Metarhizium anisopliae is a candidate for the integrated management of the disease vector mosquito Aedes aegypti. Metarhizium anisopliae is pathogenic and virulent against Ae. aegypti larvae; however, its half-life is short without employing adjuvants. Here, we investigated the use of neem oil to increase virulence and persistence of the fungus under laboratory and simulated field conditions.The entomopathogenic fungus M. anisopliae and added to recipients. Larvae were then placed in recipients at 5-day intervals for up to 50 days. Survival rates were evaluated 7 days after exposing larvae to each treatment. The effect of neem on conidial germination following exposure to ultraviolet radiation was evaluated under laboratory conditions. Statistical tests were carried out using ANOVA and regression analysis.Neem was mixed with Laboratory bioassays showed that the fungus alone reduced survival to 30% when larvae were exposed to the treatment as soon as the suspension had been prepared (time zero). A mixture of fungus + neem resulted in 11% survival at time zero. The combination of fungus + neem significantly reduced larval survival rates even when suspensions had been maintained for up to 45 days before adding larvae. For simulated-field experiments 1% neem was used, even though this concentration is insecticidal, resulting in 20% survival at time zero. However, this toxic effect was reduced over time. When used alone under simulated-field conditions the fungus rapidly lost virulence. The formulation fungus + neem effectively maintained fungal virulence, with larval survival rates significantly reduced for up to 45 days after preparation of the suspensions. The effective half-life of the fungus or neem when used separately was 6 and 13 days, respectively. The half-life of fungus formulated in 1% neem was 34 days. Conidia suspended in neem maintained high levels of germination even following a 2-h exposure to ultraviolet radiation.M. anisopliae with neem oil effectively increases the half-life and virulence of the fungus when tested against Ae. aegypti larvae, even under simulated field conditions. Neem oil also protected the fungus from the damaging effects of ultraviolet radiation.A combination of the entomopathogenic fungus The online version of this article (10.1186/s13071-019-3415-x) contains supplementary material, which is available to authorized users. Aedes aegypti, namely dengue, Zika, chikungunya and urban yellow fever, are of growing concern, not only in tropical and semi-tropical regions of the world, but also in southern Europe where it is now a threat  was placed in 700 ml black rectangular plant pots  were suspended in Tween 80 (TW 80) (0.05% v/v) and subsequently neem oil was added to a final concentration of 1% and 0.01% (v/v). Fungal suspensions were also prepared in TW 80 without neem. Three milliliters of each sample was placed in plastic Petri dishes (55 mm diameter) and exposed to a UV-B lamp , which at a distance of 70 cm was equivalent to a UV-B intensity of 1630 mW m-2. Conidia were exposed to UV radiation for 0.5, 1, 2, 3, 4 and 5 h periods. Controls for each treatment were not exposed to UV light.These experiments were carried out under laboratory conditions with the aim of investigating the possibility that neem oil protects conidia from the deleterious effects of UV radiation. Conidia  and incubating at 27 °C (12 h light: 12 h dark regime) for 12 h. At this time the number of conidia germinating was quantified using an optical microscope . Three fields of view were counted for each sample by the same researcher (AR). Conidia were considered as germinated when the germ tube was of equal size or larger than the conidia itself. Each treatment was replicated three times.post-hoc test at the 5% level was used.Mean survival rates were compared using one-way analysis of variance. When significant differences were seen between treatments or between time points within the same treatment, the data was further analyzed by Duncan’s post hoc test at the 5% level. The persistence/half-life of the different treatments was evaluated by using polynomial regression analysis, with the effective half-life given as the time (in days) that the treatments resulted in 50% larval survival/mortality. Differences in mean germination rates following exposure to UV were compared using one-way analysis of variance. When significant differences were detected, Duncan’s 9 conidia ml-1) and a combination of neem + fungus, with larvae exposed to the different treatments immediately following preparation (time zero) or following different time periods of up to 50 days. The results for these experiments are shown in Table Ae. aegypti larvae when exposed to the fungus alone was 30% at time zero , whilst for neem alone the larval survival rate was 74% at time zero. However, exposure of larvae to the formulation fungus + neem resulted in 11% survival at time zero. Fungus + neem significantly reduced survival rates when compared to the other two treatments and to the controls  = 1446.2, P < 0.01). The survival rates for larvae exposed to the fungus alone were not significantly different (P > 0.01) to the controls following 10 days under laboratory conditions, showing the low half-life of the fungus when suspended in TW 80. The slight but significant reduction in survival seen at time zero for neem alone was transitory, with no significant reduction in survival by day 5 (81%) when compared to controls (83%).Before testing fungal persistence in the field, bioassays were carried out under laboratory conditions using 0.01% neem (v/v), fungus  = 157.5, P < 0.01). When larvae were exposed to this formulation following 50 days in recipients, the survival rate was 74%, which was not significantly different to the controls (P > 0.01). The half-life of this formulation under laboratory conditions was 42.8 days as calculated by regression analysis .The survival rates were then monitored for the formulation fungus + neem maintained in recipients for different periods of up to 50 days. The mixture of fungus + neem continued to significantly reduce larval survival rates when maintained in recipients for up to 45 days, with 58% survival observed at this time (P > 0.01) following 10 days of maintaining the fungus or neem oil under field conditions  = 236.9, P < 0.01), but larvicidal activity was rapidly lost. Therefore, the next set of experiments was carried out using 0.1% (v/v) neem.These tests were similar to those described above but with recipients maintained under field conditions. Two preliminary experiments were carried out but both were stopped after 10 days. The first experiment was set up using the same concentration of neem as that used in laboratory experiments, 0.01% (v/v). However, none of the treatments were significantly different to the controls  = 441.0, P < 0.01), the experiment was stopped due to the rapid decline in larvicidal activity.However, a similar pattern was seen for the experiments using 0.1% neem, with larvicidal activity rapidly reduced after 10 days under field conditions  = 478.4, P < 0.01). At time zero, the fungus alone significantly reduced larval survival  when compared to the controls, as did neem alone , with no significant difference between these two treatments  neem  = 556.6, P < 0.01). Fifty days after maintaining this formulation under simulated field conditions, and then adding larvae to the recipients, fungus + neem was no longer effective at reducing survival rates and there was no significant difference between the treatments.The persistence of the formulations was evaluated for up to 50 days under simulated field conditions. When comparing survival rates of all treatments with controls over time, the formulation fungus + neem maintained significant larvicidal activity until day 45 , at which time the survival rates were statistically different to the other treatments and the controls (P > 0.01). Neem alone caused significant reductions in survival (72%) until day 25 when compared to the controls.The fungus when tested alone under field conditions rapidly lost virulence, with no difference to the controls at 25 days, when survival rates were 83%  = 46.07, P < 0.01), with 80.3% (± 1.69%) germination observed. However, 3 to 5 h exposures caused significant reductions in germination when compared to shorter exposure times. Neem 0.01% (v/v) was a less effective protectant, with rapid and significant reductions in conidial germination when exposing conidia to UV-B for 1 h or more  = 9270.0, P < 0.01). All treatments showed low levels of germination following a 5-h exposure to UV. The mean germination rates of all three control treatments were not significantly different (P > 0.01) and approximately 80% of the conidia had germinated when quantified 12 h after inoculating the suspensions in culture media. The complete data and statistical analysis for the germination experiments are shown in Additional file The results for the three treatments investigated here showed the significant protective characteristics of neem when considering the deleterious effects of UV radiation on conidial germination Fig. . When coF5,7 = 46.07Bacillus thuringiensis serotype israelensis (Bti). When Bti is applied in the field in recipients exposed to sunlight, control is efficient for up to four weeks. However, when used in recipients not directly exposed to sunlight, the product is effective for up to 11 weeks [Current conventional mosquito larval control measures in domestic environments involves local government agents visiting residences and treating putative breeding sites, such as drains and water tanks with synthetic chemical larvicides or products containing 11 weeks .Metarhizium conidia in mineral oil in a product known as Green Muscle®, used for the control of the desert locust [One of the most serious drawbacks of deploying entomopathogenic fungi for pest and vector control is their short effective half-life under field conditions. With the aim of increasing the persistence of these fungi, many studies have been carried out on different formulation technologies . Amongstt locust . In thist locust .M. anisopliae against Ae. aegypti larvae [In our previous study of neem oil as an adjuvant for use with entomopathogenic fungi, very low concentrations of neem oil significantly increased the virulence of i larvae , probablAe. aegypti larvae were exposed to neem oil, their development was affected, resulting in a significant reduction in adult emergence [An. stephensi and An. gambiae were reduced following exposure to neem. Pupal formation and adult emergence were significantly reduced in the case of An. stephensi and An. gambiae larvae exposed to neem [Neem oil has insecticidal properties and is currently used for pest control in organic farming systems . Howevermergence , 25. Fee to neem , 27.Ae. aegypti larvae. Neem when used at 0.01% (v/v) together with M. anisopliae enabled the fungus to significantly reduce larval survival rates for up to 45 days after the suspension had been prepared and maintained on the bench. The fungus when used alone rapidly lost virulence, with survival rates equal to that of the controls after only 10 days. The decline in virulence of M. anisopliae seen here was greater than that seen previously, when the half-life of the same fungus was estimated to be 10 days under similar conditions [In the first set of experiments here, under laboratory conditions, the concentration of neem that increased fungal persistence was not considered to be toxic to nditions .Ae. aegypti oviposition sites. The same type of plant pot is normally used as a so called “ovitrap” to collect eggs in the field. Although in the present experiments, mosquito larvae were added to the pots to test the persistence of the formulations, future experiments will be carried out using ovitraps with fungus + neem in the field as a possible control strategy against the egg stage and any larvae emerging from the eggs. However, we have yet to test if this formulation could be detected by females and act as a repellant to oviposition. Studies have shown that Ae. aegypti females are not repelled by the presence of M. anisopliae conidia on black cloths [An. gambiae, which oviposit higher numbers of eggs in the presence of bacteria than in sterile water [The persistence of the fungus when formulated with neem oil was tested under field conditions. The present field experiments were performed using black plant pots, which are highly suitable as k cloths ; howeverle water .Ae. aegypti larvae. Neem at 1% (v/v) has been previously shown to cause a 50% reduction in survival rates within two days [An. stephensi larvae [Ae. aegypti larvae when used alone, the advantage of formulating M. anisopliae with this concentration of neem was a significant increase in the fungal half-life in the field. The half-life of the fungus alone was only six days, which would seriously impair its use under field conditions. The larvicidal activity of neem also fell rapidly under these conditions, with an estimated half-life of 13 days seen here. This was probably due to bio-degradation.The initial results for field tests , exposure of conidia to UV rapidly reduced viability. However, conidial suspensions in 1% neem oil protected the conidia for up to two hours exposure to UV, with no significant reductions in germination when compared to controls. For sesame or colza oil, the protective capacity was approximately 70% following a five-hour exposure to UV-B following field trails .M. anisopliae. This work shows for the first time an important strategy for maintaining fungal virulence over extended time periods. One of the major drawbacks of using entomopathogenic fungi is their short half-life. However, the addition of neem to conidial suspensions not only increased virulence, but significantly increased the persistence of the fungus.Neem was tested in the laboratory and under field conditions as an adjuvant for the entomopathogenic fungus Additional file 1: Figure S1. Black rectangular plant pot used for field testing the virulence and persistence of the fungal and neem oil treatments against Ae. aegypti larvae. Figure S2. Field testing the persistence of the conidia against Ae. aegypti larvae. Experiments on a covered veranda were carried out within netting cages to prevent mosquitoes using the plant pots as oviposition sites. Table S1. Survival rates of Ae. aegypti larvae following exposure to different treatments under simulated field conditions. Table S2. Survival rates of Ae. aegypti larvae following exposure to different treatments under simulated field conditions. Table S3. Germination rates (mean % ± SD) of Metarhizium anisopliae conidia following exposure to UV-B when formulated in two concentration of neem oil or Tween.


Bacillus cereus food poisoning is the three-component enterotoxin hemolysin BL. It consists of the binding component B and the two lytic components L1 and L2. Studying its mode of action has been challenging, as natural culture supernatants additionally contain Nhe, the second three-component enterotoxin, and purification of recombinant (r) Hbl components has been difficult. In this study, we report on pore-forming, cytotoxic, cell binding and hemolytic activity of recently generated rHbl components expressed in E. coli. It is known that all three Hbl components are necessary for cytotoxicity and pore formation. Here we show that an excess of rHbl B enhances, while an excess of rHbl L1 hinders, the velocity of pore formation. Most rapid pore formation was observed with ratios L2:L1:B = 1:1:10 and 10:1:10. It was further verified that Hbl activity is due to sequential binding of the components B - L1 - L2. Accordingly, all bioassays proved that binding of Hbl B to the cell surface is the crucial step for pore formation and cytotoxic activity. Binding of Hbl B took place within minutes, while apposition of the following L1 and L2 occurred immediately. Further on, applying toxin components simultaneously, it seemed that Hbl L1 enhanced binding of B to the target cell surface. Overall, these data contribute significantly to the elucidation of the mode of action of Hbl, and suggest that its mechanism of pore formation differs substantially from that of Nhe, although both enterotoxin complexes are sequentially highly related.A major virulence factor involved in  Bacillus cereus is considered an emerging pathogen relevant for food poisoning worldwide [Bacillus cereus group [B. cereus strains, the hbl genes in about 45–65%. Strains bearing only hbl have not yet been found [orldwide ,2,3. Whiorldwide . The entorldwide ) and Hblorldwide ) are comus group ,7. The gen found ,9,10. hbl genes are organized in the operon hblCDA, encoding the components Hbl L2, L1 and B, respectively [hbl operon is influenced by a variety of regulatory proteins, such as the major virulence regulator PlcR [2, L1 and B were first isolated from strain F837/76, they had a molecular weight of 45, 36 and 35 kDa, respectively [B. cereus strains produce Hbl components of various sizes, and that in one strain more than one variant of each component can exist [2, L1 and B were found in one B. cereus strain, leading to the assumption that two distinct homologs of the hbl operon exist [hblB, which is located downstream of hblCDA [hblB was considered a pseudogene, as no transcription could be detected [B. cereus strain ATCC 14579 [hbl operon, named ahbl, contains only hblCDA [The ectively . Expresstor PlcR ,13, CcpAtor PlcR , ResD [1tor PlcR  and Fnr tor PlcR ,17. Whenectively . Later ian exist . Two dison exist . The morf hblCDA . Since tdetected ,21. OnlyCC 14579 . The secy hblCDA .B. cereus diarrheal enterotoxin [1 - L2 leads to cytotoxic activity [E. coli [2 and L1, which are also necessary to induce toxicity, were suggested to either stabilize Hbl B, induce conformational changes to B, or even enter the cell [The mode of action of Hbl is still not clear. The toxin is hemolytic, cytotoxic, dermonecrotic and active in vascular permeability tests ,18. Furterotoxin . A recenerotoxin . Pore foerotoxin . The samactivity . Solving[E. coli ,27,28. Tthe cell .1 or B was shown to inhibit hemolytic activity [B. cereus supernatant via anion exchange chromatography. With this approach, the purity of the single components could not be completely guaranteed. To exclude trace contaminations as well as biased results, the use of recombinant Hbl components was recommended [E. coli, which is highly advantageous, regarding more detailed studies on the mode of action of Hbl. Moreover, for the first time we demonstrated complex formation between Hbl L1 and B, as well as L1 and L2 in solution, prior to cell binding [Another characteristic feature of Hbl is the discontinuous hemolysis zone phenomenon on blood agar ,18,24. Wactivity ,24. All ommended ,18,29. J binding . In the 2; and vascular permeability tests showed minor edema with only Hbl B and L1 [It is controversially discussed in literature whether or not two Hbl components alone might be able to exhibit toxic activity, or form a pore, as has been shown for the structurally-related Nhe components B and C . In earlB and L1 . Other sB and L1 . 1 and B, L2 and B, or L2 and L1 were applied to the cells, neither toxic activity nor pore formation could be observed. We gained the same results when PI influx tests were performed under consecutive conditions, i.e., for 1 h each  to Vero cells. Increasing fluorescence was measured representing influx of propidium iodide . Superna2:L1:B required for maximum activity, PI influx tests on Vero cells were carried out. The three components were pre-mixed in the ratio 1:1:1  compared to the dilution series of pre-mixed L2, L1 and B , which is best suited for detecting Hbl B on cell surfaces, was replaced, as it cross-reacts with L1 [1  was used, together with different combinations of the rHbl components. Fluorescence was barely detected when Vero cells were incubated with only mAb 1H9 (negative control), rHbl L2+1H9, rHbl B and L2+1H9 or rHbl L1 and L2+1H9 . Shorter incubation times were first tested for the binding component B, with rHbl L1 being constantly applied for 10 min. Full PI influx was observed for 10 min incubation. To determine the time necessary for each Hbl component to bind to the target cells, further PI influx tests were performed. The rHbl components were successively applied to the cells in concentrations of 37.5 pmol/mL each. Application order was B - L1 were applied with decreasing incubation times . Interestingly, moving the tag on rHbl B from the N- to the C-terminus resulted in a significant delay of pore formation. That pore formation is decelerated became even more obvious when the Hbl components were applied consecutively with decreasing incubation times for rHbl B. While for rHbl B with an N-terminal tag (free C-terminus), incubation times as low as 4–5 min were sufficient for cell binding  and a “rapid lytic reaction by L1+L2” (within 3 min) have been observed [1 and L2 can easily be diluted by a factor of 10–20 without affecting the toxic activity.Furthermore, we showed that pore formation of Hbl occurs within minutes. Binding of Hbl Lobserved . Our datE. coli ClyA [2 and L1 induce conformational changes, stabilize B or even enter the cell similar to anthrax toxin [In our study, only Hbl B was able to bind to the target cell surface. Thus, we found no indication for a “membrane attack complex” leading to cell lysis . Furtheroli ClyA ,28, Hbl ax toxin .1 - L2. Nevertheless, rHbl B binding seemed to be enhanced in the presence of rHbl L1 D(L1)A(B)—and structurally related [B. cereus. With K 10-10 M ,33. Alth related ,34, ther1 and B) has been reported [1 complexes show less stability [1 seems to enhance the cell binding of B (this study). Furthermore, large amounts of NheC inhibit toxicity [For Nhe, formation of small “pro-pores” by NheB and C  was used in this study. To obtain toxin-rich culture supernatant, the strain was grown in CGY medium with 1% glucose for 6 h at 32 °C with shaking. According to [E. coli strain BL21 (DE3) was used for overexpression of recombinant (r) Hbl proteins.The rding to , the samE. coli strain BL21 (DE3) and purified as described earlier, resulting in rHbl B and L2 with N-terminal strep-tag as well as rHbl L1 with C-terminal strep-tag [hblC (L2), hblD (L1) and hblA (B) were amplified by PCR using pASK-IBA5+hblC, pASK-IBA3+hblD and pASK-IBA5+hblA as templates, as well as the primer pairs HblL2-fw-SacII (ATA TCC GCG GAT GCA AGC AGA AAC TCA ACA AGA AAA C) and HblL2-rev-NcoI-GC (ATA TCC ATG GGC AAA TTT ATA CAC TTG TTC TTC AAG G), HblL1-fw-KpnI (ATA TGG TAC CCG CAC AAG AAA CGA CCG CTC A) and HblL1-rev-NcoI (ATA TCC ATG GCT ACT CCT GTT TAA AAG CAA TAT CTT), and HblB-fw-SacII (ATA TCC GCG GAT GGC AAG TGA AAT TGA ACA AAC G) and HblB-rev-NcoI-GC (ATA TCC ATG GGC TTT TTG TGG AGT AAC AGT TTC TAC). The genes lacking the sequences for the N-terminal signal peptides for secretion [Hbl proteins were overexpressed in the trep-tag . In thisecretion ,42 were 2 with C-terminal strep-tag could not be purified via affinity chromatography, its concentration was estimated via enzyme immuno assay (see [E. coli cell extract was used. The cell extract applied solely did not lead to any pore formation or toxic activity (data not shown).As rHbl Lsay see ,44) and  and 2 wi2 culture flasks in a humidified incubator at 37 °C and 7% CO2 in MEM Earle’s medium  plus supplements, as recommended by the supplier.Vero cells were obtained from ECACC (European Collection of Cell Cultures). They were cultured in 80 cmB. cereus culture supernatant or rHbl proteins were placed into 96 well plates (0.1 mL per well) and subsequently, Vero cell suspensions (1 × 104 cells and 0.1 mL per well) were added. After 24 h incubation at 37 °C and 7% CO2, cell viability was determined by the addition of WST-1 . Optical density was recorded in a Tecan photometer at 450 nm using Ridawin software. Dose-response curves were calculated to obtain 50% lethal concentrations, which are shown as reciprocal titers. Cytotoxicity assays were performed as previously described ,43,45. B4 Vero cells were seeded in 200 µL MEM Earle’s medium /well in black 96 well plates and incubated for 24 h at 37 °C and 7% CO2. 100 µL medium were removed and 100 µL fresh medium were added containing 10 µg/mL PI  and B. cereus culture supernatant (1:20) or rHbl components in appropriate dilutions. Fluorescence was measured immediately in a Victor 1420 multilabel counter  for 4 h every 2.5 min . Fluorescence curves of three replicates were calculated using Microsoft Excel. The start point of PI incorporation and the slope of the fluorescence curves allow concluding on the efficiency and velocity of pore formation  influx tests were used. 4 × 10see also ,40), whiStock solutions of the rHbl components were pre-mixed in appropriate ratios and added as serial dilutions  or in concentrations of 37.5 pmol/mL each (PI) to the cells.6 cells in 500 µL EC buffer . rHbl B ± rHbl L1 or L2 were added in EC buffer in concentrations of 9.38 pmol/mL each to a total volume of 1 mL. After 1 h incubation at 37 °C with moderate agitation, 2 mL 1% BSA-PBS were added and cells were centrifuged for 5 min at 800 rpm. Cells were washed again in 2 mL 1% BSA-PBS. Samples were incubated with 5 µg/mL mAb 1G8 or 1B8 [® 488 goat anti mouse IgG  for 45 min at 4 °C. Alternatively, mAb 1G8 directly labeled with Alexa Fluor® 488 was used for detection. After two washing steps with 2 mL 1% BSA-PSB, cells were resuspended in 500 µL 1% BSA-PBS, and transferred to flow cytometry tubes. For the detection of Hbl L2, Vero cells were first incubated with rHbl B, L1 or B+L1 (4.17 pmol/mL each) and in a second step with a mixture of rHbl L2 (4.17 pmol/mL) and the neutralizing mAb 1H9 (5 µg/mL) [For flow cytometry analyses, Vero cells were counted and adjusted to 1 × 108 or 1B8 ,43 in 1 5 µg/mL) . Fluores2, L1 and B (1.5 pmol/µL each) were filled individually or as mixture in different ratios into 3.5 mm diameter stamp holes on sheep blood agar plates. After 3–6 h incubation at 32 °C hemolysis patterns were assessed and documented.10 µL of toxin containing F837/76 supernatant or rHbl L


Digital spaces, and in particular social networking sites, are becoming increasingly present and influential in the functioning of our democracies. In this paper, we propose an integrated methodology for the data collection, the reconstruction, the analysis and the visualization of the development of a country’s political landscape from Twitter data.The proposed method relies solely on the interactions between Twitter accounts and is independent of the characteristics of the shared contents such as the language of the tweets. We validate our methodology on a case study on the 2017 French presidential election (60 million Twitter exchanges between more than 2.4 million users) via two independent methods: the comparison between our automated political categorization and a human categorization based on the evaluation of a sample of 5000 profiles descriptions; the correspondence between the reconfigurations detected in the reconstructed political landscape and key political events reported in the media. This latter validation demonstrated the ability of our approach to accurately reflect the reconfigurations at play in the off-line political scene.echo chamber effect on the different types of political news  caused by the community structure and emphasize the importance of addressing the meso-structures of political networks in understanding the fake news phenomena.We built on this reconstruction to give insights into the opinion dynamics and the reconfigurations of political communities at play during a presidential election. First, we propose a quantitative description and analysis of the political engagement of members of political communities. Second, we analyze the impact of political communities on information diffusion and in particular on their role in the fake news phenomena. We measure a differential Politoscope”, a macroscope that delivers some of our results in an interactive way.Giving access to an intermediate level, between sociological surveys in the field and large statistical studies  we demonstrate that social networks data make it possible to qualify and quantify the activity of political communities in a multi-polar political environment; as well as their temporal evolution and reconfiguration, their structure, their alliance strategies and their semantic particularities during a presidential campaign through the analysis of their digital traces. We conclude this paper with a comment on the political and ethical implications of the use of social networks data in politics. We stress the importance of developing social macroscopes that will enable citizens to better understand how they collectively make society and propose as example the “ Digital spaces are becoming increasingly present and influential in the functioning of our democracies, to the point where events transpiring in these spaces can no longer be ignored when it comes to analyzing trends in the political environment. In the wake of each of the major political upheavals that have shaken western democracies in recent months , commentators have turned to the social networking sites in the search for a better understanding of these events.In the space of a few years, the use of social networking sites has become fully integrated into the strategies of political parties. Barack Obama was qualified as a “game changer” in 2012, when he became the first presidential candidate to make massive use of social networking sites for his campaign. The 2016 presidential campaign in the USA confirmed this trend, with an estimated total budget of around 500M € devoted to the use of social networking sites  Social groups in the political twittosphere: dense networks of accounts that recurrently relay content, without modification (retweet), between each other.GT =  in which each node, u ∈ V, is a Twitter account and each  ∈ E is a weighted and undirected link between nodes u and nodes v, w being proportional to the number of retweets having occurred between accounts u and v during a period of time T.In order to define user groups based on retweets, we selected the retweets from our dataset GT, we adopt an approach similar to those proposed by  Link w-strong: two accounts A and B are related by a link w-strong over the T period if they have retweeted each other at least w times, in either direction.[Definition] w-communities over period T: communities detected with the Louvain algorithm on the retweet graphs, on which only those links at least as strong as w-strong have been retained.T according to this definition are noted The political communities found over a period In the following, we have taken labels from the list of presidential candidates, and added a small number of political figures who, although they were not candidates, played an important role in this campaign . The resolution used with the Louvain algorithm was its default value.the sea”.A significant portion of accounts do not belong to any community with this approach. They correspond to accounts that, although dealing with political issues, do not fit into the informational sphere of a group of political activists. We will refer to this set of account as “It is possible to study the political environment coarsely or finely, by selecting a shorter or longer list of representative accounts, and by adjusting the resolution of the Louvain algorithm in order to identify distinct communities in the neighbourhood of these accounts. We observed that the different political currents within a party could be identified in this way. Such an analysis lies however beyond the scope of the present study and we choose to keep the default Louvain algorithm settings that made it possible to identify the major political parties and currents accurately.Socio-semantic networks evolve together: the actors are influenced by the content they are exposed to, which leads them to develop their local network, and thus to modify the nature of the content they will be exposed to in the future. As a result, it is common for individuals to change their sphere of political influence during a presidential campaign, or to be influenced by new political figures, which leads to a modification of the Twitter patterns.Ti.In order to take this phenomenon into account, we consider sets of graphs The choice of As a consequence of variations in political opinion, resulting from the overlapping of several dynamic processes with highly different time constants , it may be relevant to analyze the data using several different time scales.For example, the analysis of the 5–communities over the 5 months period August 1st to December 31 2016 cf.  depicts For the present study, we chose a time scale matching timing of Twitter and the media in the context of a political campaign. By analyzing the temporal distribution of retweets within our corpus, we observed that both current political events and Twitter activity have a weekly periodicity, and that on average the retweet period of an original tweet is of the order of one week, with 98.13% of retweets being made within a period of seven days . In the following analyses, we consider the communities computed every day over a 14 days period to assign a community to each tweet of that day. For the analysis of the dynamics of the communities, we took the subset of the communities computed every Monday, with k ∈ , |Tk| = 14 days, |Tk−1 ∩ Tk| = |Tk ∩ Tk+1| = 7 days, when the intervals are arranged according to their starting date.We thus considered seven-days intervals to be a characteristic period for political activity on Twitter, and used two-week intervals Each tweet in our dataset is thus taken into account in exactly two retweet graphs for the analysis of community reconfigurations. As shown below, this choice allows transformations within political communities and their semantic space to be followed in a reasonably continuous manner.w in the community definition, we choose to retain on average the strongest 10% retweet relationships over T for the analysis, which led us to conduct our analysis on the 3–communities. The change in the number of links and accounts of these 3–communities over time is shown in To set the value of Not all candidates have the same communication strategy and some use relatively little Twitter. We respect this choice and we have decided to present detailed statistics only for the communities which leaders have a significant presence on Twitter.Computing the number of tweets of each candidates on March 31, 2017, we observed a gap between the six most active candidates that tweeted more than 500 times and the others. Consequently, we retain these six communities for detailed investigations: Asselineau, Fillon, Hamon, Le Pen, Macron and Mélenchon. Among them, Asselineau was ranked in the “small” candidates (0.92% of the votes at the first round). The other five represent the main political trends in France: very left (Melenchon), left (Hamon), center liberal (Macron), right (Fillon), extreme right (Le Pen).All 5 other candidates received less than 1.5% of the votes at the first round except for Dupont-Aignan who received 4.7%. The total of their weight at the first round was 7.82% of the votes.On Twitter, markers of social influence related to forms of expression or opinions extend far beyond the framework of retweets. Individuals can for example influence one another in their use of expressions or hashtags, without necessarily having to retweet. Furthermore, the act of retweeting can be interpreted in several different ways, with for example a negative interpretation being common when the retweet is delayed .For the analysis of candidates’ programs we first contacted, on 1 March 2017, all the campaign teams that were about to achieve to collect the 500 signatures from French mayors required to be able to become an official candidate . We invited them to provide us with their program elements and key expressions that they would consider characteristic of their program. Unsurprisingly, we had almost no response, only the team of a “small” candidate indicated to us that given the size of the campaign team, they were not sure they could participate. We used then the programs of the eleven candidates downloaded from their websites  from which we manually extracted 980 policy measures , that implements advanced NLP methods and co-word topic detection; as well as visual analytics methods for the representation and interaction with the results.Consequently, we analyzed these two corpora using the CNRS text-mining software Gargantext uses a combination of lemmatization, post-tagging and statistical analysis like tf-idf , split [S], emergence [E], fade-out [F] or abrupt size change [C] of some of the communities and associated to press coverages . Among others, the following events can easily be identified as merge 2016/11/27-2016/12/01 [[M + F] the reconfiguration of the right after the primaries with the alliance after negotiations between Fillon and Sarkozy and the fade-out of Juppé’s community,16/12/01 : [M + F]2016/12/01 [[E] the leverage effect of Holland’s renunciation on the Macron’s campaign,16/12/01 : [E] the2017/01/17 [[S] Frictions among les Républicains after disputed legislative investitures, including that of Nathalie Kosciusko-Morizet (NKM), to whom Fillon offered an easy-to-win district,17/01/17 : [S] Fri2017/01/25 [[M + S + E] Start of the Penelope Gate, some fillionnists joint the Sarkozy community, the Alain Juppé community reappears,17/01/25 : [M + S 2017/01/29 [[C + M + S] Second round of the left-wing primary won by Hamon,17/01/29 : [C + M 2017/02/23 [[M] Bayrou rallies to Macron,17/02/23 : [M] Bay2017/02/26 [[M] Jadot rallies to Hamon,17/02/26 : [M] Jad2017/03/05-06 [[S + M + C] François Fillon organizes a rally in Trocadéro (Paris). He announces 200 000 attendees (including leading supports of Sarkozy) and presented this as a support to the upholding of his candidacy, sarkozists support Juppé come back but this latter finally publicly renounces all prospect of presidential candidacy. Confirmation of the legitimacy of Fillon by the political bureau of les Républicains, which was originally convened to ask that he be dismissed  Dupont Aignan claims that “many sarkozists are joining my movement”,17/03/23 : [M] Dup2017/04/23  Dupont-Aignan rallies to Le Pen,17/04/28 : [M] DupWe analyze these key events in more detail in Figs The reconstruction method we propose reveals the deep reconfigurations of a political landscape during a presidential campaign in multi-polar political environment like the French one. By describing the behavior of a set of activists, corresponding to a wide spectrum of political affiliations, it is positioned at an intermediate level between political leaders and their electoral base.It provides access to a level of observation that is rarely accessible in the framework of sociological studies or in political sciences. In total, between Aug 1st 2016 and May 8 2017, nearly 190,000 individual accounts have been associated to a political community, with most communities involving several tens of thousands of activists . The cumulative distribution of days spent within a community is given by Fig F in The Overall during commitment of an account a toward a candidate c be the percentage of a’s community tweets that belong to a within a within any community during the same period. This index indicates the extent to which a contributes to the circulation of ideas in the community of c when a is involved in interactions with political groups. The average commitment of a community commitments of accounts participating to Let the commitments of the accounts , we generalized these indicators at the community level by weighting them by the activity of an account a over i.e. number of tweets captured by the Politoscope). The activity of a community activity of accounts participating to the politicization of an account a over a set of periods i.e. number of tweets captured by the Politoscope during Politoscope, we estimate for every account the total number of tweets emitted during Politoscope, leading politicization to be more than 1 for some users. Consequently, we set the maximum of politicization of an account to 1. As these two biases should apply equally to all communities, we consider this estimate as a good indicator for comparing the degree of politicization of communities. The politicization of politicization of accounts participating to the political integration of an account a over a as author. An account that tweets mostly in interaction with a community will have a high political integration index. On the contrary, an account that mostly tweets political content not in relation with any structured group on Twitter (i.e. this account if often categorized as belonging to the “sea”) will have a low political integration index. The average integration of political integration of accounts participating to the These indicators makes it possible to characterize different types of political engagement that could be specific to some communities. In turn, the identification of some particularities at the level of the communities could point toward specificities in the mode of recruitment of new members.The Asselineau community is clearly an outlier in this set of political groups with by far the smallest community size, lowest commitment level and a politicization and an integration among the lowest.et al. [Excluding this atypical community and ranking the other five along a left-right axis, we observe a U shape for the commitment which indicates that people with extreme political views tend to be the most committed to their ideology. This is to be compared with the observation of Preotçiuc-Pietro et al.  in the ULe Pen and Fillon community members are most active on Twitter with an activity of 200.33 (resp. 275.52). They are also the most long-lasting ones inside communities (resp. 17.18 and 18.49 days) and the ones with the highest integration (resp. 63.40 and 60.72).While the average number of days spent within a community is directly linked to the activity level, high integration means that more than for other communities, when members of Fillon and Le Pen communities express their political views on Twitter, it is to rally some particular political group. These two communities are however distinct with respect to the commitment of their members, Le Pen community members having the highest commitment score of all communities . Combined together, high commitment and high integration of Le Pen community members means that they are the most inflexible of all political activists in their opinions and the most committed to a leader. Given the fact that Le Pen community is also one of the most active on Twitter, it is the one with the most stable community core. This probably explains why right-wing leaders are unable to attract far-right voters despite their stated will to do so; and why, as reported in , insteadk and 22.1k respectively) but also the ones with the smallest integration and politicization and quite low averages of days spent inside the community. This reflects the highly dynamics character of these both candidacies which final scores were unanticipated at the beginning of the campaign and that got momentum when getting closer to the end of the campaign. This also suggests that they were successful in recruiting a low politicization audience.The Macron and Melenchon communities are by far the ones with the largest numbers of accounts : at some point (around March), many people from the Hamon community joined either the Melenchon or the Macron communities when the collective belief ended on the idea that Hamon would not qualify for the second round. And indeed, he ended up far behind all the other candidates.Hamon community is the one with the highest politicization (6.16%) with moderate levels of commitment and integration which tends to indicate that it was a community with a strong political background that was more focused on ideas than on the support to a particular leader. This is in line with the analysis of  that demactivity, politicization, political integration and commitment allows to discriminate different types of political groups. As a perspective, we conjecture that the analysis of their evolution through time would be a good predictor of the destinies of communities.The decomposition of the online engagement of political communities members into Social systems are among the most sophisticated complex systems in terms of entanglement of levels and multi-scale dynamics. We should consequently expect their multi-level structure to be a key factor in the diffusion phenomena, social networks being a privileged place to observe their role.In this section, we look at how communities act on the dissemination and circulation of political information, defending certain themes or highlighting certain topics with a specific framing. Particular attention is paid to the phenomenon of fake news which can be studied here in all its community dimension.echo chamber effects [Given our reconstruction method, a first intuitive results is that political communities generate strong  effects : the anaecho chambers effect observation, we have selected 11 of the main topics of discussion for this election , makes it possible to explore, for each of the main candidates and each of the eleven themes, the changes in the 15 most significant keywords for each of the respective candidates. One can also observe, in addition to the semantic specificity of the communities with respect to these themes, a variation as a function of time for the different candidates, with the usage of certain keywords appearing, strengthening or disappearing over time , that has collected, throughout the year 2017, URLs of web pages and Facebook posts that include fake news [e.g. “Jean-Luc Mélenchon has a Rolex” or “More than 30% of Emmanuel Macron’s campaign was financed by Saudi Arabia”) have been grouped together in stories and associated to a debunk, a blog post in which Le Monde journalists unveil and explain why this news is a fake. There are 179 stories and 179 debunks in total (see Files A6 and A7 at DOI: 10.7910/DVN/AOGUIA for a json version).The question of how comparatively true and false information spreads on a social networking site like Twitter and which contexts influence this spread is an important issue for the “science of fake news” that should help to solve the post-true crisis , 71. To ake news . URLs reDecodex database is a good entry point to identify the stakes of the fake news phenomenon through the lens of our political landscape reconstruction.The Decodex URLs; and having double checked that the tweets mentioning a link to a fake news or a debunk have really for purpose to broadcast them  to the macro-level (fake news reach less communities than other news) is very important when addressing this issue from a societal perspective.Fake news might reach on average more people than other political news but they spread more locally. This The number of shares is not the only relevant variable. The analysis of fake news and debunks circulating inside the five major political communities  reveals some clear patterns in terms of localization and temporal distribution.i.e. from what we have called “the sea” (we remind that communities are processed each day and the community of a tweet is the community attributed to the account issuing the tweet at the time of the tweet issuance). Since globally 43.44% (25 695 888) of tweets have no community, this means that the accounts that are the least politicized or not involved into some group of activists share on average much less fake news than the most politically integrated accounts.18.9% (922) of the shares of fake news correspond to tweets with no community affiliation, There is also a significant heterogeneity in the practice of fake news sharing within communities. The communities of Fillon (right-wing) and Le Pen (extreme-right wing) are the most active with respectively 50.75% and 22.21% of the total number of fake links shares (89.9% of fake news shared within communities); while the community of Hamon is the least active with only 0.43% of the total shares cf. .early spreaders per communities defined as the users that rank in the 1/30 percentile . The estimation of this social status is out of the scope of the present paper but these accounts have sufficiently close interactions with political communities to be associated with them through our community detection method.The analysis of the tweets containing debunk links offer a radically different pattern cf. . While 3To compare the fake news and debunks sharing propensities, This preliminary study depicts a “split-brain” political landscape from the perspective of fake news: one “hemisphere” produces or spreads fake news, the other produces or spreads debunks. This dissociation between the audience that reads fake news and the one that reads their debunks must be taken into account in mitigation strategies for the fake news phenomenon.backfire effect [continued influence effect [e.g. believers in conspiracy theories). Thus exposing some communities that were previously not aware of the existence of a fake news could start a new outbreak of fake news in areas of social media that were spared until then.Fighting fake news is a very tricky task with at least two counter-intuitive effects: the e effect —repeatine effect —despite Decodex failed to identify a significant proportion of fake news, this is nevertheless a quite low proportion. This estimate is also in line with Vosoughi et al. (2018) who identified “only” 4.5M shares of stories over 10 years of Twitter records, to be compared to an estimate of 500M tweets sent per day in 2017.Unlike vaccination for which people have to be treated preventively, in the domain of fake news, people have to be debunked after exposure. In that sens, the fact that fakes news tend to spread in less communities than other news is a good news: counter measures can target only those particular communities or the media they rely on without interfering with a broader audience. The second good news, when we look closely at the figures, is that at least on Twitter, political fake news do not seems to be that much popular. Our study identified that in France, only 0.08% of political tweets contained fake news. Although it is likely that de We have demonstrated that methods involving the analysis of exchanges of political content on social networking sites such as Twitter allow automatic reconstruction of political communities which, by their semantic profile and patterns of development, provide deep insights into the dynamics at play inside the space of political activists.By applying these methods to data from the 2017 French presidential election, we reconstructed with a high accuracy the multi-polar French political space, retrieved the main events which occurred during the campaign, and revealed the multiple reconfigurations of political communities, some of which took place under relatively confidential circumstances. By using interaction data only, our approach has the advantage of being independent from the language used during these exchanges, provided the studied social activity involves a certain level of commitment from the participants, as it is the case in politics.After checking the accuracy of this reconstruction, we demonstrated how this approach can help to better characterize online political communities, both from the perspective of their structure and from point of view of the kind of information they propagate, fake news included:Political communities can be reconstructed from Twitter data:The usage of Twitter by political communities makes it possible to reconstruct the evolution of the political landscape of a country during presidential campaigns through correspondence between the different political currents and communities reconstructed algorithmically from their digital traces,Official and non official key political events correspond to identifiable mathematical structures in the reconstructed political landscape like split, merge, fade-out, fade-in or abrupt change of sizes of communities,This reconstruction gives insights into the structures of political communities and their dynamics (the case of France):Online communities are able to reconfigure themselves in a very short period of time as a result of key events such as victories at primaries or alliances between leaders,There is a generic political community structure on Twitter, comprising a core of activists who remain for several weeks within the community, a considerably more volatile peripheral membership, and an intermediate set of accounts covering a broad spectrum of participation,Political communities are heterogeneous with respect to the online activity, commitment, political integration and politicization of their members suggesting both different profiles of political commitment from one community to another and distinct dynamics for recruiting new members. The characterization from digital traces of the variety of processes sustaining the cohesion of a community is an open challenge,People with extreme political views tend to be the most committed to their ideology,This reconstruction gives insights into the impact of communities on information diffusion and in particular on their role in the fake news phenomena (the case of France):The online community structure constrains the diffusion of information among Twitters activists, most retweet cascades occur within one or two communities,The French political Twitter landscape looks like a “split-brain” from the perspective of fake news: one “hemisphere” produces or spreads fake news, the other produces or spreads debunks,Fake news seems to be more shared than their debunks in terms of number of accounts reached, but the phenomena is reversed at the community level: the spread of fake news tends to be confined to fewer communities than other political news. Consequently, in fake news impact assessment, the variety of communities they reach should be taken into account in addition to the number of people they reach.Overall, on Twitter, for French politics, the fake news phenomena seems rather limited both in terms of volume (order of magnitude of 0.1%) and in terms of the diversity of the communities concerned. 72,9% of all fake news analyzed was produced and disseminated by only two political communities. Their diffusion is therefore above all the work of political communities, not laymens.Politoscope to be cataloged into a political community), this study establishes the basis for a series of analyzes providing unprecedented insight into opinion dynamics and the reconfiguration of political communities. It provides access to an intermediate level of resolution between sociological surveys in the field and global statistical studies , from which we expect that, in the future, it will represent a major source of political and sociological knowledge supplementing the more traditional approaches.As a consequence of its 11-months duration, its temporal resolution , and its scale , and by considering an increasingly significant history based on population sample sizes that tend to be of the order of magnitude of the population size itself. Marketing and the economic sector were the first areas of activity to seize these new opportunities, in the interests of financial gains. More recently, the political sphere has adopted these new opportunities and has tried to take advantage of them to gain greater insight into the preferences and political persuasion of voters.A perfect example of this tendency can be found in the leak of sensitive personal data concerning nearly 200 million Americans, compiled for the Republican party during the Trump 2016 campaign (≈ 99% of the electorate), which occurred in July 2017 .Politoscope project to anonymize the processed data and to produce aggregate data only, thus making it impossible to identify specific individuals, with the exception of a small number of well-known political figures. However, the research presented in this paper demonstrates that it is possible, using the conspicuous trails left in the public digital domain, and with relatively accessible computational means, to catalog a significant proportion of the electorate of an entire country, according to their political orientations, even though, as we have pointed out, there is still a certain margin of uncertainty. From this observation, it is important for citizens to realize that they can be accountable for any trace they leave on the web. The open platform politoscope.org is, in particular, designed to promote this awareness. We can only count on the honesty and good faith of current and future political leaders to refrain from using, as has happened so often in the past and continues to be the case in certain countries [We took considerable care during the entire ountries –80, dataS1 FileText A: Main political events of the 2017 French presidential campaign,B.1 Collection focused on main political accounts (follow API),B.2 Collection focused on political terms (track API),B.3 List of the followed political figures (track and follow API),B.4 Platform architecture,B.5 Availability of the data,Text B: Data collection platformText C: Bots detection,Text D: Lifetime of tweet and Twitter activity patterns,Text E: Community stability,Text F: Correlation between community structure and retweets patterns: confirmation of the echo chamber effect,7.1 Definition of the themes through queries,7.2 Distribution of topics by political communities,7.3 Evolution of community vocabulary,Text G: Analysis of the communities’ semantic backgroundText H: Fake news data cleaning,Text I: Vocabulary of the political discourse of the presidential election 2017,Text J: Analysis of late retweets.Table of content:(PDF)Click here for additional data file.


CIRCADIAN CLOCK ASSOCIATED 1. Under simulated natural light and humidity cycles, humidity oscillation increases the amplitude of the circadian clock and further improves plant fitness-related traits. In addition, humidity oscillation enhances effector-triggered immunity at night possibly to counter increased pathogen virulence under high humidity. These results indicate that the humidity oscillation regulates specific circadian outputs besides those co-regulated with the light-dark cycle.Early circadian studies in plants by de Mairan and de Candolle alluded to a regulation of circadian clocks by humidity. However, this regulation has not been described in detail, nor has its influence on physiology been demonstrated. Here we report that, under constant light, circadian humidity oscillation can entrain the plant circadian clock to a period of 24 h probably through the induction of clock genes such as  Humidity has been shown to influence many aspects of plant physiology. Here Mwimba et al. show that oscillating humidity entrains the circadian clock under constant light conditions and enhances clock amplitude in simulated natural environments, while also improving immunity and overall growth. In plants, the circadian clock is a complex network of transcriptional and translational feedback loops6. At the core of the plant circadian clock are two partially redundant morning-phased transcription factors (TFs)7, CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), which repress their own expression as well as the expression of their repressors, the PSEUDO-RESPONSE REGULATORS 8. PRR1, better known as TIMING OF CAB EXPRESSION 1 (TOC1), is an evening-phased TF, which, together with CCA1 and LHY, forms the core feedback loop9. This CCA1/LHY-TOC1 core loop is further connected with the morning loop  and the evening loop  to create the complex architecture of the plant circadian clock8.Life on earth is fundamentally dictated by the daily and yearly rhythms produced as a result of Earth’s rotation and revolution around the Sun. Living organisms perceive these rhythms and use biological circadian clocks to help tell time and schedule physiological processes in anticipation of regular changes in the environment11. Yet, most studies of plant and animal circadian clocks have been conducted using only light or temperature as a clock entraining signal even though correlations between humidity changes and potential clock outputs, including metabolism15, animal behaviours  and incidence of plant diseases21 have been reported. The extent to which daily humidity oscillation directly regulates the circadian clock has not been investigated. Our study reveals that in the absence of light and temperature cues, air humidity entrains the plant circadian clock probably through high humidity-mediated induction of CCA1; whereas, in the presence of light-dark cycles, oscillating air humidity increases the amplitude of clock genes, further improves plant fitness-related traits and enhances resistance against evening-inoculated pathogens. Therefore, this study not only demonstrates humidity oscillation as a regulator of the circadian clock but also shows its specific impacts on plant physiology.Early investigations had alluded to effects of light, temperature and air humidity on circadian clocks24. Additionally, we performed our experiments in constant light (LL) to exclude the influence of the light-dark cycle. We first compared plants grown under 22 °C in either constant RH of 50% or 90% to plants grown under 22 °C in 12 h 50%/12 h 90% oscillating RH. The oscillating RH was designed to simulate weather conditions observed in Harvard Forest  during the equinoxes, which had a 24-h period and 40% RH range  plants to the clock core loop triple mutant cca1 lhy toc1. Plants were grown in LL at 22 °C under either 12 h 50%/12 h 90% oscillating RH or 70% constant RH, so that they were exposed to the same total amount of humidity. We found that WT plants accumulated more biomass and flowered earlier under oscillating humidity than under 70% RH and that these effects were dependent on core clock genes  could be used as a proxy for vapour pressure deficit (VPD), which is known to regulate plant transpirationnes Fig. . These rArabidopsis plants carrying luciferase reporters driven by promoters of the clock genes CCA1 (CCA1p:LUC), LHY (LHYp:LUC) and TOC1 (TOC1p:LUC). In order to distinguish the influence of humidity oscillations from those of temperature and light cycles on the circadian clock, we first grew plants in the humidity- and temperature-controlled Percival chambers   and the evening-phased COLD, CIRCADIAN RHYTHM and RNA BINDING 2 (CCR2) and CATALASE 3 (CAT3)  in the other chamber. After 3 weeks, plants were moved to the imaging chamber and clock gene expression was measured by luciferase reporter activities in the area around the shoot apical meristem, which displays robust and synchronous waveformsRH) Fig. . A largeRH) Fig. . In addiCCA1, LHY and CAT2 peaked at the low-to-high humidity transition, which corresponded to dusk in nature, whereas the opposite occurred to the evening-phased genes TOC1, CAT3 and CCR2. To confirm that humidity oscillation could set the phase of the clock, we grew two populations of plants from germination at 22 °C and 50% RH but under antiphasic light regimens (LD or DL). After 2 weeks, plants were temporarily moved to free-running conditions (LL and 50% RH) at their respective dawn for imaging and then moved to LL 12 h 50%/12 h 90% RH humidity oscillation conditions, which would have corresponded to LD in nature  and 50% RH, and then released to DD and 50% RH. After 54 h under DD, i.e., 6 h after the subjective dawn when humidity is unlikely to be high in nature, we exposed these plants to a treatment of 2-h 90% RH and detected an immediate induction of the morning-phased CCA1p:LUC reporter  and, as a control, to air temperature oscillation (12 h 21 °C/12 h 22.5 °C), which covers the range of ambient temperature fluctuations observed during humidity oscillation in our chambers  camera, we determined the relationship between air temperature/humidity and leaf temperature in our growth chamber  genes involved in Pseudomonas syringae (Ps) delivery of effectors into the plant cell41. We subjected Ps pv. tomato DC3000 (Pst) colonies to a 6-h treatment of 95% RH and found that high humidity induced the expression of hrpL in bacteria grown on plates made with either the King’s B medium or the hrp minimal medium (HMM), which is known to induce hrpL expression through its reduced carbon content and low pH43 , which is deficient in effector delivery  was stronger under oscillating humidity than under constant humidity in the evening-inoculated leaves , which is elicited by the recognition of pathogen effectors delivered inside the plant cell. To further test whether an influence of humidity oscillation on plant immunity is ETI-specific, we also assessed pattern-triggered immunity (PTI), which is elicited by the recognition of microbe-associated molecular patterns on the host cell surface. We pressure-infiltrated the pathogen into day Fig. . We founves Fig. . Moreoveves Fig. . These dves Fig. , suggestons Fig. . We haveons Fig. , indicat45, exposure to constant 90% RH post inoculation suppressed PCD  at night could lead to a severe loss of plant fresh weight. This was likely due to SA-mediated repression of genes in water transport, such as 9 of the 26 Arabidopsis aquaporin genes54. Water availability is not only essential for plant growth but also critical for pathogen virulence as recently demonstrated for Pst whose proliferation in the apoplast requires both establishment of aqueous conditions by its effectors and high humidity in the environment45. Therefore, humidity may function as a cue to allow scheduling of defence and growth activities with minimal fitness cost.Our study supports previous findings that the circadian clock not only allows plants to anticipate infection at the time when it is most likely to occur55. This is in line with the early observations that insect activity cycles are influenced by the diurnal humidity fluctuations reported by Colin Pittendrigh, a founder of chronobiology, in his work on malaria-transmitting mosquitoes19. We also anticipate that our study in plants may lead to more in-depth investigation of humidity influences on mammalian physiology, as multiple studies have reported a correlation between humidity and disease susceptibility in humans58.The daily oscillation of humidity is a universal natural phenomenon experienced by all terrestrial organisms. A thorough investigation of humidity as a regulator of circadian clock in other species may further advance the field of chronobiology by uncovering new clock components, or even organisms to which humidity might be a primary zeitgeber. In addition to our finding that high humidity can activate T3SS in bacteria  where light, temperature and humidity could be controlled independently. Light provided by Philips AltoII tubes was set at 100 µmol m−2 s−1 during the day or LL and 0 µmol m−2 s−1 at nights; temperature and air humidity were set according to experimental designs. Plants were grown in MetroMix 360 soil in 72-well flats (Hummert # 11–0700) and watered every 2–3 days to keep the soil volumetric water content (soil moisture) between 20% and 40% so that they did not experience drought stress. When applicable, plant genotypes were randomized to minimize position-specific effects and conditions in Percival chambers were switched between experiments  to minimize chamber-specific effects. When three humidity conditions were needed, e.g., when comparing leaf-rosette biomass accumulation  in 0.02% Triton X-100 (Sigma) at both 24 and 12 h before luciferase imaging. Plants were then placed into the custom-built imaging chamber under specified light and humidity conditions and images were obtained using a charge-coupled device camera (PIXIS 2048) at 20 min exposure time every 2 h. Throughout the luciferase imaging, temperature, soil moisture, air humidity and light intensity were recorded every minute by Vernier probes STS-BTA, SMS-BTA, RH-BTA and LS-BTA, respectively. For the humidity entrainment experiments involving luciferase imaging, 50% RH was used as the free-running condition because it was technically more plausible to maintain constant 50% RH than 70% RH in the imaging chamber. Leaf temperature was measured in the Percival chamber using a FLIR A320sc IR imaging camera.To simultaneously image luciferase reporter activities in two sets of soil-grown mature plants (up to 192) under constant and oscillating humidity, respectively, while controlling light and temperature, we custom-built an imaging chamber with two compartments. Humidity was fed to the chamber by ultrasonic humidifiers (one Holmes humidifier for each compartment) and humidity in the room was kept at 50% RH using a dehumidifier . Light was provided by red and blue light-emitting diode lights in the ratio of 2 to 1, respectively, and the fluence rate set at 100 µmol m63 with an expected period between 18 and 36 h. Rhythms with relative amplitude error ≤ 0.7 were considered rhythmic and theirs calculated amplitudes and circadian phases by averaged peaks relative to time zero were reported. Supplementary Table N the number of oscillators.To monitor clock gene expression, 3-week-old plants carrying luciferase reporters were sprayed with 2.5 mM luciferin 24 and 12 h before being moved into the imaging chamber. In the imaging chamber, plants were kept under their growth conditions for at least 1 day to stabilize gene expression before imaging at 2-h intervals. Gene expression was determined by bioluminescence emission of the area around the shoot apical meristem. For each plant, luciferase data were normalized to its mean expression value or its peak expression before humidity treatment and periodicity analysis was performed using the Biodare’s FFT-NLLS and LS toolsUBIQUITIN 5 (UBQ5) and bacterial genes gyrA and gap1 were used as internal controls. When indicated, experimental replicates were combined using a linear mixed effect model (lme4 package in CRAN) before significant differences were determined by Student’s t-test or analysis of variance (ANOVA). Results are reported as mean ± s.d. of three independent experiments.Plant RNA extraction was performed according to protocols provided by the manufacturers of the TRIZOL reagent (Ambion) and the Direct-zol (Zymo). cDNA construction was performed using superscript III reverse transcriptase enzyme (Invitrogen); and quantitative PCR  was carried out according to the manufacturers’ protocols. Total bacterial RNA was extracted using the RNeasy kit (Qiagen) and the Bacteria Protect Reagent (Qiagen) following the manufacturer’s instructions. Briefly, bacteria were re-suspended, washed twice in cold phosphate-buffered saline containing the Bacteria Protect Reagent and mechanically disrupted by 10 min of sonication Bioraptor300). RNA was purified from the extract using the RNeasy kit and DNA was removed by treating total RNA with two rounds of the Ambion DNase I enzyme. cDNA was synthesized using random hexamers (Roche) and superscript III reverse transcriptase enzyme (Invitrogen) according to the enzyme protocol. Primer pairs . For seed mass and seed count measurements, plants were watered until leaf senescence was visible to at least 50% of leaves and then left to dry. Seeds from at least 12 plants were pooled and stored in a paper bag for at least 2 weeks before seed mass was measured using an analytical balance. Per experiment and condition, three samples of dry seed pools were weighted and seeds in each pool were counted to determine the average seed mass. Experimental replicates of seed mass and seed count were combined using a linear mixed effect model (lme4 package in CRAN) before significant differences were determined by Student’s Pst (OD600nm = 0.001), Pst AvrRpt2 (OD600nm = 0.001) or Pst hrpL(−) (OD600nm = 0.01) in 10 mM MgSO4 and tissue collected 3 days after inoculation. Colony-forming units were counted according to Durrant et al.64.Whole leaves of 3-week-old soil-grown plants were infiltrated with Pst AvrRpt2 (OD600nm = 0.001) or Pf AvrRps4 (OD600nm = 0.02). Ion leakage measurements were performed at 36 h post inoculation when ETI-associated PCD started to occur. Similar to the method described in Woo et al.65, two leaves per plant were immersed in 5 mL of 400 mM mannitol with gentle shaking for 2 h, after which the sample conductivity was measured using Orion Star series meter (Thermo scientific) and normalized to total conductivity determined after boiling samples for 10 min. Six samples per growth condition were reported collectively as mean ± s.d. The experiment was repeated three times with similar results.Ion leakage was determined by measuring electrolytes leaked from leaves inoculated with Statistical analyses were performed using GraphPad Prism 6 or the R programming language (R version 3.3.1). All centre values shown in figures are means of biological or experimental replicates. When mixed linear model was used, statistical analysis was performed on the mean estimates and their standard deviations.Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1


A 76‐year‐old man with a past history of acute exacerbation (AE) and idiopathic pulmonary fibrosis (IPF) was treated with nintedanib because of decline in his forced vital capacity over time. A new small nodular lesion was visible on a computed tomography scan of the chest before initiation of nintedanib. Disease progression in IPF and change in size of the nodular lesion were not detected during administration of nintedanib. Nine months after starting nintedanib, the patient was diagnosed with acute gangrenous appendicitis, and nintedanib treatment was discontinued. The nodular lesion increased in size four months after the cessation of nintedanib. The nodular lesion was diagnosed as squamous cell carcinoma. In this case, nintedanib inhibited the disease progression of IPF and lung cancer simultaneously. Nintedanib may play an important role in the treatment of IPF‐associated lung cancer. Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive fibrosing interstitial pneumonia of uncertain origin associated with a poor prognosis A 76‐year‐old man, former smoker (80 pack‐years), with hypertension and hyperlipidaemia was referred to our hospital with a diagnosis of AE with IPF in 2013. After admission to our hospital, he was treated with steroid pulse therapy followed by systemic corticosteroid and cyclosporine therapy. His respiratory condition gradually improved, and corticosteroid dose was tapered. However, he needed 2 L/min of oxygen via a nasal cannula at the time of discharge. Corticosteroid dose was tapered gradually in the outpatient clinic. His forced vital capacity (FVC) declined by 8% in approximately 30 months after remission of AE. Nintedanib (300 mg/day) was administered in December 2015 because of the decline in FVC and a history of AE with IPF. A new small nodular lesion, measuring 13.5 mm × 11.7 mm  is being conducted to evaluate the safety and effectiveness of nintedanib in combination with carboplatin plus nab‐paclitaxel, and the results are eagerly awaited.Y. Nakano received lecture fees from Nippon Boehringer Ingelheim Co., Ltd. . All authors also received research funding from Nippon Boehringer Ingelheim Co. However, the company had no control over the interpretation, writing, or publication of this work.Appropriate written informed consent was obtained for publication of this case report and accompanying images.


With the increasing power density of electrical and electronic devices, there has been an urgent demand for the development of thermal interface materials (TIMs) with high through-plane thermal conductivity for handling the issue of thermal management. Graphene exhibited significant potential for the development of TIMs, due to its ultra-high intrinsic thermal conductivity. In this perspective, we introduce three state-of-the-art graphene-based TIMs, including dispersed graphene/polymers, graphene framework/polymers and inorganic graphene-based monoliths. The advantages and limitations of them were discussed from an application point of view. In addition, possible strategies and future research directions in the development of high-performance graphene-based TIMs are also discussed. Thermal management has become a central issue to guarantee the reliability and service life of electrical and electronic devices, such as highly integrated CPUs, high-power LED lights and energy harvesting systems . ParticuSince graphene was discovered and exhibited an ultra-high intrinsic thermal conductivity of 3500–5300 W/mK ,9, a larIn earlier research, graphene-based TIMs were prepared through a solution or melt-blending process to disperse graphene sheets into a polymer matrix a. A summIn recent years, the penetration of polymers into three-dimensional graphene frameworks has been considered as a promising solution to develop composites with improved thermal conductivity for TIM applications. So far, the approaches for the fabrication of a graphene framework can be mainly classified into two categories, the self-assembly method and the template-synthesis method ,22,23,243) [3) composed of densely packed CVD graphene can be obtained, as shown in In contrast to hard epoxy, soft PDMS with the lower compressive modulus of 2–10 MPa ,33 is a 3) , such as3 [Most recently, we reported a filtrated graphene framework composed of a horizontal and densely packed graphene structure with a density of 60–80 mg/cm3 . In orde3 , as show(i)Soft elastomers as matrix for good gap filling, such as PDMS.(ii)A relatively higher density of the graphene framework for higher graphene content in the polymer matrix, leading to high thermal conductivity.(iii)An opportune arrangement of graphene in the framework for avoiding an unsatisfactory thermal conductivity in the through-plane direction.(iv)Graphene framework with reversible deformation for maintaining high thermal conductivity while under packaging.As discussed above, graphene frameworks show great potential for the devotion of polymer composites with improved thermal conductivity. So far, however, there are few reports on graphene framework/polymer composites for TIM application with the thermal management performance in excess of commercial TIMs. Part of the reason is that the removal of heat through the interface requires TIMs to have high through-plane thermal conductivity, while the high thermal conductivity of graphene is only provided along the in-plane direction ,8. In or3) composed of vertically aligned graphene sheets, which was constructed via “rotating-reassembling” as-prepared graphene paper (2/W [2/W [3) and studied the thermal conductivity as function of compression strain (2-bonded carbon structure [Apart from the incorporation with polymers, graphene can also be used as a TIM by the fabrication of inorganic graphene-based monoliths. In 2011, Wong and co-workers reported a graphene monolith (1.6 g/cmne paper a–c. Withper (2/W . However2/W [2/W . That san strain g–i. Undetructure ,49,50. Ttructure ,52. PerhThis report briefly reviews the progress of graphene-based TIMs and discusses three types of materials, including dispersed graphene/polymers, graphene framework/polymers and inorganic graphene-based monoliths. Each material both has its advantages and disadvantages, as shown in


The control of graphene’s topography at the nanoscale level opens up the possibility to greatly improve the surface functionalization, change the doping level or create nanoscale reservoirs. However, the ability to control the modification of the topography of graphene on a wafer scale is still rather challenging. Here we present an approach to create well-defined nanowrinkles on a wafer scale using nitrocellulose as the polymer to transfer chemical vapor deposition grown graphene from the copper foil to a substrate. During the transfer process, the complex tertiary nitrocellulose structure is imprinted into the graphene area layer. When the graphene layer is put onto a substrate this will result in a well-defined nanowrinkle pattern, which can be subsequently further processed. Using atomic force and Raman microscopy, we characterized the generated nanowrinkles in graphene. The transfer of either exfoliated or chemical vapor deposition (CVD) grown graphene to another substrate is one of the key steps during the fabrication of graphene-based devices. The creation of rugae , a singlHowever, those “unwanted topographic features” can be very suitable to further explore graphene’s potential. One of the most significant changes due to the presence of these rugae is a change in the pyramidalization angle, which describes the angle between the σ- and π-orbitals of graphene, and which is a measure for the chemical reactivity of carbon allotropes ,7,8. As Besides changing these fundamental properties of graphene, a homogeneous, local deformation of graphene can act as a trap for liquids and gases, as graphene is an impenetrable membrane for most liquids and gases . BesidesAs shown recently by Hallam et al. , the strThe actual type of corrugation that is created during the transfer can be predicted if the properties of the used polymer for transfer are known. Using rugae mechanics, it can be shown that the shear modulus of both graphene and the polymer, the adhesion energy, the mismatch strain between the polymer and graphene, the orientation of graphene’s grain and defects edges and the structure of the used polymer are the dominant factors that determine the types of rugae that can be observed when the graphene is transferred to its final substrate ,17. For In this article we show that by transferring graphene using nitrocellulose as a transfer polymer, we can induce a homogeneous landscape of nanoscale wrinkles in the whole graphene sheet, in which topography is induced from the structure of the used nitrocellulose polymer. This easy and straightforward method dramatically improves the production of large homogeneous areas of graphene with nanoscale wrinkles, which are created in a controllable and reproducible way. The ability to create large areas of graphene with the desired topographical properties improves the study of the influence of rugae on the electrical, optical and chemical properties of graphene, as there is no need to extensively search for large, homogeneous areas with the desired type of rugae.2. The copper foil was exposed to 30 sccm CH4 and 50 sccm H2 for 20 min, after which the copper foil was cooled to room temperature.Graphene samples were grown by the CVD method as reported previously . In brie2 graphene/NC layer is the same as is used for the transfer with PMMA. In short; the Cu foil was etched away using FeCl3 , and the graphene/NC sample was fished using a Si/SiO2 wafer from the solution and subsequently washed several times using highly purified water. Finally, the graphene/NC layer was fished using the final substrate, which was carefully cleaned using acetone and isopropanol, and dried with nitrogen gas. Residual NC can be easily removed from the graphene by washing the sample several times with methanol. The samples were dried with nitrogen gas and no further annealing steps were performed.On top of the foil, a nitrocellulose layer (NC) , was spincoated with 2700 revolutions per minute (rpm) for 30 s and air dried, which results in a thickness of the NC of approximately 50 nm. The transfer of the 1 × 1 cm1, G2 and D’) and two (2D1 and 2D2) peak profiles were used to fit the D, G and 2D modes respectively. The G1, G2, 2D1 and 2D2 modes were used to account for the delaminated graphene, which was created by the NC transfer [Prepared samples were characterized by atomic force microscopy (AFM) and Raman spectroscopy. The Raman spectra were obtained at a WITec alpha300R spectrometer  equipped with a piezo stage. Raman spectral maps were measured with 2.33-eV (532-nm) laser excitation, a laser power of approximately 1 mW, a grating of 600 lines/mm and lateral steps of 1 μm in both directions. The laser was focused on the sample with a 100× objective. The D, G and 2D modes of the graphene monolayer were analyzed using pseudo-Voigt peak profiles, where one, three  at room temperature in air. High quality images were processed in the standard way using Gwyddion software [AFM was measured using PeakForce quantitative nano-mechanical mapping (PeakForceQNM) and imaging in tapping mode, using a Dimension Icon microscope . Images were captured using Bruker Scanasyst-Air probes , and to determine their geometry and width (wrw). Surface characterization was used for the estimation of the roughness (aR), root-mean-square roughness (qR), and surface area (SA). The profile height distribution was analyzed to determine the median peak-to-valley distance (tmR), of the wrinkle graphene layer topography, which was used as the wrinkle height. The topography AFM image was cut into individual lines using home-built procedures, in order to identify all the minima and maxima attributed to the highest parts of the wrinkles and the lowest parts in the inter-wrinkle valleys. Then, the median difference of all maxima and minima was calculated as tmR = μp − μv, where μp is the median wrinkle height and μv is the median valley depth, calculated over all the lines in the image.To determine the overall orientation of wrinkles, Gwyddion’s 2D FFT analysis  is used. Grain analysis was used to evaluate the surface coverage by the winkles  of the 2D1 mode are shown in 1 and 2D1 modes was not homogeneous, and well-defined regions with a smaller or larger Raman shift are visible. These regions were caused by the different Cu grains of the Cu foil, onto which the graphene was grown. As shown before [The Raman data were further analyzed in order to get deeper insight in the charge and strain management in the graphene monolayer with regularly distributed nanowrinkles. Raman spectral maps of the Raman shift of the Gn before , the actn before ,23. As a1 mode of the NC transferred graphene was rather homogeneous and there was no obvious dependence visible, as could be seen for the Raman shift of the G1 and 2D1 modes. This is an indication that during the NC transfer, the wrinkles as shown in the AFM images in 1 mode, which shows a narrow distribution of FWHM values around 48 cm−1, where extremely flat graphene can have a FWHM of the 2D mode well below 20 cm−1 [The FWHM of the 2D mode, which acts as a probe of the nanometer-scale flatness  is shown 20 cm−1 .1, as shown in 1 mode. For increasing compressive strain, the FWHM of the 2D mode slightly decreased, whereas for increasing tensile strain, the FWHM of the 2D mode has a larger scatter and was not increasing.However, the correlation between the calculated strain and FWHM of the 2D1 – 2D1 (colored points) and G2 – 2D2 (black points) modes, as shown in 1 mode is indicated via a color code. The attached graphene to the substrate (colored points) was relatively doped and a large strain variation was present. Interestingly, these points were clustered close to the blue line indicating graphene with a strain value close to zero, but with an increasing doping level, had a lower FWHM of the 2D1 mode than points that had some tensile strain.This trend is also visible in the correlation plot between the Raman shift of the G1 mode of the NC transferred graphene seemed to be strain dependent, this suggests that there is also the uniaxial strain component present within the graphene layer [1 mode is expected, as the FWHM did not change significantly when the doping level was changed.As the FWHM of the 2Dne layer . Althougne layer . No signThe delaminated graphene (black points) had a very low doping level and showed the same large strain variation as observed for the attached graphene.The rugae landscape in CVD grown graphene that is transferred to another substrate can be controlled by the used polymer. Up to now, the choice of polymer was often influenced by the possibility to completely remove the polymer after the transfer, so that as clean as possible graphene could be obtained. Here, we show that NC transferred graphene, besides utilizing a very clean transfer method , also reThe relatively rigid NC preserves the strain in graphene that is built up during the CVD growth as can be seen in the maps of the Raman shift of the G and 2D modes . Areas wThe structure of NC shows at the nanoscale a complex network of nanoscale wrinkles, as is visible in The observed wrinkles in the AFM images are not caused by polymer residuals. The strong asymmetric G and 2D modes indicates that a significant part of the graphene layer is delaminated . FurtherWe have shown that when NC is used as the polymer for the transfer of CVD grown graphene from the Cu foil to another substrate, randomly distributed regular nanowrinkles are imprinted in the graphene layer. Our approach opens up many new possibilities to control and exploit the properties of graphene with nanometer spatial resolution, like the doping level or chemical functionalization. Moreover, it allows to discover the potential of nanoscale reservoirs on wafer scale.


The growth conditions were systematically optimized and the as-grown doped graphene were well characterized. The growth strategy provides a controllable transfer-free route for high-quality doped graphene synthesis, which will facilitate the practical applications of graphene.Direct synthesis of high-quality doped graphene on dielectric substrates without transfer is highly desired for simplified device processing in electronic applications. However, graphene synthesis directly on substrates suitable for device applications, though highly demanded, remains unattainable and challenging. Here, a simple and transfer-free synthesis of high-quality doped graphene on the dielectric substrate has been developed using a thin Cu layer as the top catalyst and polycyclic aromatic hydrocarbons as both carbon precursors and doping sources. N-doped and N, F-co-doped graphene have been achieved using TPB and FThe online version of this article (doi:10.1007/s40820-015-0052-6) contains supplementary material, which is available to authorized users. Howeverstrength . Doping strength  and furtstrength –13.3 as N doping source) [Among all the approaches to synthesize doped graphene, chemical vapor deposition (CVD) is the most popular method to obtain high-quality doped graphene in large scale by introducing copper or nickel foil as the catalyst , 14, 15  source) , 17. Rec source) . Liu et  source) . However source) , 20, whi16CuPc) as solid carbon source and both N and F doping sources.Recently, we have developed a new transfer-free approach capable of synthesizing graphene directly on dielectric substrates using polycyclic aromatic hydrocarbons (PAHs) as carbon sources . Signifi2-layered Si (SiO2/Si) without transfer is shown in Fig. 2/Si (SiO2: 300 nm thick) substrate was ultrasonically cleaned by acetone, ethanol, and deionized water for 15 min, respectively. Then PAHs with planar structure (TPB or F16CuPc) were evaporated on the substrate as solid carbon sources by thermal evaporation system (Organic Evaporation Coating Machine ZZB-U500SA), followed by the Cu film layer deposition on the surface of PAHs as catalyst by electron-beam evaporation system . After annealing in a tube furnace under Ar gas flow at ~1.8 × 102 Pa, doped graphene was synthesized between the Cu layer and the substrate. At last, Cu layer was etched away by Marble’s reagent (CuSO4:HCl:H2O = 10 g:50 mL:50 mL), then doped graphene was obtained directly on SiO2 substrate without any transfer process.The schematic of growth process of doped graphene directly on SiOKα X-rays (1486 eV) as the excitation source. The optical transmittance spectrum and sheet resistance of doped graphene are measured by ultraviolet–visible–near-infrared spectrophotometer (LAMBDA 750) and four-point probe system (ST-2258A), respectively.The morphology of doped graphene was characterized by scanning electron microscopy (SEM) (FEI Quanta 200F). Raman spectra were recorded at room temperature using a Jobin–Yvon HR800 Raman microscope with laser excitation at 514 nm. Optical images were obtained using Fluorescence optical microscope (DM4000M). The HR-TEM images were taken by transmission electron microscope (TEM) (Tecnai G2 F20). The surface state and electron structure of the samples were studied by X-ray photoelectron spectroscopy (XPS) measurement (Kratos AXIS UltraDLD ultrahigh vacuum (UHV) surface analysis system), using Al Carbon source is an important factor in graphene synthesis. We found that planar configuration of PAHs might provide a hexagonal honeycomb skeleton structure for the graphene growth and the growth mechanism from PAHs maybe involves surface-mediated nucleation process of dehydrogenated PAHs catalyzed by Cu rather than segregation or precipitation process of small carbon species that decomposed from the precursors. Therefore, planar PAHs that contain heteroatoms  were chosen as solid carbon sources for doped graphene growth in our work. In addition to the specific structure of solid carbon sources, there are some other key factors to control the quality of doped graphene, such as the thickness of solid carbon sources, the thickness of Cu film layer, annealing time, annealing temperature, etc. Hence, in order to achieve high-quality doped graphene, optimal conditions for doped graphene growth had been investigated by rationalizing the above factors. 2, 4, 6-triphenylborazine (TPB) with planar configuration was selected as the solid carbon source to evaluate the growth conditions of the doped graphene.The thickness effect of TPB on the quality of graphene was investigated firstly. The Raman spectra shown in Fig. S1a reveal that the optimum thickness of TPB layer is 5 nm. When the thickness of TPB is less than 5 nm, the carbon source cannot form continuous film on the substrate, which could result in the formation of discontinuous graphene. While the thickness of TPB is greater than 5 nm, the excessive amount of carbon source leads to multilayer graphene or amorphous carbon formation due to the extremely low solubility of C in Cu. Different annealing temperatures were also investigated for the growth of TPB-derived doped graphene. In general, the growth temperature, in conventional CVD method, required to synthesize good-quality graphene is 1000–1050 °C. Figure S1b shows the Raman spectra of graphene synthesized at different growth temperatures, suggesting that graphene can be obtained above 950 °C. Annealing temperature below 650 °C results in the deposition of amorphous carbon, as characterized by the broad D and G bands and a very weak 2D band shown in Fig. S1b. When the annealing temperature was increased to 1050 °C, the obtained graphene layer also has a larger D band than that grown at 1000 °C in the Raman spectra. It has probably arisen from the partial evaporation of thin Cu film at 1050 °C.ID/IG and higher I2D/IG ratio can be achieved when the annealing time is 60 min. The effect of Cu film thickness on doped graphene growth was investigated as well. When Cu film thickness is above 100 nm, the graphene film can be obtained. However, when the thickness of the Cu film was decreased below 100 nm, most of the Cu was evaporated during the annealing process at 1000 °C and resulted in discontinuous doped graphene. In addition, the graphene formed on the top surface of Cu was observed when a thin Cu layer was used. When the thickness of the Cu film was increased to 1000 nm, relatively high-quality doped graphene was obtained indicated by the G/2D ratio (~0.3), D/G ratio (~1.3), and FWHM of the 2D (~42 cm−1) band in Fig. S1d. Thus the optimal growth conditions for the doped graphene growth from TPB were set at 5 nm TPB as carbon source, 1000 nm Cu film on the top surface, and annealing temperature of 1000 °C for 60 min.Subsequently, different annealing times were studied. As shown in Fig. S1c, higher quality doped graphene with lower 2/Si substrate at the optimal condition using TPB as the carbon source. The continuous film with almost no contrast indicates that the graphene is distributed uniformly on the dielectric substrate. The corresponding Raman spectrum in Fig. Figure 16CuPc of 5 nm was used as the carbon source to prepare N, F-co-doped graphene on the SiO2/Si substrate at 1000 °C for 60 min. F16CuPc is also a PAH compound with a planar structure. The Raman spectrum of the product , confirming the graphitic structure of the as-grown graphene grains. The shoulder around 285.5 and 286.6 eV can be assigned to the C–N and C–F bonding, respectively. Figure XPS investigation further verifies that N and F co-doping has been achieved in the graphene. XPS spectra of N, F-co-doped graphene are shown in Fig. rum Fig. b is assicarbon 28.4 eV, co2/Si substrate, exhibiting a high optical transmittance of ~93 % at 550 nm, even though the doped graphene film is 3–4 layers. The sheet resistance (Rs) obtained from four-point probe measurement is ∼2.5 kΩ (sq)−1, revealing that the as-grown N, F-co-doped graphene film is of high conductivity.Figure 16CuPc as solid carbon sources, respectively. The properties of the as-grown samples were well studied and N, F-co-doped graphene exhibits a high optical transmittance and low sheet resistance. The present growth strategy provides a controllable transfer-free route for high-quality doped graphene growth, which will facilitate the practical electronic applications of graphene.In summary, a facile method for high-quality synthesis of doped graphene film on the dielectric substrate has been developed. PAHs containing dopant elements with planar configuration were used as both carbon feedstocks and doping sources and a layer of Cu film as the catalyst. The thickness of Cu layer and PAHs, the annealing time, and temperature are optimized for high-quality graphene growth. N-doped and N, F-co-doped graphene have been synthesized using TPB and FSupplementary material 1 (PDF 473 kb)Below is the link to the electronic supplementary material.


Some lesions have presenting inflammatory signs. The pathological anatomy study of a skin biopsy revealed a granuloma with epithelioid and giant cells without caseous necrosis, suggesting the diagnosis of sarcoidosis first. A chest x-ray showed mediastinal lymphadenopathy and bilateral interstitial syndrome confirmed on thoracic computed tomography. The exploration of respiratory function reveals a restrictive ventilatory disorder supporting pulmonary parenchymal involvement. Ophthalmological examination shows anterior uveitis. The angiotensin converting enzyme assay is high and the tuberculin skin test reveals an anergy. The patient was treated by corticosteroids; synthetic antimalarials will be prescribed when no response or insufficient response happens. Subcutaneous sarcoidosis, also known as Darier-Roussy, and lupus pernio, are uncommon but specific of sarcoidosis. Their presence is often associated with systemic involvement, particularly thoracic.