the CD4+ and CD8+ T cell population when we grouped cancer cell expression in four groups. We did not observe a dependence of CD4+ or CD8+ TILs density and expression of PD-1 or PD-L1 in cancer cells (p < 0.0001), as well as with CD3+, PD-1+, and PD-L1+ TILs . There was a trend towards a positive correlation between PD-1 mRNA and PD-1 expression in cancer cells , however not between PD-1 mRNA and PD-L1 expression in cancer cells or between PD-L1 mRNA and PD-1/PD-L1 expression in cancer cells (p > 0.1 each).Informative mRNA expression data were available for 200 cases for PD-1 and 204 cases for PD-L1. Both markers showed a rather low expression with PD-1 40-deltaCT values ranging from 24.31 to 32.98 (median 29.49) and PD-L1 data ranging from 25.39 to 33.33 40-deltaCT , while for PD-1 expression only a trend was seen for OS  and OS (p = 0.051) were longest, in cases with only one positive marker were intermediate, and in double negative cases were worst, however as only two tumors were PD-1-/PD-L1- the data for the latter group must be interpreted with caution .Both PD-1 and PD-L1 expressions in cancer cells were significantly linked to a better PFS , p = 0.002. This significant positive impact on prognosis was also seen for PD-L1+ TILs density for both PFS (HR = 0.75 (95% CI 0.58-0.97), p = 0.026) and OS (HR = 0.72 (95% CI 0.55-0.96), p = 0.025), as well as for PD-1+ TILs density for PFS (HR = 0.78 (95% CI 0.62-0.99), p = 0.044), with a lack of significance for OS .First we investigated the prognostic impact of TILs using a cutoff-free approach. Due to the fact that TILs distribution was positively skewed, we logarithmized the data and performed Cox regression analysis using continuous data. A high density of CD3+ intratumoral TILs was a significant positive prognostic marker for PFS and OS on the continuous scale: PFS: HR = 0.65 (95% CI 0.49-0.85), 2, respectively. Using this optimal cutoff, Kaplan-Meier analysis showed that CD3+ TILs had a significantly favorable impact on PFS and OS , with borderline significance in multivariate analysis .As all TIL subpopulations in our study were highly correlated with each other, we wondered if the prognostic effect of PD-1+ and PD-L1+ TILs might only be a bystander effect of the impact of T cell infiltration by itself. We therefore investigated the prognostic effect of combined CD3+ and PD-1+/PD-L1+ TILs density. Surprisingly, tumors that had a high PD-1+ TILs or PD-L1 TILs density in addition to high CD3+ TILs had a better prognosis (both PFS and OS) than tumors with low PD-1+ or PD-L1+ TILs counts despite of a high CD3 infiltration Figure . Thus, Pp = 0.012), but PD-L1 mRNA was not (p = 0.609). Significant cutoff points - similarly to TILs density - were found over a rather large range: for PD-1 mRNA 39 out of 128 cutoff points (39.5%), and for PD-L1 mRNA 49 out of 128 cutoff points (38.3%) showed significant positive prognostic impact  as well as in the subgroup with residual tumor after surgery , and furthermore in cancers without residual tumor after surgery . PD-1 expression (syn. PDCD1 in TCGA) was also a positive prognostic factor for the total cohort, however its prognostic value was of reduced robustness as only few cutoffs were significant: Agilent: 14 out of 460 cutoffs significant (3.0%), optimal cutoff p = 0.02, Figure p = 0.013, not shown), however missed significance in RNA seq data . In the subgroup of tumors with residual tumor after surgery a trend for better OS was seen for PD-1 expression . A partially significant effect was also seen in the subgroup without residual tumor after surgery .Gene expression datasets form The Cancer Genome Atlas project on primary high-grade serous ovarian carcinomas  were anavs > 60 years) and stage (II vs III/IV). Similar results were obtained for OS (not shown).We additionally investigated the prognostic value of PD-1 and PD-L1 expression only for patients that had documented platinum-based chemotherapy. PD-1 and PD-L1 expression, both in cancer cells and TILs as well as mRNA expression were significant prognostic factors for PFS in this well-defined subgroup, too, and significance was retained for all but PD-L1+ TILs and PD-L1/CD3 TILs combination in multivariate analysis . Data foIn this study, we systematically investigated PD-1 and PD-L1 expression in primary high-grade serous ovarian carcinoma. High expression in cancer cells and mRNA expression were favorable prognostic factors, as was the density of PD-1+ and PD-L1+ TILs, moreover, this favorable prognostic effect of TILs was independent from the T cell infiltrate in general. We validated our findings on the mRNA level in the independent cohort of high-grade serous carcinomas from TCGA.n = 12 PD-1+ TILs/mm2 (tumor and stroma not separated), our study: n = 12 PD-1+ TILs/mm2 , and it is remarkable that this small subpopulation of T cells obviously has a biological significance as measured by patient prognosis. This rather surprising finding prompted us to investigate whether the positive prognostic effect of PD-1+ and PD-L1 TILs might be a bystander effect of the intratumoral T cell infiltration in general. The absolute counts of PD-1 and PD-L1 TILs in high-grade serous carcinoma were strongly associated with CD3+ TILs, but only a small fraction of T cells actually expressed PD-1 and PD-L1 and their amount seemed to increase proportionally to the intratumoral immune reaction - as reflected by the density of the lymphocyte infiltrate - potentially in terms of a feedback activation. We have made similar observations in breast cancer, where we found that mRNA levels of a plethora of immune-related genes, PD-1 and PD-L1 included, were positively correlated with each other as well as with the density of TILs, irrespective of their immune-promoting or immune-inhibitory function, and were all strong indicators of a higher response rate to neoadjuvant chemotherapy. [Our findings are in line with a recent report on a positive prognostic effect of PD-1+ TILs in a cohort of 195 high-grade serous carcinomas.  This stutherapy.  However,therapy. , 17, 18 therapy.  as well therapy.  play an vs γδ cells, an issue, which would be worth further investigations.An impact of PD-L1 on γδ T cells has been reported. One study investigated the expression and function of PD-1 in human γδ T cells that recognize phosphoantigens.  The resuThe localization of TILs seems to be of major relevance as to their prognostic impact. Our data showing a consistent positive impact of intratumoral  TILs on survival is in line with several previous reports. , 24, 25 Currently, the expression of PD-L1 on tumor cells is regarded as an immune-escape mechanism of the tumor, as it attracts PD-1 expressing immune-inhibitory TILs. However, this mechanism is expected to rather result into a negative impact of tumorcellular PD-L1 expression on survival, and this is reported, e.g. for breast cancer, NSCLC, renal cell carcinoma , osteosToday it can only be speculated why components of the PD-1 pathway are in some instances (such as in ovarian carcinoma) linked to a favorable prognosis. A positive impact of PD-L1 expression of tumor cells as seen in our study might be explained by a compensatory up-regulation of this marker in a microenvironment that threatens the tumor by an active immune response. An association between PD-L1 on tumor cells and a high TILs density would be an argument for this hypothesis, and has as well been described by Hamanishi et al. in their study on various ovarian cancer histotypes  as well vs any expression), although we are aware of the fact that the cutoff point for response prediction might be different. A recent phase I study on pembrolizumab in NSCLC for the first time validated a previously defined cutoff point of 50% for response prediction, however responses were also seen among patients with tumors with expression below the cutoff indicating that tumoral PD-L1 expression might not constitute the definite predictive marker. [n = 1). [The fact that PD-1 and PD-L1 are expressed in two tumoral compartments (cancer cells and TILs) and that this expression has prognostic impact strongly suggest that the PD-1/PD-L1 axis has a biological relevance in high-grade serous ovarian carcinoma. This entity therefore appears as a candidate malignancy in which PD-1/PD-L1 targeting drugs should be tested. Although these agents have shown remarkable effects in subsets of patients with in poor-prognosis and therapy-resistant cancers such as NSCLC  and rena marker.  For the  marker.  as well [n = 1).  Numbers Our study has several strengths and weaknesses: We only investigated primary tumors, but the current use of immune checkpoint inhibitors is focused on metastatic or recurrent tumors to date. However, the data of Powels et al. indicate that the immunological microenvironment of a tumor might be temporally stable in terms of its predictive value.  A comparTo summarize, we report a significant prognostic impact of PD-1 and PD-L1 expression in primary high-grade serous ovarian carcinoma. PD-1+ and PD-L1+ TILs increase proportionally to the general lymphocytic infiltrate, however carry independent prognostic information. PD-1 and PD-L1 expression in cancer cells as well as mRNA expression are favorable prognostic markers. Our data indicate that PD-1 and PD-L1 molecules are biologically relevant regulators of immune response in high-grade serous ovarian carcinoma, and that the evaluation of immune checkpoint-inhibiting drugs might be of value in this poor-prognosis cancer type, for which only limited options for targeted therapy are available to date.n = 165) patients had received surgery in the Department of Gynecology of the Charité and had been included into the TOC project . Scientific use of TOC and non-TOC cases has been approved by the ethics committee of the Charité. Data on residual tumor mass after surgery (applies for stage II-IV) were available for 152 patients (76.4%). The majority of patients had been treated with standard adjuvant platinum-based chemotherapy . Data on overall survival (OS) were available for all patients. Median OS was 37.9 months, 113 patients died during follow-up (52.6%). Data on progression-free survival (PFS) were available for 185 patients .Formalin-fixed and paraffin-embedded (FFPE) surgical specimens from 215 patients with primary ovarian high-grade serous carcinoma were used (Table +), as a chromogen. CD3, PD-1, and PD-L1 were stained on consecutive TMA sections enabling the evaluation of corresponding tumor areas. Specificity of the PD-1 and PD-L1 antibodies had been validated before by evaluation of the staining patterns in normal lymphoid tissue and lymphoproliferative diseases, for which a PD-1 or PD-L1 expression had been described . Stained slides were scanned and evaluated on screen by an experienced pathologist (CAK); doubtful cases were discussed with a senior hematopathologist (KJ) until consensus was achieved.Staining was performed on tissue microarrays (TMAs) with two cores for each case, according to standard procedures. In brief, a mouse monoclonal antibody against PD-1  was used in a dilution of 1:50. For PD-L1 detection, a rabbit monoclonal antibody was used in a dilution of 1:300 (clone EPR1161(2), Abcam plc., Cambridge, UK). PD-1 and PD-L1 staining was performed using the BondMax™ device . Antigen retrieval and visualisation of bound antibodies were performed employing the manufacturer's protocols and reagents . CD3 staining was performed using a rabbit polyclonal antibody in a dilution of 1:100 , CD4 was stained with a mouse monoclonal antibody in a 1:20 dilution  and CD8 was detected with a mouse monoclonal antibody , using the BenchMark XT device  and 3,3′-diaminoenzidine peroxide substrate . Intensity and rate of stained cells were determined by two pathologists (CAK in support of KJ) on screen for both membranous and cytoplasmic staining and were combined to a semi-quantitative immuno-reactivity score (IRS).  The scor2) could be assessed. For PD-1+ and PD-L1+ TILs the same tumor areas as for CD3+ TILs were evaluated accordingly. A screen-shot of CD3 evaluation in a representative case is shown in CD3+ TILs were evaluated first: 5 tumor areas in a 400x magnification (high power fields (HPF)) were screen-shotted with the use of VM Slide Explorer 2.2 (VMscope) considering preferentially areas with higher intratumoral TILs density. Microphotographs were subsequently evaluated using the ROI Manager software (CognitionMaster) . In eachp < 0.0001), PD-L1+ , as well as for PD-1+ TILs density . Membranous PD-L1 expression in tumor cells  also correlated in TMA and large sections . In the group of cases that were scored as positive on large sections  all had been classified as positive on TMAs, too. Two of the three cases that were scored as negative on large sections  had been scored positive on TMA, one with an IRS of 2, the second with an IRS of 3, which both are in the low-expression range. Using the novel PD-1 antibody in large sections, there was no PD-1 staining in cancer cells, therefore a comparison between TMA and large sections results as to cancer cell expression was not feasible for PD-1.10 cases with high and low TILs density, respectively, were stained on paired TMAs and large sections. Large sections were stained and evaluated in exactly the same way as TMA spots had been treated before. An exception was PD-1, for which another antibody was used  because the antibody we used before was not commercially available any more. There was a strong correlation between paired TMA and large section data for CD3+  in combination with a liquid handling robot . Two 5-μm thick sections were cut from each paraffin block and transferred to a 1.5-ml tube. All tumor samples included in the study contained at least 30% tumor tissue as evaluated by H&E staining (median tumor content 60%). To achieve this tumor content, manual microdissection of the tumor area was performed if necessary. Expression of PD-1, PD-L1 as well as the normalization gene RPL37A was assessed in triplicate using a ViiA™ 7 Real-Time PCR device . Sequences of primers and probes have been published earlier. deltaCT 2) were logarithmized using the formula: log10(TILs density + 1). For the determination of cutoff points, the Cutoff Finder online tool was applied (molpath.charite.de/cutoff) [Statistical analyses were performed with IBM SPSS Statistics 22 , and GraphPad Prism v.5 . Associations were tested by Spearman, Chi square, Kruskal-Wallis or Mann-Whitney test, as indicated. Survival analysis was performed using the Kaplan-Meier method or Cox regression. All p-values were calculated two-tailed and p values < 0.05 were considered as significant. For Cox regression using continuous TILs data, TILs density  .https://tcga-data.nci.nih.gov/tcga) and investigated for the prognostic impact of PD-1 and PD-L1 gene expression using the software package R. Inclusion criteria for the analysis were: serous histology, and a G2 to G4 grading.Gene expression datasets  from TCGA  were dow


People make a city, making each city as unique as the combination of its inhabitants. However, some cities are similar and some cities are inimitable. We examine the social structure of 10 different cities using Twitter data. Each city is decomposed to its communities. We show that in many cases one city can be thought of as an amalgamation of communities from another city. For example, we find the social network of Manchester is very similar to the social network of a virtual city of the same size, where the virtual city is composed of communities from the Bristol network. However, we cannot create Bristol from Manchester since Bristol contains communities with a social structure that are not present in Manchester. Some cities, such as Leeds, are outliers. That is, Leeds contains a particularly wide range of communities, meaning we cannot build a similar city from communities outside of Leeds. Comparing communities from different cities, and building virtual cities that are comparable to real cities, is a novel approach to understand social networks. This has implications when using social media to inform or advise residents of a city. Yet, despite a century of effort, our understanding of how cities evolve is still woefully inadequate . BeneathA city has many networks, for example the road, the gas, the electric and the main drain networks. However when we think of the character of a city, these physical networks are unlikely to be our immediate thought. Instead we characterize a city by the people within the city, and the social network that they form—the essence of any society. Despite previous fears that cities impair communities , the risOne can think of each city as a unique amalgamation of social communities that differentiate it from other cities. But how unique is each city? Would an amalgamation of social communities from one city resemble another city? This paper resamples the communities from one city to create virtual cities of any size. By comparing these virtual cities to real cities of a corresponding size, we establish that many cities do not conflict with the proposition that they are indeed made up of the same social building blocks. Alternatively, we may identify which cities are inimitable.Insight into the social structure of cities may be used to ensure success of media campaigns, such as those which aim to bring social cohesion or to inform, or other types of behavioural intervention. If a social campaign was successful in one city, it is also likely to be successful in cities comprising similar social building blocks. Moreover, it may prove economical to test a campaign in a smaller city before transposing the learnings gained to a larger city.Generally physical networks evolve alongside the development of a city. Cities develop from the bottom up, and grow to facilitate a division of labour that generates scale economies . This giSize is the major determinant of most characteristics of a typical city; history, geography and design have secondary roles ,9. UsingCommunity structure within communities has been studied for a long time (e.g. ), yet thet al.  of a graph defines how easily a graph can be separated into communities, where each node belongs to only one community. A modularity close to 1 indicates clear community structure, where as modularity close to 0 indicates a complete graph, which cannot be divided into communities. The authors are unaware of any work which relates the modularity of the communities of a network to the modularity of the network as a whole.The modularity R≥1 separate networks, say {Gs)( into communities. By considering the Gs)( is the ns×ns adjacency matrix for Gs) and we may write this asConsider a set of notation , with a G has exactly G, Q, must satisfyGs) within G, thereby connecting up some communities across the Gs). Thus, equation . When α>0 we have growth which follows a power law, and when α<0 the system becomes saturated.We might have deduced the form of equation  directlyNP hard problem, there may be circumstances where it is simpler to calculate the modularity of numerous smaller networks than one large network. We now use Erdos–Rényi graphs to test the lower bound (equation (Equation  is a gooequation ).3.Gs), for some probability p. Then G is just the union of these relatively highly connected ‘ghettos’ (the Gs). If we wish we could also add a very small number of edges so as to connect up a few vertices within the separate ghettos. Such a ghetto-model gives rise to a joint distribution for the triple . Suppose we generate R such graphs and calculate the modularity Q of the union G, using a chosen standard method, in this case one based on the successive bisection of communities [Let us assume that each munities ,16, as iR we may observe how Q increases. In fact we may generate an ensemble of such increasing virtual cities G, made up from R independent ghetto graphs, Gs). We may contrast equation . The envelope is at its widest at small and intermediate values where R<20 and is not so large as to provide a reliable sampling of the distribution assumed for the ghettos. For larger R, we see convergence of Q towards R copies of the averaged value for the s-dependent terms ).Then as we increase equation , by plugequation  are veryA, we may thus generate other virtual cities (by sampling from A’s communities with repetition) that are made of the same stuff, the same underlying distribution of component communities as City A, yet are of any size we chose. In particular, we may match the size of any other observed city, City B. So we shall be able to ask whether City B is similar or not to one made of the same underlying components as City A, while allowing for their differences in scale.After introducing some suitable dataset representing the digital social networks within distinct British cities in the next section, we shall apply the theory developed in this section. By using the community sub-structure observed from any particular city, City 4.We defined a reciprocated (Twitter) mentions network, over 10 UK cities and a four week period, as follows.We identified all those Twitter accounts that were active during 1–28 October 2014 (four full weeks), having biographical information indicating that they are associated with one of 10 cities: London, Manchester, Leeds, Sheffield, Nottingham, Birmingham, Bristol, Cardiff, Glasgow and Edinburgh. The data were collected, using the DataSift Twitter Firehose access, by selecting individuals who had one of the 10 cities stated in their user description field. (We did not look for geo-coded Tweets because the coverage would be too sparse for it to be useful.) We trapped all Tweets during that period . This resulted in 14 946 171 original Tweets made by 321 255 individual accounts.The giant components from using 14 days of data are not sufficiently different from the corresponding giant components using 28 days of data, and a diminishing returns effect becomes apparent. Nonetheless, when analysing a social network a longer time period is advantageous. However, for our purposes of demonstrating our method, 28 days is sufficient.Next we identified a subset consisting 7 241 148 of the Tweets that contained a mention of another account (@name). These Tweets were sent by 260 439 accounts.Finally, we selected the subset of accounts that mentioned any other of the accounts for which there was also a reciprocal mention. That is, those accounts, say Xname, that sent at least one Tweet containing a mention of another account, say Yname (a mention @Yname), where there is at least one reciprocal Tweet made by Yname containing a mention of Xname (a mention @Xname). This resulted in the selection of 59 789 accounts, with each having a reciprocated mention of at least one other account. Representing those accounts as vertices, and the reciprocated mentions as undirected, unweighted edges, we thus obtained a 10 city-based Twitter reciprocated mentions network defined for October 2014. It has average degree of 2.36.The employment of reciprocated mentions  shows that the accounts not only are aware of each other but have actively engaged with each other during the prescribed interval of time (28 days). In that sense the undirected network contains validated (active), two-way connections. The full dataset for this network is available in the electronic supplementary material, where the first column relates to the city , and the other two columns indicate an edge between the two listed nodes.Each of the accounts is associated with at least one city. In many cases, they may be associated with more than one city. For example, they might state ‘I am from Manchester, working in London’ implying they may be genuinely connected into more than one city-based social network. In fact out of the 59 789 accounts we have, 29 814, 16 935, 10 680, 6236, 3949, 5890, 6043, 5644, 4830 and 4061 accounts were associated with London, Manchester, Leeds, Sheffield, Birmingham, Nottingham, Cardiff, Bristol, Glasgow and Edinburgh, respectively. So on average each account claims to be associated with 1.57 cities.We may restrict the mentions network to each separate city as follows. First, we examine the subnetwork containing those accounts associated with each city. We only considered accounts that are active within a particular city, so if an account has reciprocated mentions with another account within that city, it is included within the corresponding network. This results in 10 city-based reciprocated mentions networks. Each of the city-based mentions networks contains a single giant connected component and a very large number of smaller connected components (with less than a handful of vertices).In this paper, we shall focus on these giant components as being representative of the nature of the digital social network for each city, as represented by the corresponding reciprocated Twitter mentions network. Details of the giant component for each city are described in Q. These values are relatively high compared with the modularities of random graphs conditioned solely on the corresponding degree distributions. In line with intuition, London is exceptional. The lower bound for modularity (equation (Each city may be partitioned into separate communities by the optimization of modularity. In equation ) is consQ) [There is a number of heuristic methodologies that have been designed to produce the optimal partition  –17. In aR=74. We show both the modularity and the Shannon partition entropy  as a fraction of their optimal values obtained at 74 communities. The behaviour is very similar to that observed in Onnela et al. [Q was taken while introducing a degree of freedom to control the relative scale of discrimination . The modularity is almost optimal when k=20 or so, with the remaining work, in splitting off smaller communities, representing some fine-tuning. Since modularity is not very sensitive, assuming equality within equations . If we resample each one of them exactly once and then take the union we arrive at a city very similar to City A itself, that is just missing the relatively small number of edges connecting across sub-communities . Now let us resample from X, independently with repetition for as many times, R, as we wish. For each value of R, we may obtain an ensemble of virtual cities (an ensemble where each such city contains exactly R sub-communities), that are made up of the same types and distribution of communities as we observed in City A. As with bootstrapping, we use the observed distribution, X, to represent our best sampled estimate for the underlying (and unobserved) distribution A are composed.The idea employed here is very similar to that of bootstrapping . We haveA has very few identifiable communities (a low resolution partition) forming up X, then it will be a poor representation of the underlying possibilities in X increases, we may see much more diverse behaviour inherited from Of course, if City R sub-communities from X and take their union to form-up a new city. The sth sub-community that we sample has associated observables . Let X. ThenSuppose that we independently resample exactly R=|X| then we are constructing virtual cities of a similar size to the conditioning City A, and we may create an ensemble of such virtual cities that will include the conditioning city. Now consider another city, say City B, that is observed to have nB vertices. Set R draws from X, randomly with replacement, we may construct an ensemble of virtual cities that are all approximately the same size as City B, yet that are made up of components from the same distribution, X, as City A. Thus, the deviation or otherwise of measures for B, such as Q, from the distribution for the same measure obtained from this virtual ensemble can indicate whether or not it is reasonable to assume that B may be made up of the same distribution of sub-communities as A.From , assuminA. On top of the ensemble envelope of Q-values obtained for all of the virtual cities we show the other observed cities. The size of the envelope, which is generally proportional to the number of communities R, is a guide as to the variety of communities within a city. For example, Glasgow and Edinburgh have small envelopes indicating fairly homogeneous communities. Therefore, a city with a wider envelope, such as Manchester, contains social structures not seen in Glasgow and Edinburgh.In Consider Bristol as the conditioning city. It is clear that while Manchester, Nottingham, Edinburgh and Birmingham are within the equivalent Bristol-like envelope, Leeds, especially, is not. We would contend that the social structure observed within Leeds is not consistent with that observed within Bristol; even when we allow for changes of scale, there must be some specific elements that are present or absent on one or another. Thus, the performance of any interventions or other activities designed to work within such social networks cannot be extrapolated from Bristol to Leeds. However, since Leeds contains a wider variety of social structures, a Bristol-like city could be created from Leeds communities. The transitive properties from one city to another, excluding London, are summed up in 6.NP hard problem. Therefore, representing the modularity of a graph in terms of the modularity of the communities is computationally advantageous.We have presented a way to estimate the approximate maximum modularity of a large network based on the maximum modularities of the communities within the network (independent of the method used to define communities).The lower bound estimate presented here is useful because modularity lacks sensitivity near the optimal resolution. Maximizing modularity is an We presented the null model where we built a large city from ghettos represented as a combination of Erdos–Rényi graphs. We showed that the modularity converges after the city had been divided into approximately 20 communities, irrespective of the total number of communities.We represented cities as combinations of communities from different cities, the ‘conditioning city’. This showed many cities, such as Bristol, Nottingham, Edinburgh, Manchester and Birmingham, comprise communities with similar structure—despite their size and location varying considerably. Conversely, it also identified cities like Leeds, which are not made up of similar communities to other cities we sampled. Leeds, despite being smaller than Manchester, contains social structures not observed in Manchester. At the other end of the variance spectrum, Edinburgh and Glasgow have very little variance between their communities. Therefore, recreating cities with greater community variance is challenging from these limited pools of Scottish communities. This type of analysis can aid decisions about social media activities and interventions. For example, a campaign that is successful in Bristol should also be successful in Manchester (but not Leeds). Since Bristol is considerably smaller, it may prove useful to test the campaign in Bristol first.The method is not limited to Twitter data. One could apply the same method to any form of network that could define a city, such as Facebook or e-mail. Furthermore, we are not restricted to cities. It would be interesting to compare universities to each other, or countries, or the social structure in different governments. The method is only limited by our imaginations and the data available.


The transactivation response element DNA-binding protein 43 kDa (TDP-43) is a major component of the ubiquitin-positive and tau-negative inclusions in frontotemporal lobar degeneration and sporadic amyotrophic lateral sclerosis (ALS). TDP-43 may accumulate in cases of Alzheimer’s disease (AD), Lewy body disease (LBD), and argyrophilic grain disease (AGD). However, few studies have focused on the incidence and extent of TDP-43 deposition in aging.We analyzed 286 consecutive autopsy brains neuropathologically. Of these, 136 brains with pathologically minimal senile changes were designated as control elderly brains (78.5 ± 9.7 y). For comparison, we selected 29 AD, 11 LBD, and 11 AGD patients from this series of autopsy brains. Sections of the hippocampus, amygdala, medulla oblongata, and lumbar spinal cord were immunostained with anti-phosphorylated TDP-43 antibody (PSer409/410). TDP-43 immunoreactive structures were classified into four types: dystrophic neurites (DNs), neuronal or glial cytoplasmic inclusions, and intranuclear inclusions. TDP-43 immunoreactive structures were observed in 55/136 control elderly (40.0 %), 21/29 AD (72.4 %), 8/11 LBD (72.7 %), and 6/11 AGD (54.5 %) brains. TDP-43 immunoreactive structures in control elderly brains were mostly DNs. These DNs were predominantly present in the uncus of the anterior hippocampus over age 65. The frequency of cases with DNs in the amygdala of control elderly brains was less than that of AD, LBD, and AGD brains. The mean age at death was significantly higher in cases with TDP-43 immunoreactive structures than cases without them.In conclusion, TDP-43 immunoreactive DNs may develop as a consequence of aging processes in the human brain. In particular, the uncus of the anterior hippocampus is an area highly susceptible to TDP-43 accumulation over age 65. The transactivation response element DNA-binding protein 43 kDa (TDP-43) is a major component of the ubiquitin-positive and tau-negative inclusions in frontotemporal lobar degeneration (FTLD-TDP) and sporadic amyotrophic lateral sclerosis (ALS) –3. HowevPrevious studies have indicated that TDP-43 immunoreactive inclusion bodies can be detected in other neurodegenerative disorders as well, including Alzheimer’s disease (AD) –9, Lewy Tissue samples were collected at the Tokyo Metropolitan Geriatric Hospital, which provides community-based medical service to the elderly population. From February 2008 to July 2012, we obtained the 286 consecutive autopsy brains (165 men and 121 women) used for this study. Patient ages ranged from 43 to 104 years . Clinical information was retrospectively obtained from the medical chart. The Mini-Mental State examination (MMSE)  was usedThe brains and spinal cords were examined according to routine protocols used in our laboratory . BrieflyThe case selection procedure in this study is shown in Fig. As to the cases with comorbid pathologies and other neurological disorders, we also provided the results of TDP-43 pathology. However, they were not used for comparison with control elderly cases because they were limited in number: AD plus LBD (n = 6), AD plus AGD (n = 3), AD plus progressive supranuclear palsy (PSP)  (n = 1),The presence and severity of TDP-43 immunoreactive structures were assessed in four brain sections including the anterior hippocampus, amygdala, medulla oblongata, and lumbar spinal cord. In fact, the anterior hippocampus is key anatomical structures of FTLD-TDP. In addition, TDP-43 immunoreactive deposits have been well analyzed in AD, DLB and AGD in the limbic regions , 6, 8–11U test was used to compare the age at death and brain weight between TDP-43–positive and TDP-43–negative groups. Fisher’s exact test was used to compare the male-to-female ratio and number of TDP-43 structures. A p value of less than 0.05 was considered statistically significant.The Mann–Whitney The demographics of the cases in this study are shown in Table TDP-43 immunoreactive structures were found in 55/136 (40.0 %) cases (Table TDP-43–positive structures were found in 21/29 (72.2 %) AD brains and were widely distributed in the amygdala and hippocampus Tables . These sTDP-43 immunoreactive structures were found in 8/11 (72.7 %) LBD brains and predominantly observed in the uncus of the anterior hippocampus and amygdala Tables . To a leTDP-43 immunoreactive structures were found in 6/11 54.5 %) AGD brains and observed in the uncus and amygdaloid nuclei  among cases with and without TDP-43 immunoreactive structures were 38.3 %  and 34.3 % , respectively.In addition, we examined the influence of cerebrovascular pathology. The rates of cases with cerebrovascular pathology among cases with and without TDP-43 immunoreactive structures were 61.2 % (34/55 cases) and 67.9 % (55/81 cases), respectively. There were no significant differences between cases with and without TDP-43 immunoreacrive structures.TDP-43 immunoreactive structures were also found in the following cases: AD plus LBD ; AD plus AGD ; AD plus PSP ; AD plus HS ; LBD plus AGD ; LBD plus PSP ; AGD plus HS ; NFTD ; CJD ; ALS/ALS with dementia ; MSA ; SCA6 ; SCA3 ; PSP ; HS ; DRPLA ; CBD ; FTLD-TDP ; MELAS ; NFTC ; ADC ; and PSC .Our study is the first neuropathologic analysis that has clarified the anatomical distribution of TDP-43 immunoreactive structures in elderly brains with minimal senile neuropathologic changes in large numbers. We revealed the following findings. First, TDP-43 immunoreactive structures were present in 40 % of control elderly. Second, these structures were predominantly observed at the uncus of the anterior hippocampus as DNs over age 65. Third, the number of cases with DNs in the amygdala of control elderly was less than that of AD, LBD, or AGD. Fourth, no NCIs or GCIs were found in the medulla oblongata and lumbar spinal cord in all four groups. Finally, the mean age at death was significantly higher in cases with TDP-43 immunoreactive structures than those without such structures.et al. reported that 2/63 (3 %) were positive for TDP-43 in neurologically normal individuals ranging from 23 to 94 years old at death [A few studies have assessed the frequency of TDP-43 immunoreactive structures in normal brain. Wilson at death . Anotherat death . In yet at death . The widThe distribution of TDP-43 immunoreactive structures in control elderly was different from those in AD. The structures in AD were widely present in the amygdala and hippocampus. Furthermore, in most cases, the TDP-43 immunoreactive structures in AD took the form of NCIs/GCIs, whereas only two cases of control elderly had GCIs or NCIs. These findings suggest that the pathological mechanism of TDP-43 accumulation in control elderly is different from that in AD. Although the amygdala was considered to be the first affected region of TDP-43 deposition in AD , TDP-43 Previous studies that used a small number of cases also reported that TDP-43 immunoreactive structures were frequently observed in the amygdala in AD, LBD, and AGD , 6, 8–11We found TDP-43 immunoreactive DNs in the medulla oblongata and the spinal cord in control elderly brains and there were no NCIs or GCIs in all groups. These structures were predominantly located in the inferior olivary nuclei and the white matter of the spinal cord. The cases of AD, LBD, and AGD brains also showed TDP-43 immunoreactive DNs in those anatomical regions. We believe that the TDP-43 immunoreactive DNs in those specific anatomical regions may be associated with human physiological aging. In fact, the distribution pattern of TDP-43 deposits in this study clearly differs from that in ALS and FTLD-TDP brains that show many NCIs .Our study has some limitations. Because many aging brains have comorbid pathologies, the numbers of pure AD, LBD, and AGD brains in this study were relatively small. In addition, a part of TDP-43 accumulation of in AD, LBD, and AGD brains may be influenced by the aging process. In fact, for AD, LBD, and AGD, the mean age of death was significantly higher than that for control elderly.Although TDP-43 has been associated with memory loss and medial temporal atrophy in AD , the rolIn summary, our study clearly revealed that TDP-43 immunoreactive structures may develop as a consequence of aging processes in the human brain. Most of them were DNs not NCIs and GCIs, and were observed in the uncus of anterior hippocampus. Additional studies may be important to understand the role of TDP-43 accumulation in normal human aging. Besides those control elderly cases, we believe that the cases without TDP-43 immunoreactive deposits are also substantial for analysis of pathomechanism of TDP-43 accumulation.The data sets supporting the results of this article are included within the article and its additional files.


Large size graphene (LSG) and multiwall carbon nanotubes (MWCNTs) on LSG were synthesized on a copper surface via chemical vapor deposition (CVD) at low temperature and normal pressure. The LSG were formed through an easy chemical cyclic reaction in which liquid benzene was heated to a temperature below its boiling point to create benzene vapors as graphene precursor material. The reaction mechanism was observed, and the time-dependent analysis of the reaction revealed that mounds of the carbon nanotubes had grown as a result of the island that was found on the LSG sheet. The implications of the mechanism that we have introduced were investigated by coating a titanium sheet on the MWCNTs/LSG and LSG on the semiconductor electronic device. The photonic response was observed to be markedly high, which can be attributed to the positive synergetic effect between the Ti and LSG sheet of our prepared composites. Graphene consists of a single layer of carbon atoms that can be obtained from highly-oriented pyrolytic graphite through mechanical exfoliation, and it has been shown to have outstanding electronic, optical, and mechanical properties. Although various techniques have been developed to produce graphene on a solid material substrate1234678911et al. studied the growth of epitaxial graphene (EG) on insulating silicon carbide (SiC) surfaces by developing a promising route to produce large-scale high-quality graphene. The amorphous carbide precursor has been transformed to graphene nanosheets through chlorination at low temperature and ambient pressure, and some exceptional outcomes have been obtained. These results suggest the importance of the size effect when synthesizing CVD grown graphene on metal substrates16171819CVD is a relatively simple technique that can be used to obtain graphene with the desired features. CVD involves the decomposition of carbon precursor materials, that is, hydrocarbons or polymeric components, by adding heat and some metallic catalysts, such as Ni, Pt, Cu, etc., that have been found to improve the growth of graphene. The optimum conditions to produce large size graphene through CVD have yet to be determined, and therefore, various approaches have been proposed14In this study, we investigate a novel CVD growth process for LSG/MWCNTs on copper foil at low temperature of 500 °C. This procedure consists of two steps. First, we observe a large size micrometer graphene sheet on Cu foil at the appropriate temperature, and in the second step, we develop the growth mechanism for LSG and MWCNTs/LSG at very low temperature. We thus suggest a novel growth mechanism for LSG and MWCNTs/LSG through chemical vapor deposition on a copper foil at low temperature. The novel mass production of LSG and MWCNTs/LSG on Cu foil may be very useful in the fabrication of ultrafast charge transfer for next generation optical and electronic devices. Finally, we suggest a potential application by coating Ti on the MWCNTs/LSG on Cu foil and coating LSG on electronic devices.FESEM images of the as-prepared CVD-grown LSG and MWCNTs/LSG assembly are shown in When the reaction time increases to 2 and half minutes by keeping the temperature at 500 °C, a small dot appears on the surface of the LSG, and these dots are referred to as carbon nanotube seeds, as shown in The production of large area graphene and the number of layers of the G1 sample were investigated through the TEM image shown in The production of MWCNTs on the LSG sheet was verified through a transmission electron microscopy (TEM) analysis of the G4 sample. −1 from the excitation line are associated with the Cu present in the sample. Cu arises as a result of scratching the graphene composite from the Cu foil.Raman spectroscopy is a powerful technique that can be used to study the structure and electronic properties of carbon materials, including graphene/graphite and carbon nanotubes. −1 that corresponds to the 2D peak in graphene. This 2D peak was further split into four Raman peaks, and the dispersion of the peak yielded information on the number of layers and the stacking order in the graphene system. 2D peaks in bilayer graphene have been reported to be split into four components, which further increases in the layer and bulk area of graphene and leads to lower intensity peaks22The results of our study indicate that by increasing the reaction time, the Raman band intensities of the G band and D band shift towards a lower frequency. This allows the MWCNTs to start growing on the LSG sheet. The other reason for the variation in intensity is the large number of MWCNTs that roll together, encouraging phonon confinement and thus reducing the intensity24−1 while the D band frequency was observed at 1352 cm−1. For the MWCNTs, the large number of CNTs with different diameters affects the characteristic G band, and therefore theses are found close to the G band frequency of graphite−1 were attributed to the Cu that was used as a substrate3132The laser excitation also has an effect on the Raman band shift and the variation in intensity. In the MWCNTs, the bands could be attributed to the Raman resonance condition, and the incident laser on the carbon nanotubes can yield an optical resonance profile for a given mode at a vibrating frequency of the respective modes2627283435We then used AFM topography images to determine the thickness and the height of the LSG and MWCNTs/LSG composites. The LSG and MWCNTs/LSG composites are shown in The surface growth of vertically-aligned CNTs on the substrate material at a micrometer scale with nonmetric effect is very important for next generation devices. The AFM images revealed the surface construction of MWCNTs leading towards nanohill formation of carbon nanotubes, which is unique, and this formation is a complex phenomenon that may be the result of the different competing factors, such as temperature, reducing atmosphere, oxide reduction during the CVD process, and benzene decomposition. This elusive, complex phenomenon will require focused investigation in the future to explore the outstanding physical and chemical properties of the MWCNTs/LSG composites.The The scheme to form the LSG and MWCNTs is depicted in The samples were coated by titanium through the same process that was explained above by using TNB as a precursor material. During the CVD process, we observed that the Ti attached as a sheet similar to graphene that covered the maximum surface of the graphene samples. −1. The peak at around 1100 cm−1 can be attributed to the Ti lattice td symmetryThe Raman spectrum of the Ti coated G4 sample is shown in The photo-catalytic properties of the LSG/MWCNTs and Ti-MWCNTs/LSG that were obtained can be examined by using Rh.B degradation under visible light. The scratch sample from the copper foil was used as powder for the photocatalytic experiment. The Rh.B photodegradation rate was assessed under visible light . The photo activities of the LSG, MWCNTs/LSG and Ti-MWCNTs/LSG were determined according to the degradation rate of Rh.B as a function of time under visible irradiation. In order to attain adsorption/desorption equilibrium, the solution was kept in the dark for 2 h. After adsorption/desorption equilibrium had been achieved, the solution was irradiated with visible light for 30 minutes. For the analysis, the sample was periodically withdrawn, and the solids were removed via centrifugation.The dye concentration of the sample was then analyzed using a UV-visible spectrometer. Under visible light, the energy state of the electrons changed from a valance band to a conduction band, and the excited electrons reacted with the adsorbed oxygen, resulting in the formation of free radicals. These free radicals reacted with the dye, resulting in the mineralization of the dye into simple molecules. During the experiments, a decrease in the color intensity of the solution was gradually observed over time due to the adsorption and degradation of Rh.B. The Rh.B concentration in the solution was determined to be a function of the irradiation time from the change in absorbance at a wavelength of 554 nm. The results of the photo degradation  are shown in the app). The Kapp values provide the degradation rate of the Rh.B molecules by the photocatalyst materials under the influence of visible light. The values of the Kapp rate constant are listed in −3 min−1 under visible light, which was much higher than the G1 and G4 sample in our nanocomposites. These results further confirm that the Ti-MWCNTs/LSG nanocomposites are a much more effective catalyst material than the other nanocomposites in the CVD-grown LSG/MWCNTs nanocomposites.Kinetic studies were performed on the basis of the degradation rate of the organic dye, and the reactions between the dye molecules and the catalyst materials can be expressed according to the Langmuir- Hinshelwood model, as shown in et al. synthesized CNTs-Fe/Ni-TiO2 composites, and an improvement was observed in the catalytic effect due to the role of the CNTs in the compositeset al. studied the properties of CNTs-TiO2/Al2O3 membranes in terms of their photocatalytic function and provided a significant description of the use of CNTs-based materials for purification systemsThe photocatalytic properties of the CNT composites with other semiconductor materials have been extensively studied over the last two decades, and the effect of the CNTs has been made quite clear from these investigations. For example, Lei To further demonstrate the photo-stability and cyclic performance of the Ti-MWCNTs/LSG composite photo-catalysts, cyclic experiments were carried out with Rh.B as an organic dye. A semiconductor index device was considered for coating with our proposed CVD method. The actual image of the device is shown in 4344452. This high level activity may be a result of the positive synergy and high charge mobility of the MWCNTs between the Ti and graphene sheet. Thus, we believe that our low-temperature, normal-pressure CVD process provides a simple and reproducible way to produce high quality Ti-LSG/MWCNTs for the large-scale integration of next generation graphene-based devices.In summary, we have investigated a new, short route to produce large area graphene. This method can be used to produce large area graphene for industry at bulk by further controlling the experimental parameters to determine the optimum values. We demonstrate the production of large area graphene with a very short reaction time in an argon environment by using chemical vapor deposition. An increase in the reaction time to 5 minutes provides interesting results due to the production of ~350 nm long, vertically-aligned MWCNTs that are homogenously distributed through the copper foil, as shown in the AFM images. The FESEM images show that that the island found on the graphene can further give rise to vertical CNTs. The valleys that are observed between the graphene domains may be a result of the use and handling of a very thin copper foil. The UV-visible spectrum clearly shows an enhanced catalytic effect for the Ti-MWCNTs/LSG nanocomposite under visible light, suggesting an optimal loading effect of graphene to TiO16H36O4Ti) was used as a titanium precursor and was purchased from Samchun Pure Chemical Co. Ltd., Korea. The Cu foil (99.9%) was annealed and uncoated and was used as the substrate material to grow LSG/MWCNTs. Argon and Nitrogen gas were purchased from Samchun Pure Chemical Co. Ltd., Korea. A specially-designed split Si tube furnace was divided into two parts: an inner tube and an outer tube. The dimensions of the outer tube were 30 cm length and 4 cm with two nozzles, one for the inlet of benzene vapors and the other for the inlet of Ar and N2 gas. The inner furnace, which is also called the heating zone, had a 10-cm length and a 5-cm diameter and was used to grow the MWCNTs/LSG on the Cu foil. Benzene was first heated below its boiling temperature to produce vapors, and these vapors were then simultaneously transferred to the tube with Ar gas, which also acts as a carrier for the benzene vapors and prevents the Cu foil from reacting with water molecules. The vapor flow and the Ar gas ratio were controlled by using control valve, and the furnace temperature increased to 500 °C in the first step, and Ar gas was released into the Si furnace. After reaching a temperature of around 500 °C, a Cu foil was inserted in the inner tube, followed by a controlled amount of Ar gas and benzene vapors. The growth of the MWCNTs/LSG was verified at different reaction times  at 500 °C, and the samples that were obtained were labeled as G1, G2, G3 and G4, respectively.Benzene was used as a carbon precursor material and was purchased from Dae-Jung Chemical and Metals Co. Ltd Korea. Titanium (IV) n-butoxide -5200 JOEL, Japan) was used because it offers expanded imaging capabilities and is customizable in terms of the performance requirements for the analysis. High-resolution transmission electron microscopy (HRTEM) JEOL, JEM-2012, Japan was used to determine the state and the sheet morphology of the prepared graphene. A transmission electron microscope (TEM) at an acceleration voltage of 200 kV was used to investigate the number and the stacking state of the graphene layers on the various samples. Atomic force microscopy (AFM) was then carried out using Park NX 20, Korea.How to cite this article: Ullah, K. and Oh, W.-C. Fabrication of large size graphene and Ti- MWCNTs/ large size graphene composites: their photocatalytic properties and potential application. Sci. Rep.5, 14242; doi: 10.1038/srep14242 (2015).