Drosophila system where we have engineered the genome to replace the endogenous TDP-43 orthologue with wild type or mutant human TDP-43(hTDP-43). In contrast to other models, these flies express both mutant and wild type hTDP-43 at similar levels to those of the endogenous gene and importantly, no age-related TDP-43 accumulation observed among all the transgenic fly lines. Immunoprecipitation of TDP-43 showed that flies with hTDP-43 mutations had increased levels of ubiquitination and phosphorylation of the hTDP-43 protein. Furthermore, histologically, flies expressing hTDP-43 M337V showed global, robust neuronal staining for phospho-TDP. All three lines: wild type hTDP-43, -G294A and -M337V were homozygous viable, with no defects in development, life span or behaviors observed. The primary behavioral defect was that flies expressing either hTDP-43 G294A or M337V showed a faster decline with age in negative geotaxis. Together, these observations implied that neurons could handle these TDP-43 mutations by phosphorylation- and ubiquitin-dependent proteasome systems, even in a background without the wild type TDP-43. Our findings suggest that these two specific TDP-43 mutations are not inherently toxic, but may require additional environmental or genetic factors to affect longevity or survival.Mutations in TDP-43 are associated with proteinaceous inclusions in neurons and are believed to be causative in neurodegenerative diseases such as frontotemporal dementia or amyotrophic lateral sclerosis. Here we describe a  Cytoplasmic protein aggregates are a common pathological feature of many neurodegenerative diseases . The TARTDP-43 is a member of the heterogeneous nuclear ribonucleoprotein family , is exprtbph gene. No significant phenotypes were observed in flies expressing wild type hTDP-43 compared to control flies, suggesting hTDP-43 can fully replace the functions of the Drosophila gene. Also in contrast to TDP-43 over-expression models, the fly lines carrying ALS-associated mutations were viable and only exhibited mild behavioral phenotypes. These results suggest that these two TDP-43 mutations, G294A and M337V, are not inherently toxic, but might require additional genetic and/or environmental factors to exhibit disease-like phenotypes.Using CRISPR/Cas9 genome editing techniques, we generated three fly lines where the coding region of the endogenous TDP-43 orthologue (TBPH) was replaced with either wild type human TDP-43 or one of two mutations; hTDP-43 G294A (identified as a sporadic mutation) and hTDP-43 M337V . In contrast to other studies, in which mutated TDP-43 is expressed in addition to the endogenous TDP-43, these three fly lines express wild type or mutant hTDP-43 in the absence of expression of the fly Drosophila stocks were reared at 25°C using standard procedures [http://flystocks.bio.indiana.edu/): y[1] M{vas-Cas9.RFP-}ZH-2A w[1118] was the parental line used for CRISPR/Cas9 injections and y[1] w[67c23] P{y[+mDint2] = Crey}1b; sna[Sco]/CyO carrying the Cre recombinase was used to remove the DsRed cassette in the transgenic flies. The hTDP-43 transgenic flies and the parental flies were crossed with w1118 at least five times for minimizing the nonspecific effects caused by CRISPR/Cas9  as described previously [Negative geotaxis assays were performed by placing flies (ranged from 1~28 days-old of age) in separate vials , one day before the assays. Flies were transferred to adapted 25 ml tubes to be used as vertical climbing columns. Flies were placed in a 25°C incubator for recovery for 30 min before the experiment and then tapped to the bottom of the tubes and allowed to climb for 60 s and a digital image captured of the entire tube. The whole climbing tubes was captured as an image. The number of flies in the top one tenth  and aged (~ 28 days post eclosion) flies. For both young and aged flies, these results clearly show that there was no difference in the levels of TBPH/hTDP-43 transcript between control flies (expressing TBPH) and flies expressing wild type hTDP-43. By contrast, both mutant lines had slightly reduced transcript levels in young flies , howeverTo assess the levels of hTDP-43 protein expressed in these lines we separated the total CNS proteins of all fly lines into two fractions, Tris-soluble and SDS-soluble fractions, and then analyzed these fractions on immuno-blots using an antiserum against hTDP-43. The Tris-soluble fraction revealed a single immunoreactive band at the expected size for hTDP-43 in the three lines expressing hTDP-43 . No immuTo characterize the molecular phenotypes further, we used antiserum specific to phosphorylated TDP (pTDP) and examined immunoblots of head lysates of the fly lines. In addition, we used an ubiquitination linkage-specific K48 antibody to examine immunoprecipitated protein, pulled-down by a TDP-43 antibody. When the lysates were blotted with the pTDP-43 antibody, a band of~53 kDa was visible in all three of the transgenic fly lines and notable was increased in flies expressing hTDP-43 M337V . When thWe also examined the expression of hTDP-43 in neurons in the adult CNS using immuno-fluorescence to localize the FLAG-tag, fused at the N-terminus of hTDP-43 . These rDrosophila fragile X mental retardation 1 (FMR1), a component of stress granules in Drosophila [The punctate nature of the cytoplasmic TDP-43 in the flies expressing mutant hTDP-43 was reminiscent of stress granules. To determine whether the observed cytoplasmic puncta shared characteristics of stress granules, we co-labeled the CNS using anti-FLAG antiserum as well as an antiserum against osophila –52. AlthNeurodegenerative diseases that include TDP-43 proteinopathies are usually characterized with phosphorylated TDP-43 and ubiquitin positive inclusions in the cytoplasm or the nuclei of neurons . OccasioAll three of the fly lines were viable as homozygotes and to determine whether there were any defects in development, we measured the length of time between hatching and adult emergence (eclosion). No significant differences were observed between control flies and those expressing wild type hTDP-43 and no delays in development were observed with flies expressing either mutant form of hTDP-43 , indicatrd instar larva crawled during a 5 minute observation period. No differences were detected between any of the lines  expressing mutant hTDP-43, however, there was no difference in transcript levels among all the older flies (4 week-old), suggesting that the mutant TDP-43 does not induce the overexpression of the gene itself. We also detected an increase in the levels of soluble hTDP-43 protein in young flies expressing mutant hTDP-43, but these differences were diminished in aged flies, as did the levels of wild-type hTDP-43. It is interesting that in young flies we detected reciprocal changes in transcript and protein levels of hTDP-43. The levels of TDP-43 protein are known to be autoregulated by binding of the TDP-43 protein to the 3’UTR of the TDP-43 transcript [tbph UTR to maintain the transcript-protein homeostasis seen in cell- and mammal models. If the mutant protein has a slower turnover rate compared to wild type protein it would result in slightly decreased transcripts in the mutant fly lines as we have observed. Our observation that hTDP-43 protein levels decrease with age is consistent with previous findings reported from both Drosophila and rodent models [In the current study, we have developed a anscript . We hypot models , 75. Intt models , 76, migt models . In addit models , 78, 79.In contrast to the relatively small differences in the proteins or transcripts among all the fly lines, the proteins immunoprecipitated with TDP-43 serum showed more substantial changes. Although the input levels of TDP-43 were equivalent across the three lines, the levels of TDP-43 immunoprecipitated were highly enriched in samples from the two mutant lines compared to the wild type hTDP-43 line, as assessed by FLAG immunoblots. This result suggests that the epitope of the wild type hTDP-43 is masked, possibly by its partner proteins, since TDP-43 usually functions as a protein complex . This woUnexpectedly, there were only mild behavioral defects associated with expression of mutant hTDP-43 in our lines. The lack of any differences between the flies expressing wild type hTDP-43 and control flies expressing TBPH suggests that hTDP-43 can fully fulfill all of the essential functions of TBPH. However, we expected to observe defects in flies expressing mutant hTDP-43. Development and longevity were not diminished and only minor defects in fecundity and negative geotaxis were observed. We did observe slightly increased longevity in the males expressing hTDP-43 M337V, although we do not know whether this is due to the presence of the mutant protein or as a result of differences in the genetic background of the lines, despite outcrossing all the transgenic lines with the control line. These results, nevertheless, stand in marked contrast to results from over-expression of mutant hTDP-43 in a variety of systems where it has been shown to be highly toxic , 81–85. In summary, we have described the preliminary characterization of fly lines designed to allow the examination of the effects of TDP-43 mutations in the absence of endogenous TBPH. Flies that express mutant forms of hTDP-43 display only mild behavioral and developmental phenotypes yet show a number of cellular and molecular characteristics that appear to parallel the histopathological hallmarks of human neurodegenerative disease. These flies will be invaluable tools to identify interacting genes and the downstream components resulting from TDP-43 dysfunction. Interestingly, a recent report describes using similar CRSIPR/Cas9 technology to introduce point mutations into the TDP-43 orthologue in zebra fish, but at this time no phenotypes were reported .S1 FigAdult fly heads from each genotype were homogenized in Tris buffer see , centrif(TIF)Click here for additional data file.S2 FigA. Wild type hTDP-43. FMR1 positive cytoplasmic puncta are clearly visible (A’). B. hTDP-43 M337V. Both hTDP-43 positive puncta (B) and FMR1 positive cytoplasmic puncta are clearly visible (B’) but no overlap of the two types of puncta were observed (B”). Arrows indicate the hTDP-43 containing puncta in flies expressing hTDP-43 M337V (B) and arrowheads indicate the FMR1-containing puncta. Scale bar is 2μm.Adult brains were fixed and stained with an anti-FLAG (green) and anti-FMR1(magenta) antiserum to label FLAG-hTDP-43  and FMR1 . (TIF)Click here for additional data file.S3 FigA. Only low levels of ubiquitin staining was visible in flies expressing wild type hTDP-43. B&C. Flies expressing mutant hTDP-43 showed sporadic ubiquitin nuclear inclusions. Representative images from flies expressing hTDP-43 G294A (B) and hTDP-43 M337V (C) showing ubiquitin positive puncta overlapping with DAPI staining (arrows). In flies expressing hTDP-43 M337V, ubiquitin-positive puncta were also present in region not labeled with DAPI (boxed area in C”). These preparations were also stained with anti-FLAG antiserum (arrow heads in D). Examination of the merged images (D”) for both ubiquitin (arrows in D’) showed little overlap of TDP-43 and ubiquitin in these preparations. Scale bar is 2μm.Adult brains of flies expressing wild type (A) and mutant hTDP-43 (B&C) were fixed and stained with an anti-ubiquitin (gray) antiserum to label ubiquitin  and DAPI to label nuclei . Merged images are shown in the third column. (TIF)Click here for additional data file.


Over the last decades, sequencing technologies have transformed our ability to investigate the composition and functional capacity of microbial communities. Even so, critical questions remain about these complex systems that cannot be addressed by the bulk, community-averaged data typically provided by sequencing methods. Over the last decades, sequencing technologies have transformed our ability to investigate the composition and functional capacity of microbial communities. Even so, critical questions remain about these complex systems that cannot be addressed by the bulk, community-averaged data typically provided by sequencing methods. In this Perspective, I propose that future advances in microbiome research will emerge from considering “the lives of microbes”: we need to create methods to explicitly interrogate how microbes exist and interact in native-setting-like microenvironments. This approach includes developing approaches that expose the phenotypic heterogeneity of microbes; exploring the effects of coculture cues on cellular differentiation and metabolite production; and designing visualization systems that capture features of native microbial environments while permitting the nondestructive observation of microbial interactions over space and time with single-cell resolution. This is an exciting time for those of us interested in microbial communities. The technologies available to us now (many hardly imaginable even a decade ago) are allowing new scientific questions to be asked and driving the field forward. For instance, sequencing technologies have provided us with fantastic insights into which microbes are present in specific environments as well as into what their transcriptional states are. However, as important as sequencing approaches remain, these methods typically provide only broad snapshots of the bulk behavior of microbial communities. Developing a deeper appreciation of the factors driving the establishment and stability of microbial communities, as well as a predictive framework to enable their rational perturbation, will require a better understanding of the causal relationships between microbial community members. As I illustrate here, a productive approach to do so is to consider “the lives of microbes.” In other words, we need to develop methods that capture the native features of the environments that these tiny organisms experience in the natural world and that allow us to explicitly interrogate how microbes exist and interact with one another at the appropriate spatial scale: the microscale.Bacillus subtilis  and are developing single-cell imaging systems to address the question of how cells physically partition themselves within microbial communities .In their native environments, microbes most frequently live as members of complex multispecies communities. They are therefore surrounded not only by the neighboring cells that are physically present but also by the metabolic signals that these microbes have secreted. These secreted metabolites are primary drivers of microbial community interactions . More exBacillus cereus, has two structurally distinct activities: killing and biofilm induction , which aims to promote a community of similarly minded researchers who are interested in creating controlled model ecosystems to study microbial communities in response to perturbations.In “thinking like a microbe,” we must remember that microbes typically exist at significantly lower densities and overall cell numbers than are usually examined in laboratory culture. Thus, we must establish methods to investigate populations of cells interacting at the spatial scale at which microbes natively exist: the microscale. Ideally, these study systems would capture some of the physical and chemical complexity of natural systems while still allowing the nondestructive visualization of the spatiotemporal dynamics of individual cells over time. Since my laboratory is focused on soil microbial communities, we are creating such native-setting-like microcosms using an optically transparent soil-like substrate ; in someIn addition to the areas outlined above, technological advances in several areas would dramatically advance our understanding of microbial systems. Our ability to understand microbial interactions would be revolutionized by the ability to detect and identify “wild” and genetically intractable microbes, for instance, by using their distinct autofluorescence signatures. Although this would require significant advances in our optical detection capabilities, it would also substantially increase our ability to examine natural, nonmodel microbial systems. Other revolutionary advances will likely result from our improved ability to combine multiple imaging modalities  to simultaneously detect the spatiotemporal distributions of microbes and their metabolites at the microscale level within microcosms or natural environments. Such methods would allow us to dissect not only which cells are producing metabolites within microbial communities but also which microbes are altering their physiology and metabolism in response to these cues.


Implementation fidelity refers to the extent to which a proposed intervention is enacted as designed and is necessary to determine how much the intervention in question is the primary mechanism in any changes observed. Start2quit was a randomised controlled trial that aimed to improve attendance at the English Stop Smoking Service (SSS). The complex intervention combining computer-tailored personal risk letters and no-commitment (“taster”) sessions aimed at encouraging attendance at the SSS doubled attendance at the SSS and significantly increased abstinence rates, although attendance and abstinence varied between participating SSSs. Assessment of the fidelity of the delivery of the taster sessions to the protocol was embedded into the trial and is the focus of this study.Eighteen SSSs participated in the study. Taster sessions were delivered by SSS advisors in the area. Of the 131 sessions delivered, 93 (71 %) were recorded and 41 (31.3 %) were selected for transcription and analysis. The taster session protocol contained 73 specified behaviours, which were independently classified into component behaviour change techniques (BCTs) using an established taxonomy for smoking cessation. All transcripts were coded by two authors with 25 % additionally coded by a third. The fidelity of each taster session was expressed as the percentage of overall protocol-specified behaviours that were delivered. Adherence to each BCT was measured as the number of behaviours applied by the advisors within each BCT divided by the total number classified within each.Adherence of protocol-specified behaviours was relatively high (median 71.23 %), though there was considerable variation (28.76 to 95.89 %) in individual sessions. Median fidelity to specific BCTs across sessions also varied from 50 to 100 %. Shorter sessions, sessions run jointly by two advisors, by female advisors, or by advisors aged 45 to 54 were associated with higher levels of adherence. There was no association between adherence and subsequent attendance at the SSS.These results suggest that the delivery of the intervention of this study is not likely to have been impacted by issues of fidelity. As such, we can have greater confidence that variability in the main outcome is not due to variability in SSS advisor adherence to the protocol of the taster sessions.ISRCTN76561916Current Controlled Trials  Implementation fidelity refers to the extent to which a proposed intervention is enacted as designed . Three cThe assessment of treatment receipt and treatment enactment is a complex process involving the verification of participant understanding of intervention content and the monitoring of participant use of treatment-related strategies in everyday life . MeasureDespite this, a systematic review of psychosocial treatments by Perepletchikova and colleagues  found thRecent research has begun to address this issue and to propose methods to monitor the fidelity of delivery within study interventions . In partIn addition to identifying the extent of intervention delivery fidelity, it is also important to understand the ‘essential’ components of an intervention , 12. BehIn this paper, we report on an assessment of the fidelity to the protocol of one part of a complex intervention of a randomised controlled trial. The Start2quit trial  aimed top < 0.001). Seven-day point-prevalent abstinence at the 6-month follow-up, validated by salivary cotinine was also significantly higher in the intervention group . The number completing the 6-week SSS course was also significantly higher in the intervention than that in the control group . However, some variation was found in both attendance and in 7-day validated abstinence between SSSs. Overall attendance between SSSs varied from 2.1 to 23.1 % and validated 7-day abstinence from 2.1 to 13.4 % [Participants of the Start2quit trial randomised to the intervention group received a computer-tailored personal risk letter and an invitation to attend a no-commitment ‘taster session’ to find out more about the support provided by the SSS. These taster sessions were delivered by trained SSS advisors and were based on a detailed treatment manual. Results from the trial showed that this intervention more than doubled the odds of attendance at the SSS during the 6-month period between randomisation and follow-up as measured by SSS records, compared to the control group who received a standard generic invitation to contact the service  . This stn = 106,819) were sent an invitation to participate in the study along with a Smoking Behaviour Questionnaire (SBQ). Smokers returning the questionnaire and giving consent (n = 4384) were randomly allocated to the control group (n = 1748) or to the intervention group (n = 2636). Those in the control group were sent a generic letter advertising the SSS in their area and those in the intervention group were sent a computer-tailored letter signed by their GP containing personalised risk information and an invitation to a no-commitment ‘taster session’ to find out more about the SSS.Eighteen SSSs across England and 99 general practices within the SSS areas were recruited into the trial between February 2011 and October 2013. All current cigarette and roll-up smokers over the age of 16 identified in participating practices . If there was more than one recorded session available for a lead advisor, one session was selected at random.Of the 131 sessions delivered, 93 were recorded (71 %). The remainder were not recorded due to forgotten recording equipment, equipment failure or no consent for recording by one or more participants attending the session. Due to the quantity and length of the recordings, sessions to be transcribed and analysed were purposively selected to ensure one session by each lead advisor (where available) was included .n = 222).Attendees in this study were defined as those participants of Start2quit who were randomised to receive the intervention and who also attended a taster session included in this analysis  or instructions that they should follow (e.g. ask attendees how many of them enjoy smoking). A coding frame was developed by the first author based on this protocol. Two additional researchers verified the frame by checking that it contained all behaviours specified in the taster session protocol.The behaviours specified in the coding frame were independently classified by two of the authors into component BCTs using an established taxonomy of 45 smoking cessation BCTs . This waTaster session characteristics included the structure of the session (one advisor or two providing content), the length of the session and number of attendees.All advisors were recruited on the basis that they were employed by the SSS and had some previous experience of assisting people to quit smoking. Advisors also completed a short questionnaire at the time of training. Data gathered included gender, age, highest educational qualification, type of smoking cessation training, time since smoking cessation training, employer, job title and number of patients seen in the previous 6 months.The proportion of attendees at each session who subsequently attended the SSS, and the proportion of attendees at each session who were found to be 7-day point-prevalent abstinent at the 6-month follow-up, biochemically validated by salivary cotinine, were measured. It is important to note that attendance at the SSS was the behaviour targeted by the taster sessions and the one hypothesised to be related to the fidelity of delivery of the intervention. However, 7-day point-prevalent abstinence data was also included to test the hypothesis that there was no link between this outcome and adherence given the potentially complex pathway between the taster sessions and cessation.All transcripts were anonymised for SSS area and advisor. Each transcript was coded by a minimum of two authors; specifically MSM coded all transcripts and DK and AH coded 50 % each. The remaining authors (HG and SG) coded 25 % of the transcripts between them, chosen at random, to provide an extra level of reliability checking. Average inter-rater reliability for coding was 86 % (68–99 %) across sessions. All disagreements were resolved through discussion between researchers. Data from the coding frames were double entered into Excel and discrepancies corrected.The fidelity of each taster session was expressed as the percentage of overall protocol-specified behaviours that were delivered, that is the number of protocol specified behaviours applied by the advisor divided by the total number of behaviours . Adherence to each BCT was measured as the number of behaviours applied by the advisors within each BCT divided by the total number classified within each.t tests and analysis of variance were used to assess differences in adherence to the protocol by advisor characteristics and advising structure. Correlations were computed to explore the association between adherence to protocol-specified behaviours and the length of session. To assess whether adherence was related to the main outcome measures of attendance at the SSS and 7-day point-prevalent abstinence, high and low adherers were split into two groups (high adherers were those sessions that reached the median and above), and t tests used to assess differences in outcomes between these groups. Data were analysed in SPSS (v22).Mean and median adherence to protocol specified behaviours and for each BCT was calculated. The study was approved by the by the South West London Research Ethics Committee, and R & D approval was obtained from all participating SSSs.Mean session duration was 43 min and 4 s and ranged from 14 min to 1 h and 18 min. Twenty-seven sessions (65.85 %) were facilitated by one lead advisor with minimal administrative support by an additional advisor, and 14 (34.15 %) were split between two advisors with one taking the lead. The number of smokers attending a session ranged from 1 to 17 .Only lead advisor characteristics were assessed due to their leading role in the taster sessions and the significantly greater proportion of the information that they communicated to attendees. The majority of advisors were female (73.17 %) and 51.22 % were aged between 45 and 54. The majority were educated to degree level or higher (73.17 %), 41.46 % had received smoking cessation training to NCSCT  stages 1 and 2 with various additional training and 60.98 % had received training within the previous 3 years. All were employed by the SSS and 70.73 % were employed as SSS advisors. Most (90.24 %) had seen 21 or more patients in the last six months (Table m = 68.53 %) of protocol-specified behaviours was delivered across sessions, though there was considerable variation. Adherence in individual sessions varied from 28.76 to 95.89 %  to 100 % in five of the BCTs Fig. . Howeverp = 0.044). There was a negative correlation between session length and adherence, thus shorter session length was associated with increased adherence to protocol-specified content .Sessions where the advisor was assisted by a second advisor were found to have significantly higher adherence levels than sessions run by one advisor alone . Level of adherence also varied with advisors’ age, those aged between 45 and 54 were significantly more adherent (75.41 %) than advisors who were younger (61.87 %) or older (60.45 %) (p = 0.021). Full details including CIs are reported in Table Female advisors had significantly higher levels of adherence to protocol-specified behaviours than male advisors , and the mean proportion of attendees who were found to be validated 7-day point-prevalent abstinent at the 6-month follow-up was 20.21 % (range 0 to 66.67 %).The proportion of attendees who subsequently attended the SSS ranged from 0 to 100 %  or in the proportion who were 7-day point-prevalent abstinent .This study represents a thorough assessment of intervention delivery fidelity to the taster session protocol of the Start2quit trial. The median adherence to specified behaviours of 71.23 % was high relative to similar evaluations , 22. HowThis study has some important implications for future fidelity assessments of novel interventions. Firstly, the finding that more specific techniques were more likely to be delivered suggests the importance of considering the previous training and expertise of those delivering an intervention. While it has been reported elsewhere that more specific BCTs are less likely to be delivered reliably , this waThe finding that sessions run by two advisors had higher levels of adherence than those run by a single advisor has potentially significant implications for future implementation assessments. One possible explanation for this result is that the second advisor acted as a safety net, delivering key items that the first advisor failed to present. Research in the field of sociology has suggested that co-facilitation of training sessions can be beneficial due to shared responsibility and mutual support , 25. ThuThis study also examined specific advisor characteristics. Being female and being aged between 45 and 54 were significantly associated with adherence to the protocol. These findings should be treated with caution due to the small sample size, the fact that not all advisors were included in the analysed sessions, and the omission from the analysis of the contribution of secondary advisors who delivered a minority of the content. However, they do indicate a need for more research into how advisor characteristics potentially influence adherence to study manuals and how this influence can be mediated through training. To date, the exploration of the reasons for variation in adherence has been largely ignored with the exception of some work on the impact of practical issues such as lack of time .While the median adherence to specified behaviours was high, there was considerable variability across study areas suggesting some modifications be made to the training provided to advisors if this intervention were to be adopted. Specifically, it may be beneficial to tailor training to advisor experience levels in delivering behaviour change interventions. In addition, given that the two BCTs with lowest adherence were related to providing information on the SSS itself, greater emphasis on the importance of communicating this practical information may be needed.However, the lack of association between adherence to BCTs and the outcomes of attending the SSS and 7-day abstinence suggests that absolute fidelity may not be necessary for this intervention to be effective. Absolute fidelity is rarely realistic in real-world settings, and in this intervention, it is important that advisors have some freedom to adapt to the specific needs of the people to whom they are presenting. As discussed by Dusenbury et al. , key to Finally, the lack of association between adherence and study outcomes may have been influenced by the generally high levels of motivation of attendees. Responding to an invitation to a smoking cessation study and then attending a session to find out more about the SSS indicates a high level of interest in both quitting smoking and availing of SSS support to do so. Perhaps this enthusiasm meant that the presentation of protocol-specified content was less important than theorised as attendees were already inclined to sign up for an SSS course. In addition, receiving the first half of the intervention, a personalised risk letter, may have meant that the likelihood of participants changing their behaviour had already increased and as such they were more amenable to attending the SSS or quitting smoking.A key strength of this study is the robust methodology. All sessions were double-coded and 25 % were triple-coded. Sessions were anonymised for area and advisor to remove potential bias and inter-rater agreement was high for all sessions. The protocols were highly specific and all advisors underwent a 2-h training session. In addition, as previously mentioned, all advisors were experienced in delivering smoking-related behaviour change interventions.A significant limitation of the study is its evaluation of only a sample of the taster sessions delivered as part of the Start2quit study, due to the prohibitive resources required to transcribe and analyse the additional 47 sessions recorded. However, the 41 sessions analysed represent 31.3 % of the total session number, well over the 20 % minimum recommended by Schlosser , 28. OthWe did not assess the other key components of implementation fidelity, intervention receipt and enactment. These additional measurements were outside the scope of this study but could have provided clarity on the efficacy of specific BCTs. Finally, the high rate of unrecorded sessions is an issue and it is possible that when this was due to tape failure, it was not random. For example, less well-organised and prepared advisors may have been less likely to remember to record and operate the machine correctly.Assessing implementation fidelity is essential in ensuring that research studies are actually measuring that which they claim to be measuring , 7. This


In recent years, the demand for high data rate wireless communications has increased dramatically, which requires larger bandwidth to sustain multi-user accessibility and quality of services. This can be achieved at millimeter wave frequencies. Graphene is a promising material for the development of millimeter-wave electronics because of its outstanding electron transport properties. Up to now, due to the lack of high quality material and process technology, the operating frequency of demonstrated circuits has been far below the potential of graphene. Here, we present monolithic integrated circuits based on epitaxial graphene operating at unprecedented high frequencies (80–100 GHz). The demonstrated circuits are capable of encoding/decoding of multi-gigabit-per-second information into/from the amplitude or phase of the carrier signal. The developed fabrication process is scalable to large wafer sizes. To meet the fast growing demand for telecommunication services, developing high-data-rate communication links in the range of multi-gigabit per second (Gbps) is necessary. The high speed data links can be implemented using either wireless or fiber optic technologies. Wireless technology, particularly in urban areas, has several advantages over fiber optics such as mobility, universal deployment, short installation time and cost effectiveness. However, to achieve data rates comparable to that of the fiber optics, there is a need to develop wireless systems with a very large bandwidth ∼10 GHz). This may be achieved by operating at millimeter wave (mm-wave) frequencies (30–300 GHz)0 GHz. ThT) of 400 GHz and maximum oscillation frequency (fMAX) of 100 GHz have been demonstrated3567691011131415171819212223In this regard, graphene is a promising material for the development of mm-wave electronics due to its excellent electron transport properties. Recently, there is a rapid progress in the development of graphene field effect transistors (G-FETs). G-FETs with intrinsic current-gain cutoff frequencies  based on epitaxial graphene in high data rate applications in the 90 GHz band. The developed process is scalable up to full wafer sizes. Currently the limiting factor is not the wafer size but the uniformity of available epitaxial graphene resulting in about 70% yield. The fabricated MMIC has different circuits elements capable of receiving and retrieving information embedded in the amplitude and phase of the carrier signal at the rate of 4 Gbps. Furthermore, the developed circuits are highly linear allowing to generate modulated signal up to the rate of 8 Gbps at 90 GHz band with a bit-error-rate (BER) below 102/V and 150–250 Ω/Vs respectively. More details of graphene growth and its properties can be found in the method and Typically, the term “epitaxial graphene” refers to graphene grown on SiC which is in fact not done by epitaxy but by sublimation of silicon. In this study graphene is grown by traditional Chemical Vapor Deposition (CVD) epitaxy using carbon precursor or more accurately by Vapor Phase Epitaxy (VPE)To achieve complete wireless communication systems based on graphene it is needed to realize all the above mentioned components with graphene technology. Transistors are versatile elements and by choosing proper operating points and passive components, all needed functionalities are attainable.Therefore, G-FETs are the only needed graphene based active elements for these circuits. In addition, it is desirable to have whole transmitter and receiver chain on a single chip which facilitates the very broadband performance needed for the high data rate applications. Hence, developing G-FET based MMICs is a key requirement to obtain graphene based high speed wireless systems.MAX more than 200 GHz is needed. Therefore, more development in G-FET technology is needed to realize G-FET based amplifiers at this frequency band. However, for other applications where the G-FETs operate in other modes, the existing G-FETs technology based on hydrogen intercalated graphene is sufficient. This is because this type of graphene exhibits a high carrier mobility with a very low sheet resistance. It results in a reduction of channel resistance and consequently achieving small time constants in G-FETs needed for higher frequency operations. Developing microstrip MMIC process on SiC substrates is a laborious task especially at high frequencies where the parasitic elements and unwanted propagation modes can significantly deteriorate the circuit performance. In addition, graphene compatibility is extremely important and because of that many existing circuit fabrication technologies cannot be directly applied for the development of graphene based MMICs. These process have several steps that can potentially degrade graphene. One way to avoid this problem is to fabricated the passive elements first and then transfer graphene and perform G-FETs fabrication2O3 deposited by the atomic layer deposition (ALD) method2O3 exhibits a poor thermal conductance and a high tensile stress. Since SiC via hole etching is performed at very high powers, the generated heat causes the Al2O3 layer to crack. This completely damages G-FETs as well. To avoid it, a thin layer (15 nm) of ALD Al2O3 is deposited and it is followed by 100 nm SiN sputtered at a very low density plasma. This process does not damage the graphene. In addition, the new encapsulated layer can completely protect graphene from the remaining plasma steps. Furthermore, the new layer has a better thermal conductance and a lower stress and can survive the via hole etching process. In this process the SiC substrate is thinned down to 70-μm providing a platform to develop circuits up to 250 GHz. By using our device and circuit technologies we are able to realize needed RF modules except amplifiers at 90 GHz band. RF signal in mm-wave can be generated by using G-FET based frequency multipliers. However, since the output power of these frequency multipliers are very low67Circuit performance depends on both device and circuit technology. Device (G-FET) technology has recently shown a rapid progress. However, to have G-FET based amplifiers operating at 90 GHz band, G-FETs with fn layer 1 nm of ALThe most fundamental digital modulation techniques are binary amplitude-shift keying (BASK) and binary phase-shift keying (BPSK) where binary information is coded in the amplitude and phase of the carrier signal respectively . A mixerTo evaluate the performance of the power detector based demodulator MMIC, a BASK signal at 90 GHz with modulation rates of 1, 2 and 4 Gbps is used. Due to the band width limitation of our arbitrary waveform generator, the BASK signals have triangular-shaped pulses . The eyeThe performance of the demonstrated G-FET based demodulators and this work is shown in −6 can be estimated. The experiment is repeated by 16-QAM signals modulated at the rate of 4, 8 and 16 Gbps. The reconstructed constellation diagrams are plotted in −6, 10−5 and 10−4 respectively. It can be seen that the achieved constellation diagrams are not distorted meaning that the mixer is highly linear. The MMIC mixer circuit can be used for signal modulation as well. As shown in 29To achieve high frequency devices base on graphene having a high carrier mobility is not sufficient. Graphene films should have high carrier density as well in order to obtain low sheet resistance levels needed for high frequency applications. These properties exist in hydrogen intercalated epitaxial graphene and thereby it is more beneficial for RF devices and circuits. The development of G-FET MMICs that can perform multi-Gbps signal modulation and demodulation at mm-wave makes a start on the realization of complete high data rate transceivers based on graphene. Prior to this work, the operating frequency and data rate of the demonstrated graphene based ICs are far below the performance of the existing technologies. This is mainly due to the lack of high quality process technology and material. For high data rate communication, the linearity of the RF components plays a key role. The fabricated MMIC mixer is highly linear allowing to generate and retrieve multi-Gbps data at 90 GHz atmospheric window. This work demonstrates G-FETs’ great potential for high data rate communication systems. To achieve complete wireless communication systems based on graphene, more development in G-FET technology is needed to realize amplifiers at mm-wave band.2 nominally on-axis 4H-SiC (0001) or 6H-SiC (0001), Si-face semi-insulating chemo-mechanically polished substrates. Graphene layers are grown under an argon laminar flow at hot-wall Aixtron VP508 and Aixtron G5 reactors where graphene growth on 4″ SiC substrates is under development. The process depends critically on the creation of the flow conditions in the reactor that control Si sublimation rate and enable mass transport of hydrocarbon to the SiC surface. Reynolds number (Re) measures the ratio of inertial forces to viscous forces and consequently quantifies the relative importance of these two forces in a given gas flow. Tuning the value of the Re number allows to form a thick enough Ar boundary layer to prevent Si sublimation. This lets the diffusion of hydrocarbon to the SiC surface and growth of epitaxial graphene. The intercalation of hydrogen is obtained in situ at the temperature of 1100 °C and 900 mbar pressure. In our samples the presence of hydrogen atoms is maintained up to 700 °C, high enough to meet the requirements of high-speed electronics and high-temperature sensing.Epitaxial graphene is grown at 1600 °C and 30 mbar pressure by CV technique on (15 × 15) mm2O3 (15 nm) and sputtered SiN (100 nm) layers are utilized. The rest of the fabrication process are based on photolithography. For forming thin film resistors, tantalum Nitride (TaN) is sputtered. The metal-insulator-metal (MIM) capacitors are based on SiN. After the fabrication of MIM capacitors, dielectric via etching is performed to reach to the G-FET metal pads for the deposition of the large pads and transmission lines. Gold electroplating is done to reduce the metal resistivity and form air-bridges. Then a thick layer of resist is used to protect the surface and the chip is glowed to a carrier wafer for the back side processing. The substrate is thinned down to 70 μm and polished by a CMP process. For via hole formation, a thick layer of nickel is plated as a hard mask and high power ICP is used to etch via holes. The nickel mask is wet striped and a seed layer is sputtered on the back side of the chip. Finally, gold is plated for the metallization of the ground plane and via holes.The fabrication process has about 30 different steps including 3 ebeam lithography and 13 photolithography steps. Ebeam-lithography is used for the fabrication of G-FETs since ebeam resists are more compatible with graphene. For G-FET passivation a combination of ALD AlAn Agilent signal generator and an OML S10MS W-band source module are used to provide the LO signal. The digital modulated signals at baseband and intermediate frequency are generated by a Keysight M8195A 65-Gsps sampling rate 8-bit arbitrary waveform generator. The millimeter wave output is measured by a Lecroy Labmaster 10-100Zi real time oscilloscope which has 100 GHz input bandwidth and 240 Gsps sampling rate. High data rate BASK (on off keying) signal is generated by an in-house fabricated W-band frequency multiplier module based on InP DHBT MMIC. The data captured by the real time oscilloscope is processed using Matlab code.How to cite this article: Habibpour, O. et al. Wafer scale millimeter-wave integrated circuits based on epitaxial graphene in high data rate communication. Sci. Rep.7, 41828; doi: 10.1038/srep41828 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The adsorption state was probed by analysing the G-peak position in the measured in-situ Raman spectrum during electrochemical experiments. We conclude that electrochemical Raman spectroscopy on graphene is a valuable tool to obtain in-situ information on adsorbed species on graphene, isolated from the rest of the electrochemical behaviour.Electrochemistry on graphene is of particular interest due to graphene’s high surface area, high electrical conductivity and low interfacial capacitance. Because the graphene Fermi level can be probed by its strong Raman signal, information on the graphene doping can be obtained which in turn can provide information on adsorbed atoms or molecules. For this paper, the adsorption analysis was successfully performed using three electroactive substances with different electrode interaction mechanisms: hexaammineruthenium(III) chloride (RuHex), ferrocenemethanol (FcMeOH) and potassium ferricyanide/potassium ferrocyanide (Fe(CN) Graphene has received much attention during the last decade because of its unique properties such as mechanical strength and impermeability to gasses12341101112141516171819202119232526Here we will exploit the technique of Raman spectroelectrochemistry to obtain information about, among others, the reaction kinetics during Faradaic reactions on the surface of graphene. Often the desorption phase is the rate-determining step in nano- and microfluidic systems, where mass transport by diffusion is relatively fast compared to large scale systems. E.g. in redox cycling experiments slow desorption results in a lower effective diffusion coefficient and thereby in reduced signal levelsIn the pathway of electrochemical reactions several steps can be distinguished as shown in 6 (potassium ferricyanide/potassium ferrocyanide), which is known to have different reaction kinetics on monolayer graphene and pyrolytic graphiteTo explain redox reactions for complex molecules two mechanisms are recognized: inner-sphere and outer-sphere electrode reactions121010Sm will then be a superposition of two spectra: the graphene with adsorbed molecules Sad and the bare graphene Sgr. For the ratio between both we define an occupation factor β as followsWhenever molecules adsorb to the graphene surface, the graphene Fermi level is shifted by local doping (changing the carrier concentration), which can be probed by Raman spectroscopy19when this factor is low β ≈ 0 solely the bare graphene spectrum is measured, indicating no interaction between the graphene and the molecules in solution. For a large factor β ≈ 1 the graphene is entirely occupied by adsorbed molecules which interact with the graphene and therefore change the Raman spectrum.−1) is analysed, since this is the most sensitive indicator for the graphene Fermi level and therefore the adsorption level8CNP , which is the applied gate voltage corresponding to a minimum in the G-peak position curve, probing the graphene Fermi level. This will cause the valley to move horizontally A typical Raman spectrum measured for our graphene is shown in in-situ Raman spectroscopy experiments the adsorption (and possibly the approach) of redox species can be probed independently of the remainder of the electrochemical behaviour. To explain the electrochemical voltammogram data, we will thus distinguish adsorption and electron exchange at reactive sites 6 was measured using the second graphene device.To test our hypotheses, the adsorption of the three redox active species during electrochemical experiments using k0, therefore we conclude that the electrode kinetics of RuHex are such that it is a fast reactionin-situ recorded G-peak position curves are displayed, which are at about the same position as the baseline curve. Both curves are constant over several measurements, showing no change over time. This dataset indicates that RuHex adsorption to the graphene electrode was absent or below our detection limit (β ≈ 0). As expected for an outer-sphere redox couple that does not need adsorption to react, a sizeable redox current was still observed despite this lack of adsorption.The cyclic voltammogram of RuHex at the graphene electrode is shown in k0 . Both effects can be explained by the adsorption of FcMeOH molecules. Furthermore, the valley  than for RuHex (0.10 V) and FcMeOH (0.14 V), indicating a much lower standard rate constant k0 (see The spectroelectrochemistry of Fe(CN)t k0 see  and thus6 adsorption, whereby the difference between the first and the subsequent measurements suggest that two adsorption states exist. In addition the slope becomes shallower. The observations are as expected for the inner-sphere redox couple Fe(CN)6, that needs adsorption before reacting.In 6 can be found, the reservoir was three times rinsed with background electrolyte and subsequently the measurements were performed. The results are shown in 6. The voltammogram shows a quite different behaviour: the peak separation voltage has reduced from approximately 0.9 V to approximately 0.4 V and the current has decreased 50-fold. The decrease in peak separation voltage could be explained by a difference in standard rate constant resulting in different overpotentials. The voltammogram in 6 on graphite, where reaction takes place at reactive edge sites with low overpotential. The traces in 6. In the first measurement the minimum in the G-peak position curve is still quite similar to the position in 296 that is hard to desorb, similar to FcMeOH.To test whether also evidence for desorption of Fe(CN)6 activity was verified in the Fe(CN)6 solution with the results as shown in 6 can be found, showing to what extent the device history influences its behaviour.Finally, a measurement was performed to establish whether the signal shown in β, as visible from the Raman spectrum, and the amount of reacting species were directly coupled. The data for RuHex, an outer sphere redox substance, were not in conflict with this model, as for such substances redox activity is independent of adsorption. For FcMeOH, another outer sphere redox substance, adsorption was still found. This, however, is in accordance with literature reports, whereby it could not be established whether this adsorption contributes to the redox activity. The results for Fe(CN)6 were also in line with expectations, as we observed both adsorption and electroactivity for this inner sphere redox couple. We however observed in the Fe(CN)6 desorption experiments that two type of reactions occur, with low and high overpotential. The latter one has a major contribution to the current. The former one is persistent and remains when the solution is flushed.In the model, the occupation factor CNP shifted to a more negative voltage and for Fe(CN)6 (−3 and −4 charge) to a more positive voltage, suggesting that the charge of the molecule determines whether n- or p-doping is induced. Secondly, graphene electron mobility is reduced by grain boundaries 3739A further complication in the analysis of the cyclic voltammograms is that the graphene conductivity is influenced by adsorbed species in two ways. Firstly, the adsorbed molecules on the basal plane  influence the conduction by changing the carrier concentration. For FcMeOH (uncharged) the charge neutrality point Vin-situ Raman spectrum during electrochemical experiments. Prior to every experiment a G-peak position curve was recorded for the bare graphene, serving as a baseline for subsequent measurements. For RuHex no change in the G-peak position curve was found during the electrochemical experiment compared to the baseline in solely background electrolyte, which was as expected. In case of FcMeOH a clear stable negative shift in the G-peak position curve was observed, indicating a strong adsorption state to the graphene, potentially caused by π-interactions or electrostatic interactions. For Fe(CN)6 a clear positive shift in the G-peak position curve could be seen, however for this species the measurement required some time to settle towards a constant voltammogram and G-peak position curve. This is suggesting that FcMeOH and Fe(CN)6 interact differently with the graphene. Furthermore, when measuring Fe(CN)6 on the graphene electrode after FcMeOH we found no clear difference in the voltammogram compared to Fe(CN)6, while the G-peak position curve indicated that both species were adsorbed.Evidence for the adsorption state during redox reactions at graphene in the Raman spectrum was found. The adsorption state was probed by analysing the G-peak position in the measured in-situ information on adsorbed species on graphene, independent of the cyclic voltammograms measured. This makes graphene an even more interesting electrode material for electrochemical sensing as it allows a fundamental investigation of detailed adsorption mechanisms by Raman spectrometry. We found indications that the adsorption of species for the larger part takes place on the less reactive basal plane, while local reactive sites mainly determine the electrochemical behaviour. We also observed additional effects of the adsorbed species to the graphene surface, where a slight change in the electrochemical behaviour is most probably caused by changes in graphene charge carrier density and passivation of the surface.Based on these findings we conclude that Raman spectroelectrochemistry on graphene is a powerful tool to obtain et al.−10 m2/s. Current is calculated by integrating the normal flux at the electrode boundary over its area and multiplying by F. On t = 0 is the concentration of the reduced species 1 mM and the oxidized species are absent. The transient behaviour is simulated by scanning the overpotential between −1 V and 1 V with 25 mV/s.The peak potential separation as function of the standard rate constant is simulated in COMSOL Multiphysics 5.1 using the ‘Transport of Diluted Species’ physic module. A round electrode is modelled in a cylindrical coordinate system with boundary condition: 2 for both devices.The schematic of the fabrication and measurement setup of the graphene devices are shown in fcutoff = 5 Hz). To the background electrolyte, 0.1 M KNO3, the reactive species 6) were added in a concentration of 1 mM. Subsequently, 100 μL of solution was applied to the graphene device. All potentials were measured against an Ag/AgCl reference electrode (WPI Dri-Ref-450). The spectra are recorded using a WITec alpha 300 system with a 532 nm laser at 1 mW with an immersion objective for measuring in liquid (Nikon Fluor 40x 0.8 W DIC). Analysis of the recorded Raman spectra is performed using a MATLAB script, which accounts for an offset in the spectrometer by fitting the Rayleigh peak. Next Lorentzian curves are fitted to the graphene peaks to obtain their positions and intensities. The voltammetric measurement protocol was as follows. In each measurement three scans were performed in series (whereby the results of the last two scans are displayed). Measurements in a series were performed with a time spacing of approximately 1 minute.The setup used in this experiment as shown schematically in How to cite this article: van den Beld, W. T. E. et al. In-situ Raman spectroscopy to elucidate the influence of adsorption in graphene electrochemistry. Sci. Rep.7, 45080; doi: 10.1038/srep45080 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


We describe how mechanical strain is able to control the flow of heat on a graphene sheet, since this material can either absorb or expel heat from/to a thermal reservoir, depending on the strain energy. In a similar fashion as the magneto- and electro-caloric effects, the present case considers the fact that a mechanical strain produces a pseudo-magnetic field that, on its turn, is responsible for the barocaloric effect. This result pushes graphene to the list of multicaloric materials. Just to name a few, we can cite its electrical, mechanical and thermal features, like a high electronic mobility2, strong mechanical resistance3 and, in addition, tunable thermal conductivity - from ultra-high (for heat-sinking applications), down to ultra-low values (for thermoelectric applications)4.Graphene has outstanding physical properties5, optoelectronics6, electro-7 and magneto-caloric9 effects. These last two are of great importance for the present article. Few years ago, one of us reported the interesting caloric properties of a graphene sheet due to a change of either a magnetic or an electric field; and the results were the oscillating magnetocaloric8 (OMCE) and electrocaloric7 (OECE) effects, respectively. Few years later, a follow-up of these papers appeared, discussing the properties of the OMCE of a multilayer graphene9 and applications into a six-stages thermodynamic cycle10.However, one special reason to justify the always fresh and renewed interest of the scientific community on graphene is the possibility of interplay of many of those outstanding, extraordinary properties. For instance, we can mention graphene plasmonics11), to exchange heat with a thermal reservoir ; or change its temperature  due to a change of an external parameter, say, either a magnetic or an electric field. The amount of heat exchanged with a thermal reservoir, considering an isothermal process, is given by 11; but, surprisingly, diamagnetic materials also present this effect (much reduced in magnitude in comparison to the former case), presenting, on the other hands, an oscillatory pattern13. These oscillations are based on the same physical mechanism as the de Haas-van Alphen14 and Shubnikov-de Haas effects15.In more details, those caloric effects are the ability of a material  stands for the electrons (holes) band; We have considered a graphene sheet on the 19. The remaining parameters above used were defined on the Methods section, when describing the Hamiltonian of the system. It is worth to note that equation th LL.However, the strain energy mentclass2pt{minimGrand potential 9. Thus, as described on the Methods section:From this energy spectra, one can obtain the 2 is the magnetic flux quantum; −2 is the density of charge carriers; It is worth to note that the strain contributes with an additional term to the Fermi energy. By contrast, the zero-strain relationship between the Fermi level and the number of charge carrier Grand potentials, it is possible to obtain the entropy due to the strain; that can be evaluated using a standard thermodynamic relationship: From these equation , is temp11, and can be achieved measuring the difference between field- and zero-field entropies: S = 0 and therefore the barocaloric effect on graphene reads as 8. For the sake of simplicity, as already done in other works8, we will only consider the The caloric effect can be defined by means of the entropy change, considering the isothermal process9 and the electrocaloric7 effects. In addition, this effect can be either normal or inverse, i.e., the material can either absorb or expel heat from/to a thermal reservoir, depending on the value of the pseudo-magnetic field, that, on its turn, depends on the strain energy. In other words, this (in/out) flow of heat can be managed by a simple handle of compression. In fact, the barocaloric effect largely overlaps the (zero-strain) oscillating magnetocaloric effect8 at low magnetic field, previously described by one of us, as can be seen on Fig. The barocaloric effect is drawn on Fig. mentclass2pt{minimmentclass2pt{minim18. One can say that the strain is able to both, either to mimic the magnetocaloric effect at low magnetic field and to produce a robust barocaloric effect. These properties can be regarded as unique advantages of strain gauge field. Based on this, more promising applications are thus eagerly expected to be realized in practice.From another perspective, the real magnetic field hardly achieves high value as c.a. 300 T at present, but the strain pseudo-magnetic field really does19. Although the degeneracy of the valleys in zero-An important point shall be addressed on Fig. Bs =  300 T at room temperature18.After a deeper looking on equation 9- and electro7-caloric effects previously proposed for graphene, moving therefore this material to the list of multicaloric substances - a fresh issue on the literature.We present the barocaloric effect (BCE) on graphene: the capacity of this material to exchange heat with a thermal reservoir due to a mechanical strain, like nanobubbles and even standard compression. In practice, strain produces a pseudo-magnetic field that, on its turn, changes the entropy of the material. Remarkable are the barocaloric oscillations, a pure quantum effect that allow to control the BCE as either normal or inverse, i.e., the material can either absorb or expel heat from/to a thermal reservoir, depending on the value of the pseudo-magnetic field. In other words, the flow of heat can be managed by a simple handle of compression. This effect is completely analogous to the oscillating magnetox–y plane and subject to a strain that gives rise to a pseudo-magnetic field et al.18. The 2D Dirac Hamiltonian for this model reads as:For the present work we have considered a graphene sheet on the et al.18: a value much higher than any other obtained in a laboratory, being therefore a unique opportunity to study a graphene sheet under an extremely high (pseudo-)magnetic field.The first term of the above equation represents the kinetic energy, where: Grand potential, represented on equations Grand potential calculation.The aim of this subsection is to provide further details on how to obtain the The total energy of the system at zero temperature reads as:20:Considering the generalised Zeta functionmentclasspt{minima2020The second contribution to the total energy, 20 it is known that:However, from ref.A comparison of these two equations leads to:20:20:The above term can be further developed. Considering now The term developed above has a cosine function and, for this reason, it is named as oscillatory contribution; while the other contribution (on equation 22. The procedure is to obtain the Grand potential following the equation:22:Above we presented our results on how to obtain the total energy, at zero temperature, of the system under consideration. From now on, let us consider the temperature effect, following the methodology of refsLet us first consider the non-oscillatory contribution, and, for this purpose, we need to insert equation Interestingly, the approximate result above for finite temperature is the same as equation 20:Grand potential can be obtained by inserting the above equation into equation 20:Analogously to above, let us now consider the oscillatory contribution, by considering the integral representation for Grand potential:Finally, we obtain the oscillatory term of the


Bone marrow sarcoidosis is extremely rare. The association between sarcoidosis and lymphoproliferative disorders has been previously speculated, although the diagnosis of sarcoidosis often precedes any hematological derangements.Here, we report for the first time, a case of a 57-year-old Caucasian woman with a previous diagnosis of monoclonal gammopathy of undetermined significance (MGUS) developing hypercalcemia and renal failure with workup notable for isolated bone marrow sarcoidosis and not multiple myeloma as expected. The patient was successfully managed with prednisone taper therapy with resolution of her hypercalcemia and repeat bone marrow biopsies demonstrating resolving granulomas.Our case illustrates the diagnostic challenges associated with bone marrow sarcoidosis and suggest that chronic immune stimulation in the bone marrow in the setting of MGUS may be associated with the development of localized sarcoidosis. The long term consequences of steroid therapy targeting sarcoidosis in this patient with underlying MGUS remain unknown. Greater surveillance and closer followup is planned in light of the increased risk of malignant transformation of MGUS into multiple myeloma in the setting of bone marrow sarcoidosis. Extrapulmonary manifestations of sarcoidosis are uncommon. Bone marrow sarcoidosis is infrequently clinically encountered. In the few cases reported, bone marrow involvement most often occurs in the setting of systemic sarcoidosis –3. IsolaA 57-year-old Caucasian woman with a four-year history of monoclonal gammopathy of undetermined significance (MGUS) complained of several weeks of dry mouth, polydipsia and episodes of confusion. Her serum calcium was 14.4 mg/dL when tested at the office of her primary care physician (PCP) and sent to the emergency department (ED) where her exam was remarkable only for dry mucous membranes. Initial labs were notable for Ca 14.4 mg/dL, albumin 5.0 g/dL, PTH 7 pg/mL, and Cr at 1.94. CBC was notable for mild pancytopenia with WBC 3.5 (th/cmm) with normal differential, Hgb 11.7 g/dL, Hct 34.2 (%), and PLT 145,000 (th/cmm). She received a bolus of normal saline and was subsequently admitted to inpatient service. Initially, the patient’s hypercalcemia was attributed to milk-alkali syndrome and her calcium supplements were discontinued. The patient was managed only with IV hydration. The patient’s serum calcium trended down to 10 mg/dL at the time of discharge.There was a concern for a neoplastic process, particularly myeloma, given her history of MGUS. The patient underwent bone marrow biopsy at the time of discharge, which was remarkable for non-necrotizing granulomas with no findings suggestive of plasma cell dyscrasia or other neoplasms. Fig.  Two weekThe patient underwent a rapid prednisone taper over 9 days. After discontinuation of prednisone, she developed hypercalcemia again. She was then treated with a slow prednisone taper for four months with normalization of serum calcium levels. A follow-up bone marrow biopsy 5 months later (1 month after 4-month course of slow prednisone taper) showed partial resolution of granulomas without malignancies. Fig.  At her fBone marrow sarcoidosis is rare and previously reported cases mostly involved patients with severe, chronic, and widespread sarcoidosis. Bone marrow biopsies are not routinely performed in the standard workup of sarcoidosis, thus the overall incidence of bone marrow involvement is unknown. One small cohort study  estimateOur case illustrates the diagnostic challenges associated with an atypical and rare manifestation of sarcoidosis. The clinical presentation of bone marrow sarcoidosis is variable, non-specific, and may overlap with those of systemic sarcoidosis which include fever, fatigue, malaise, weight loss, and night sweats. Patients with bone marrow involvement may have associated hematological abnormalities including anemia, leukopenia, and thrombocytopenia –5.Although the diagnosis of sarcoidosis seemed unlikely during the early workup of this patient, three lab abnormalities had some clinical utility and diagnostic value in support of sarcoidosis: elevated ACE serum level, hypercalcemia, and hypercalciuria.. ACE levels are elevated in 75% of untreated patients with sarcoidosis and levels tend to correlate with disease activity . One stuGiven the rare occurrence of bone marrow sarcoidosis, there are currently no randomized controlled trials comparing treatment modalities. However, a review of the relevant literature presents four case reports of patients with biopsy-proven bone marrow sarcoidosis demonstrating various approaches to treatment. One case report by Saliba et al.  involvedThese case reports highlight that the therapeutic approach to patients with bone marrow sarcoidosis depend on clinical presentation. Patients with symptomatic hypercalcemia appear to respond well to IV saline hydration and in severe cases may require steroid taper therapy. Ackermann et al.  recommenAn intriguing aspect of this case report involves the potential relationship with the patient’s underlying MGUS. The association between sarcoidosis and the increased risk of malignancy particularly lymphoproliferative disorders has been previously examined –8 and caSarcoidosis has been observed to develop in patients with hematologic malignancies with some investigators attributing this to the use of immunosuppressive therapy targeted at the underlying malignancy , 18. AsiIn conclusion, isolated bone marrow sarcoidosis is rare and can be a diagnostic challenge. The clinical suspicion for sarcoidosis should be raised in patients with underlying hematologic disorders or malignancies. Due to the rarity of the condition, long terms outcomes in such patients are unclear. Surveillance and regular follow-up is planned in view of the possibility of progression to multiple myeloma or another lymphoproliferative disorder.


The aqueous concentration can be as high as 5.0 mg mL−1, which is an order of magnitude higher than previously reports. The water soluble graphene can be directly spray-coated on various substrates, and the back-gated field effect transistor give hole and electron mobility of ~496 and ~676 cm2 V−1 s−1, respectively. These results achieved are expected to expedite various applications of graphene.Despite significant progresses made on mass production of chemically exfoliated graphene, the quality, cost and environmental friendliness remain major challenges for its market penetration. Here, we present a fast and green exfoliation strategy for large scale production of high quality water dispersible few layer graphene through a controllable edge oxidation and localized gas bubbling process. Mild edge oxidation guarantees that the pristine  Oxidation-reduction is the earliest wet process developed for the scalable fabrication of chemically exfoliated graphene, and it remains to be one of the most mature approaches ever developed, due to its versatility, scalability and controllability in terms of surface area, size and thickness1235et al. demonstrated high yield graphene production by electrochemical exfoliation of graphite with weak oxidationWeak oxidation or oxidation-free process is very helpful in preserving graphene’s lattice integrity, thus retaining its performance advantages. Extensive efforts were devoted towards this research direction, with many novel processes developed7891011et al. developed a method, using remnant structure defects after reduction of graphene oxide, to fabricate water-dispersable graphene−1, this method remains currently the most common method to obtain aqueous graphene dispersions.Water dispersibility is another key performance index for the development of graphene211−1, one order of magnitude higher than published results. The aqueous graphene can be directly spray-coated on flexible or rigid substrates, and the high quality was confirmed by carrier mobility measurements on back-gated field effect transistors (FETs). This process developed features good cost-effectiveness and environmental friendliness at the same time. It may be applicable for fabricating other two-dimensional materials with uncompromised quality and water solubility.The pursuit of high quality and environmental friendliness leads us to a novel strategy which is to mildly oxidize the graphite only at the edges and to realize exfoliation by bubbles generated by the catalytic reaction at the edges. Weak oxidation only at the edges minimizes lattice damage, while the oxidized edge brings in excellent solubility in water without any surfactants or additives. The water solubility reaches 5 mg mL4) facilitates the wetting of graphite in water and the edge opening of layered graphite sheets, while keeping most basal planes of graphite intact; Then, the exfoliation is accomplished by interlayer gas bubbling process of the precursor in the mixture of hydrogen peroxide (H2O2) and ammonia (NH3). The weak oxidation on the graphite edges was realized through controlling mass ratios between oxidant KMnO4 and natural graphite. The typical mass ratio of KMnO4 and natural graphite is 1:1, and the reaction temperature and time is 25 °C and 2 h, respectively. Compared with generally reported Hummers or modified Hummers, as shown in 4 for a graphite flake with 10 μm thickness, and the surface atomic layers will protect the inner layers from further oxidation while the edges have more defects as “weak” points to be attacked, and the space between graphite layers provides possibility for intercalation from the edges.Our approach is clearly illustrated in the schematic diagram of sp2 carbon structure)ID/IG, on the edge of the precursor is 0.8–1.5 while its most basal plane shows very low ID/IG , which proves that most parts of the sample has intact sp2 structure, and confirms the efficient and reliable edge oxidation on graphite. The edge oxidation can also be directly confirmed by SEM on the graphite edges before and after before oxidation. As shown in 4 addition. 4 and natural graphite. With the more KMnO4 addition, the weak oxidized area (ID/IG ~ 0) shrunk, and when the mass ratio of KMnO4 and natural graphite is 10:1, the oxidation became uniform for the whole flake, which further confirms that the oxidation of a graphite flake stars from the edges and goes into the inner parts gradually.The controllable edge oxidation of graphite was verified through Raman spectrum and scanning electron microscopy (SEM) measurements. 3 and H2O2. A lot of bubbles around precursor came into being, and as a result the graphite sheets floated on water although its density is much higher than that of water, as shown in −1 by just controlling the water amount during the exfoliation or re-dispersion. Especially, the aqueous concentration can be as high as 5.0 mg mL−1, which is an order of magnitude higher than previously reports2133 and H2O2 can be removed in the liquid before graphene collection, or by heating the dry products. The freeze-dried water re-dispersed graphene show excellent re-dispersibility in water, N,N-Dimethylformamide (DMF) and N-methyl-2-pyrrolidone (NMP)  , keepingID/IG ratio (0.13) in Raman data on the central region of a graphene basal plane indicates the highly crystallized sp2 structure of the products. Compared with the ID/IG ratio of precursor (0.12), the slight change implies no additional oxygen-containing groups formed in exfoliation progress. Moreover, the symmetrical 2D band (ca. 2700 cm−1) suggests the sample is the complete few layer graphene rather than the multilayered graphite 15ca. 284.2 eV (83.75 at. %), along with an O 1s peak at ca. 532 eV (16.25 at. %), as shown in sp2-hybridised carbon , epoxy/hydroxyls  and carboxyl/carbonyl groups To evaluate the quality of bubbling-exfoliated graphene, the samples were characterized by SEM, transmission electron microscope (TEM), atomic force microscopy (AFM), X-Ray Diffraction (XRD), Raman, XPS and Fourier transform infrared spectroscopy (FT-IR). The SEM image of 1614cursor 0., the sliAccording to the synthesis procedure and measuring results, the bubbling exfoliated graphene should have good quality without the needs of reduction since most part of the basal plane is close to pristine graphene, differentiated from the uniform oxidation of graphite through the typical Hummers method. We further demonstrate the advantages of our preparing strategy through good water solubility, conductive film on polymer, and FET performance. 22232 (10 cm × 5 cm) polyethylene terephthalate (PET) substrate through a commercial liquid sprayer (−1 with a transmittance of 65.9%. SEM image (−1) and close to CVD graphene (500–1500 ohms ◻−1), as summarized in −1 when the transmittance is 65.9%, 42.9%, 32.5%, 29.3% and 5.56%, respectively, which means that the graphene can be easily spray coated onto polymer substrate, and can be used to construct the flexible conductive film. The inset in The water-dispersable graphene can be directly spray coated on several substrates. 1.0 mL of water-dispersable graphene ink can be spray coated on a 50 cm sprayer . After tEM image  shows th2/Si substrates. The inset of DS-VG) of the FETs measured at room temperature under ambient conditions. The typical IDS-VG curve measured at a VDS of 150 mV shows that the gate can cause either hole or electron conduction. The Dirac point of the FETs shifts slightly to a positive gate at VG ~ 25 V, demonstrating light p-type hole doping performance. According to the two slopes of the linear regions on both sides of the V-shaped curve, the hole mobility is μh ~ 496 cm2 V−1 s−1 and the electron mobility is μe ~ 676 cm2 V−1 s−1. As shown in 25262729303132333435363738To determine the transport properties of the synthesized graphene films, back-gated graphene field effect transistors FETs were fabricated on 300 nm SiO2O23+ as an example as shown in It is necessary to discuss the exfoliation mechanism to further understand this efficient approach. According to 4 as oxidant to avoid the Mn impurities. Then, we added graphite oxide, graphite oxide + MnSO4 (0.5 g + 10 mg), graphite oxide + MnO2 (0.5 g + 10 mg), graphite oxide + KMnO4 (0.5 g + 10 mg) into the 80 mL H2O2/NH3 solutions, respectively, as shown in 4 were added, as well as graphite oxide + MnO2 and graphite oxide + KMnO4, large amount of bubbles generated immediately, and the color of the mixture gradually changed to black. These results confirm that the Mn is a key factor for the bubbling exfoliation, no matter what its valence state.In fact, all kinds of Mn salts can catalytically accelerate the bubbling. To verify this, we fabricate graphite oxide through the Staudenmaire method using KClO2O2 requires the alkaline medium41423 also plays an important role in bubbling progress. NH3, not NaOH or KOH, was chosen since it can be easily removed in the obtained products. The exfoliation progressed in different mediums confirmed the importance of NH3 intuitively. As shown in 2O2 is 5, 4, 3, 2, 1, 1, 1, 1 and 0 units, respectively from left to right. At the same time, the volume of NH3 is 0, 1, 1, 1, 1, 2, 3, 4 and 5 units, respectively from left to right (1 unit volume is 10 mL). The drastic bubbling process can be observed only with the present of both H2O2 and NH3. However, no bubble can be observed without the present of NH3, which demonstrated its assisted catalysis effect. Thus, Mn ions, H2O2 and NH3 are the key factors for the bubbling exfoliation. The H2O2 is the most important factor, with the assistance of Mn ions and NH3. The O2 bubbles continually generate in the interlamination of graphite edges, which facilitates continuous and fast exfoliation of the precursor into water-dispersable graphene , the bubbles generated immediately at the edges and HOPG expanded lengthways, as shown in 2O2 with the help of NH3 to in-situ continuously generate bubbles, and that the bubbling exfoliation takes place between all the graphite layers from the edges, not a layer-by-layer model. It is the exfoliation model that helps the fast spontaneous exfoliation of edge-oxidized precursor into high quality graphene in 15 minutes.In order to further demonstrate the bubbling exfoliation mechanism intuitively, highly oriented pyrolytic graphite (HOPG) was selected as the raw material. HOPG was oxidized under the same reaction conditions as natural graphite to obtained edge oxidized HOPG. Before the bubbling exfoliation, the HOPG appearance had no obvious change after the weak oxidation. When it was put into the mixture of H4 and the in-situ continuous bubbling exfoliation with the catalytic H2O2 decomposition. The unique graphene, with edge oxidized and most basal plane intact, has excellent water solubility with extremely high concentration of 5 mg mL−1, good conductivity without the need of reduction, large lateral size of several microns and 1–6 atomic layer in thickness. This approach not only provides a new strategy for graphene synthesis, but also paves the way for large scale application of graphene in aqueous mediums, such as water-based green or functional coatings.For graphite to graphene, we employed a new, simple, mild, mild, fast and scalable spontaneous exfoliation approach. It is based on the weak edge oxidation with the controllable dosage of KMnO2SO4 (98%), H2O2 (30.0 wt. %) and NH3 (25 wt. %) were purchased from Lingfeng (Shanghai China). KMnO4 (99%) was purchased from Jianglaibio (Shanghai China). Highly oriented pyrolytic graphite (HOPG) was purchased from Shanghai NTI Co. Ltd (Shanghai China). The water used throughout all experiments was purified through a Millipore system.Natural graphite powder (45 μm) was purchased from Huatai Lubricant sealing S&T Co.Ltd (Qingdao China). H2SO4 for 30 minutes at 25 °C in 500 mL double-layer beaker with a cooling system based on Julabo F20. 10 g potassium permanganate (KMnO4) was added slowly to the mixture in 30 minutes. After the whole KMnO4 was added, the reaction was kept for 2 hours. Next, 100 mL deionized water was added and stirred continually for 2 hours. Finally, 10 mL (H2O2) was added to the mixture and reacted for 30 minutes. The final liquid was washed with the deionized water by a vacuum filtration and frozen drying in order to obtain the edge-oxidized graphite. The precursor was put into the mixing liquid of NH3:H2O2:H2O (1:1:5) for 30 minutes to finish the bubbling exfoliation. The exfoliated graphene was centrifuged, filtered and frozen dried. The yield was obtained by calculating the mass ratio of exfoliation graphene after frozen dried and the raw natural graphite.For the preparation of weak oxidized graphite, 10 g graphite was mixed and agitated with 200 mL concentrated H2SO4, KMnO4) with same dosages except various graphite additions . The mass ratio of KMnO4 and graphite is 10:1, 2:1 and 1:1, respectively.To control the edge oxidation, we used the same chemicals . Next, potassium permanganate (0.3 g) is added slowly to the mixture during 30 minutes (note: some ice is placed out of the beaker for ensuring the reaction in room temperature). After the whole KMnO4 is added, the reaction is kept for 2 hours and then deionized water (3.0 L) is added and stirred continually for 2 hours. Finally, H2O2 (0.6 mL) also is add to the mixture reacting for 30 minutes. The final liquid is washed with the deionized water by a vacuum filtration and frozen drying in order to obtain the dry product. The edge oxidized HOPG (0.3 g) is put into the mixing liquid of NH3:H2O2:H2O (1:1:5) and frozen drying. The high quality soluble graphene sheet would be obtained.HOPG (0.3 g) is mixed and agitated with concentrated H2SO4 . Next, potassium permanganate (500 g) is added slowly to the mixture during 30 minutes (note: some ice is placed out of the beaker for ensuring the reaction in room temperature). After the whole KMnO4 is added, the reaction is kept for 2 hours and then deionized water (5 L) is added and stirred continually for 2 hours. Finally, H2O2 (500 mL) also is add to the mixture reacting for 30 minutes. The final liquid is wash with the deionized water by a vacuum filtration and frozen drying in order to obtain the dry product. The 500 g edge oxidized graphite is put into the mixed solution of NH3:H2O2:H2O (1:1:5) to exfoliate the water soluble graphene. After 0.5 h, the homogeneous dispersion liquid of water soluble graphene is obtained. The yield is 52.2% .For the preparation of weak graphite oxide, the graphite (G) (500 g) is mixed and agitated with concentrated HAtomic Force Microscopy (AFM) data were obtained in a Bruker Dimension Icon with a Nanoscope 8.15 in tapping mode. Scanning electron microscopy  was used to image the morphology of the samples. Transmission electron microscopy (TEM) measurements were obtained on a Hitachi H-8100 EM  with an accelerating voltage of 200 kV. X-ray photoelectron spectroscopy (XPS) measurements were carried out using a Thermo ESCALAB 250Xi spectrometer. Raman Spectroscopy with the excitation laser line of 532 nm was performed using a Thermo Fisher DXR Raman Microscope. X-ray diffraction (XRD) patterns were obtained from a X-Ray Diffractometer (Bruker D8 ADVANCE) with a monochromatized source of Cu Kα1 radiation (λ = 0.15405 nm) at 1.6 kW . The sheet resistances were measured by a Hall measurement system (Accent HL5500). Ultraviolet–visible spectra were obtained using a Varian Cary 300 Bio UV-visible spectrophotometer. Fourier transform infrared spectra were recorded on a PE Paragon 1000 spectromer (film or KBr disc).2 (10 sccm) at 300 °C for 8 h in a tube furnace. The back-gated GFETs were characterized under ambient conditions using the Agilent (B1500A) semiconductor parameter analyzer. The mobility was extracted using the following equation (S1):L and W are the channel length and width, respectively, Cox is the gate oxide capacitance (11 nF cm−2), VDS is the source drain voltage, IDS is the source drain current, and VG is the gate voltage. The linear regime of the transfer characteristics was used to obtain dIDS/dVG.The graphene films were spray coated to highly doped p-Si substrate with a 300 nm thick thermal oxide, followed by deposition of source and drain electrodes with Au/Ti (50/10 nm) by electron beam evaporation. Afterwards, another standard photolithographic step employing inductively coupled plasma (ICP) was used to pattern the graphene to form a field-effect transistor with a channel length of 8 μm and width of 2 μm. To impove the contact of the back-gated GFETs device, annealing was performed in Ar (500 sccm) and HHow to cite this article: Tian, S. et al. Controllable Edge Oxidation and Bubbling Exfoliation Enable the Fabrication of High Quality Water Dispersible Graphene. Sci. Rep.6, 34127; doi: 10.1038/srep34127 (2016).