Pomatostomus ruficeps), a cooperatively breeding Australian bird with variable number of carers. Our analysis suggests that turn-taking occurs across a range of group sizes (2–6), with individual birds being more likely to visit following other individuals than to make repeat visits. We show using a randomization test that some of this apparent turn-taking arises as a by-product of the distribution of individual inter-visit intervals (“passive” turn-taking) but that individuals also respond actively to the investment of others over and above this effect (“active” turn-taking). We conclude that turn-taking in babblers is a consequence of both their individual provisioning behavior and deliberate response rules, with the former effect arising through a minimum interval required to forage and travel to and from the nest. Our results reinforce the importance of considering fine-scale investment dynamics when studying parental care and suggest that behavioral rules such as turn-taking may be more common than previously thought.For individuals collaborating to rear offspring, effective organization of resource delivery is difficult because each carer benefits when the others provide a greater share of the total investment required. When investment is provided in discrete events, one possible solution is to adopt a turn-taking strategy whereby each individual reduces its contribution rate after investing, only increasing its rate again once another carer contributes. To test whether turn-taking occurs in a natural cooperative care system, here we use a continuous time Markov model to deduce the provisioning behavior of the chestnut-crowned babbler  whether this a deliberate strategy or simply a by-product of independent care behavior. We find that babblers indeed take turns and conclude that part of the observed turn-taking is due to deliberate responsiveness, with the rest arising from the species’ breeding ecology.The online version of this article (10.1007/s00265-017-2391-4) contains supplementary material, which is available to authorized users. Individuals cooperating to rear offspring face several problems when attempting to share provisioning effort efficiently. Firstly, care is costly Williams , so eachExisting theory on offspring care has focused on the total amount of care delivered and on the response of carers to a change in the amount delivered by others. Models have typically adopted either “sealed bid”  has provided evidence that such reciprocal investment rules are used to regulate provisioning of young ; higher degrees of turn-taking are associated with higher food delivery rates and lower predation risk , which exhibit a strong tendency to alternate visits , which do not , a medium-sized (50 g) endemic Australian passerine that breeds in groups of 2–15 (mean ≈ 6), with helpers provisioning at the nest in addition to the breeding pair  in their propensity for turn-taking. To this end, we generate and fit models of provisioning behavior using a continuous-time Markov-Chain Monte-Carlo (MCMC) approach Bremaud  and applProvisioning data were collected between July and November in both 2007 and 2008 at Fowlers Gap Arid Zone Research Station, NSW, Australia . Adults were caught using mist-nets, ringed, tagged with Passive Integrated Transponder (PIT) tags , and had a small (< 100 μl) blood sample taken from the ulnar vein. Chicks were similarly blood-sampled, ringed, and PIT-tagged at around 15 days old. Chestnut-crowned babblers are sexually monomorphic, so identification of parentage and sex was primarily obtained through molecular analysis  visited more than ten times each. We also discounted data from the last day in the nest prior to fledging  and any data from periods of disturbance (such as periodic measuring of chicks or changing of data loggers). We grouped multiple PIT records by the same individual into a single visit when they occurred within 2 min, an approach validated by previous studies on babbler provisioning  carers, six with three carers, five with four carers, three with five carers, and two with six carers; between zero and five, individuals in each group were excluded for failing to classify as a significant carer (mean = 1.1). The amount of data required to fit our models grows rapidly as group size increases, because the number of transitions to be estimated is equal to the square of the carer number. This escalating need for data at large group sizes meant that two groups with good provisioning data were excluded from the final analysis because they contained too many significant carers (eight and nine) to fit our models.Among the groups used, brood size ranged from 1 to 5 chicks (mean 3.3), and chick age from 10 to 19 days old; previous work suggests that babbler provisioning rate is relatively static between day 10 and day 20  of each individual is highly consistent, near-perfect turn-taking  might be expected even if individuals do not monitor or respond to one another’s behavior in any way. This turn-taking will be less precise if this “refractory period” is variable, but even with highly variable intervals, any significant refractory period will introduce some additional passive turn-taking. Furthermore, continuous-time Markov models assume that events  are approximately exponentially distributed, an assumption violated when significant refractory periods exist. One possible solution is to fit a more complex semi-Markov model explicitly defining the event distribution; however, we lacked sufficient data on individual, group, and environmental variation in IVIs to fit this model effectively. To circumvent these problems and fully distinguish between passive turn-taking and active turn-taking, we therefore employed a randomization test similar to that of Johnstone et al. .Our randomization test removes any potential active turn-taking from the data by eliminating relationships between the visit times of different individuals. We first calculated all of an individual’s IVIs in a given day, then randomly re-ordered these intervals within each individual and day. From these randomized intervals, we reconstructed a new list of artificial visit times for each individual on each day. The artificial visit times for all individuals were then combined to form an artificial provisioning day, which could then be analyzed in the same way as the original data. We re-ran the above dissociation 1000 times on the data from each breeding attempt to generate distributions of expected (passive) turn-taking given the structure of nest visits within each attempt. By comparing the extent of turn-taking observed in the natural data to the distribution of values obtained from the dissociated data, we can determine the degree to which turn-taking is passive, i.e., simply attributable to group size, unequal visit rates, and a lack of immediate re-visits, or active, being caused by individuals responding to one another’s behavior. This type of randomization test can be biased if individual IVIs are ordered across the observation period  provision in synchronous bouts, their IVIs will tend to cluster, with more consecutive short IVIs than would be expected by chance. Whether short IVIs are significantly clustered can hence be tested using a standard Wald-Wolfowitz runs test, after categorizing each IVI as either short or long based on a threshold value. Fewer runs than expected would indicate clustering and suggest some degree of group-level provisioning.c carers featured 2c states and c(c + 1) possible transitions between states, including reset transitions. The best-fit model calculated by msm specifies the rate at which each possible transition occurs (which determines how likely particular birds are to follow particular other birds). We assume that individuals may react differently to each of the other carers visiting the nest and thus estimate, for each individual, as many different visit rates as there are carers, giving c2 transition (visit) rates to be estimated .For each breeding unit, we fitted a continuous-time Markov chain model to the nest visits using the R package msm Jackson . Each viλi for each individual and the visit rate when an individual makes a repeat visit (“following self”) as μi. To fit this model, we constrain the analysis such that visit rates when following any other bird are equivalent and compare whether this reduced model (with 2c unique transitions) explains the observed data similarly well compared to the full model. Similarly, we can further constrain the analysis to model all individuals visiting at the same rate, giving only two possible transition rates regardless of group size: any bird following any other bird (λ0) and a bird following itself (μ0). Comparisons between models were made using likelihood ratio tests, which follow a chi-squared distribution with degrees of freedom equal to the difference in free parameters between the full and constrained models.Owing to the numerous possible transitions in larger groups, it is also useful to define an individual’s visit rate when they follow any bird other than themselves. Following Johnstone et al. , we defin = 73) and group sizes (2–6), individuals alternated visits  more often than they visited twice in a row, and as expected, the proportion of alternated visits increased with group size  .Provisioning data were analyzed for 20 breeding attempts by 19 different breeding groups . Across all carers (λ = 123.7 ± 43.4) was much higher than the rate at which they made repeated visits (μ = 50.5 ± 18.2) . One breeding attempt was excluded from further analysis due to insufficient data to fit individual-specific models; for a majority (17/19) of the remaining groups, models in which individuals visited at different rates following particular other individuals were a better fit  than models that assumed each individual had only two possible visit rates (λi and μi). In all groups, both of the above models were much better fits to the data  than a null model assuming all individuals had the same two possible rates (λ0 and μ0). Despite the significance of individual differences in transition rates within groups, across all groups, neither within-individual nor between-individual visit rates were significantly different between breeding males, breeding females, and the primary (most helpful) non-breeding helpers in a group , although this may be attributable to small sample sizes and our restricted window of analysis, as some of the above factors are known to influence babbler provisioning behavior when the entire rearing period is considered , forming an approximately geometric distribution with a long tail of larger intervals consistent with the absence of a refractory period . Approximately 97% of all IVIs were less than 1 h, suggesting that all individuals visited throughout the day. As might be expected, the intervals between any one carer visiting the nest were smaller in larger groups . Histograms of individual IVIs for different group sizes are included in supplementary material  in fitted Markov models was far lower under our randomization test than in the natural data , suggesting that individual IVIs generate some degrees of visit structure. Individual IVIs were randomly ordered for every individual in our dataset  or any group visit patterns inconsistent with a broadly random distribution of nest arrival times under occasional disturbance , beyond the apparent turn-taking arising from combination of group size effects, unequal visit rates, and the distribution of individual inter-visit intervals (passive turn-taking). Babblers exhibited active turn-taking across all group sizes, and all carers with similar visit rates adopted similar provisioning rules. We found no evidence that the observed levels of active turn-taking could be attributed to synchronous nest visits by the care group.Both our run tests and Markov analyses suggested that some of the tendencies for individuals to take turns could not be explained by group size effects alone. Refractory periods are a likely cause for some of this additional passive turn-taking, as individual carers may take some well-defined minimum period to travel to and from the foraging site and another minimum period to find food while foraging. Consequently, individual IVIs will have a well-defined minimum value, and longer minimum IVIs will arise whenever carers cease provisioning chicks to collect food for themselves. Under these circumstances, some degrees of turn-taking will always occur because during the refractory period, other carers can visit while the focal individual cannot. For the above reason, some passive turn-taking was inevitable in our data due to the way the raw PIT-tag data was processed: no individual IVI could be less than 2 min, as PIT readings from the same individual were grouped into a single visit if they occurred within 2 min of each other. While the choice of a 2-min grouping window has been validated using nest video data and nest-watches  are suggested by their breeding behavior and ecology. Compared to great- and long-tailed tits, babblers forage more distantly from the nest  conflict (Mariette and Griffith ESM 1(PDF 333 kb)


CD3+ T cell was more infiltrated in PD-1 positive, tumors with MSI and were associated with better OS, indicating an adaptive immune resistance may be occurring. Further research into the cancer immunotherapy markers of SRCC immune microenvironment may highlight targets for immunotherapy.Recent data supports a potentially significant role for immune checkpoint inhibitors in the treatment of gastric cancer. However, there are few data on the clinical implications of immunotherapy markers in gastric signet-ring cell carcinoma (SRCC). We evaluated the expression of programmed cell death protein-1 (PD-1), programmed cell death ligand 1(PD-L1), infiltration by CD3+ T cell, microsatellite instability (MSI), and Epstein-Barr Virus (EBV), and the relationship of each factor to survival in 89 advanced SRCC patients. All patients received 5-FU-based first-line chemotherapy. PD-L1 and PD-1 were expressed in 40.4% and 18.0% of the patients, respectively. There was a significant correlation between PD-L1 and PD-1 expression . There was loss of at least 1 of the 4 DNA mismatch repair (DNA-MMR) gene proteins in 32.6% of samples. Only 1 case out of 89 was EBV positive, with concurrent PD-L1 positivity, a high degree of CD3+ T cell infiltration and MSI. Increased CD3+ T cells numbers was associated with increased PD-1 expression  and MSI status . High CD3+ T cell infiltration was related to better OS (23.7 months, 95% CI: 19.0-38.0  Gastric cancer (GC) consists 8% of general tumor cases and 10% of tumor-associated deaths worldwide, having an estimated 989,600 new occurred incidence and 738,000 deaths annually. Approximately 40% of GC in China, with patients commonly presenting with advanced disease . ChemothAmong recent years, immunotherapeutic agents targeting immunosuppressive proteins have attracted more and more attention in cancer therapy, especially those targeting the PD-1 pathway . Immune Tumor-infiltrating lymphocytes (TILs) have been detected among several kinds of tumors, thought to be a convincing prognostic biomarker in cancer –18. ImmuUsing data from The Cancer Genome Atlas (TCGA) , a recenEBV+ GCs have thought to be a special subgroup with distinct clinicopathologic characteristics and better prognosis , resultsMSI tumors have attracted much attention on account of favorable outcomes compared with those MSS tumors . MSI tumThe immune response seems to be important in the progression of GC. However, few studies have evaluated the prognostic value of PD-L1, PD-1, CD3+T cells, MSI, and EBV in SRCC patients.Given the therapeutic potential of PD-L1/PD-1-based therapies, we sought to evaluate PD-L1 and PD-1 expression in SRCC. We examined the CD3+ TILs infiltration in the tumor microenvironment, as well as EBV infection and MSI phenotype. Finally, the prognostic values of PD-L1, PD-1, CD3+ TILs, and MSI phenotype were evaluated.Of the 89 patients sampled, 75.3% of the patients were male. The median age was 57 years . At the time of initial diagnosis, 80 (89.9%) were classified as stage III-13 (13.5%) were stage IIIA, 47 (52.8%) were stage IIIB, 21 (23.6%) were stage IIIC. Nine patients (10.1%) were classified as stage IV. Patients at stage III were all administrated first-line 5-FU-based adjuvant chemotherapy after D2 gastrectomy, while patients at stage IV were treated by first-line 5-FU-based palliative chemotherapy. Overall survival data was available in 86 (96.6%) cases. The mean follow-up period was 28.0 months . To our best knowledge, there is no research on the relationship of biomarkers and chemotherapy efficacy. Therefore, in the present study, we didn't estimate correlation between markers and chemotherapy efficacy.The expression levels of PD-L1 and PD-1 were evaluated by immunohistochemical staining in all 89 samples. The staining of PD-L1 and PD-1 was detected in membrane and cytoplasm of cells. PD-L1-positive staining was visible in tumor, stromal, and immune cells, but not in non-neoplastic gastric epithelium, while PD-1-positive staining was seen in TILs  samples had a decreased, missing, or not evaluable immunostain of any of the 4 DNA-MMR proteins. Among the 29 samples, there was a loss of MLH1 and/or PMS2 in 25 (86.2%) cases, and 8 (27.6%) cases had complete loss of expression of MSH2 and/or MSH6  in all 86 patients. When OS was separated by cancer stage, the OS was 21.0 months (95%CI: 17.5-26.5 months) for those at stage III and 4.2 months (95%CI: 3.0-19.8 months) for patients at stage IV (Table The median OS was 21.0 months (95% CI: 18.0-32.6 months) in PD-L1-positive cases and 17.5 months in PD-L1-negative cases (95% CI: 13.5-24.8 months). The expression of PD-L1 was related with better OS in SRCC patients, but did not have significant difference (p=0.516) Figure . SimilarTo test the independent prognostic effect of these clinical factors, Cox proportional hazard models were applied, adjusting for age, gender, PD-1 and PD-1 expression, CD3+ TILs infiltration, and MSI phenotype. As shown in Table In European Society for Medical Oncology annual meeting of 2014, the anti-PD-L1 antibody, pembrolizumab, attracted lots of attention because of its safety and efficacy in treatment for GC . It has In present study, PD-L1 and PD-1 expressed in 40.4% and 18.0 % of the cases , respectively. This rate of PD-L1 positivity is similar to that found in a report informed in the American Society of Clinical Oncology of 2015, in which PD-L1 was detected in 40% of advanced GC received the treatment of the PD-1 monoclonal antibody, pembrolizumab  . PD-L1-pIn breast cancer, it has been reported that the PD-L1 expression was closely related to the number of infiltrated regulatory T-cells (T-regs). Therefore, PD-L1 and T-regs build a suppressive immune microenvironment . In melaRecent research has showed a daedal association between immune cells and tumor cells for tumor development . InfiltrSome previous reports have shown that EBV+ GCs have particularly abundant PD-L1-positive tumor cells and TILs. Derks et al.  discoverMSI tumors are known as abundant infiltration of dense immune cell and cytokine-rich microenvironment –46, and In conclusion, this study demonstrated that PD-L1 and PD-1 are expressed in some patients with SRCC; however, no significant differences had been showed in clinicopathological characteristics or clinical outcomes based on PD-L1, PD-1 expression. Our findings also suggest that CD3+ TIL levels in the tumor microenvironment predict a significant difference in OS for patients with SRCC. Interestingly, we detected more infiltration of CD3+ TILs in patients with PD-L1, PD-1 positive, EBV-infected, and MSI SRCC. To our best knowledge, few studies have evaluated the prognostic value of these biomarkers in SRCC patients. Our current findings support that TILs can stimulate host immune response and suppress tumor progression. Thus, TILs in SRCC may be a biomarker for predicting patient outcome. These biomarkers, including PD-1/PD-L1, EBV, TILs, and MSI status, showed more information about the state of immune system in SRCC. These histological characteristics should be considered when selecting subgroup of patients which might benefit from PD-L1 inhibition treatment.We identified 89 patients with SRCC from over 400 patients with gastric cancer. Specimens with histology of SRCC were collected at the Nanjing Drum Tower Hospital. None of the patients had radiotherapy, chemotherapy or other medical intervention before specimen collection. Patients were excluded if they had a history of an autoimmune disease or were taking immunosuppressive drugs. This study was approved by the Ethics Committee of Nanjing Drum Tower Hospital. Written informed consent was obtained from each individual.The expression of PD-1, PD-L1, CD3, mutL homologue 1 (MLH1), mutS homologue 2 (MSH2), mutS homologue 6 (MSH6) and postmeiotic segregation increased 2 (PMS2) in tumors was evaluated via immunohistochemical analysis. Briefly, 2-μm-thick sections were deparaffinized in xylene and rehydrated in graded alcohol. Endogenous peroxidase activity was blocked by absolute methanol containing 3% hydrogen peroxide for 15 min. The sections were microwaved for 10 min with citrate buffer for antigen retrieval, and then incubated in 1 % horse serum in Tris-buffered saline for blocking nonspecific binding. After blocking, sections were incubated overnight at 4°C with Commercially available primary antibodies using the manufacturer's instructions . Samples were then incubated with secondary antibodies for 30 min at room temperature. Then signals were visualized by incubation with 3-3′-diaminobenzidine for 5 min. The slides were then counterstained with hematoxylin before mounting.The PD-1, PD-L1 is observable in the cytoplasm or on the membrane of the tumor cell or the TILs. The immunoreactivity of PD-1, PD-L1 was evaluated semi-quantitatively according to the percentage and intensity of positive cells. Four grades were used to differentiate the intensity of the staining: negative, weak, moderate, or strong. Specimens in which PD-1 or PD-L1 were observed in more than 1% of tumor cells or immune cells were considered PD-1 or PD-L1 positive.CD3 was detected in the nuclei of the TILs. The distribution of CD3+ TILs was observed in the areas with the highest density of TILs first at low magnification. The amount of positive TILs was then recorded at high magnification (HPF 400×magnification). The number of CD3+ TILs was determined in 30 random high power fields in each section.Staining for MLH1, PMS2, MSH2, and MSH6 was regarded as positive when the tumor nuclei stained positively with the same intensity as the control tissue . To achieve a high sensitivity for detecting MSI in GC, any case with loss or absence of nuclear immunostaining or reduced protein expression, when compared with normal tissue, was submitted for molecular analysis of microsatellite status. Nuclear negativity and a nonconvincing immunostain of the internal positive control tissue were classified as a “missing value” (nonimmunoreactivity of single antigens).Two independent observers carried out the immunohistochemistry analysis; both observers were blinded to any prior information on clinical or pathological characteristics of the patients' samples. If there was discrepancy between the analyses performed by the two observers, these slides were reinvestigated by both investigators using a multiheaded microscope and a consensual decision was reached.in-situ hybridization using fluorescein-labeled oligonucleotide probes . When >20% of the tumor cells showed staining for EBER, the case was defined as EBER positive.EBV-encoded RNA (EBER) was detected via chromogenic Pearson's chi-square test and Fisher's exact test were used to determine the correlation between PD-1, PD-L1 expression, infiltration by CD3+TIL, MSI phenotype, and other clinicopathological characteristics. Overall survival (OS) was measured from the time of the operation to death from any cause or the last follow-up. Survival curves were obtained using the Kaplan–Meier method and compared with the log-rank test. A univariate analysis was performed to evaluate for survival differences based on gender, age, tumor stage, the expression levels of PD-L1, PD-1, infiltrating CD3+ TILs, and MSI phenotype. A multivariate analysis was performed. The hazard ratios (HR) and 95% confidence intervals (CIs) were estimated with the use of a forward and backward Cox proportional hazards model. The correlations between the expression levels of PD-1, PD-L1, CD3+ T cells infiltration, and MSI phenotype were analyzed by Pearson correlation analysis. A p-value<0.05 was considered significant. Statistical analysis was performed using the Statistical Package for the Social Sciences for Windows version 19 .


Biodeterioration is a major problem for the conservation of cultural heritage materials. We provide a new and original approach to analyzing changes in patterns of colonization  by biological agents responsible for the deterioration of outdoor stone materials. Here we analyzed BPs of four Khmer temples in Angkor (Cambodia) exposed to variable environmental conditions, using qualitative ecological assessments and statistical approaches. The statistical analyses supported the findings obtained with the qualitative approach. Both approaches provided additional information not otherwise available using one single method. Our results indicate that studies on biodeterioration can benefit from integrating diverse methods so that conservation efforts might become more precise and effective. Different biological agents can colonize stone artifacts and monuments in archaeological sites causing their biodeterioration12356788Research on colonization dynamics and BPs is generally qualitative, with little attention to ecological considerations 1114within biological communities. Here we show how it can be successfully applied also to investigate non-random relationships between biological communities.Most studies have used the co-occurrence analysis mainly to investigate the importance of ecological competition in the establishment of biological communities .Here we combine a qualitative assessment with various statistical approaches using temples in the archaeological site of Angkor (Cambodia) as test sites. We test the possibility of obtaining a model suitable to predict the changes occurring in BPs in relation with changes in environmental factors using an ecological analysis of the main biological communities and related BPs that are detectable in exposed stone surfaces of four temples   with difThe ecological niche of colonizing species determines the colonization dynamics. The ecological requirements that shape species niches can be seen as limiting factors, i.e., those environmental conditions and/or resources present in a quantity proximal to the upper or lower tolerance limit of an organism25122930The communities responsible for biological colonization on specific substrata tend to be similar among sites with similar environmental/climatic conditions. In fact, cyanobacteria, algae, and lichens, and sometimes also mosses and vascular plants form colonizing communities that tend to dominate on exposed stones in favorable climates, with changes in species that depend on the surrounding environmental factors. Previous research has shown that BPs of photoautotrophic communities vary mostly in relation to shadowing by forest canopy, which in turn might affect water availability (humidity and precipitation) and solar radiation, but can vary also in relation to exposure and inclination of stone surfacesIn the analyzed temples in Angkor, the distribution of various colonizing communities is strongly affected by light and water availability . In the Scytonema and Gloeocapsa) and the lichen community with Pyxine (B-E), which resulted in the co-occurrence analysis as marginally significant . Also the transitions between the two lichen communities of Pyxine-Cryptothecia (E-F) and the Lepraria lichen-mosses (D-G) both showed a non-significant p value. The identified associations among communities were then used to build a weighted network of interactions that provided an effective way to visualize the ecological relationships between transitions   being slightly more informative than water (SC = 0.10).Our data showed that, for the temples in Angkor, light and water are the most critical factors in explaining the distribution of the colonizing communities. In particular, we found that the combination among these environmental factors had a very high explanatory power, having an ‘overall score’ (Pyxine sp. and Cryptothecia sp. (E-F), we noted in the field that they often showed a certain overlapping due to their similar ecological spaces (niches) and this might have altered the analysis of their co-occurrence.The statistical analyses supported the findings obtained with the qualitative assessment in the field. For instance, the fact that the two transitions (D-G and E-F) had non-significant p values  is coherEndocarpon and the lichen community of Lepraria (C-D) and possibly also other two transitions in Trentepohlia - Cryptothecia (A-F); Trentepohlia- Pyxine (A-E); see also The consistency between the transitions identified in the field and those identified by the co-occurrence analysis indicates that both the ecological qualitative assessments and the co-occurrence analysis can provide robust, complementary results. This is because even if some transitions were not observed in the field, they appear to be ecologically possible, and probably correlated to changes in additional ecological factors. For instance, some of the additional transitions determined by the co-occurrence analysis could also indicate some unusual ecological conditions, as in the case of the transition between the cyanobacteria community with The statistical analyses supported some of the findings of the qualitative assessment but also provided some additional complementary information. Our results indicated that biodeterioration studies can benefit from integrating ecological qualitative observations with quantitative ecology analytical techniques. This is true whether we consider simple presence-absence data of colonizing communities or the evaluation of ecological niches of colonizing species.Our results indicated that the spatial distribution of colonizing communities and the transitions among them are significantly affected by light and water availability. However, considering the wide range of ecological factors that could affect BPs, it would be worth exploring further ecological niche modeling techniques. This could help defining BPs at a larger scale of bioclimatic regions, i.e., by combining high-resolution climatic data and other environmental and ecological factors, creating a wider model for each bioclimatic area related to different stone materials. This research could be valuable for optimizing conservation efforts by identifying conditions with high susceptibility to BPs.The proposed ecological scheme can be a useful tool for the conservation of monuments. This scheme could allow predicting the changes of BPs when ecological factors change. Furthermore, it represents a useful tool for evaluating the possible use of indirect methods for biodeterioration control . In this way, it would be possible to fine-tune interventions creating more suitable or suboptimal microclimatic conditions for stone conservation, balancing between conditions that would cause chemical weathering and biodeterioration phenomena.In the Angkor archaeological area, we selected four temples  to have a representative sample of different forest coverage (and hence of different microclimatic conditions) . In partWe noted the environmental conditions (macroclimate and microclimate) of the archaeological site and of the examined temples. The macroclimate of the study area shows tropical monsoon characteristics. The area receives an average amount of rainfall of approximately 1,200 mm and is characterized by a wet season that starts in May and ends in November and a dry season from December to March36In total, we carried out about 200 field observations (test areas). These test areas were approximately 30 cm × 30 cm using as a centroid the transition between two communities. We randomly selected the test areas keeping in mind the variety of environmental conditions across the analyzed temples. At each test area, we visually assessed light and water availability, using synthetic indicators, as usually carried out in vegetation surveys40et al.et al.Trentepohlia sp. community, a pioneer community creating a reddish biofilm; B) Cyanobacteria biofilms of Scytonema sp. and Gloeocapsa sp., forming black patinas; C) Cyanobacteria biofilm in combination with the lichen Endocarpon sp.; various communities of lichens dominated by: D) Lepraria sp., forming greenish and powdery crusts with diffuse margins; E) Pyxine coralligera Malme forming green-greyish crusts with irregular to radiating margins; F) Cryptothecia subnidulans Stirt. forming whitish crusts; mosses (G); and vascular plant communities (H). As regards the vascular plant communities we found both perennial herbaceous ferns , and flowering plants (herbaceous and woody). The most representative annual herbaceous flowering plant was Peperomia pellucida (L.) Kunth. This species has a shallow root apparatus and it is native to American and African tropical regions (http://www.ipni.org). Among the tree species, Tetrameles nudiflora R. Br. and Ficus altissima Blume are the most representative and potentially harmful species since their roots can severely damage the masonry structure of buildings.For each temple, we identified the different colonizing communities and their BPs according to the classification used by Caneva For any colonizing community and related BPs identified on the stone surface of the temples, we took note of the environmental factors that could possibly affect themTo investigate the ecological structure for each colonizing community and related BPs observed in the field, we repeated the following procedure at each site:(a) Identification of an area without any obvious biological growth (point 0);(b) observation of the pioneer community in adjacent stone surfaces;(c) identification of the differences in environmental factor/s between the point 0 and the adjacent area with colonization;(d) observation of the transitions (ecotones) among communities, if present;(e) observation of the environmental factor/s associated with that ecotone stage.In this way, we identified the communities and related BPs, and the transitions among them. We noted all the potential differences in environmental changes for transitions found at each temple in a binary matrix. We performed at least 10 observations for each transition (if present). Observational data were then organized in a general scheme synthesizing how environmental factors affect the distribution of communities and related BPs along ecological gradients. An accurate photographic documentation complemented field notes and observations.P(j) that two communities respectively occurring at N1 and N2 sites co-occur at exactly j sites  using simple combinatorics. This probability P(j) is computed as the ratio between the number of ways in which the two units can co-occur at exactly j sites, and the total number of unique ways in which the two units can be arranged among all sites. Furthermore, this approach allows estimating the expected value of co-occurrence of two communities, i.e., to identify how many sites are expected to host both communities. Since the maximum possible value of co-occurrence is equal to the minimum of N1 and N2, the expected co-occurrence value can be obtained by simply summing up P(j) × j from j = 0 to j = min . The difference between the expected and the observed co-occurrence value can then be standardized  and used to compare co-occurrence between different pairs14To confirm the potential transitions between colonizing communities, we assessed their degree of pairwise co-occurrence using the probabilistic approach introduced by Veechet al.To assess the relative importance of water and light availability in determining the ecological requirements of the colonizing communities and related BPs we used the statistical approach described in Strona - for each community, we generated 1000 simulated associations, each occurring at an hypothetical site having a value of light exposure randomly extracted from the range min (light) – max (light), and a value of humidity (as a proxy for water availability) randomly extracted from the range min (humidity) – max (humidity).sc) value- We compared each one of these simulated associations with the light and humidity ranges of any  association, computing, for each simulated association, an “individual score” (SC) value, as the total number of random values we generated, divided by the sum of all sc values. SC can vary between 1  and 1/T, where T is the total number of considered associations .- After this procedure was replicated for each association, we computed an “overall score” , the higher is the importance of that factor for the survival of communities, and hence in determining their distribution. A high SC score indicates also the suitability of a factor for predictive models.Finally, we compared the How to cite this article: Caneva, G. et al. Combining Statistical Tools and Ecological Assessments in the Study of Biodeterioration Patterns of Stone Temples in Angkor (Cambodia). Sci. Rep.6, 32601; doi: 10.1038/srep32601 (2016).


Sir,DNAJB2) is capable of resolving aggregates caused by TDP-43 (Heat shock proteins (HSPs) are protein chaperones that play important roles in maintaining protein homeostasis. Ours and others’ research have shown that DNAJB2a .ALS is the neurodegenerative disease that most prominently affects motor neurons. It is a heterogeneous disorder where the majority of affected individuals have no family history of ALS. Nevertheless, TDP-43 proteinopathy  is found as a common phenotype in affected tissues in ALS with or without mutations in TDP-43. This pathological feature is also seen in ∼60% of FTD which supports the idea that FTD and ALS are on the same disease spectrum. We agree with Frasquetper seis sufficient to trigger TDP-43 aggregation. On the contrary, our data show that HSF1(+) still refolds TDP-43 when DNAJB2a is knocked down (unpublished data). This result suggests that there are some functional redundancy of HSP, and DNAJB2a, although is capable in refolding TDP-43, is not the only HSP for this task. Thereby, a simply loss of DNAJB2 is unlikely to cause TDP-43 proteinopathy that is associated with ALS and FTD. However, as we proposed in our original paper, it does appear that the heat shock response as a whole is compromised in ALS due to a yet to be determined cause. It will be of great interest to investigate the genetic contribution from not only DNAJB2a, but any HSPs and also HSF1.The known DNAJB2a mutations in association with dHMN are c.352 + 1G > A  to CES with additional support from The Motor Neuron Disease Association, Heaton Ellis Trust, Psychiatry Research Trust and American Amyotrophic Lateral Sclerosis Association.


Graphene-spaced magnetic systems with antiferromagnetic exchange-coupling offer exciting opportunities for emerging technologies. Unfortunately, the in-plane graphene-mediated exchange-coupling found so far is not appropriate for realistic exploitation, due to being weak, being of complex nature, or requiring low temperatures. Here we establish that ultra-thin Fe/graphene/Co films grown on Ir(111) exhibit robust perpendicular antiferromagnetic exchange-coupling, and gather a collection of magnetic properties well-suited for applications. Remarkably, the observed exchange coupling is thermally stable above room temperature, strong but field controllable, and occurs in perpendicular orientation with opposite remanent layer magnetizations. Atomistic first-principles simulations provide further ground for the feasibility of graphene-spaced antiferromagnetic coupled structures, confirming graphene’s direct role in sustaining antiferromagnetic superexchange-coupling between the magnetic films. These results provide a path for the realization of graphene-based perpendicular synthetic antiferromagnetic systems, which seem exciting for fundamental nanoscience or potential use in spintronic devices. Antiferromagnetic spintronics may pave the way to innovative information storage devices with perpendicular coupling, however experimental demonstrations are still sparse. Here, the authors demonstrate a graphene-mediated perpendicular antiferromagnetic coupling between Fe and Co layers in a Fe/graphene/Co sandwich structure. SAF structures based on metallic multilayers were initially developed in the 90s, inspired by the discovery of exchange coupling in multilayers and oscillatory magnetic interactions5. A prototypical SAF structure is composed by two ferromagnetic films that are antiferromagnetically exchange-coupled through a non-magnetic spacing material due to the Ruderman–Kittel–Kasuya–Yosida (RKKY) interaction, and have been broadly used to improve the thermal and magnetic properties of spin valves8. Furthermore, exchange-coupled magnetic layers with perpendicular magnetic anisotropy (PMA) are intensively researched for the realization of vertical magnetic tunnel junctions (MTJs)9, in views of potential application in future high-density and low current-induced magnetization-switching spintronic devices10.Antiferromagnetic (AF) systems, among which synthetic ferrimagnetic and antiferromagnetic (SFiM/SAF) structures, are receiving renewed attention for spintronic applications and magnetic information storage11, hybrid 2D-material magnetic systems are presently intensively investigated to enable new fundamental and applied developments. Remarkably, graphene/magnetic structures display among other characteristics long spin-lifetimes at room-temperature12, spin filtering14, and tunnel magneto-resistance17, which are appealing properties for a range of innovative graphene-based spintronic technologies19. Moreover, graphene has been reported to promote large PMA at the interface with magnetic thin-films21, thus possibly serving as a building-block for perpendicular spintronic devices incorporating a spacing layer with weak spin–orbit coupling. In this context, assessing the possibility to realize exchange-coupled PMA magnetic thin-films across a single-layer graphene (Gr) is of primary importance towards the realization of graphene spintronic devices. This has been recently stressed by a study of Yang et al.21 with a theoretical prediction of strong PMA and AF exchange-coupling (AFC) in Gr{Con/Gr}m multilayers. However, the graphene-mediated exchange coupling playground remains largely unexplored, and graphene-spaced systems with large PMA and robust AFC have not been yet experimentally realized, in spite of their interest in future spintronic information processing technologies. In-plane AFC through a graphene layer was early observed between a Ni thin-film and Co-porphyrin molecules at temperatures below ~200 K by Hermanns et al.22, as well as in small-area MTJ devices with a graphene tunnel barrier and ferromagnetic metal electrodes by Li et al.16. More recently, graphene-mediated AFC between magnetic bulk single crystals and magnetic adatoms was studied, and it was shown to weaken or evolve onto a complex coupling when adatoms assemble to form small clusters23. Under these conditions, up-scaling for realistic applications is challenging.With the emergence of graphene and other two-dimensional (2D) atomic-crystals with unique electronic and structural properties25. In contrast, the presence of a bulk magnetic layer dilutes the effect of interfacial contributions and eventually adds the complexity of magnetic surface closure domains. One suitable path to shift away from bulk magnetic materials is the intercalation approach widely used in the 70s–80s to tune graphite’s physical properties26. In the last few years, it has been demonstrated that a number of elements can be intercalated between a graphene layer and its supporting substrate28, forming 2D layers that are well localized, chemically, and mechanically protected29. This approach was exploited to grow ultra-thin magnetic layers below graphene, which displayed a remarkable PMA enhanced by the interaction between graphene and the underlying magnetic metal thin-film31.Moving away from bulk magnetic materials into thin film structures or nanostructures is key to achieve the development of graphene-based robust AFC systems. The implementation of magnetic ultra-thin films allows to achieve strong magnetic anisotropies and exchange coupling via interfacial engineering, and also enables the introduction of symmetry breaking Dzyaloshinskii-Moriya and Rashba interactionsHere, we show the realization of hybrid graphene-ferromagnet thin-film structures exhibiting strong perpendicular AF exchange-coupling. The Fe/Gr/Co/Ir(111) ultra-thin films investigated display a robust perpendicular AF exchange-coupling between the ferromagnetic layers mediated by the single-layer graphene spacer. Remarkably, the perpendicular AF coupling is stable above room temperature and field controllable, and displays a tunable remanent net magnetization as a function of the Fe layer thickness. Our first principle theoretical simulations confirm graphene’s direct role in establishing the AF coupling via a graphene-mediated super-exchange mechanism. These results demonstrate the realization of graphene-based synthetic AF systems, which appear of fundamental interest in nanoscience and that possess an ensemble of properties well-suited for potential applications in spintronic devices.μ0Hc = 0.27 T, together with a 92% magnetic remanence along the out-of-plane easy-axis. The monolayer (ML) Co coverage was determined on the basis of Auger electron spectroscopy measurements  sample. This is an exchange coupling strength of the same order of magnitude than that reported for conventional RKKY SAF multilayers5.A trilayer composed by an intercalated Gr/Co/Ir(111) ultra-thin structure Fig.  coupled 32.Even at the maximum available external field of 6 T, the magnetic saturation of the Fe layer is not completely reached, as evidenced by the non-horizontal slope of the Fe magnetization curve. One likely hypothesis would be that this behavior is related to considerable 3D-film morphology, giving place to a distribution of switching fields and/or weekly coupled magnetic regions or grains. Other possibilities should not be excluded, taking into account that the magnetization reversal of AF exchange-coupled systems has been demonstrated to yield complex phenomenologies30, which were e-beam deposited on top of a single high-quality layer of Gr CVD-grown in-situ on Ir(111)  Keff for the Gr/Co[1.9 ML]/Ir(111) system is estimated employing the saturation field along the hard axis Hhard and assuming a simple uniaxial anisotropy term, according to Hhard = 2Keff/μ0Msat24. This yields Keff ≲ 9MJ m−3 and a MAE per-unit-area of Keff ·t ≲ 2.6 mJ m−2, values in good agreement with previous findigs for the interfacial anisotropy of Co/Ir(111) and Gr/Co in the Gr/Co/Ir(111) system30. An enhancement of the PMA of the cobalt-graphene interface has been early reported by Vo-Van et al.20 and later confirmed for Co-intercalated graphene systems30. The intercalation process has been previously shown to take place preferentially at graphene point defects and wrinkles34, and in correspondence with Ir(111) step edges31, remarkably preserving the structural integrity of the graphene layer. Our characterization by high-resolution scanning electron microscopy (SEM) (see Supplementary Note It is worth describing here in detail the Fe/Gr/Co/Ir111) multilayer fabrication and optimization, as this path seems applicable to obtain further related FM/Gr hybrid structures with tailored properties. The growth of the Co film below the graphene layer was realized by the thermally activated intercalation of Co adatomse Gr/Co1. ML/Ir(111 multila35. The Fe/Gr/Co/Ir(111) LEED patterns analysis  due to long-range repulsive interactions37. Our computed adsorption energies for Fe on Gr/Co/Ir(111) (see below) being comparable to the Fe on Fe ones, should equally favor the low-coverage nucleation of Fe islands over the incorporation of deposited atoms atop of the already-nucleated islands, favoring a large surface coverage. Summarizing the results of our LEED data analysis together with the adsorption-energy interpretations, it seems reasonable to expect that the Fe films with equivalent thickness ranging between 2 to 4 MLs have a 3D thin film growth with a full or almost-full surface coverage, coexisting with 3D multilevel islands. It is interesting to mention that, as a result of the balance of adatom-adatom and adatom-substrate interactions, the growth morphology of Fe (and Co) layers on graphene appears controllable by deposition temperature and deposited thickness. Indeed, atomically-flat growth of magnetic overlayers on graphene has been demonstrated by pulsed laser assisted growth20, and can be ascribed to a larger instantaneous growth rate favoring islands nucleation and reducing cluster formation. These considerations suggest that the morphology of such kind of AFC Gr-based multilayers might be tailored according to the requirements set by the desired application, which can be relevant for properties such as the coercive field or the exchange coupling38.The LEED patterns provide also information on the growth morphology of the Fe overlayer in the Fe/Gr/Co/Ir(111) systems. This approach does not offer the capability of local probes with atomic resolution, but a unique statistically averaged information over a large area39. These properties can be controlled to a considerable extent by the thermal annealing temperature of the intercalation process and the total Co layer thickness. Thermally activated magnetic hardening in Co/Ir(111) films has been interpreted as a result of the partial interfacial alloying, with maximum coercive fields observed for annealing temperatures around 700 K40, in close proximity to the intercalation temperatures used here. It is thus expected that a certain amount of Co atoms have diffused and intermixed at the very Co/Ir interface, as recently evidenced by a related surface X-ray diffraction investigation of the Co intercalation on partially Gr-covered Ir(111)41. On the other hand, the preserved integrity of the graphene layer after Co intercalation , Co/Pt(111), and other related systems such as Co/Ir(111) can display strong PMA with high coercivity and remanence42. Rather than simulating this superstructure , we consider a computationally more efficient approach, where commensurability is assumed, and different stacking configurations are used to model the three principal Moiré domains sketched in the figure. On top of these, a Fe ML is considered, with Fe atoms placed on the possible A/B/C sites of the underlying Ir(111) lattice Fig. 43, as reported in Supplementary Table zC–Co interlayer distances Fig. −2, seem in excellent agreement with our experimental observations.To investigate the nature of the coupling in the graphene-based AFC structure, we performed atomistic calculations based on density functional theory, and analyzed the role played by the single-layer carbon spacer. Our calculations are performed on a model for a Fe/Gr/Co ultra-thin film stacked on a Ir111) surface, and reproduce both the AFC and the strong PMA observed in experiments. Due to the lattice mismatch between graphene and Ir(111), a Moiré pattern is expected in the heterostructure , with a lattice parameter of ~2.5 nm Fig. 42. Rathe11 surfac43, who reported a strong effect of graphene on the anisotropy of ML Co on Ir(111), with an overall reduction of the MAE and a tendency for in-plane magnetization when carbon sits on top of Co. On the contrary, our calculations show that perpendicular magnetization is preferred for both Co/Ir(111) and Gr/Co/Ir(111) heterostructures, and although the strength shows some dependence with the stacking of carbon atoms , this affects marginally the already high MAE of ML Co on Ir (1.7 mJ m−2). The MAE values resulting from our simulations are not far from the experimental values by Rougemaille et al.30 , or Vo-Van et al.20 . A graphene-induced enhancement of the PMA has been reported for graphene-covered Co trilayers by Yang et al.21, although their study do not include a Co/Ir(111) interface, nor different C stacks due to the underlying Moiré pattern. Our simulations for the case of ML Co on Ir(111) do not show a clear enhancement of the PMA due to graphene, but indicate a systematic increase in the computed PMAs when the Fe ML is placed on top of graphene, no matter what stacking (A/B/C) is considered.The origin of the large PMA in Fe/Gr/Co/Ir(111) heterostructure is analyzed from the study of the magnetic anisotropy energy (MAE) at the different interfaces. The Co/Ir(111) and Gr/Co/Ir(111) interfaces were studied by Shick et al.AB/CoB/Ir stacking, that has a short interlayer distance and a significant charge accumulation on graphene, suggesting that clustering of Fe adatoms in this region would be preferred during film growth44 (similar results are obtained for the GrBC/CoB/Ir stack). The computed Fe-ML adsorption energies, Eads, evaluated after subtracting from the total energy of each configuration the energy of the clean Gr/Co/Ir slab and the Fe ML, conform to this assumption, with GrAC being on average less favorable than GrAB or GrBC.In the following, we focus our attention on the GrJ, defined as the difference between the energies of parallel (FM) and antiparallel (AF) alignments of magnetizations in Fe and Co (J = EFM − EAF). Resulting exchange coupling energies depend on the stacking and Fe adsorption site on top of graphene, with values between 15 and 277 mJ m−2 as indicated in Supplementary Table 45. Similarly strong couplings are obtained for GrBC/CoB stacks, while the coupling is substantially reduced (even switched to FM) for GrAC/CoB, where the interlayer distance is larger, and the charge accumulation on graphene suppressed. We note that a fair comparison with the experimental estimate requires an average over the whole Moiré pattern, considering temperature effects, and taking into account possible impurities and irregularities at the interface, all of which would result in a significantly reduced value.For the three possible Fe-stacks considered, we obtain a strong interlayer exchange coupling, d states that point out of the layer plane (dyz/xz). It is known that hybridization with graphene’s 2pz states strongly affects the energy position of Co-3d states46. A J couplings. Notably, if we take the same fixed structures but removing the graphene ML  sample grown at room temperature. As the temperature is lowered, an increase in the out-of-plane Fe layer remanent magnetization, together with a notable increase in the Co coercive field, is observed. The exchange coupling Jex and μ0HAFC field  films. The AFC remains stable among the different coverages, as evidenced by the XMCD-sign inversion between Fe and Co atomic edges. Most notably, the X-ray absorption (XAS)-normalized Fe XMCD signal measured at μ0H = 0 increases monotonically with Fe coverage up to a 3.8 ML Fe layer thickness. This indicates that the remanent Fe magnetization depends on the Fe layer thickness, as evidenced by the element-specific hysteresis loops shown in Fig. 33. The XMCD sum rules can give quantitative information on the atomic magnetic moment of the probed atom. We can estimate the relative Fe and Co magnetization for the Fe/Gr/Co/Ir(111) system as MFe/MCo = tFeμFe/tCoμCo, where tFe, tCo are the layer thicknesses, and μFe, μCo are the atomic moments of Fe and Co obtained via the XMCD sum rules. In Fig. MFe/MCo ratio as a function of the Fe layer thickness. The data points are relative to the spectra reported in Fig. MFe/MCo room-temperature ratio shows a clear dependence on the Fe film thickness for thicknesses below ~10 ML’s and notably crosses the magnetic compensation condition (MFe/MCo = 1) for values close to Fe ~4 ML’s. This result provides an indication that the total magnetization can be controlled by tuning the Fe layer thickness, demonstrating that a Fe/Gr/Co/Ir(111) magnetically compensated system can be realized at room temperature. A strong variation of the MFe/MCo ratio is also observed as a function of sample temperature, as reported in Fig. To assess the feasibility of a magnetically compensated graphene-based SAF/SFiM system, we analyzed the room temperature dependence of the magnetic properties of a Fe/Gr/Co/Ir(111) heterostructure as a function of Fe top-layer thickness. We take special care in investigating two scenarios: the first, a magnetically compensated system at room temperature; and the second, a structure whose Fe top-layer magnetic moment exceeds that of the Co layer, providing an inverse scenario to that of an AF structure with a dominant PMA Co layer so far discussed. In Fig. MFe/MCo ratio takes values higher than unity, indicating that the out-of-plane remanent magnetization of Fe layer exceeds that of the Co layer. The Fe out-of-plane remanent magnetization /CoFeB MTJs48, graphene-based SAF systems present analogies but also differentiating characteristics and potential advantages by merging appealing magnetic properties with graphene unique electronic, mechanical, or thermal properties. First, the Gr spacing layer can contribute to the definition or preservation of the chemical/magnetic interfaces, acting as a barrier for diffusion preventing alloying of two neighboring materials or protecting a magnetic material14, in analogy to MgO(1–2 nm) or Al2O3 barriers6. Second, a single Gr spacing layer has been shown to perform as an effective pinhole-free spacing layer for the fabrication of MTJs, in spite of its single atom-layer character16. Also, the graphene-mediated AFC between two layers allows a SFiM or SAF structure avoiding the need for a high spin–orbit metallic barrier such as Ruthenium, or the need of a third layer for the biasing as in MTJs with MgO or Al2O3 barriers. And finally, graphene-based perpendicular SAF hybrid structures could exploit graphene unique properties such as its outstanding mechanical and thermal characteristics in the design of flexible electronic devices49 or improving thermal management strategies in devices50.When compared to commonly used in-plane SAF spin valve structures such as CoFe/Ru/CoFe51. Hence, a YSZ/Si(111) platform appears a relevant and feasible path for the scalability and monolithic silicon integration of the SAF structures investigated in this work. Regarding compatibility with device fabrication processes such as complementary metal-oxide-semiconductor (CMOS), the high CVD Gr growth temperature here used  exceed the thermal budget of CMOS manufacturing, being high enough to produce issues by diffusion or melting. Alternative graphene-growth methods such as plasma assisted52, CVD reactors14, or polycyclic aromatic hydrocarbons precursors54 enable a significant reduction of graphene-growth temperatures (700–900 K). Alternatively, in past years an increasing number of solutions have been proposed for heterogeneous Gr-Si integration, employing the clean transfer of Gr grown on other supports directly onto SiO2/Si or silicon-on-insulator substrates56. This has enabled roll-to-roll production of 30-inch graphene films for transparent electrodes57, graphene Si-CMOS hybrid Hall integrated circuits58 or Gr/CMOS integrated image sensors59, among others. It is also noteworthy that Gr-based flexible devices have been demonstrated by alternative fabrication routes to CMOS60. The field is in continuous development as a result of the strong efforts being deployed for addressing the challenges towards graphene spintronic devices19.It is worth briefly discussing potential paths and challenges for the eventual technological introduction of these structures on devices. The monolithic integration of graphene on silicon has been recently reported via an Yttrium-Stabilized-Zirconia (YSZ) buffer approach. Remarkably, the growth of Gr on Ir(111)/YSZ/Si(111) substrate is scalable in wafer size up to 4-inches and the Gr layer quality is comparable to that obtained for the CVD growth on Ir(111) single crystals9, or in all-optical switching magnetic materials61. We expect that these results will help to spark interest towards the search and discovery of related perpendicular AFC Gr-based magnetic multilayers, a class of materials largely unexplored and unexploited at present, but which could enable developments in the field of graphene spintronics.In summary, we have addressed the feasibility of Fe/Gr/Co/Ir(111) structures displaying a strong perpendicular AFC that is robust on temperature and Fe layer thickness. Atomistic calculations confirm graphene’s direct role in sustaining AF superexchange-coupling between the magnetic layers, and are in good correspondence to the experimental findings. These results demonstrate an additional class of synthetic-AF multilayered materials that, while being of fundamental interest, appear capable of providing practical magnetic devices with PMA which are potentially relevant for perpendicular magnetic recording media, perpendicular spin valves, MTJ structures−9 mbar. The Ir(111) single crystal was prepared by repeated cycles of Ar+ sputtering at 2 KeV followed by annealing at T = 1000 K. The quality of the surface was checked by LEED, giving a sharp six-fold hexagonal pattern without any presence of reconstructions or diffuse background. The Gr/Ir(111) was then prepared exposing the clean substrate held at T = 1300 K to a C2H4 residual gas atmosphere at a pressure of 2.0 × 10−6 mbar for 10 min. This procedure leads to the formation of large single-domain single-layer graphene over the whole surface area, as has been reported42 and as deduced from the Moiré LEED pattern  to molecular oxygen at a pressure of 1 × 10−6 mbar for 5 min an then checking the XAS at the Co L2,3 edge. In case of non-completed intercalation the sample was showing definite signs of Co oxidation  rod.The samples were all prepared in situ in the preparation chamber available at the XMCD magnet endstation of the BOREAS beamline at the ALBA synchrotron, with a base pressure better than 1 × 1062. All measurements were performed in situ immediately after sample preparation. The base pressure during measurements was ~1×10−10 mbar. The X-ray beam was focused to about 500 × 500 μm2, and a gold mesh has been used for incident flux signal normalization. The XAS signal was measured with a Keythley 428 current amplifier via the sample-to-ground drain current . The magnetic field was generated collinearly with the incoming X-ray direction by a superconducting vector-cryomagnet (Scientific Magnetics). The magnetization loops were measured sweeping continuously the magnetic field at a fixed speed and acquiring the absorption TEY current at the maximum of the L3 XMCD signal and at a pre-edge position in order to cancel-out any field-induced artifact in the measurements. We recall that such XMCD measurements at the Co and Fe L2,3 absorption edges provide direct element-specific information on the magnitude and sign of the projection of Co and Fe magnetizations along the beam and field direction.The XAS and magnetic circular dichroism experiments were carried out at the BOREAS beamline of the ALBA synchrotron using the fully circularly polarized X-ray beam produced by an apple-II type undulator63. The generalized gradient approximation64 for the exchange-correlation (XC) potential was considered. We used norm-conserving pseudopotentials in the separate Kleinman-Bylander65 form under the Troullier-Martins parametrization66, and to address a better description of the magnetic behavior, nonlinear core corrections were included in the XC terms67. The geometry optimizations were carried out using the conjugate gradient method at spin-polarized scalar relativistic level. A double-ζ polarized with strictly localized numerical atomic orbitals was used as basis set, and the electronic temperature-kBT in the Fermi-Dirac distribution-was set to 5 meV. The relaxed lattice parameter for the Ir(111) surface was 2.715 Å, in very good agreement with previous experimental reports. After the relaxation process the forces per atom were less than 0.01 eV Å−1. Based on the locality of the fully relativistic contribution, there are two different levels of approximations when the spin–orbit (SO) is taken into account. We have used in the present work the off-site approach68, that takes into account not only the local SO contributions to the total energy but also the neighboring interactions between atoms to obtain the total self-consistent energy. As usual, the MAE is defined as the difference in the total self-consistent energy between hard and easy magnetization directions. Within the present work, we performed an exhaustive analysis of the MAE convergence in order to achieve a tolerance below 10−5 eV. We employed around 1000 k-points in the calculations for each geometric configuration, which was sufficient to achieve the stated accuracy (Supplementary Note Our density functional based calculations were performed using the SIESTA codeThe data sets generated during and/or analyzed during the current study are available from the corresponding authors on reasonable request.Supplementary InformationPeer Review File


A high degree of crystallinity was confirmed for patterned nanoribbons before and after the in-situ experiment by selected area electron diffraction (SAED) patterns. However, the maximum local true strain of the nanoribbons was determined to be only about 3%. The simultaneously recorded low-loss electron energy loss spectrum (EELS) on the stretched nanoribbons did not reveal any bandgap opening. Density Functional Based Tight Binding (DFTB) simulation was conducted to predict a feasible bandgap opening as a function of width in GNRs at low strain. The bandgap of unstrained armchair graphene nanoribbons (AGNRs) vanished for a width of about 14.75 nm, and this critical width was reduced to 11.21 nm for a strain level of 2.2%. The measured low tensile failure strain may limit the practical capability of tuning the bandgap of patterned graphene nanostructures by strain engineering, and therefore, it should be considered in bandgap design for graphene-based electronic devices by strain engineering.The mechanical response of patterned graphene nanoribbons (GNRs) with a width less than 100 nm was studied  Graphene shows great potential in many areas due to the unique properties, particular the intriguing electronic properties7. One of the most exciting applications is in novel electronic devices, e. g., flexible electronics and high-frequency transistors10. However, the absence of a bandgap is a serious limitation for its application in transistors as a planar channel material, since switching off any kind of graphene-based field-effect transistor would be extremely difficult, not to mention the required value of ~106 for the on/off ratio in logic transistors. Therefore, bandgap engineering and increasing the on/off ratio are essential for its application in electronics. Strain engineering has been proposed to open the bandgap of graphene for a few years17, since the very strong bonding between carbon atoms is supposed to offer remarkable mechanical properties of graphene. Its intrinsic strain of 25% calculated from the experimental data (Young’s modulus E and third-order elastic modulus D) in ref. 19. Therefore, the investigation of strain engineering on patterned graphene-based nanostructures is a necessary step towards its practical application for future electronics.Graphene is an allotrope of carbon, and it is the first isolated two-dimensional crystalline materialin-situ stretched in the TEM at 80 keV. The maximum local true strain of patterned graphene nanoribbons (GNRs) is determined to be about 3%, which is much less than theoretically expected. Since such a low tensile failure strain would limit the freedom of tuning the bandgap of patterned graphene nanostructures by strain engineering, it has to be considered in designing a bandgap for graphene-based electronic devices by strain engineering. Density Functional Based Tight Binding (DFTB) simulation is conducted to predict a feasible bandgap opening as a function of width in GNRs at low strain levels.In this study, graphene is patterned to nanoribbons with a width less than 100 nm in a transmission electron microscope (TEM) at an acceleration voltage of 200 keV, and subsequently studied and 21. The high quality of the single layer graphene after transfer to SiO2/Si and to the TEM lacey grid was proven by Raman spectroscopy, selected area electron diffraction (SAED) pattern and high resolution transmission electron microscopy (HRTEM)  furnace system using copper as the catalyst and the substrateEM) Fig. . To tranEM) Fig. . The PTP−1 visible in the Raman spectrum, it is proven that the PMMA was removed by acetone and IPA. The D peak (defect peak) at about 1345 cm−1 is negligible in the measured spectrum, which demonstrates the high quality of the graphene. The intensity ratio of the 2D (about 2671 cm−1) and G (about 1582 cm−1) peaks is higher than 2, which indicates that the transferred specimen is single layer graphene.A typical Raman spectrum acquired from the transferred graphene on the target gap is shown in Fig. 22. Focused ion beam (FIB) was abandoned due to the wide lateral damage observed in our previous study23. A graphene ribbon (GR) patterned by FIB was severely damaged, as shown in Fig. The graphene transferred on the PTP device was patterned into nanoribbons Fig.  by the fin-situ stretching of GNRs was performed inside a Carl Zeiss LIBRA 200 MC Cs STEM tool at an acceleration voltage of 80 kV to reduce the knock-on damage in the graphene24, and to avoid the Cherenkov radiation in the electron energy loss spectrum (EELS)26. A Hysitron PI 95 TEM PicoIndenter with a conductive diamond flat punch indenter was used to compress the semi-circular part of the PTP device , as shown in Table in-situ test, and prove that the patterning by the focused electron beam in the TEM should not affect the mechanical response of GNRs to a significant amount. The force-engineering strain curve of the in-situ tensile test is shown in Fig. 23). Nevertheless, a strain was applied to the patterned GNRs. Representative images from the in-situ stretching test are shown in Fig. in-situ video information  to minimize the effect from the electron beam on the mechanical response of GNRs in the in-situ stretching experiment. The graphene membrane was tested to be very stable even after a few hours’ illumination using the electron beam with an illumination step 4. Therefore, the illumination of less than 5 min of high-keV electron flux should not introduce significant defects into the graphene, and consequently, it should not affect the tensile strain value of GNRs during the in-situ stretching experiment in the TEM. SAED patterns of the same sample  if a change of magnetic lenses has to be performed.One interesting phenomenon is worth to mention, i.e., that any change of magnetic lenses  in the TEM tends to break the strained GNRs and/or graphene membrane. This behavior was typically observed when the GNRs and the graphene membrane were strained by more than 1.5%. It is speculated that the change of magnetic lenses in the used TEM generates a fluctuation of the magnetic field, resulting in a current pulse in the used capacitive force/displacement transducer (PI 95 TEM PicoIndenter), which may create a displacement jump of the conductive tip. This jump would lead to an additional strain on the sample and it would break it. Therefore, any changes of magnetic lenses during the 35. Despite this effect, we did not find any fracture of the ribbon at these small strain levels. However, by extrapolating the width dependence of the bandgap  is detected when comparing to the results obtained for pure armchair edges, and a totally different non-monotonic fluctuating behavior of the bandgap after increasing the strain in small and large GNRs is observed. Similar to AGNRs, those ribbons did not break for strain levels up to 4.3%. It is speculated that the early breaking of the ribbons at low strain level observed in the in-situ experiment above is due to the presence of tiny structural defects, whose study goes beyond the scope of the present work. These tiny structural defects are inevitable in the producing process of CVD graphene and in the subsequent direct patterning process by the electron beam, which are also expected in the production line of future graphene electronics unless more appropriate approaches are developed.In addition, the strain dependence of the bandgap for GNRs with mixed zigzag and armchair edges see Fig.  were comin-situ using quantitative tensile testing in the TEM at 80 kV. A high degree of crystallinity of the patterned GNRs before and after the in-situ experiment was confirmed by SAED patterns, however, the maximum strain of the ribbons was determined to be only about 3%. The recorded low-loss EELS on the stretched ribbons did not reveal any bandgap opening for the studied GNRs. DFTB simulation was conducted to predict a feasible bandgap opening as a function of width in GNRs with low strain. The bandgap of unstrained AGNRs vanished for a width of about 14.75 nm, and this critical width reduced to 11.21 nm for a strain level of 2.2%. These data explain that no bandgap opening in strained nanoribbons with a width of 40–75 nm is detectable. In order to open a reasonable bandgap > 0.7 eV at a strain level of 2.2%, graphene should be patterned into AGNRs with a width less than 2 nm. The measured low tensile failure strain may limit the practical capability of tuning the bandgap of patterned graphene nanostructures by strain engineering, and should be considered in the bandgap design for graphene-based electronic devices by strain engineering.In summary, CVD graphene was transferred to a PTP device for graphene stretching by the modified PMMA transfer method, and it was successfully patterned into GNRs with widths less than 100 nm using a 200 keV focused electron beam in a TEM. The mechanical response of patterned GNRs was studied Supplementary informationVideo S1


Humidity measurement is an important technique for the agricultural, foods, pharmaceuticals, and chemical industries. For the sake of convenience, electrical relative humidity (RH) sensors have been widely used. These sensors need to be calibrated to ensure their accuracy and the uncertainty measurement of these sensors has become a major concern. In this study, a self-made divided-flow generator was established to calibrate two types of electrical humidity sensors. The standard reference humidity was calculated from dew-point temperature and air dry-bulb temperature measured by a chilled mirror monitor. This divided-flow generator could produce consistent result of RH measurement results. The uncertainty of the reference standard increased with the increase of RH values. The combined uncertainty with the adequate calibration equations were ranged from 0.82% to 1.45% RH for resistive humidity sensors and 0.63% to 1.4% for capacitive humidity sensors, respectively. This self-made, divided-flow generator, and calibration method are cheap, time-saving, and easy to be used. Thus, the proposed approach can easily be applied in research laboratories. Humidity measurement is an important technique for various industries. Humidity affects the air properties and materials that come into contact with air. The processes of manufacturing, storing, and testing are related to humidity . AccuratAir humidity can be expressed as dew point temperature, absolute humidity and relative humidity. Relative humidity (RH) is the common term used to express the amount of the vapor contained in a moist air. Because they offer convenient and real-time measurement, electrical RH sensors have been widely used in various industries . These sThere are four types of humidity standard generator systems: fixed-point humidity systems, two-pressure humidity generators, two-temperature humidity generators, and two-flow humidity generators .The fixed relative humidity point can easily be made with a saturated salt solution . SeveralIn the two-pressure method, an air stream at higher pressure than atmosphere air is saturated at a pre-set temperature and then be thermally expanded into a test chamber at that air pressure. The relative humidity is calculated by the pressure and temperature values of the test chamber. The uncertainty analysis of this kind of humidity generator has been introduced . HeinoneThe theory of the two-temperature method is to saturate the air with water vapor at a fixed temperature and then to increase the air temperature to a presetting value. Heinonen  discusseIn the two-flow method, two streams are mixed in the test chamber to produce the different humidity levels. One stream is absolute dry gas and the other is saturated with water. The reference humidity in the test chamber is calculated from the flow rates of the two gas streams. The key of the technique is to control the ratio of the two flow rates. Takahashi and Kitano  proposed2 ceramic nanofibers have been fabricated and served as the humidity sensing element to observe the effect of relative humidity on electrical resistance of sensing element in the humidity ranged from 0 to 100% RH [The accuracy of commercial electrical sensors is usually evaluated by the secondary standard humidity value. The dew-point temperature is the common standard. Recently, the measurement uncertainty of commercial electrical sensors has become a matter of concern . The def 100% RH .The two-flow humidity generator method is widely used in many testing and calibration laboratories. However, the cost is still expensive and this system is complicated, especially when it comes to precisely controlling the flow rates of dry and saturated stream. In this study, a self-made divided-flow humidity generator system was established to calibrate two types of electrical humidity sensors. A dew-point hygrometer was served as the reference standard. The measurement uncertainties of these humidity sensors were evaluated with the ISO GUM.d and air temperature, Ta serve as the standard values to establish the calibration equations for resistive-type and capacitance-type sensors.d) is the saturated vapor pressure of Td and Pvs(Ta) is the air temperature Ta.The RH values calculated from the dew point temperature, TThe unit of T is Kelvin temperatureref) is calculated as follows:a) and u(Td) are the uncertainly of Ta and Td.The uncertainty of the reference standard, u was used as the RH standard reference for calibration. The air temperature sensor used a platinum resistive thermometer. The accuracy of this thermometer was 0.1 °C after calibration. Two types of hygrometers, a THT-B11 resistive hygrometer  and a H51P-143A capacitive humidity transmitter , were used to establish its calibration equations and calculate their measurement uncertainties. The specifications of these sensors are listed in The calibration of two types of electrical hygrometers at the temperatures ranging from 20 °C to 25 °C was carried out. The mass flow controllers were manually adjusted. As the humidity environment in the calibrated chamber was stable, the reading values of the resistive RH sensor, capacitive RH sensor, and dew-point temperature of a chilled mirror monitor were recorded. Then two mass flow controllers were adjusted to the next RH level. It took three to four hours to finish the whole calibration process from low to high RH level. Three repetitions were performed for the calibration.i is the standard RH values and xi is the reading values of sensors.The establishment of the calibration equation builds the relationship between the standard humidity values and the reading values of sensors. There are two statistical ways to establish the calibration equations: the classical method and inverse method. Because the predicted uncertainty of the inverse method is significantly lower than that of the classical method , the invi and xi have a linear relationship, then0 and b1 are constants.If yi and xi have a polynomial relationship, then0, c1, …,cn are constants.If yR2 and standard of the estimated value s. The suitable order of the polynomial equation was tested by the t-value of the parameter values of each equation. The qualitative criteria are the residual plots. For a suitable equation, the data distribution of residual values should be a horizontal band centered on zero.The calibration equation was analyzed by using of SigmaPlot v12.2 . The quantitative criteria to compare the fitting performance of calibration equations were the coefficient of determination The relationship between reading values of resistive humidity sensors and the standard reference values calculated from the dew-point temperature and air temperature measured by chilled mirror monitor are presented in The calibration equation is established by regression analysis.a.Linear equationR2 was found, the residual plots indicated that the linear calibration equation was not a suitable equation.The residual plots of this linear equation are presented in b.3rd order polynomial equationt-tests of parameters and residual plots, this equation was adequate.The result for the second order polynomial equation presented a systematic pattern (not presented in the figure). The residual plot for the 3rd order polynomial equation is shown in The relationship between the reading values of capacitive humidity sensors and the standard reference values is shown in a.Linear equationb.3rd order polynomial equationThe residual plots of the linear equation for the capacitive humidity sensor shown in t-tests of parameters and residual plots  and (4). The uref values at different temperatures have the relationships with relative humidity :res is the reading RH values of the resistive sensor.The uncertainty of the resistive humidity sensor for calibration equations, uThe type B uncertainty analysis for resistive humidity sensor is computed by Equations (6) and (7).a.Uncertainty from nonlinearity and repeatabilityb.Uncertainty from resolutionc-res, is calculated by Equation (9). The uc-res value for the restive humidity sensors used two calibration equations are presented in ref, reaches higher values in high RH ranges :cap is the reading RH values of the capacitive sensor.The uncertainty of the capacitive humidity sensor, uThe type B uncertainty analysis for the capacitive humidity sensor is computed as follows:Uncertainty from nonlinearity and repeatabilityUncertainty from resolutionc-cap, as calculated by Equation (9), is shown in The combined standard uncertainty of the capacitive humidity sensor, uref calculated by Equations (3)–(5) are shown in ref value was < 0.7% when the RH value < 80% and less than 1.0% in the RH range higher than 80%. In this condition, the combined standard uncertainty for the two calibration equations and for two types of electrical humidity sensors are presented in c-ref could be < 1.2% and uc-cap < 1.05% at 90% RH. At 70% RH, uc-ref could be < 1.0% and uc-cap < 0.8%.The main sources of the combined uncertainty were the standard reference and calibration equations. In this study, the reading values of a commercial chilled-mirror monitor were used as the standard reference. The accuracy of this dew-point temperature was ±0.2 °C, according to the specifications of the manufacture. For a dew-point temperature monitor with ±0.1 °C accuracy, the uA previous study reported the uncertainty of humidity sensors at 60% RH, as evaluated by a home-made divided-flow generator . The estIn another works, two types of electrical humidity sensors were calibrated by several saturated salt solutions . The comOrlando et al.  mentioneIn this study, the uncertainty of the reference standard increased with the increase of RH value. Therefore, the combined measurement uncertainty of the two types of electrical hygrometers was > 1.0% RH when the RH reading values > 70%. The uncertainty of the humidity environment produced by saturated salt solutions were < 0.55% . HoweverIn this study, a self-made divided-flow generator was established to produce a humidity environment for the calibration of two types of electrical humidity sensors. The dew-point temperature and air dry-bulb temperature of a chilled mirror monitor was measured to calculate the relative humidity value. This value then served as the standard reference humidity for calibration. The uncertainties of two types of electrical humidity sensors were evaluated and compared.The consistency of the data distribution between the reading values of resistive and capacitive humidity sensors and standard reference values showed the usefulness of this divided-flow generator. The uncertainty of the reference standard increased with the increase of RH values. The combined uncertainty of the adequate calibration equations were ranged from 0.82% to 1.45 % RH for the resistive humidity sensor and 0.63% to 1.4% for the capacitive humidity sensor, respectively. When we used a more accurate dew-point temperature monitor, the combined uncertainty of the two types of electrical humidity sensors could be reduced to 1.0% RH. Because the self-made divided-flow generator and calibration method are cheap, time-saving, and easy to be used, therefore, the proposed approach can easily be established and applied in a research laboratories.