Social coordination can provide optimal solutions to many kinds of group dilemmas, and non-human subjects have been shown to perform single actions successively or simultaneously with partners to maximize food rewards in a variety of experimental settings. Less attention has been given to showing how animals are able to produce multiple (rather than single) intermixed and co-regulated actions, even though many species’ signal transmissions and social interactions rely on extended bouts of coordinated turn-taking. Here we report on coordination behaviour in three pairs of chimpanzees (mother/offspring dyads) during an experimentally induced turn-taking scenario. Participants were given a “shared” version of a computer-based serial ordering task that they had previously mastered individually. We found that minimal trial-and-error learning was necessary for the participants to solve the new social version of the task, and that information flow was more pronounced from mothers toward offspring than the reverse, mirroring characteristics of social learning in wild chimpanzees. Our experiment introduces a novel paradigm for studying behavioural coordination in non-humans, able to yield insights into the evolution of turn-taking which underlies a range of social interactions, including communication and language. Conversations require participants to take turns by temporally modulating their behaviour in accordance with others’ speaking and listening cues4. Turn-taking behaviours are also suggested to be an important feature of early language development5 and the acquisition of a theory of mind7. In addition to humans, many social species are known to rely on turn-taking processes to facilitate their signal transmissions and other collective activities15. Given the ecological importance of coordination in animal communication, as well as in human cognitive development and routine social interaction, a key challenge for comparative psychology is to determine the proximate mechanisms that underlie turn-taking behaviours across different species.If “two heads are better than one”, it is primarily because those heads are capable of effectively coordinating their behaviour to achieve a common goal. Coordination between humans is known to be the optimal solution to many kinds of social dilemmas18, form coalitions to guard potential mates19, jointly patrol their territories20, and coordinate risky road crossings with dominant males acting as crossing guards21. In laboratory settings, chimpanzees have been shown to coordinate their behaviours to solve a variety of social dilemmas that require pairs of participants to perform one action each simultaneously23 or successively28. To examine coordinated turn-taking, additional interaction scenarios are needed that aim to require participants to make an extended series of inter-dependent actions in response to the behaviour of their partner.Among species that display a suite of coordinated behaviours, chimpanzees, the closest extant relatives of humans, are known in the wild to hunt cooperatively30, involving stimulus sets comprised of consecutive as well as successive but non-consecutive numerals, but they had always completed them individually and had never experienced the shared version of the task.We therefore developed a multiple-action task to test chimpanzees’ ability to coordinate actions in an experimentally induced turn-taking scenario. Three chimpanzee mother/offspring dyads were given a novel “shared” serial ordering task that required them to alternately press numerical stimuli in ascending order on a touch-panel screen. All participants were experts at such numerical ordering tasks from their prior experimental historyOur results showed that the chimpanzees succeeded already in the earliest phases of the shared task with minimal trial-and-error corrections, suggesting that a propensity for coordinated turn-taking was already part of their social cognitive repertoire rather than the result of instrumental conditioning. Furthermore, offspring were quicker to take cues marking their turn from their mothers than vice versa, suggestive of asymmetries in attending to a partner’s behaviour as a function of dyadic role.31. Numeral sets were presented at random locations across the whole of the screen, thereby requiring the subjects to alternately touch them in ascending order . It was hypothesized that had the subjects been ignoring their partner’s behaviour and relying instead on the mere disappearance of the numerals during the social task, their accuracy rates, types of errors, and response latencies would not change significantly when confronted with the automated partner.Was this truly a “social” task? Without any real social coordination, animals can be trained to behave as if they modify their behaviour based on another individual’s response through simple conditional associations1,90 = 19.64, P = 0.007). Examining the data in Fig. Contrary to the predictions of the non-social hypothesis, all subjects experienced a decrement in performance during the initial presentation of the automated condition Fig. . A repeaTo determine if the timing of touching behaviour was affected by the partner’s direct involvement in the task, the mean response latencies of touch types (stay and switch) by each subject in both conditions  were analysed statistically. A LMM on offspring response latency revealed a significant interaction between touch type and condition , and a second LMM on mothers also revealed an interaction between touch type and condition  but in a different direction, as evidenced by the opposite signs for the coefficients provided by the two models for the interaction term Fig. . In both36. Efforts were made to mitigate the impact of such effects by keeping the partner during the automated condition situated in the immediate vicinity of the subject , albeit not in the adjacent touch-panel booth due to logistical constraints . Future studies that elaborate on the computerized partner method developed in this study may help to elucidate the impact of these potential confounds.The differences in response latencies and performance rates between the social and automated conditions suggest that for both mothers and offspring the presence of a partner had significant, yet different, effects on their strategy for solving the task. An alternative explanation is that the differences may have largely been due to an unexpected change in procedure , and that such a change was likely to have been enhanced by the higher amount of training and exposure to the social task compared to the automated task. This explanation cannot be ruled out, although steps were taken to keep the task itself as consistent as possible between the social and automated conditions , so that the major procedural change was limited to the absence of the partner’s behavioural cues. Along these same lines, a further consideration involving the partner’s absence was the possibility of confounding social facilitation effects, which would have posed an interference if the presence of the partner in the adjacent touch-panel booth had enhanced the ability of subjects to solve the task by increasing their arousal, motivation, attention, and engagement levels39, as well as in a recent study using chimpanzee pairs by Fletcher and colleagues40 that focused on the topic of role-comprehension. In that study, pairs of individuals alternated actions that caused a marble to roll between them through a shared puzzle box. The distributions of actions were always strictly alternating (self-other-self-other), and the location of the marble served as a salient cue for when each individual should act, so an examination of social turn-taking was limited. In the current study, there were seventy different permutations of self-other action sequences, and there was no simple external cue to guide behaviour, because the decision to press a given numeral at a given time needed to come from an associative link between the visual input of the numerals remaining on the screen, and the subjects’ internal representations of the numerals’ order.Shared instrumental actions over multiple bouts of behaviour have been previously examined in studies on other non-human primate species41 or specific rope pulls42 for achieving optimal food rewards. Chimpanzees in these studies failed to jointly establish a robust turn-taking heuristic across multiple trials, though it should be noted that rewards were delivered after each individual action. In the current experiment multiple actions were required of each individual before any food was dispensed.Turn-taking in chimpanzees has also been examined within the context of reciprocal food sharing, with tasks involving a requisite alternation of token exchanges44. Findings from these studies point to a set of turn-taking characteristics that are generally consistent across cultures4, such as the temporal distributions of verbal turns and the gaps between them43, as well as neural evidence of complex multitasking44, since during conversation the production of a response temporally overlaps with the comprehension of a turn. Such findings are suggestive of an innate interactional framework that underpins language use and development, and suggest that communicative turn-taking behaviours may be an evolutionary precursor to language44. Furthermore, recent work has begun to emphasize the importance of considering multimodal signal production and reception for a fuller understanding of turn-taking during social interaction in animal communication45.For humans, informal verbal interactions constitute a core aspect of social life, and it follows that the empirical study of “turn-taking” has its origins in the fields of sociology and linguistics. Prior research in these fields has modelled the structure of conversational exchanges, compared them across cultures, and examined their neural and cognitive underpinnings46 presented evidence for turn-taking in wild chimpanzee and bonobo mother/offspring dyads prior to joint travel episodes. The chimpanzees and bonobos observed used bidirectional gestural signals in immediate, overlapping, and delayed sequences during exchanges, and their cooperative turn-taking exhibited many similarities to human conversational exchanges. Extended bouts of alternating actions have also been observed during routine social activities of chimpanzees including reciprocal grooming, play chasing and wrestling48. Many other animal species whose communications are transmitted in a back-and-forth manner also display turn-taking coordination14. Within the primate lineage, it has been suggested by Takahashi and colleagues14 that the capacity for vocal turn-taking found in humans and marmosets may have resulted from a highly pro-social temperament composed of cooperative breeding habits, strong social bonding, high intragroup tolerance, cooperative problem-solving, and active food sharing. Chimpanzees display many of these prosocial tendencies49 , with the major exception being cooperative breeding, though some cases of alloparenting have been observed in the wild52. In light of a “by-product of selection for temperament” hypothesis14, the presence in chimpanzees of turn-taking for shared instrumental actions is consistent with a temperament that does not involve cooperative breeding habits but which is otherwise highly prosocial.Phylogenetic comparisons offer to further elucidate the evolutionary roots of turn taking in a communicative context. A recent study by Fröhlich and colleagues53 and the wild54. Indeed, they are also in line with patterns of communicative turn-taking in wild chimpanzees and bonobos, where overlapping responses were found to be more common in infants and delayed responses were more frequent in mothers46. Furthermore, the findings may provide insights into both the proximate mechanism underlying the turn-taking behaviour itself, and its implications for group-level characteristics of social information flow in wild chimpanzee communities. First, in regards to the former, while the quick response latency of offspring during the social task may suggest that young chimpanzees were comparatively more adept at solving cognitive puzzles, the latency results from the automated condition (in which mothers were quicker than offspring on switch touches) oppose this notion. Instead, we suggest that this particular discrepancy might be related to the mothers’ longer experimental history with non-shared numerical sequencing tasks, which might have interfered with their ability to adapt to the shared version. A recent study55 on the neural mechanisms underlying human turn-taking behaviour during piano duets showed that transcranial magnetic stimulation to the dorsal premotor cortex disrupted players’ performance only if they had rehearsed their partner’s role beforehand, but not if no prior rehearsal had taken place. The authors suggest that coordinated turn-taking might therefore involve acting on predictions of what a partner will do based on one’s own experience with carrying out the action sequence in question . In the current study, mothers with more experience than their offspring at completing numerical sequencing tasks may have been assigning a similar prediction heuristic to their offspring’s behaviour, and experienced greater temporal disruptions when their partners’ behaviour did not match the timing of their own extensively rehearsed actions.Our findings that offspring were particularly responsive to the behaviour of their mothers (more so than the reverse) echo previous observations on the existence of attentional asymmetries both in captivity58. Specifically, the “master-apprenticeship” hypothesis states that young chimpanzees acquire tool use behaviours through continual observation of their mothers, who perform those behaviours in sight of their offspring without actively teaching57. Results from the current study may shed light on how information flows in a similar manner through mother-offspring dyads that are not engaged in social learning per se, but are rather engaging in a joint endeavour that requires coordination over a sequence of actions. Possible future studies involving a greater variety of subject pairings  should provide more conclusive insights into the precision, speed, and direction of social information flow within chimpanzee groups.Second, it is possible that the asymmetry in tracking social cues we documented is also an inherent property of age-related differences in chimpanzee social attention and cognition, and that its implications thus include complex community-level processes. For example, prior studies of wild chimpanzee material cultures have posited that directed social learning underlies the maintenance of community-specific tool use traditionsPan troglodytes) served as subjects: three mothers named Ai (age 35), Chloe (age 32), Pan (age 29), and their respective offspring, named Ayumu , Cleo , and Pal . None of the mothers had any other offspring, and none of the offspring were themselves mothers. Mothers were always paired with their own offspring throughout all experimental sessions. We used all available mother/infant pairs at the facility that were able to perform the task. We limited pairing to mother-offspring dyads for logistical reasons, as it best ensured that subjects would be willing to perform a joint task. While our sample size was small, it nonetheless provided three independent replicates for a highly sophisticated task that required substantial pre-existing competence at handling numerical sequences.Six chimpanzees . A single 22-inch touch-panel (3M C2254PW) was located along the joint wall of the twin booth such that each subject had tactile access to half of the screen, and visual access to the whole screen. Food rewards for correct trials (8 mm apple cubes) were delivered automatically by two universal feeders . The experimental task required the participants to touch between two and eight numerals in ascending order. During the eight-numeral task, each subject was given four numerals to press, and every possible way of dividing the eight numerals into two groups of four and spreading them across the two halves of the screen was accounted for, resulting in seventy possible permutations or “trial types”. Some of these trial types followed a strictly alternating “back and forth” pattern, while others required subjects to touch multiple numerals in sequence before it was the other subject’s turn. The locations of numerals on each half of the screen were distributed randomly inside a 5-by-10 grid. For the eight-numeral task, each subject pair participated in 32 sessions of 72 trials each , followed by eight sessions with only one member of the pair (automated condition). In the automated condition, each subject participated individually, and numerals disappeared from the partner’s side automatically, according to a pre-programmed schedule. This schedule was based on the partner’s average response latency during the social condition for each number in each of the seventy possible trial types. All methods involving the use of chimpanzee participants for this study were carried out in accordance with the 3rd edition of the lme4 package59. The outputs of all models are included in the Supplementary Information (Supplementary Table The effects of dyadic role (mother or offspring), response type (switch or stay) and experimental condition  on subjects’ response latencies were analysed using Linear Mixed-effects Models (LMMs). Dyadic role, response type, numeral, pair, and condition were included as fixed effects, and subject ID was included as a random effect, nested in dyadic role. When models included interaction terms , the significance of these terms were further assessed through maximum-likelihood comparisons to models containing the main effects but not the interactions. A repeated measures ANOVA was used to test for differences in subjects’ accuracy between the last eight blocks of the social condition and the first eight blocks of the automated condition. Response latency data were log-transformed and accuracy data were arcsine transformed to meet assumptions of parametric analysis; homogeneity of variance and normality of error were confirmed for all models following the transformations. All statistical analyses were performed in R , using the Supplementary InformationVideoS1


Renal cell carcinoma is one of the most common urological tumors. The role of programmed cell death 1 ligand 1 (PD-L1) in renal cell carcinomas in predicting outcome of the patients is yet unclear. We analyzed the clinical and RNA-seq data of 522 kidney clear cell cancer, 259 kidney papillary cell carcinoma and 66 kidney chromophobe patients from The Cancer Genome Atlas (TCGA) database. In kidney clear cell cancer patients with high PD-L1 mRNA level and low PD-L1 mRNA level in tumors, the median overall survival periods were 45.0 and 37.1 months respectively (p=0.002). Multivariate Cox regression tests found that PD-L1 mRNA level in tumor was an independent predictor for overall survival status in kidney clear cell cancer patients . However, no significant difference in overall survival status was found between high and low PD-L1 groups in kidney papillary cell carcinoma and kidney chromophobe cohorts. Gene-set enrichment analysis on the data from databases of TCGA and GSE53757 dataset in Gene Expression Omnibus databases showed that several pathways relating to immunological functions were activated in kidney clear cell cancers with high PD-L1 mRNA expression, and glycolysis and epithelial-mesenchymal transition pathways relating to tumor progression and metastasis were increased in kidney clear cell cancers with low PD-L1 mRNA level. In conclusion, higher PD-L1 mRNA level in kidney clear cell cancer tissues was associated with a favorable outcome due to the higher immunological responses in tumor tissues. PBRM1, BAP1 and SETD2 are identified to be the molecular biomarkers for the prognosis of RCC [Renal cell carcinoma (RCC) is estimated to be the ninth leading cause of cancers in the US . Three ss of RCC , 5. BeyoProgrammed cell death 1 ligand 1  expressed on antigen presenting cells, B cells and other tissue cells can bind its receptor PD-1 on T cells, B cells and myeloid cells to negatively regulate immune responses . In RCC The prognosis of locally advanced or metastatic RCC is poor. Targeting therapy directly inhibiting the specific molecules such as tyrosine kinase or mammalian target of rapamycin (mTOR) has better clinical responses than cytokine therapy, but many patients become refractory to these therapies after a period of the treatment . RecentlIn this study, we aimed to investigate the role of PD-L1 mRNA expression in tumors in predicting the outcome of RCC based on the analyses of the clinical and RNA-seq data presented in The Cancer Genome Atlas (TCGA) database. Gene-set enrichment analysis (GSEA) on the data in TCGA and Gene Expression Omnibus (GEO) databases contributes to comprehend the immunological changes in RCC and to provide potential strategies for systemic therapy of RCC.The integrated data of 522 KIRC, 259 KIRP and 66 KICH patients in TCGA were enrolled for analyses (Supplementary data 1). Demographic, clinical, follow-up and tumor pathological features of the three RCC subtypes are listed in Table PD-L1 mRNA levels of the three RCC subtype cohorts were extracted from the RNA-seq2 data, which displayed continuous variables with a wide range of 0 to 5,361.1. The three RCC subtype cohorts were further divided into high PD-L1 group and low PD-L1 group based on the median PD-L1 mRNA value  longer than the median overall survival of 37.1 months (0-133.6 months) in low PD-L1 group. The overall survival status is significantly different between high PD-L1 group and low PD-L1 group  gene positively correlated with PD-L1 expression in KIRC cells but not in KIRP and KICH cells [Previous studies using immunohistochemistry and ELISA to measure PD-L1 protein in tumors and sera reached the conclusion that higher PD-L1 level was associated with poor prognosis of the three subtypes of RCC , 8, 17. CH cells , suggestIt seems paradoxical that higher expression of immunosuppressive PD-L1 correlated with improved outcomes. This will be resolved if PD-L1 expression is viewed as a reflection of the presence of endogenous antitumor immunity . In otheTreatment strategies for tumors with different status of endogenous immune responses should be different . RecentlIn conclusion, our study provides a new insight into the significance of PD-L1 in KIRC. Higher PD-L1 mRNA level was associated with a better outcome of the patients. The underlying mechanism may be the higher antitumor immune responses in the microenvironment of KIRC. PD-L1 mRNA level in tumor may be one of the factors affecting the outcome of KIRC patients, and may also be a reference for drug treatment strategy for these patients.http://cancergenome.nih.gov/publications/publicationguidelines).Clinical, follow-up and RNA-seq data of the 536 KIRC, 291 KIRP and 66 KICH patients were obtained from TCGA by cBioportal platform and TCGA-Assembler , 24. TheGene-set enrichment analysis (GSEA) was used to identify the pathways in two different PD-L1 mRNA level groups . RNA-seqKIRC, KIRP and KICH patients were divided into two groups according to the median value of PD-L1 mRNA level in tumors. Comparisons of demographic, clinical and pathological features between the two PD-L1 mRNA level groups were conducted using chi-square test or Fisher exact test. Overall survival was assessed using Kaplan-Meier method and log-rank test. Hazard ratio (HR) was calculated by Cox regression model and the result was provided as HR value and 95% confidence interval of the HR. In order to investigate whether PD-L1 level was an independent predictor for outcome in KIRC cohort, we included all the variables in multivariate Cox regression test using a backward conditional approach and eliminated the variables that the p value was >0.05. FDR q value was used for the evaluation of different pathways in different groups. Statistical tests were analyzed using SPSS 19.0 software . The p value of <0.05 was considered to be statistically significant.


In critically sick adults, sustained low efficiency dialysis [SLED] appears to be better tolerated hemodynamically and outcomes seem to be comparable to CRRT. However, there is paucity of data in critically sick children. In children, two recent studies from Taiwan (n = 11) and India (n = 68) showed benefits of SLED in critically sick children.The objective of the study was to look at the feasibility and tolerability of sustained low efficiency daily dialysis-filtration [SLEDD-f] in critically sick pediatric patients.Design: Retrospective study Inclusion criteria: All pediatric patients who had undergone heparin free SLEDD-f from January 2012 to October 2017. Measurements: Data collected included demographic details, vital signs, PRISM III at admission, ventilator parameters (where applicable), number of inotropes, blood gas and electrolytes before, during, and on conclusion of SLED therapy. Technical information was gathered regarding SLEDD-f prescription and complications.Between 2012–2017, a total of 242 sessions of SLEDD-f were performed on 70 patients, out of which 40 children survived. The median age of patients in years was 12 (range 0.8–17 years), and the median weight was 39 kg (range 8.5–66 kg). The mean PRISM score at admission was 8.77±7.22. SLEDD-f sessions were well tolerated, with marked improvement in fluid status and acidosis. Premature terminations had to be done in 23 (9.5%) of the sessions. There were 21 sessions (8.6%) terminated due to hypotension and 2 sessions (0.8%) terminated due to circuit clotting. Post- SLEDD-f hypocalcemia occurred in 15 sessions (6.2%), post- SLEDD-f hypophosphatemia occurred in 1 session (0.4%), and post- SLEDD-f hypokalemia occurred in 17 sessions (7.0%).This study is the largest compiled data on pediatric SLEDD-f use in critically ill patients. Our study confirms the feasibility of heparin free SLEDD-f in a larger pediatric population, and even in children weighing <20 kg on inotropic support. Acute kidney injury (AKI) in critical care is often secondary to sepsis or shock, and children are usually hypotensive with vasopressor support , 2. StanHybrid therapies providing RRT over an extended period but on an intermittent basis  using the standard HD machines includes the best of two worlds–the slow sustained modality of CVVH/D, ensuring haemodynamic stability, and better biochemical clearance along with the cost effectiveness of conventional IHD. Evidence accumulated in its favor has been primarily among adults, with pediatric literature still limited , 13.SLED first performed for a patient with AKI in 1945 as an alternative modality of RRT instead of CRRT, is a special form of intermittent dialysis with low dialysate and blood flow rates and prolonged duration . In receet al., it was concluded that SLEDD-f therapy was only available in 20% of the centers of the developed world and in 25% of the centers in the developing world  in critically sick pediatric patients. SLEDD-f is the first modality in all critically sick children at this center.Decisions to initiate SLEDD-f and duration (6–12 hours) were taken by both the attending pediatric intensivist and nephrologist according to indications, such as oliguria, positive fluid balance, persistent metabolic acidosis, or electrolyte imbalance refractory to other therapy. Percent Fluid overload was calculated as (fluid in-fluid out)/ICU admission weight x 100. Decision to start dialysis in fluid overload was taken combined by nephrologist and intensivist based on degree of fluid overload (>15%) and presence of other complications including oliguria, metabolic acidosis and dyselectrolytemia.All pediatric patients who had undergone SLEDD-f from January 2012-October 2017.SLEDD-f was defined as any hemodialysis session with convection; with blood flow rates ≤5ml/kg/minute and dialysate flow rates not more than twice the blood flow rates, and duration ≥6 hours. For children more than 30 kg, blood and dialysate flow rates were 150ml/minute and 300 ml/min. respectively. According to blood flow (Qb) and filtration rate (Qf), total pre-dilution replacement fluid was calculated as (QfxQb)/(Qb-Qf)x60xtreatment duration (hours).Definition of AKI was based on AKIN criteria .et al consensus paper ; FX 50 [children body surface area 1m2] and FX 60 [children body surface area 1.4m2] were used.Vascular access. The access used was femoral site or internal jugular vein. Standard hemodialysis catheters were used.Ultrafiltration rate. Initial ultrafiltration rate was kept at 0.5-1ml/kg/hour, and was increased as per the hemodynamic stability of the patient.Data collected included demographic details, vital signs, ventilator parameters (where applicable), number and dose of inotropes, blood gas and electrolytes before, during, and on conclusion of SLEDD-f therapy. Pediatric Risk of Mortality III (PRISM III) score at time of admission to PICU was also noted. Technical information gathered regarding SLEDD-f prescription included blood flow rate, dialysate flow rate, replacement flow rate prescribed and achieved duration, ultrafiltration volume, heparin dose and complications if any.All values in figures and tables are expressed as mean ± standard error. Student’s t tests  were used for intergroup comparison, with p value <0.05 indicating statistical significance. Analysis was performed with SPSS software.. Between January 2012-June 2016, 22 sessions of SLEDD-f were performed on 10 children, which were not consecutive. Intermittently, children also received SLED without convection during the period 2012-June 2016.From 2012–2017, a total of 242 sessions of SLEDD-f were performed on 70 patients, out of which 40 children survived After June 2016-October 2017, all sessions of SLEDD-f were consecutively done on next 60 patients (220 sessions). Since June 2016, all representative pediatric ICU patients received SLEDD-f.The median age of patients in years was 12 (range 0.8–17 years), and the median weight was 39 kg (range 8.5–66 kg). The mean PRISM score at admission was 8.77±7.22. A total of 46 patients (65.7%) were ventilated for a median of 68 hours (range 0–489). Sepsis occurred in 29 patients (41.4%), of which 10 patients (14.3%) had fungal sepsis. MODS occurred in 52 patients (74.3%). The median length of hospital stay was 14.5 days (range of 1–46).of SLEDD-f: SLEDD-f was done for combined metabolic acidosis and fluid overload in 134 sessions, persistent metabolic acidosis (pH<7.1) and hyperkalemia (serum potassium>6meq/L) in 34 sessions, persistent fluid overload in 37 sessions, persistent metabolic acidosis only in 16 sessions, combined metabolic acidosis, fluid overload and hyperkalemia in 20 sessions, and hyperammonemia in 1 session.Blood flow rate was 4.25±0.82 ml/min, and dialysate flow rates were 9.2±6.2 ml/minute. Replacement fluid administration rate was modified to keep filtration fraction less than 30%. Duration planned was 6.8±1.54 hours and achieved was 6.3±1.6 hours.All children less than 20kg were dialysed with pediatric blood lines . Whenever the extra corporeal blood volume exceeded 10% of total blood volume (n = 17) the circuit was primed with saline / 5% albumin or packed red blood cell (if haemoglobin <7 gm/dl).The ultrafiltration rate was 8.75±6.75 ml/kg/hr (mean±SD).Pre- SLEDD-f urea (mean ± SD) was 141 ± 104 mg/dL and post- SLEDD-f urea (mean ± SD) was 94.3 ± 84.2 mg/dL (p < 0.05). Pre- SLEDD-f creatinine (mean ± SD) was 4.1 ± 2.9 mg/dL and post- SLEDD-f creatinine (mean ± SD) was 2.9 ± 2.5 mg/dL (p < 0.05).Out of 242 sessions, children were on more than 1 inotrope in 154 sessions , and more than 2 inotropes in 101 sessions (41.7%). The total number of inotropes was 1.08+1.06 [mean+SD] (non survivors 1.01+0.98 and survivors 1.32+1.10 p = 0.04). Mean number of inotropes pre-SLEDD-f was 1.02+0.98 and post-SLEDD-f was 1.03+0.96; p = 0.40. Pre- SLEDD-f blood pressure (mean ± SD) was 86.3 mmHg and post- SLEDD-f blood pressure (mean ± SD) was 83.5 ± 14.7 mmHg with a p value > 0.05, thus indicating no hemodynamic compromise post the dialysis sessions.Table 2. There was, however, significant improvement in acidosis, as noted by the post- SLEDD-f serum bicarbonate level. Pre- SLEDD-f bicarbonate (mean ± SD) was 19.8 ± 4.7 mEq/L and post- SLEDD-f bicarbonate (mean ± SD) was 21.9 ± 4 mEq/L (p < 0.05). On the other hand, the oxygenation index did not show any statistical improvement; with the pre- SLEDD-f oxygenation index (mean ± SD) being 17.35 ± 14.5 and the post- SLEDD-f oxygenation index (mean ± SD) being 15.45 ± 14.6 (p > 0.05).Pre and Post-SLEDD-f metabolic and clinical parameters have been shown in Premature terminations had to be done in 23 (9.5%) of the sessions. There were 21 sessions (8.6%) terminated due to hypotension and 2 sessions (0.8%) terminated due to circuit clotting.Post- SLEDD-f hypocalcemia occurred in 15 sessions (6.2%), post- SLEDD-f hypophosphatemia occurred in 1 session (0.4%), and post-SLED hypokalemia occurred in 17 sessions (7.0%).Out of the forty children who survived (40/70), ten children with CKD or end stage renal disease being treated in the intensive care for acute deterioration. Rest thirty children all recovered their renal function on follow up ..A total of 30 patients died during this study. Those who died had MODS, a higher PRISM score and were ventilated et al reviewed their experience on SLED-f  among 14 critically ill children totalling 60 sessions. The standard prescription included blood flow 5 ml/kg/min, dialysate flow 260 ml/min, hemofiltration 35 ml/kg/h for 8–10 h daily delivered by Fresenius 5008 system. The study concluded that in their cohort of sick children, SLED-f provided good hemodynamic tolerance and correction of fluid overload, pH, and electrolyte imbalance. In addition they also showed a significant drop in inflammatory markers such as adiponectin, interleukin 17 A (IL-17A) and IL 16, post SLED-f session. Despite a relatively high PRISM score (16.8±23.3) they reported an overall 28 day survival of 71.4% [SLED is a common dialysis modality in critically ill adults in intensive care units. In view of only two paediatric studies, there is a need for more studies in critically ill children to identify feasibility and tolerability of SLED in this unique population. In the first pediatric study known on the effects of SLEDD-f on this population, Lee et al, from India showed feasibility of SLED in a retrospective record review from January 2010 to June 2016 from four tertiary pediatric nephrology centers in India. During the study period a total of 68 children received 211 sessions of SLED. Fifty-seven patients were ventilated (84%). Most of the patients had one or more organ system involved in addition to renal failure . Heparin free sessions were achievable in 153 sessions (72%). Out of 211 sessions, 148 sessions were on at least one inotrope (70.1%). Intradialytic hypotension or need for inotrope escalation was seen in 31 (15%) sessions but termination of the session for drop in BP was required in only 20 (9%) sessions [Sethi sessions .et al., a total of 68 critically ill pediatric patients who required renal replacement therapy received 211 sessions of SLEDD-f and of those, 153 sessions (72%) were done completely heparin free [et al. reported that in 14 treatments without heparin, one episode of circuit clotting  occurred which ultimately led to premature termination of SLEDD-f treatment [The decreased need for anticoagulation with either unfractionated heparin or citrate when compared to CRRT has been identified as a major advantage of SLEDD-f with the postulated benefit of decreased risk of bleeding and its associated decrease in morbidity and mortality. However, a potential risk associated with not using anti-coagulation is circuit clotting. In a study done by Sethi rin free . In prevrin free . Lee et reatment . In a streatment . In a streatment . While treatment , 24.Additionally, our study differed in the fact that it was more representative of the standard PICU population as we did not exclude children who weighed less than 20 kg, thus demonstrating the feasibility of SLEDD-f in the small pediatric population. In our study, 17 children were less than 20 kg, with the smallest child being 8.5kg. In this study, we analyze the feasibility and tolerability of heparin free SLEDD-f use in critically ill pediatric patients with AKI. There was no statistical difference in mean blood pressure, number of inotropes, and oxygenation index measured before and after the use of SLEDD-f sessions showing the tolerability of SLEDD-f in the sick subset of pediatric patients.There are several reports supporting a relationship between increased small solute clearance and improved patient outcomes in critically ill population –28. A grThe cost-efficacy of SLEDD-f treatment is another major advantage when compared to IHD and CRRT . The filComplications associated with SLEDD-f treatment were similar to those associated with IHD and CRRT; these include hypotension, filter clotting, hypokalemia, and hypophosphatemia. Some adult studies have shown however that there is no difference in hemodynamic stability between SLEDD-f and CRRT therapy . For theThis study has both strengths and weaknesses; this current study is the largest compiled data on pediatric SLEDD-f use in critically ill patients. Our study confirms the feasibility of SLEDD-f in a larger pediatric population and goes even further to demonstrate that it can be safely performed in children less than 20 kg on inotropic support and without the use of anticoagulation. Being a single center, retrospective study is our study’s limitation. The lack of need of anticoagulation can prove to be of extreme importance as this patient population can often have deranged coagulation, which puts them at an increased risk for anticoagulation related complications.


The ability to understand and control the electronic properties of individual molecules in a device environment is crucial for developing future technologies at the nanometre scale and below. Achieving this, however, requires the creation of three-terminal devices that allow single molecules to be both gated and imaged at the atomic scale. We have accomplished this by integrating a graphene field effect transistor with a scanning tunnelling microscope, thus allowing gate-controlled charging and spectroscopic interrogation of individual tetrafluoro-tetracyanoquinodimethane molecules. We observe a non-rigid shift in the molecule's lowest unoccupied molecular orbital energy (relative to the Dirac point) as a function of gate voltage due to graphene polarization effects. Our results show that electron–electron interactions play an important role in how molecular energy levels align to the graphene Dirac point, and may significantly influence charge transport through individual molecules incorporated in graphene-based nanodevices. The development of single-molecule electronics calls for precise tuning of the electronic properties of individual molecules that go beyond two-terminal control. Here, Wickenburg et al. show gate-tunable switch of charge states of an isolated molecule using a graphene-based field-effect transistor. This system allows the substrate Fermi energy (EF) to be continuously tuned all the way through the lowest unoccupied molecular orbital (LUMO) energy of a single F4TCNQ molecule. Using STM spectroscopy we have determined the gate-dependent energetic evolution of the LUMO level (EL) and its associated vibronic modes relative to the graphene Dirac point (ED). We show that the energy alignment between EL and ED changes as the substrate charge carrier density is tuned by gating, indicating the presence of electron–electron interactions that renormalize the molecular quasiparticle energy. This is attributed to gate-tunable image-charge screening in graphene and is corroborated by ab initio calculations. Our findings reveal that such tunable electronic correlation effects significantly renormalize the electron addition and removal energies for individual molecules incorporated into graphene devices.Here we combine STM and non-contact atomic force microscopy (nc-AFM) to demonstrate gate-tunable control of the charge state of individual, well-characterized tetrafluoro-tetracyanoquinodimethane (F2 layer at the surface of a doped Si wafer (the doped Si provides the FET back-gate)234TCNQ 234TCNQ  was sele4TCNQ molecules by using electronically inert 10,12-pentacosadiynoic acid (PCDA) as a molecular anchor 353637To overcome this problem, we devised an anchoring strategy to immobilize individual Fr anchor . We evapht edges , consistht edges . Individ4TCNQ molecules anchored to PCDA on our graphene/BN devices through the use of STM spectroscopy (the F4TCNQ electronic structure was not significantly affected by the nearby PCDA (I/dV spectrum measured over the range −0.4 V<VS<0.4 V (VS is the sample voltage with respect to the tip) with the back-gate held at VG=0 V. Spectroscopy was acquired on the outer edges of the molecule to avoid inelastic tunnelling effectsVS>0 V range, marked L and L′. These peaks are asymmetric, as seen in the adjacent high-resolution dI/dV spectrum in VS<0 V only one peak is seen, marked ‘charging', which is significantly sharper than the VS>0 V peaks2140L peak (VS>0) is derived from the F4TCNQ LUMO state. The charging peak (VS<0), on the other hand, does not directly indicate a feature in the molecular density of states but rather occurs due to tip-induced band bending as the tip electric field pulls the LUMO state below the Fermi energy (VS=0 V), causing it to fill with charge21404142L and L′ peaks to vibronic sidebands, as has been observed in other systems2339444TCNQ spectrum to a sum of Gaussian peaks. The L feature is fit well by a peak located at EL=61±6 meV (blue dashed line in L′ feature is similarly fit well by a peak located at EL′=288±23 meV (purple dashed line) with three additional satellite peaks evenly spaced every 37±7 meV (orange dashed lines) . The inL and L′ peaks, we calculated the hybridized molecular orbitals and vibrational modes of an F4TCNQ molecule on graphene, as well as the associated electron–phonon coupling, from first principles. As shown in ω1≈34 meV and ω2≈183 meV (Ag symmetry (ω1=34 meV) and a breathing mode of the inner carbon ring also having Ag symmetry (ω2=183 meV). The calculated energies agree reasonably well with the experimental energy spacing of the peaks extracted from within the L and L′ features of EL′–EL=227±24 meV. This shows that L′ is a vibronic satellite of L due to phonons having energy ω2 and that the internal structure of both L and L′ represents vibronic satellites due to phonons having energy ω1. A more detailed analysis involving a cumulant expansion to calculate the spectral function including vibronic modes also agrees with our measured spectra, supporting the vibronic interpretation of the spectral lineshape . Here we label the empty and occupied LUMO orbital as LUMO0 and LUMO−, respectively. The blue curve acquired at VG=−50 V shows the LUMO level well above EF and thus empty (LUMO0), resulting in a neutral molecule at this gate voltage. In contrast, the red dI/dV trace acquired at VG=+30 V shows the LUMO well below EF and thus filled by an electron, causing the molecule to become negatively charged (LUMO−). A notable difference between the red and blue curves is that the vibrational sidebands for the LUMO− state extend downwards to more negative VS values compared with the sidebands for LUMO0 which extend upwards to more positive VS values. This can be explained by the fact that higher-energy electron-like vibronic excitations (for LUMO0) occur at higher values of VS, whereas higher-energy hole-like vibronic excitations (for LUMO−) occur at lower values of VS. Our first-principles calculation of the spectral function using a cumulant expansionA unique aspect of this study is that we are able to reversibly control the charge state of a single FEF is swept by the gate. In the absence of electron–electron interactions we expect the position of the molecular level relative to ED to be independent of EF. If, on the other hand, electron–electron interactions play a significant role then we expect the LUMO energy (EL) to shift relative to ED as EF is tuned. We measured this effect by acquiring dI/dV spectra both on a single F4TCNQ molecule (to obtain EL) and off the molecule (to obtain ED) as a function of gate voltage. I/dV spectra as a function of gate voltage (−40 V<VG<+40 V), while 0) for VG<0 V to a filled orbital state (LUMO−) for VG>0 V. Similarly, the off-molecule spectra show the graphene Dirac point sweeping from above EF for VG<0 V(the p-doped regime) to below EF for VG>0 V(the n-doped regime). To extract the experimental gate-dependent values of EL, we fit the dI/dV spectra in ED values were obtained from the spectra of Our ability to gate the substrate of an adsorbed molecule allows us to address the fundamental issue of the importance of electron–electron interactions for molecule/substrate systems. We do this by measuring how the energy level of the molecular orbital changes relative to the graphene band structure as ED and EL values as a function of gate voltage  however, the values diverge, separating by as much as 100 meV at the highest gate voltage (corresponding to a charge carrier density of ∼3 × 1012 cm−2 at VG=60 V). The energy difference between ED and EL (ED–EL) is seen to increase monotonically with increasing charge carrier density for both electrons and holes, suggesting that electron–electron interactions play a role in determining EL for this adsorbate system. The observation that ED–EL does not depend on the polarity of graphene charge carriers rules out simple band bending as an explanation for the energy shift, since band bending would shift EL to higher energies when EL–EF>0, opposite to what is observed21404TCNQ molecular orbital (via tunnelling), and the electronic polarization that it induces in the graphene substrate. This many-electron interaction always lowers the energy of a system, since the interaction between charge added to an adsorbate and its image charge in the substrate is always attractive. Such effects are well known to reduce the energy gap between affinity and ionization levels for adsorbates on conventional substrates134849EF, which is readily changed by gating a graphene device.We are able to explain this behaviour as the result of Coulomb interaction between charge added to an F4TCNQ LUMO energy level is renormalized via screening within many-body perturbation theory . This was accomplished by modelling an electron that has tunnelled into the empty LUMO level as a point charge located a distance z* above the graphene plane , which induces screening charge density, nind(r), in the graphene. We calculated nind(r) and the change it causes to the electrostatic potential, ϕind(r), within linear response theory using the RPA dielectric function of grapheneΔEL, was estimated by evaluating ϕind(r) at the location of the point charge above the graphene surface thus yielding ΔEL values as a function of EF. For EF=ED the energy correction is smallest since graphene has no density of states at the Fermi level when EF is aligned to the Dirac point. As EF shifts away from ED, the energy correction increases equally for both electron and hole-doping since the carrier RPA dielectric function for graphene is electron–hole symmetric. The near-linear dependence of ΔEL on EF stems from the linear graphene density of states, ρ(E), and the fact that the graphene electronic susceptibility is proportional to ρ(EF).To estimate the expected magnitude of this effect for comparison to our data, we calculated how the Furements ). The pong  ref. . The insΔEL to spectroscopy taken on individual F4TCNQ molecules, we can add it to our gate-dependent measurements of ED . In the absence of any screening effects (ΔEL=0 eV), this would result in EL≈ED at every gate voltage (the simple ‘rigid shift' case). The Coulomb-induced renormalization effects described above, however, cause this procedure to result in a nontrivial shift of EL with respect to ED as a function of gate voltage. EL value (blue curve) as a function of gate voltage compared with the experimental EL values (red dots). For small gate voltages the renormalized EL value coincides with ED, but as the gate voltage (and carrier density) magnitude is raised the calculated EL values fall increasingly downward compared with ED, just as seen for the experimental EL values. The model thus captures both the observed independence of the LUMO renormalization on the graphene carrier type (electrons or holes) and also reproduces the general magnitude of the molecular orbital energy change. The match is not perfect , but is reasonable considering that the calculation has no adjustable parameters. One possible source for the discrepancy between theory and experiment are intra-molecular electron–electron interactions, which are not accounted for in the simple image charge model. We have examined the effect of these interactions on the LUMO energy using an Anderson model approach and find that they do lead to additional LUMO energy renormalization, but smaller in magnitude than the image charge effects  flakes exfoliated onto a SiO2/Si substrate. h-BN flakes were exfoliated onto heavily doped silicon wafers and annealed at 500 °C for several hours in air prior to graphene transfer. The graphene was grown on copper foil by the CVD method2 substrate via a poly methyl methacrylate stampA back-gated graphene/BN/SiO2 gas at 350 °C and then annealed subsequently in UHV at T∼350 °C for several hours until an atomically clean surface was achieved before the deposition of molecules. PCDA and F4TCNQ were deposited consecutively onto the clean graphene substrate at room temperature using Knudsen cell evaporators in the UHV chamber.The graphene device was first annealed in flowing Ar/HT=5 K using tungsten tips. STM topography was obtained in constant-current mode. STM tips were calibrated on a Au(111) surface by measuring the Au(111) Shockley surface state before all STS measurements. STS was performed under open feedback conditions by lock-in detection of an alternating current tunnel current with a bias modulation of 6–16 mV (r.m.s.) at 400 Hz added to the tunnelling bias. The tips were functionalized for nc-AFM imaging by picking up individual CO molecules on a Au(111) surfacef0=28.7 kHz, Q=90 k, A=60 pm). WSxM software was used to process all STM/nc-AFM imagesSTM/nc-AFM measurements were performed using a qPlus-equipped commercial Omicron LT-STM/AFM under UHV conditions at The data that support the findings of this study are available from the corresponding author on reasonable request.How to cite this article: Wickenburg, S. et al. Tuning charge and correlation effects for a single molecule on a graphene device. Nat. Commun.7, 13553 doi: 10.1038/ncomms13553 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures 1-11, Supplementary Notes 1-4 and Supplementary References


Graphene has a promising future in applications such as disease diagnosis, cancer therapy, drug/gene delivery, bio-imaging and antibacterial approaches owing to graphene's unique physical, chemical and mechanical properties alongside minimal toxicity to normal cells, and photo-stability. However, these unique features and bioavailability of graphene are fraught with uncertainties and concerns for environmental and occupational exposure. Changes in the physicochemical properties of graphene affect biological responses including reactive oxygen species (ROS) production. Lower production of ROS by currently available theranostic agents, e.g. magnetic nanoparticles, carbon nanotubes, gold nanostructures or polymeric nanoparticles, restricts their clinical application in cancer therapy. Oxidative stress induced by graphene accumulated in living organs is due to acellular factors which may affect physiological interactions between graphene and target tissues and cells. Acellular factors include particle size, shape, surface charge, surface containing functional groups, and light activation. Cellular responses such as mitochondrial respiration, graphene-cell interactions and pH of the medium are also determinants of ROS production. The mechanisms of ROS production by graphene and the role of ROS for cancer treatment, are poorly understood. The aim of this review is to set the theoretical basis for further research in developing graphene-based theranostic platforms. An excellent review of targeted diagnostic imaging has recently been contributed by Cope et al. (2016) A key feature of PDT is to exploit the light source for selective activation of the photosensitizer within the tumor cells. A light source of appropriate wavelength (visible or near-infrared) is used to activate a photosensitizer that generates and releases ROS, for the selective killing of tumors Historically, the development of photosensitizers has resulted in three eminent generations of photosensitizer types. The first generation is porphyrins 2-bonded carbon atoms packed into a honeycomb lattice. This material has gained significant attention in many disciplines of life science, owing to its electronic, optical and structural properties. Graphene has been applied as a drug carrier in chemotherapy, and as a photosensitizer for photothermal therapy and PDT. A graphene nanohybrid showed improved anticancer PDT effects compared to the conventional photosensitizers Recently, novel photosensitizers have been fabricated to improve the selective tissue penetration of incident light, and to improve the clinical efficacy of PDT. Among such novel developments, graphene has also recently been fabricated and utilized as a photosensitizer and theranostic agent The therapeutic responses of different derivatives of graphene such as GO and graphene quantum dots revealed them as promising treatment agents and showed the possibility of exploiting ROS in cancer treatment. A better understanding of the role of ROS in the therapeutic mode of action of graphene, in cancer treatment, will facilitate the development of improved graphene-based theranostic platforms.22+ homeostasis ROS generation by nanoparticles has been considered as the primary source of their toxicity 3The proof-of-concept investigations of graphene in cancer theranostics are still at a preclinical stage. An early report on GO as theranostic agent was published by Cho and his group 1O2 production rate compared with porphyrin alone. Wei et al. vβ3 as a mechanism for tumor targeting. The authors demonstrated that the phototoxicity of GO-bound pyropheophorbide can be switched on and off in both organic and aqueous environments after the conjugation of pyropheophorbide with polyethylene-glycol. The functionalized GO efficiently targets the cancer cells’ surface ligand (i.e. integrin αvβ3). Once endocytozed by the cells, and having then escaped from lysosomes, the functionalized GO subsequently moves to the mitochondria. The two-fold on/off switching of this functionalized GO considerably increases the intrinsic pathway of apoptosis.Cancer cells possess an inherent nature of survival and re-growth 2O2-induced apoptosis usually occurs in lymphoma cells via activation of cysteine proteases such as caspase-3 2O2 is a precursor of highly reactive hydroxyl radicals, while H2O2 itself has relatively low reactivity. H2O2 produced by nanoparticles has shown great potential to initiate apoptosis in the cells of osteosarcomas, and in breast, bladder, and lung cancer cells. He et al. 2O2 (formed from superoxide) then reacted with Fe(II) and gave an improved yield of hydroxyl radicals. Excessive generation of ROS is known to trigger oxidative damage to various biomolecules, such as DNA, proteins and lipids, which in turn may lead to mitochondrial membrane permeabilization 1O2, and by which the hybrid of folic acid polyethylene glycol and C60  conjugated to GO (FA-GO-PEG/C60) achieves the combined synergistic effects of photothermal therapy and PDT.H4In summary, recent studies underpinning the application potential of graphene in the theranostic field have been reviewed. Many groups have utilized graphene in PDT, photothermal therapy and fluorescent imaging for cancer treatment. The combination of imaging and therapy could produce synergistic effects to increase the targeted killing with minimal side effects and with the maintenance of biocompatibility. The scope for functionalization and conjugation of graphene can potentially generate a promising array of theranostic agents. Further in vivo studies will be required to better understand the real-world applications of graphene. Moreover, the aspects of ROS generation, toxicity and potential cancer theranostic approaches for other derivatives of graphene such as graphene nanoribbons, graphene nanoplatelets, three dimensional graphene foams, graphene nanopores, and porous graphene nanosheets will need to be studied. Oxidative stress induced by graphene accumulated in living organs is due to acellular factors such as particle size, particle shape, surface charge, surface functional groups, and light activation, while cellular responses such as mitochondrial respiration, immune cell activation, pH of the medium and physiological redox-regulated functions are critical determinants affecting the production of ROS. To date, the mechanisms and roles of ROS production by most forms of graphene in relation to cancer treatment, are not understood. A basic understanding of graphene-cell interactions , as well as the optimal conditions for their proper use, will provide new theranostic platforms in the future.


Graphene oxide (GO) was functionalized and reduced simultaneously by a new reductant, 4-hydrazinobenzenesulfonic acid (HBS), with a one-step and environmentally friendly process. The hydrophilic sulfonic acid group in HBS was grafted onto the surface of GO through a covalent bond. The successful preparation of HBS reduced GO (HBS-rGO) was testified by scanning electron microscope (SEM), X-ray diffraction (XRD), Raman spectroscopy, Fourier transform infrared spectra (FTIR), X-ray photoelectron spectroscopic (XPS) and thermogravimetric analysis (TGA). The interlayer space of HBS-rGO was increased to 1.478 nm from 0.751 nm for GO, resulting in a subdued Van der Waals’ force between layers and less possibility to form aggregations. The aqueous dispersibility of graphene was improved to 13.49 mg/mL from 0.58 mg/mL after the functionalization. The viscosity of the epoxy resin based HBS-rGO composite could be regulated by an adjustment of the content of HBS-rGO. This study provides a new and applicable approach for the preparation of hydrophilic functionalized graphene, and makes it possible for the application of graphene in some functional polymer nanocomposites, such as specialty water-based coatings. Thesend gases ,4,5. Hownd gases ,7,8.et al. functionalized GO with glycidol, and reduced the functionalized graphene oxide by sodium borohydride [et al. utilized 6-amino-4-hydroxy-2-naphthalenesulfonic acid to functionalize GO, and chose hydrazine monohydrate as reductant [et al. prepared stable aqueous dispersions of graphene sheets using 1-pyrenebutyrate and then reduced the functionalized graphene oxide by hydrazine monohydrate [2 structure of graphene are anhydrous hydrazine, hydrazine monohydrate, sodium borohydride, and hydrogen sulfide, which are highly toxic and harmful to both living organisms and the environment [The chemical functionalization of graphene oxide (GO) has attracted a great deal of attention because of its wide application ,10,11,12ohydride . Kuila eeductant . Xu et aohydrate . These mohydrate ,27,28. Wironment ,30,31,32In this study, GO was functionalized and reduced simultaneously by 4-hydrazinobenzenesulfonic acid (HBS). Two functional groups are included in HBS, which are the sulfonic acid group and hydrazine group. The sulfonic acid group, which is hydrophilic, was grafted onto the surface of GO through a covalent bond, and oxygen-containing groups such as hydroxy and carboxyl were reduced by the hydrazine group at the same time. Moreover, HBS is hardly toxic to the human body. Therefore, this one-step method is easier to operate, achieves better mass production than the two-step one, and is more friendly to the environment. The successful reduction and functionalization of GO by HBS was verified by several characterization methods. The epoxy based HBS reduced GO (HBS-rGO) nanocomposites were prepared and their viscosity behavior was verified. This study makes it possible to compound graphene with the hydrophilic matrix, and has broad application prospects in specialty water-based coatings.HBS was purchased from Sigma-Adrich . Graphene and Graphite oxide fine powder were supplied from Nanjing XF NANO Materials Tech Co., Ltd., Nanjing, China. Epoxy was purchased from Nantong Xingchen Synthetic Material Co., Ltd., Nantong, China. All other chemical materials were purchased from Sigma-Adrich and used as received.By the epoxy ring-opening reaction, graphene surface was grafted with HBS layer with hydroxyl groups. The preparation procedure of HBS-rGO is described in The epoxy based HBS-rGO composite was prepared by solution mixing method ,34,35. TThe functionalization and reduction of graphene oxide by HBS was characterized by SEM, XRD, Raman spectroscopy, FTIR, XPS spectra and TGA. SEM images were taken by ZEISS MERLIN Compact . XRD tests were conducted on Rigaku Corporation SmartLab . The Raman spectra were excited with a laser of 488 nm and recorded on solid powder samples using a LabRAM HR800 spectrometer . FTIR spectra were collected on a Perkin-Elmer spectrometer  using KBr pellets. XPS measurements were performed on Thermo Fisher ESCALAB 250Xi . TGA measurements were carried out on a Q5000 TGA of TA instruments  at a heating rate of 10 °C/min from 30 °C to 700 °C in nitrogen.Morphology of the GO and HBS-rGO samples are characterized by SEM. As shown in The XRD patterns of GO and HBS-rGO are shown in  results ,37. The −1 and 1350 cm−1 were observed, which were generally assigned as the D band (at about 1350 cm−1) and G band (at about 1590 cm−1), corresponding to the structural defects and vibration of sp2-hybridized graphitic domains, respectively [D/IG) proved the graphitization degree of carbon solids and a lower value represented a higher degree of graphitization. The ratio of ID/IG of HBS-rGO was 0.29, which was much smaller than that of GO and very close to the value of graphene. Meanwhile, the G peak of HBS-rGO was red-shifted to 1582 cm−1, getting close to that of graphene (1563 cm−1), implying the restoration of the graphitic sp2 network. These results clearly indicated that the GO was well reduced by HBS.The Raman spectroscopy results of GO, HBS-rGO and graphene are shown in ectively ,39. The −1 (C–O) [−1, S–O at 1130 cm−1 and S-phenyl at 1042 cm−1 in HBS-rGO appeared and confirmed the presence of sulfonic groups. Besides, C–H at 1008 and 853 cm−1 in HBS-rGO showed that the benzene ring in HBS was para-substituted by –NH and –SO3H. These results indicated GO was successfully reduced and functionalized by HBS.−1 (C–O) ,41. Thes3H was the main form of S element, the O content in other oxygen-containing groups of HBS-rGO was 7.23%. The dropping of O content from 17.19% for GO to 7.23% for HBS-rGO (except the O in –SO3H group) was owing to the successful reduction of GO by HBS. 3H was the main form of S element. These results demonstrated that GO was successfully reduced by HBS and the hydrazine group, and sulfonate group was successfully grafted onto the graphene.The XPS spectra of GO and HBS-rGO are shown in TGA measurement provides further proof of the reduction of GO by the functionalization of HBS. As shown in The dispersibility of graphene in water was greatly improved after the functionalization due to the emergence of the hydrophilic sulfonic acid group. The aqueous solution of graphene formed sediments in less than 30 s, while a uniformly dispersed 1.5 mg/mL aqueous solution of HBS-rGO could be kept for weeks without any extra additives, as shown in In order to verify the viscosity behavior of epoxy resin based HBS-rGO composite, graphene, GO and HBS-rGO/epoxy nanocomposites with certain contents of filler were prepared by solution mixing, and the relation between viscosity and temperature was studied. The weight percentage of graphene/epoxy and GO/epoxy composite were both 0.50 wt %. The weight percentage of HBS-rGO/epoxy composite was determined to be 0.50 wt %, 0.75 wt % and 1.00 wt %, respectively. The results are shown in 3H group) owing to the successful reduction of GO by HBS. The aqueous dispersibility of graphene was improved to 13.49 mg/mL from 0.58 mg/mL after the functionalization. The viscosity of the epoxy resin based HBS-rGO composite can be regulated by an adjustment of the content of HBS-rGO. This study shows HBS-rGO is a potential filler material for some functional polymer nanocomposites, such as specialty water-based coatings.In summary, graphene oxide was functionalized and reduced simultaneously by a new reductant, 4-hydrazinobenzenesulfonic acid, with a one-step and environmentally friendly process. The hydrophilic sulfonic acid group in HBS was grafted onto the surface of GO through a covalent bond. The successful functionalization and reduction was testified by SEM, XRD, Raman spectroscopy, FTIR, XPS and TGA. The interlayer space of HBS-rGO was increased to 1.478 nm from 0.751 nm for GO, resulting in the subdued Van der Waals’ force between layers and less possibility to form aggregations. The O content dropped from 17.19% for GO to 7.23% for HBS-rGO (except the O in –SO


Propionibacterium acnes are considered the most implicated etiological agent for sarcoidosis. To investigate whether P. acnes is an etiological agent for sarcoid uveitis, we analyzed the frequency of P. acnes detected within the biopsied retinas from patients with ocular sarcoidosis by immunohistochemistry with a P. acnes-specific monoclonal antibody (PAB antibody). Eleven patients (12 eyes) with sarcoid uveitis were enrolled in this study. Eight patients with rhegmatogenous retinal detachment, two patients with non-sarcoid uveitis, and two patients with vitreoretinal lymphoma were enrolled as controls. In the sarcoidosis group, granulomas were mainly observed in the inner retinal layer filled with CD4+ cells and CD68+ cells, indicating the Th1 immune response. P. acnes, identified as round bodies that reacted with the PAB antibody, were present in 10/12 samples (83%) from 9/11 patients (82%) with sarcoidosis. These round bodies were scattered within the retinal granulomas mainly in the inner retinal layer. In the control group, no round bodies were detected. Our results suggested that P. acnes could be associated with sarcoid uveitis. We hypothesize that sarcoid granulomas may be formed by a Th1 immune response to P. acnes hematogenously transmitted to the retina.The etiology of sarcoidosis is still obscure; however, Mycobacteria and  This disorder commonly affects young and middle-aged adults who present with bilateral hilar lymphadenopathy, pulmonary infiltration, and ocular and skin lesions2. The liver, spleen, lymph nodes, heart, salivary glands, nervous system, muscles, and other organs may also be involved.Sarcoidosis is a multisystemic granulomatous disorder characterized by the presence of noncaseating granulomas in the involved organsHLA-DRB1 gene7. Mycobacteria are considered to play a major etiologic role in sarcoidosis in the United States and Europe14. On the other hand, Propionibacterium acnes is considered the most implicated etiological agent for sarcoidosis in Japan because it has been isolated at a high ratio from systemic sarcoid lesions by bacterial culture16. Bacterial culture has shown that P. acnes is present in up to 78% of biopsied lymph nodes from Japanese patients with sarcoidosis, while only 21% of non-sarcoidosis patients showed positive results16. Moreover, higher number of P. acnes genomes has been detected in sarcoid lymph nodes than in control samples using quantitative polymerase chain reaction (PCR)18. Furthermore, Negi, et al. reported that P. acnes was present within sarcoid granulomas in 74% of video-assisted thoracic surgery lung samples, 48% of transbronchial lung biopsy samples, 88% of Japanese lymph node samples, and 89% of German lymph node samples by immunohistochemical analysis using a P. acnes-specific monoclonal antibody (PAB antibody)19. Lipoteichoic acids are membrane-anchored molecules in the cell envelopes of gram-positive bacteria24. PAB antibody was developed to react specifically with the cell-membrane-bound lipoteichoic acid of P. acnes19. In addition, P. acnes can induce granulomatous inflammation in the murine lung25. These data imply that P. acnes is a pathogenic bacterium that may cause systemic sarcoidosis. However, only a few reports have demonstrated the presence of P. acnes in ocular tissue in patients with ocular sarcoidosis27. For example, Yasuhara et al. showed that P. acnes or P. granulosum DNA was present in the vitreous fluid of patients with sarcoidosis-associated uveitis26. To the best of our knowledge, no reports have clearly demonstrated the presence of P. acnes in the retina.The etiology of sarcoidosis is still obscure; however, sarcoidosis is thought to be triggered by either infectious agents or exposure to environmental substances in patients with various genetic factors, such as the 30. Moreover, severe vitreous opacity, macular edema, and/or epiretinal membrane are often accompanied by posterior uveitis with sarcoidosis as the cause of impaired visual acuity32. For these cases, pars plana vitrectomy (PPV) is beneficial for improving visual acuity. We have some cases performed a retinal biopsy during PPV to diagnose sarcoidosis.To diagnose systemic sarcoidosis, the most reliable method is analysis of biopsied specimens; the same is thought to be true in ocular sarcoidosis. Indeed, retinal biopsy or chorioretinal biopsy is thought to provide useful information for the diagnosis of uveitisP. acnes detected within the biopsied retinas from patients with ocular sarcoidosis by immunohistochemistry (IHC) with PAB antibody in order to prove possible involvement of P. acnes in the pathogenesis of ocular sarcoidosis.In the present study, we analyzed the frequency of Although the retinal samples were extremely small, all samples were sufficiently enough to be examined by HE staining. Histopathological examination of retinal specimens in this study confirmed the diagnosis in all 11 cases with sarcoidosis. In the sarcoidosis group, noncaseating epithelioid cell granuloma was observed in all cases. Granulomas were mainly located in the inner retinal layer  and/or large-sized (3~5 µm in diameter) forms of round bodies. In three of ten samples (30%) had round bodies detected by PAB antibody, large spheroidal bodies were sparsely detected using PAB antibody. The numbers of round bodies in the retinal sample varied among samples. In six of ten samples (60%), round bodies were sparsely detected. Similar to lung granulomas, immature granulomas tended to include more round bodies than did mature granulomas, suggesting that P. acnes may be related to the early stages of granuloma formation. In five of ten samples (50%), some round bodies were aggregated as though some granuloma-forming cells were filled with the bacterium. In contrast to the sarcoidosis group, P. acnes was undetectable in all control samples .Round bodies immunohistochemically detected by PAB antibody were observed in 10/12 samples (83%) from the 9/11 patients (82%) with sarcoidosis, demonstrating the presence of Granulomas were mainly comprised of CD4+ cells and CD68+ cells Fig. , indicatP. acnes detection (82%) in retinal biopsy specimen in patients with ocular sarcoidosis, whereas P. acnes was not detected (0%) in the control group. This marked difference strongly indicated that P. acnes may be associated with sarcoid uveitis. To the best of our knowledge, this is the first study to detect P. acnes within the retinal granulomas in patients with ocular sarcoidosis.The pathogenesis of ocular sarcoidosis is not yet fully understood. In the present study, we clearly demonstrated a high ratio of P. acnes as a promising etiological agent of sarcoidosis19. Unlike tissues in contact with external air, such as the lungs and skin, the normal intraocular environment is completely aseptic and does not contain indigenous bacteria. On the other hand, P. acnes is the most common commensal bacterium in the peripheral lung tissue and mediastinal lymph nodes identified in subjects, even those without sarcoidosis. Indeed, in a previous study, P. acnes was isolated from half of 43 lungs and 8 of 11 mediastinal lymph nodes, mostly in pure culture. Moreover, the strains of P. acnes isolated from sarcoid lymph nodes were not specific to sarcoidosis33. Even IHC with PAB antibody, the same method as used in our current study, revealed the presence of P. acnes in 18% of surgically obtained samples and 22% of lymph node samples in patients without sarcoidosis19. Given that the retina is an aseptic environment, the presence of P. acnes within the retina is extremely unusual. In fact, no P. acnes was detected within retinas in the control group in the current study. Therefore, detecting P. acnes within retinas is a critical finding for confirming the role of P. acnes as an etiological agent of sarcoidosis.Many Japanese studies have supported the potential of P. acnes is associated with sarcoid uveitis in young patients.Sarcoidosis commonly occurs between the ages of 20 to 30 and 50 to 60 years. In the present study, young patients with sarcoidosis were not included, because the patients who needed surgery were older than the average age for sarcoidosis. Further studies are needed to confirm that P. acnes using PAB antibody may depend on the sizes of samples in lung granulomas; the detection frequency in surgically obtained lung samples was reported to be 74%, whereas that in lung biopsy samples was only 48%19. Surprisingly, the detection rate of P. acnes in retinal samples, which are extremely small, was 82%, which is more than that in surgically obtained comparatively large lung samples (74%). One possible explanation for the higher detection frequency of P. acnes within retinal granulomas than in lung granulomas is the lower grade of P. acnes degradation in the retina compared with that in the lung. Negi et al. suggested that the failure to detect P. acnes within lung granulomas of some sarcoid samples may be explained by the complete degradation of P. acnes antigens by granuloma cells19. This hypothesis is supported by the findings that PAB antibody reactivity was observed more frequently in immature granulomas than in mature granulomas, showing that the P. acnes antigen in lung granuloma cells may have been degraded and abolished as the granuloma matured. Because the respiratory tract is always in contact with external air, the function to remove and degrade pathogens in the respiratory track is innately developed. In contrast, intraocular tissue is not normally exposed to any pathogens. Under these specific conditions, P. acnes may be degraded more slowly in intraocular granulomas than in lung granulomas, thus leading to higher detection of P. acnes, even within extremely small retinal samples.The detection frequency of P. acnes, and occasionally contained a few large spheroidal bodies detected with PAB antibody. These large spheroidal bodies were previously identified as Hamazaki-Wesenberg (HW) bodies in lung and lymph node samples19. HW bodies were initially found in lymph nodes as a nonspecific but frequently seen feature in sarcoidosis36. HW bodies are currently thought to be cell-wall-deficient P. acnes38. We also think that these large spheroidal bodies in retinal samples in the present study are identical to the HW bodies seen in lung or lymph node samples, because of the similarity of sizes, shapes, and staining pattern.In the present study, retinal granulomas commonly contained small-sized and/or large-sized forms of P. acnes is associated with ocular sarcoidosis, then why were all retinas not positive for P. acnes in the present study? One explanation is the size of the biopsied retinas. The biopsied samples must be extremely small because they should ideally be collected from the narrow areas of invisible retina. It may occur when one granuloma within an extremely small retinal sample is negative for P. acnes; the sample may then be considered negative, even if other granulomas react with the anti-P. acnes antibody. Alternatively, there may be other causes of ocular sarcoidosis. Indeed, Ishige et al. revealed that many P. acnes genomes were found in 80% (12/15) of lung lymph node samples from patients with sarcoidosis without coexistence of P. granulosum genomes, whereas in the remaining 20% (3/15), P. granulosum genomes were found without coexistence of P. acnes, suggesting that both bacteria may independently induce sarcoidosis17. In this present study, P. acnes was undetected in 2 of 11 (18%) of retinal sarcoid granulomas. In these patients, the failure to detect P. acnes may be explained by the presence of sarcoidosis arising from other pathogenic factors, such as P. granulosum, Mycobacterium species, or unknown environmental substances. Indeed, in the one of two patients, M. intracellulare was isolated from bronchoalveolar lavage fluid by bacterial culture, implying that the cause of sarcoidosis in this patient may be M. intracellulare.If P. acnes is not normally present in the retina. However, the mechanism through which P. acnes enters the intraocular tissue is not clear. In the present study, P. acnes was mainly detected in the granulomas in the inner retinal layer, where retinal vessels are mainly distributed. Moreover, inflammatory cells were infiltrated conspicuously in the perivascular area. Furthermore, large granulomas were detected in both the inner and outer retinal layers in some severe cases; however, there were no cases with granuloma presenting only in the outer retinal layer. Taken together, these findings implied that granulomas were caused by hematogenous transmission of P. acnes.As described above, 40. In fact, we have previously reported that high CD4/CD8 ratios in vitreous-infiltrating lymphocytes were detected, and Th-1-related cytokines were upregulated in vitreous samples from patients with ocular sarcoidosis42. In the present study, retinal granulomas were comprised of T lymphocytes and macrophages. Because these inflammatory cells produce Th-1-related cytokines and can induce retinitis and vitritis, our findings are consistent with previous studies showing that sarcoidosis is caused by a Th1-driven inflammatory process.Sarcoidosis is characterized by an increased number of activated macrophage and CD4+ T cells and is therefore thought to be a Th1-driven inflammatory processP. acnes may facilitate the development of novel treatments for ocular sarcoidosis, i.e., a combination of curative treatment, such as antibiotics to reduce P. acnes, and symptomatic therapy.Currently, the treatment of ocular sarcoidosis mainly involves local and additional systemic steroids. Although these treatments can successfully reduce the inflammation caused by sarcoidosis on clinical examination, they only treat inflammatory responses as symptomatic therapy. However, identification of the etiologic agent as Our retinal biopsy procedure was performed safely with no complications in all cases. Moreover, the mean best-corrected visual acuity improved significantly after the PPV.P. acnes, characterized by accumulation of CD68+ macrophage and CD4+ T lymphocytes, and that P. acnes may be hematogenously transmitted from the lung or another location to the retina in individuals with hereditary or acquired abnormalities in the immune system. Thus, P. acnes is associated with sarcoid uveitis. Because the retina is typically an aseptic organ, the high frequency of P. acnes in this study provides important insights into the pathogenesis of sarcoidosis. Further studies are needed to determine the precise mechanisms through which sarcoidosis is developed, particularly to overcome the limitation of the small sample size in this study.In conclusion, our findings suggested the possibility that sarcoid granulomas are induced by the Th1 immune response against This was a prospective, case-control study performed in accordance with the tenets of the Declaration of Helsinki. All procedures were approved by the Institutional Review Board of the Kyoto Prefectural University of Medicine Hospital (# RBMR-C-864). The trial was registered in the University Hospital Medical Information Network Clinical Trial Registry. Written informed consent was obtained from all participants before enrollment.2. As non-sarcoidosis controls, eight patients with rhegmatogenous retinal detachment (RRD), two patients with retinal detachment due to non-sarcoid uveitis , and two patients with vitreoretinal lymphoma were enrolled. The mean patient ages were 69.54 ± 4.50 years (range: 63–75 years) in the sarcoidosis group and 61.73 ± 10.01 years (range: 45–77 years) in the control group. In the sarcoidosis group, all cases had multiple retinal exudates located inferiorly in the fundus. All cases had severe vitreous opacities that were likely to cause decreased visual acuity.A total 24 eyes of 23 patients were enrolled in this study. All patients were Japanese. Of those 24 eyes, 12 eyes of 11 patients were diagnosed with biopsy-proven sarcoidosis. Of the 11 patients with sarcoidosis, six were also proven positive histopathological manifestations in organs other than eye by skin biopsy (two patients), transbronchial lung biopsy (two patient), and transbronchial needle aspiration of lymph nodes in the mediastinum (two patients) after retinal biopsy. These biopsies were performed at the time the skin, lung, and lymph node lesions appeared. The other five patients were comprehensively diagnosed by retinal biopsy and clinical examinations according to the statement on sarcoidosis of the ATS/ERS/WASOGFor sample collection, 25-gauge pars plana vitrectomy was performed using a Constellation Vision System  under a wide-angle viewing system. In the sarcoidosis group, local retinal detachment was manually created by infusing a balanced salt solution  with a subretinal needle attached to a syringe. Retinectomy was performed using a bimanual technique with vitreoretinal forceps and curved scissors. Retinal samples were then collected using an indwelling needle. The nasal lower or temporal lower part of the retina with exudates was selected for collecting retinal samples. Retinal biopsies were safely performed without intraoperative complications in all cases. All collected retinas could be biopsied from the peripheral region to minimize postoperative visual field loss. In patients with RRD, a retinal flap from the retinal tear was obtained by retinectomy, as described above, performed during vitrectomy. In patients with acute retinal necrosis and ocular toxoplasmosis, detached retinas to be excised due to inflammation were collected. In patients with vitreoretinal lymphoma, retinas with intraretinal lesions were collected.P. acnes according to previously reported methods19. In brief, BALB/c mice  were immunized with the sonicated whole-bacterial lysate of P. acnes (ATCC 11828). Hybridoma cell lines producing anti-P. acnes antibodies were checked by enzyme-linked immunosorbent assay with the bacterial lysate. Hybridoma cell lines with positive results were screened by IHC with formalin-fixed and paraffin-embedded tissue sections of the P. acnes-injected rat liver, which was obtained by intravenous injection of 30 mg of heat-killed P. acnes into female Sprague–Dawley rats (CLEA Japan) 1 h before killing the rat. Similarly-prepared liver tissue sections of rats injected with each strain of other control bacteria were also examined to confirm the specificity of the antibody to P. acnes. Hybridoma cell lines producing the antibody that generated a strong reaction specific to P. acnes on rat liver sections were selected and further screened by IHC with formalin-fixed and paraffin-embedded tissue sections of the sarcoid lymph nodes, in which a large number of P. acnes genomes were detected. The hybridoma producing the antibody that generated the most specific reaction product on human tissue sections was selected and cloned by two rounds of limiting dilution. A single hybridoma clone was then implanted into the intraperitoneal space of severe combined immunodeficiency mice (CLEA Japan). At 1 or 2 weeks after implantation, ascites was collected and used as an undiluted antibody without further purification.The PAB monoclonal antibody was generated in mice against whole-bacterial lysates of 19. The PAB antibody reacted with all strains of P. acnes, irrespective of the serotype, strain type, or clinical isolate, and there was no cross-reactivity with other propionibacteria including P. granulosum and P. avidum or other control bacteria. The PAB antibody recognizes lipoteichoid acid because it was found to react with the lipoteichoic acid purified from P. acnes. It is unlikely that the purified fraction of lipoteichoic acid was contaminated with another structure such as lipoglycan, because the band on the western blot membrane was slightly broad but nevertheless clearly visible as a single band. Reaction products of PAB antibody were localized at the peripheral area of the bacterial body, consistent with the localization of cell-membrane-bound lipoteichoic acid  in 10 mmol/l citrate buffer (pH 6.0) for 40 min at 97 °C. The sections were then treated with 3% hydrogen peroxide in methanol for 10 min. The sections were first incubated with normal horse serum  and then incubated overnight at room temperature with the diluted PAB antibody  in a humidified chamber. The sections were further incubated for 30 min with the biotinylated secondary antibody, followed by a 30-min incubation with streptavidin-peroxidase complex , at room temperature. Before and after each step, the sections were washed in phosphate-buffered saline containing 0.5% Tween-20. The signal was developed as a brown reaction product using the peroxidase substrate diaminobenzidine . Anti-human CD4 mouse monoclonal antibodies , anti-human CD8 mouse monoclonal antibodies , and anti-human macrophage CD68 mouse monoclonal antibodies  were also used. All sections were counterstained with hematoxylin.Biopsied retinal samples from the sarcoidosis group and corresponding control samples were examined. Sections were fixed in Zamboni solution, embedded in paraffin, cut, and mounted on silane-coated slides. Deparaffinized sections were stained with hematoxylin-eosin (HE). Adjacent sections were also examined by IHC as described previouslyp value of less than 0.01 was considered significant.Fisher’s exact test was used to evaluate statistical differences between groups. Difference with a The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.Supplemantal Figure 1


Atomically thin boron nitride (BN) nanosheets are important two-dimensional nanomaterials with many unique properties distinct from those of graphene, but investigation into their mechanical properties remains incomplete. Here we report that high-quality single-crystalline mono- and few-layer BN nanosheets are one of the strongest electrically insulating materials. More intriguingly, few-layer BN shows mechanical behaviours quite different from those of few-layer graphene under indentation. In striking contrast to graphene, whose strength decreases by more than 30% when the number of layers increases from 1 to 8, the mechanical strength of BN nanosheets is not sensitive to increasing thickness. We attribute this difference to the distinct interlayer interactions and hence sliding tendencies in these two materials under indentation. The significantly better interlayer integrity of BN nanosheets makes them a more attractive candidate than graphene for several applications, for example, as mechanical reinforcements. Atomically thin boron nitride remains undercharacterized in terms of their mechanical properties. Here authors test high-quality mono- and few-layer BN and show it to be one of the strongest electrically insulating materials and dramatically better in interlayer integrity than graphene under indentation. Investigations on the mechanical properties of these nanomaterials are, therefore, essential. In this regard, the mechanical properties of monolayer (1L) graphene have been systematically studied. Although the reported experimental values of the elastic modulus of high-quality graphene vary between 0.5 and 2.4 TPa (refs E2D) of ∼342 N m−1 with an effective thickness of 0.335 nm, consistent with many theoretical calculations671010121391516171920212223222, tungsten disulfide (WS2) and phosphorene, have also been studied262728Two-dimensional 2D) nanomaterials, such as graphene, boron nitride (BN) and molybdenum disulfide , have a structure similar to graphene but possess many distinguished propertieset al. first reported that the elastic modulus of CVD-grown bilayer BN nanosheets was 0.334±0.024 TPa , and their fracture strength was 26.3 GPa E2D=239–326 N m−1 with an effective thickness of 0.334 nm), while its breaking strength fell in the wide range of 68–215 GPa 424344454647484950et al. were attributed to the presence of defects and grain boundaries52et al. measured the Young’s modulus of ∼15 nm-thick  BN nanosheets produced by CVD to be 1.16±0.1 TPa  BN nanosheets5657There have been a few measurements on few-layer BN produced by chemical vapour deposition (CVD), but the mechanical properties of monolayer BN have never been experimentally examined. Song Here, the mechanical properties of high-quality mono- and few-layer BN are experimentally revealed, to our knowledge, for the first time. The monolayer BN is found to have a Young’s modulus of 0.865±0.073 TPa, and fracture strength of 70.5±5.5 GPa. In contrast to graphene, whose strength decreases dramatically with an increase in thickness, few-layer BN nanosheets (at least up to 9L) have a strength similar to that of 1L BN. Detailed theoretical and experimental investigations indicate that the difference is caused by the distinct interlayer interactions in these two nanomaterials under large in-plane strain and out-of-plane compression. This study suggests that BN nanosheets are one of the strongest insulating materials, and more importantly, the strong interlayer interaction in BN nanosheets, along with their thermal stability, make them ideal for mechanical reinforcement applications.2/Si) substrates with pre-fabricated micro-wells of 650 nm in radius. −1, which is very close to that of bulk hBN The BN nanosheets were mechanically exfoliated from high-quality hBN single crystalsδ) of the nanosheets, as follows:The mechanical properties of the mono- and few-layer graphene and BN nanosheets were studied by indentation at the centre of the suspended regions using AFM. To obtain load–displacement curves, the AFM displacements were converted into the deflection  is a dimensionless constant; and ν is Poisson’s ratio. E2D is the 2D effective Young’s modulus of the nanosheet, which can be converted to the conventional bulk  modulus (E) by dividing it by the thickness of the nanosheet. For BN, we used an effective thickness of 0.334 nm, and a Poisson ratio of 0.211 , 645±16 N m−1 (N=13) and 985±10 N m−1 (N=6), respectively. These values are consistent with those obtained by previous studies using AFM1E2D of 1L BN was 289±24 N m−1 (N=11). This result is in agreement with a few theoretical predictions43444546E2D of 2L and 3L BN nanosheets were 590±38 N m−1 (N=14) and 822±44 N m−1 (N=6), respectively. The dashed lines in E2D of graphene and BN nanosheets with increased thickness, which were obtained by multiplying the E2D values of their monolayers by the number of layers. In other words, the difference in the experimental data and dashed lines indicates the relative changes of E2D with the increased thickness of graphene and BN. It can be seen that the E2D of graphene deviated more than that of BN as thickness increased. This can be shown more clearly by plotting the (volumetric) Young’s moduli of graphene and BN at different thicknesses  of graphene and BN of different thicknesses are shown in −1), 107.7±4.3 GPa (72.1±2.9 N m−1), 105.6±6.0 GPa (106.2±6.0 N m−1), and 85.3±5.4 GPa (228.6±14.5 N m−1) for monolayer, bilayer, trilayer, and eight layers, respectively. Again, these values are in agreement with those reported previously19−1), 68.0±6.8 GPa (45.4±4.5 N m−1), and 76.9±2.3 GPa (77.0±2.3 N m−1), respectively. Previous theoretical calculations yielded a quite different breaking strength for 1L BN, and our experimental results match well the value calculated by Peng et al. using density functional theory (DFT)et al. and Mortazavi et al., both of which used molecular dynamics simulations43The strengths of graphene and BN nanosheets of different thicknesses were calculated based on load–displacement curves and fracture loads using finite element simulation. The fracture loads . This difference results in opposite changes in sliding energy in the two materials under strain and pressure. These results indicate that the BN layers close to the indentation centre were strongly glued and very unlikely to develop interlayer sliding. In striking contrast, the graphene layers could spontaneously slide between each other as the AB stacking was no longer stable.In standard or equilibrium crystal lattices , the sliding energy from the AA′ to AA′ stacking in 2L BN was only slightly larger than that from the pre-defined zero sliding energy of the AB to AB stacking in 2L graphene, that is, 7.22 versus 5.12 meV per unit cell . These r56616263F′). In the isotropic elastic limit, the shear strain energy in the core (Ushear) of the sandwich beam structures can be given as:For simplicity, hypothetical sandwich beam geometries were used to explore the sliding tendencies in 2L graphene and BN. Note that such an estimation did not consider the nonlinear deformation in the structures under indentation. The two surface layers of the 2L graphene and BN can be defined as faces, and the interlayer interactions including van der Waals interactions can be viewed as a core. Such designation meets the basic requirement for a sandwich structure where the faces are much stiffer than the core. In addition, the core in graphene and BN nanosheets satisfies the concept of an ‘antiplane’ core, which has no contribution to the bending stiffness of the structure but can sustain a finite shear stress. The beams with a length of 1,300 nm and width of the unit cell of graphite and hBN have both ends clamped and are under central loads ; x is the distance to the central point of load (F′); ws is shear displacement, which is equal to F′x/4AG. AG is the shear stiffness of the sandwich structure. The shear stiffness of graphene and BN was linearly approximated based on the vdW-DFT-deduced sliding energy from the AB to AB stacking in graphene, and from the AA′ to AA′ stacking in BN. When no strain or compression was applied, the G values of graphene and BN were 5.11 and 6.61 GPa, respectively . Loading/unloading curves with no obvious hysteresis were used for fitting and calculations; curves showing large hystereses were excluded. The loading/unloading curves of few-layer graphene could not be reproduced by equation Mechanical exfoliation by Scotch tape was used to prepare suspended graphene and BN nanosheets32N nm and 0.334 × N nm, respectively, where N is the number of layers. A total of 1,663 two-node linear axisymmetric membrane elements (MAX1) were employed with mesh densities varying linearly from 0.1 nm (centre) to 1.0 nm (outermost). The interactions between the indenter tip and nanosheet were modelled by a frictionless contact algorithm. An indentation depth of 100 nm was applied to a prescribed displacement of 0.1 nm per load step. The constitutive behaviours of both graphene and BN were assumed to be nonlinear elastic, and thus expressed as:Computational simulations were performed using the commercial nonlinear finite element code ABAQUS. The diamond tips were modelled as rigid spheres. The nanosheets were modelled as axisymmetric membranes with a radius of 650 nm. The initial thicknesses for graphene and BN nanosheets were assigned as 0.335 × E is Young’s modulus and D is the third-order elastic constant. The Young’s moduli of graphene and BN were set to 1,000 GPa and 865 GPa, respectively. The value of D for graphene was −2,000 GPa .Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures and Supplementary References