P = 4.1e-03, P = 1.9e-05, respectively), waist circumference , body fat percentage , insulin resistance , fasting concentrations of triglycerides  and leptin . Tauro- and glycohyocholic acids, and tauro-a-muricholic acid were directly linked with a distinct gut microbial community primarily composed of Clostridia species . We conclude that serum conjugated C-6-hydroxylated BAs associate with measures of human metabolic health and gut communities of Clostridia species. The findings merit preclinical interventions and human feasibility studies to explore the therapeutic potential of these BAs in obesity and type 2 diabetes.Knowledge about in vivo effects of human circulating C-6 hydroxylated bile acids (BAs), also called muricholic acids, is sparse. It is unsettled if the gut microbiome might contribute to their biosynthesis. Here, we measured a range of serum BAs and related them to markers of human metabolic health and the gut microbiome. We examined 283 non-obese and obese Danish adults from the MetaHit study. Fasting concentrations of serum BAs were quantified using ultra-performance liquid chromatography-tandem mass-spectrometry. The gut microbiome was characterized with shotgun metagenomic sequencing and genome-scale metabolic modeling. We find that tauro- and glycohyocholic acid correlated inversely with body mass index ( BAs emulsify dietary fats in the small intestine and facilitate their absorption2. At the terminal ileum, the majority of BAs are absorbed by the apical sodium dependent BA transporters and enter the hepatic portal circulation. In total, about 95% of BAs are reabsorbed into intestinal enterocytes, whilst > 5% of BAs enter the systemic circulation and act on multiple organs throughout the body via several types of receptors3. The most prominent of these are the farnesoid X receptor (FXR) and the Takeda G protein-coupled membrane receptor 5 (TGR5) which are expressed in a variety of peripheral tissues7. Through these receptors, BAs regulate biological processes such as immunity, neuroprotection, metabolism, and energy expenditure12.Bile acids (BAs) are a class of steroids produced from cholesterol in the liver where they are conjugated with either glycine or taurine and released into the bile. These primary BAs, which in humans predominantly comprise of cholic acid (CA) and chenodeoxycholic acid (CDCA) are absorbed by different intestinal bacteria, and conjugates of glycine or taurine are removed with subsequent alteration of hydroxyl groups to form secondary BAs14. A large part of primary BAs in pigs and rodents consists of C-6 hydroxylated hyocholic acid (HCA) and hyodeoxycholic acid (HDCA) and their conjugated derivatives, also called γ-muricholic acid15. In pigs, these C-6 hydroxylated BAs may comprise about 5% of the circulating BA pool and there is growing evidence that these specific BAs may contribute to maintain glucose homeostasis in domesticated pigs that are resistant to development of type 2 diabetes despite living in an obesogenic environment15. The dominating BAs in humans are different. Here, the primary BAs consist of cholic acids (CA) and chenodeoxycholic acids (CDCA), which are not hydroxylated at the C-6 position15.At present, we have sparse knowledge about the biosynthesis and the functions of a considerable part of the human blood profile of BAs due to the biological differences between human and commonly used animal models like pigs and rodents13. Here, we tested two related hypotheses, (i) a variety of C-6 hydroxylated BAs are measurable in human serum and are associated with human metabolic health, and (ii) the serum concentration of these C-6-hydroxylated BAs are associated with distinct co-abundant microbial species in the human gut microbiota.Recent studies have demonstrated, however, a previously underappreciated diversity of BAs in human blood including small amounts of HCA and HDCA and their derivatives16. In the present study, we measured serum BAs in 283 Danes of whom 106 were non-obese (BMI < 27 kg/m2) and 177 were obese (BMI > 27 kg/m2). None of the volunteers had undergone bariatric surgery, and none had taken antibiotics two months prior to their inclusion in the study protocol. Median age of the study population was 56.4 ± (standard deviation (SD) = 7.3) years, mean BMI was (29.5 ± 5.99 kg/m2); 133 were male and 150 were female. None of the participants were diagnosed with diabetes mellites (Type 1 or 2). See electronic supplementary notes (ESM) for gender-stratified statistics. 34 of the study participants were taking statins at the time of study. We compared the BA concentrations between the group of subjects with or without administration of statins by Mann–Whitney U test, and found no significant differences (P < 0.05).Study participants were recruited from the Danish Inter99 study and were originally selected for the MetaHit study on microbiota gene richness16. The study was approved by the local Ethical Committees of the Capital Region of Denmark (HC-2008-017), and was in accordance with the principals of the Declaration of Helsinki. All individuals gave written informed consent before participation in the study.Details of clinical and biochemical phenotyping, stool sampling and microbial DNA purification, library preparation, Illumina shotgun sequencing of microbial DNA and quality assurance of sequencing reads have been reported17. Details on sample preparation and molecular analysis of BAs are given in (ESM).Blood samples were obtained in the morning after a minimum 10 h of overnight fasting. BA levels were measured in serum by ultra-performance liquid chromatography-tandem mass-spectrometry (UPLC-QqQMS)18. Reads that mapped onto several genes were attributed with the smart shared read method. The read count matrix was then downsized to 8 M reads. Gene abundances were normalized by the gene length and the total number of reads (Reads Per Kilobase Per Million mapped reads (RPKM) normalization).Illumina reads were mapped to the Integrated Gene Catalogue version 9.9 to construct a gene count matrixet. al19. We assembled 3,463 co-abundant Cluster Genes (CAG) having 50 genes or more. In the present study, 1,437 CAGs were composed of more than 500 genes and were considered as MGS. Taxonomic annotation of MGS was based on the aggregation of its gene annotation applying ‘BLASTn’ as previously reported19.The gene count matrix was used to cluster metagenomic species (MGS) based on co-occurrence by use of the canopy clustering software developed by Nielsen ‘momr’ R package (https://cran.r-project.org/web/packages/momr/index.html). MGS abundance was defined as the mean abundance of the 50 MGS genes that correlated strongest to the median gene abundance. We considered the MGS abundance in a sample to be null, if fewer than 10% of its genes were present.We computed MGS abundance using the https://github.com/hjulienne/Gleesso_Pipeline) pipeline to adapt the SPIEC-EASI pipeline to shotgun MGS abundances and to construct gut bacterial communities. A detailed step-by-step description of the applied community construction approach is given in (ESM).We implemented the Gleesso . (2) Only species assigned to the same community on more than 60% of subsets were included in the distal analyses. Abundance was computed on the robust community composition. We defined the stability index of a species as the fraction of the subset where it was assigned to the same community. Details about the correlation analysis are given in (ESM).To assess the robustness of the gut community composition detected by the Gleesso pipeline we applied the following: (1) The Gleesso pipeline to 100 different subsets of 80% of the study sample stratified for bacterial gene richness groupscikit-learn’ library: (http://scikit-learn.org/stable/modules/linear_model.html#ridge-regression). Serum levels of BAs were scaled prior to model fitting, so the coefficients would be comparable to one another. The target variables were BMI, HOMA-IR and fasting serum levels of leptin. The latter variables were binarized around their median values. The BMI target was binarized around 27 kg/m2. Models were fitted separately for women and men. We drew 10,000 partitions of the study sample by randomly splitting the 80% of study samples in a training set and 20% in a test set. For more detail, please see (ESM).We constructed logistic ridge regressions with the python ‘20. AGORA is a comprehensive resource of semi-automatically reconstructed GEMs for the human gut microbiota. A detailed methodology of GSMM applied to BA metabolism can be found in the (ESM). We extended the GSMM framework by adding a common compartment simulating the gut epithelial cells  of gut microbiota to achieve insights into the BA biosynthetic and biotransformation potentials / pathway(s). MGS with more than 500 genes were listed and mapped to the AGORA version 1.02) compendium, comprising 773 gut bacteria (including 205 genera and 605 species).02 compe21. Transporters for HCA, particularly THCA  have been identified and reported in Recon3D22, a generic human metabolic reconstruction. The BA transporters obtained from Recon3D and several other pieces of bibliographic evidence were added to the gut epithelial cell model developed in this study (Supplementary Dataset 24. A report on the AGORA approach deployed in the study can be found in the (ESM).In the intestinal epithelial cells, some BA transporters (for e.g. ABC transporter) have been previously identifiedSTRING v10.5, a resource that curates the relatedness of COGs and proteins25. Connections with one intermediary COG was allowed, in which case the sum product of connection strengths was evaluated as the connectivity.We attempted to identify intestinal bacterial genes with the potential to produce C-6 hydroxylated BAs. We collapsed 2,250,421 microbial genes into 3,812 Clusters of Orthologous Groups (COGs) of proteins. We hypothesized that structurally similar proteins carry similar functions, and certain COGs could confer the ability to modify certain BAs. We grouped the BAs by their hydroxylation patterns, irrespective of their conjugation type(s) for a specific BA group (BAG), e.g. by collapsing GCDCA, TCDCA and CDCA into CDCAs. The concentration of a BAG was computed as the sum of the constituent BA concentrations. Pairs of BAGs that differed in only one hydroxylation position by presence/absence of steric orientation were identified, and ratios between the concentrations of these BAGs were computed for each individual. Next, these ratios were correlated with each MGS by using Spearman’s correlation analyses. Finally, by applying a Wilcoxon test, we compared the correlation strengths between the MGSs that did or did not contain a given COG. Associated COGs were deemed to be positively or inversely associated with a BAG ratio of interest based on the median correlation coefficient of the MGS that contained the COG. Further, these associated COGs for each BAG ratio were mapped onto 2 test was employed to determine if there was any significant degree of dependency between MGS correlation to a BAG ratio of interest and the presence of a COG. BAG was correlated by Spearman’s correlation to each BAG and correlation strengths were saved. For each COG, we identified which MGSs did and did not contain a gene corresponding to that given COG. In order to investigate whether the presence of a given COG conferred the ability to shift the ratio of BAGs, MGSs were indexed on two conditions; whether or not they contained a given COG and whether they correlated positively or inversely with a given BAG ratio of interest. A χ2 test was employed to determine dependency between these two traits.Additionally, a χFigure Serum BAs present in at least 50% of the study participants were considered for the data analysis. 20 BAs with known chemical structure and eight with unresolved stereochemistry structures were included in the final dataset Fig. .Figure 2P < 0.05) with BMI, waist circumference, body fat percentage, insulin resistance (HOMA-IR), fasting circulating levels of triglycerides and leptin . THCA was inversely correlated with BMI, body fat percentage, and leptin   . Despite only being significantly correlated with one adiposity trait, this BA clustered with THCA and GHCA for their similar correlations. In addition, several conjugated C-6 hydroxylated BAs with unresolved stereochemistry  were inversely associated with adiposity traits, similar to associations observed with THCA and GHCA. By contrast, serum concentrations of DCA, UDCA, and their conjugates were positively associated with fasting circulating levels of glucose, insulin and C-peptide, adiposity measures and HOMA-IR  Fig. . TαMCA, 2  modeling to assess the impact of various BA concentrations on key indicators of metabolic health: BMI, HOMA-IR and fasting serum concentration of leptin. We binarized targets around the median for HOMA-IR and serum concentrations of leptin, while BMI was binarized around 27 kg/mAs shown in  between abundance of bacterial communities and fasting serum concentration of BAs  MGSs were organized into 17 bacterial communities . The finding may indicate that the species of this community benefit from a gut ecology rich in sulfur.Community K, which was composed of pp. Fig. a, correlP = 0.012, 0.04, 0.0056, respectively, not shown in Fig. Several bacterial communities were inversely associated with the ratio of secondary to primary BAs , namely communities E, K and L , we applied GSMM. We identified 26 abundant microbial species (see Methods) that have metabolic reconstructions or genome-scale metabolic models (GEMs) in the AGORA compendium  vs. non-obese n = 106) individuals (BMI binarized around 27 kg/m individu77 vs. noP < 0.05) with one or more BAG ratios  dependencies. Additional information on the applied approach is given in (ESM).We attempted to identify Clusters of Orthologous Groups (COGs) that correlated to a ratio of two closely related sets of BAs, ignoring conjugation, termed ‘BAGs’, as described in (Methods). Ratios of closely related BAGs were correlated to MGSs by Spearman’s correlation and differences in correlation strength were evaluated by Wilcoxon test. One thousand four hundred and twenty three COGs were associated  correlated inversely with BMI and HOMA-IR26. Similarly, in a Chinese study comprising 20 healthy, 18 prediabetic and 17 drug-naïve type 2 diabetes individuals, the authors reported inverse correlations between C-6 hydroxylated BAs and BMI and plasma glucose levels after an oral glucose load27. In studies of pigs and mice supplemented with experiments in enteroendocrine L-cells, the same investigators showed that HCA, HDCA and conjugated derivatives add to promote metabolic health via an enhanced GLP-1 biosynthesis and secretion involving an activation of TGR5 and an inhibition of FXR27. Hence, given the critical role of C-6-hydroxylated BAs in maintaining metabolic homeostasis, the novel insights call for future testing of potential therapeutic applications of these BAs.Our findings of inverse relationships between C-6-hydroxylated BAs and markers of dysmetabolism and adiposity in 283 nondiabetic Danes extend previous findings in both humans and pigs. Thus, in a US study of 31 morbidly obese individuals who had undergone sleeve gastrectomy, fasting and post lipid meal increase in plasma concentration of glycine- conjugated HCA correlated positively with the surgical weight loss at 12 weeks of follow-up3.Our study has limitations. The study design is observational and no causality can be inferred. Moreover, the present study is like epidemiological studies of gut microbiota in general limited by not being able to sample microbiota specimens at the optimal intestinal localization. The gut microbiota in the small intestine has the first exposure to BAs, while our microbiota analyses were performed on faecal samples as a proxy for the overall intestinal microbiota. Additionally, serum BAs are subject to first-pass liver metabolism, which is likely to affect circulating BAs to various extents, adding another element of variabilityClostridia in the human gut microbiota are positively associated with fasting serum of C-6 hydroxylated BAs, which in turn are inversely associated with adiposity traits and markers of dysmetabolism. In perspective, our findings merit testing in future preclinical interventions and human feasibility studies exploring the potential therapeutic role of C-6 hydroxylated BAs in states of obesity and its metabolic co-morbidities. In addition, when human gut metagenome databases become considerably more complete, further search for a bacterial contribution to the biosynthesis of C-6-hydroxylated BAs merits consideration.Co-abundant species of Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.


Appraise the evidence on the outcomes of Leg Clubs on ulcer healing, psychosocial outcomes, patient safety, cost and experiences of Leg Club members.The Leg Club is a community-based social model of care in 30 UK locations and nine overseas for treating patients with chronic leg wounds. However, its cumulative effectiveness has not been reviewed to-date.Systematic review of primary research relating to the impact and quality of care of Leg Clubs treating patients with leg ulcers. Six electronic databases were systematically searched using the MeSH term ‘leg ulcer’, including other representative terms, in combination with ‘Leg Club’. The quality of individual studies was assessed using appraisal tools. The confidence in the quantitative evidence was evaluated using Grading of Recommendations Assessment, Development and Evaluation (GRADE); and the Confidence in the Evidence from Reviews of Qualitative Research  assessed the quality of qualitative findings.A total of 17 relevant publications were identified. Out of the 17 articles, four publications represent findings from randomised controlled trial (RCT). Thus, evidence from 14 independent studies involving at least 532 participants were included in the synthesis of this review. The quality of the evidence varied across the different outcomes and were mostly low or of very low quality. Findings from one underpowered RCT from Australia reporting on clinical, patient-reported outcomes and economic outcomes were evaluated as moderate quality. Studies indicate that the Leg Club model has a positive impact on ulcer healing and recurrence, mood, sleep, quality of life and pain. Moreover, only three studies assessed wound infections and reported no infections had occurred during treatment at the Leg Clubs. Economic evaluations find Leg Clubs to be probably more cost-effective than usual care. Both patients and nurses projected positive views about the Leg Clubs, with particular emphasis on improved social interactions and delivery of patient-centred care. Ten of these explored the views of UK patients  from two different Leg Clubs in the United Kingdom  during a three-month period (Gordon This review considered the evidence from a wide range of study designs developed in two countries supporting the Leg Club model of care. The quality of findings ranged from moderate to very low across the different quantitative outcomes, and the confidence in qualitative findings was moderate to low primarily due to concerns in methodological design and data adequacy as assessed by CERQual. The main limiting factor that downgraded quality in most quantitative outcomes in accordance to the GRADE framework was the imprecision of results due to the small number of participants included in the analysis. Moreover, the lack of a comparator to evaluate the effectiveness of the Leg Club interventions is another limitation associated with most studies. Although, it can be argued that the evidence for clinical outcomes and patient-reported outcomes from the one RCT could be evaluated as moderate quality since a large magnitude effect and dose-response effect were illustrated. However, blinding of participants and outcome assessors were not possible in this RCT, and as such increases the probability of performance bias and detection bias. Moreover, inclusion of data from non-RCT studies exposes further risk of bias affecting the credibility of the overall results. Therefore, the evidence was assessed as low or very low quality for most outcomes.et al., et al., et al. (Although the evidence is rich in diversity of outcomes, it is weak in providing robust scientific direction for healthcare commissioners who may wish to explore this model in community settings. The higher quality evidence showing improvements in ulcer healing and other patient-reported outcomes was from one underpowered RCT conducted in Australia. No experimental evidence exists for the majority of Leg Clubs operating in the United Kingdom. Australian findings may not be mirrored in the United Kingdom, such that healing rates in the United Kingdom were nearly two-thirds of that achieved in the Australian context (Edwards , et al. , and as et al., Two studies found that treatment concordance improved during attendance at the Leg Club Lindsay, . Howeveret al., et al., et al., Although our review identified relevant articles systematically, it was difficult to draw conclusive inferences due to variability in study designs and assessment tools. There were numerous limitations related to the studies, including minimal detail of sampling strategies, allocation concealment, blinding of participants and assessors, small sample sizes, and lack of clarity on study and analytic methods. Therefore, it was difficult to evaluate the validity and rigour of the findings. Furthermore, due to the nature of the methodology of surveys, audits and case reports, no comparators were included; thus rendering their data as inconclusive. In addition, the discrepancy in the different sample sizes reported from the same RCT could not be resolved despite attempts in contacting the authors. Consequently, we believe that the evidence published at different time points represent findings from preparatory work that includes single feasibility (Edwards et al., This review highlights the potential of the Leg Club social model of care to make contributions to reducing the burden for people with chronic leg wounds and the costs associated with these conditions. A few Leg Clubs are starting to be commissioned by NHS local commissioning groups who believe that the cost-savings and improvement in care demonstrated by local audits are convincing (Lindsay, The Leg Club holds potential for providing cost-effective specialist community-based wound care. A fully powered UK RCT is needed with appropriate comparator groups to ascertain the value and contribution of Leg Clubs to the UK NHS.


Dear Editor,Medicine is a complex system of continuously evolving knowledge. Patients characterized within this knowledge system are heterogenous, and have contextual complexity evading even the most robust algorithm. Due to this complexity and evolution, even the very best artificial intelligence  systems when making present and future predictions will inevitably be wrong some of the time. Empowering clinicians to recognize and reject incorrect predictions is of utmost importance.Translational Psychiatry reveals that explainability may not live up to its ethical ideal.‘Explainability’  has received a lot of attention both inside and outside of the ML field. Some ethical AI guidelines have proposed explainability as a core principle akin to autonomy or beneficence  and streJacobs et al.  conducteThese compelling findings are important additions to the accumulating evidence highlighting the risks of over-trust (defined herein as reliance on incorrect ML predictions) in the context of clinical decisions informed by ML –6. The iYet, such comparisons may not always be as simple as quantifying individual decisions as ‘correct’ or ‘incorrect.’ As Jacobs et al.  note, drInitiating antidepressant treatment is a shared decision between patients and clinicians, better guided recently by the accumulated evidence from a plethora of randomized controlled trials , 9. AntiTo the contrary, consider accuracy in the context of diagnosis. A diagnosis is generally made according to the presence of signs, symptoms, and other biomarkers that indicate the presence of a medical condition. Patient preferences are not central to determining a diagnosis as they are for making treatment decisions. In this case, clinician accuracy is scored on their ability to detect the true presence of a disease just as it would be for an ML model.It is particularly interesting that Jacobs et al.  found thA hint that this may be happening is in Jacobs and et al. particularly intriguing finding that clinicians with the most knowledge about ML relied on it less but were more confident and ranked it more useful than their less-experienced peers . PerhapsGuided by moral commitments to patient autonomy and best interests, clinical decision-making incorporates medical evidence as but one factor in a larger picture in which accuracy is but one metric. ML contributes to the evidence base by operationalizing a particular axis of decision-making. The key to preventing over-reliance is perhaps not in providing explanations, but in compartmentalizing these axes with clinicians accountable for the moral goal not of accuracy, but of helping patients.


Several species can detect when they are uncertain about what decision to make—revealed by opting out of the choice, or by seeking more information before deciding. However, we do not know whether any nonhuman animals recognize when they need more information to make a decision because new evidence contradicts an already-formed belief. Here, we explore this ability in great apes and human children. First, we show that after great apes saw new evidence contradicting their belief about which of two rewards was greater, they stopped to recheck the evidence for their belief before deciding. This indicates the ability to keep track of the reasons for their decisions, or 'rational monitoring' of the decision-making process. Children did the same at 5 years of age, but not at 3 years. In a second study, participants formed a belief about a reward's location, but then a social partner contradicted them, by picking the opposite location. This time even 3-year-old children rechecked the evidence, while apes ignored the disagreement. While apes were sensitive only to the conflict in physical evidence, the youngest children were more sensitive to peer disagreement than conflicting physical evidence. SomeWhile these studies reveal the ability to detect when one has no knowledge, they do not reveal an ability to think about what one already believes, which is a different kind of metacognition. Thinking about what one believes is sometimes elicited when one encounters new evidence that calls a prior belief into question. Suppose you believe it is sunny out and plan to go to the beach. Now the dog comes into the house soaking wet. This may prompt you to look out the window to recheck your reason for believing it is sunny before deciding to leave. In such a scenario, we are aware of what we believe, and we check the grounds or reason for that belief against what the new evidence indicates. Following terminology used by philosophers , this caHere, we devised a new methodology to explore this ability. In a first study, we tasked participants with evaluating two contradictory pieces of physical evidence. Apes and children were presented with an initial piece of evidence leading them to form a belief about which of two rewards was greater, which they indicated with a choice. Then, before being rewarded, new equally good evidence appeared, indicating the opposite reward was greater. Now participants could stop and look for more information before making a final decision, or they could choose again without looking. We expected that participants that recognized the contradiction between their prior belief and the new evidence would seek more information before choosing.For humans, such contradictions arise more commonly via disagreements with a social partner—one person believes it is sunny but another says it is raining. Our second study explored this kind of contradiction. After participants formed a belief about the location of a reward, an onlooking social partner gestured towards an opposite location as the site of the reward. Here, treating the conflict as grounds for uncertainty requires understanding not only that our own belief might be false, but also that our partner may be mistaken –18. Beca. 2n = 18), 3-year-old (n = 64) and 5-year-old children (n = 66). We chose these age groups because children's understanding of belief develops substantially between 3 and 5 years [figure 1d; electronic supplementary material, movie S1). Or, they could make a final choice without peeking .In our first study, were apes https://osf.io/ey5f9/). A full-null comparison was significant , where the null model lacked the terms ‘population’  and ‘condition’ (consistent and conflicting), and their interaction. Model comparison revealed no significant interaction between population and condition . Therefore, we removed the interaction term and tested for an effect of ‘condition’. This revealed a significant overall effect of condition . It appears, however, that the effect of condition is driven by the apes and 5-year olds, rather than the 3-year olds (who peeked slightly more in the consistent condition)  with binomial error structure to test the data  .Figure When presented with new evidence that conflicts with a prior belief based on equally good evidence, great apes sought additional information before making a decision . This ability appears to have emerged by the age of 5 years in human children, but not clearly by the age of 3 .. 3n = 17), 3-year-old (n = 72) and 5-year-old children (n = 42). Participants faced each other and between them were two boxes on a slider that could be moved back and forth (a). In ‘consistent’ trials, the non-target picks what the target expects, but in ‘conflicting’ trials  or can choose without peeking (figure 3c).In a second study, participants were faced with conflicting evidence that came from the opinion of a social partner, rather than new physical evidence. Participating in this study were apes (nd forth . At the nd forth a. In ‘co (a)χ2 = 14.867, p = 0.01), allowing us to reject the null hypothesis. A comparison of the full model to a reduced model lacking the interaction term showed no significant improvement . We therefore removed the interaction term and compared models with and without the term ‘condition’, finding a significant overall effect : subjects peeked more in response to a contradictory opinion than one that was consistent with their prior belief . Our next analysis further supports this interpretation.χ2 = 11.408, p = 0.003). To isolate the source of the three-way interaction, we tested for an interaction between study and condition in each population separately. We found an interaction between condition and study in the apes : apes distinguished the conditions more in the physical study than in the social study. We also found an interaction between study and condition in the 3-year olds : 3-year olds distinguished the conditions more in the social study than in the physical study. There was no interaction in the 5-year olds, but rather a main effect of condition: the 5-year olds peeked significantly more when faced with conflicting evidence in both studies  . These results support our original hypothesis: apes were more sensitive to conflicting physical evidence than social evidence, while the youngest children were more sensitive to conflicting social than physical evidence.Overall we had predicted that great apes would be more sensitive to conflicting physical rather than social information, while young children would be more sensitive to conflicting social information. To test this, we pooled all data and tested for a three-way interaction of population, condition and study—to see if different groups were more sensitive to the distinction between conditions from one study to the other. This was significant  . This is hard to explain if participants were not thinking of their prior belief and the new evidence at once.One challenge raised for studies like this one is that participants may be considering just one piece of evidence at a time, rather than considering old and new evidence together (Leahy & Carey ). Here tp < 0.0001; 3 year olds mean = 0.88, p < 0.0001; apes mean = 0.63, p < 0.001) . This rules out the possibility that the apes were not paying attention to their partners in the social study. Apes knew what choices their partners were making, but peer disagreement was not enough to get them to doubt their prior belief.Another concern is that in the social study, the apes may not be attending to their partner's choices. This would explain why they were slower to recheck evidence in light of conflicting opinions than conflicting physical evidence. To rule this out, we conducted an ‘ignorance-knowledge’ post-test. Here, participants received no prior information about the location of the reward, but could see that their partner could see where it was. Now participants significantly followed their partners' choices . Here, rational animal does not easily demarcate humans from all other species. One of the hallmarks of rationality—the ability to weigh contradictory reasons against one another—is shared by humans and great apes. On the other hand, these studies taken together suggest that the major distinction between human and great ape decision-making is in its sociality. Younger children are more likely to look for more information given peer disagreement than conflicting physical evidence, while apes are more likely to double-check given a conflict in physical evidence rather than peer disagreement. This fits with the view that human rationality is adapted for social purposes, including solving peer disputes, argumentation and knowledge transmission [good at problem-solving in social contexts, but better at solving problems socially than individually—and that the distinguishing mark of human cognition is its sociality.Recognizing that reliable decisions cannot be made on the basis of contradictory reasons has long been considered to be the cornerstone of rationality. Aristotle argued that only creatures capable of recognizing contradictory reasons as poor grounds for a decision could count as rational animals . The phismission –21,31,32Click here for additional data file.


Subtalar dislocations are uncommon injuries that involve disruption of the talocalcaneal and talonavicular joints. Whereas medial subtalar dislocations are usually caused by low-energy mechanisms and are reducible by closed means, lateral subtalar dislocations occur due to high-energy trauma, have associated foot injuries, and may require open reduction. Good outcomes can be expected for low-energy medial dislocations, whereas high-energy dislocations have guarded outcomes. Hindfoot deformity and chronic instability can result from nonanatomic reduction and inadequate stabilization. Arthrosis of the subtalar joint can occur despite anatomic reduction and is attributable to the cartilage damage at the time of injury. These injuries can be broadly grouped into two distinct patterns, although other, lesser common variants have also been reported. Medial subtalar dislocations are more common, occur due to low-energy trauma, and generally have good functional outcomes. In stark contrast, lateral subtalar dislocations are less common, occur due to high-energy trauma, and have guarded outcomes.3 From an anatomic perspective, three distinct components of the STJ complex can be identified.4 The posterior part of the STJ complex consists of the saddle-shaped articulation between the posterior facets of the talus and calcaneus; this is termed the talocalcaneal joint (TCJ). The anterior part, termed the talocalcaneonavicular joint (TCNJ), is formed by the articulations between the anterior and middle facets of the talus and calcaneus, the talar head, and the medial aspect of the calcaneonavicular ligament. The TCNJ is also called the coxa or the acetabulum pedis because of its anatomic and developmental similarities with the hip joint. The convex talar head resembles the femoral head and concave articular surfaces of navicular and anterior and middle facets of the calcaneus, the acetabulum. The interosseous canal between the talus and calcaneus forms the middle part of the STJ complex and separates the anterior and posterior parts. This canal is conical and consists of the broad sinus tarsi anterolaterally and the narrow canalis tarsi posteromedially. The interosseous canal houses the interosseous ligament complex, which is a key stabilizer of the STJ complex.3The literature on the anatomy of the STJ complex is variable. Whereas a section of the literature considers the posterior articulation between the talus and calcaneus as the STJ, other reports include the anterior talocalcaneonavicular and even the calcaneocuboid joints as a part of the STJ complex.Stability of the STJ is provided by its bony architecture and dense ligament complex. The ball and socket nature of the TCNJ imparts inherent osseous stability. Stabilizers of the STJ complex can be grouped into medial, lateral, anterior, and interosseous ligaments for simplicity.3 In addition to the ligaments, stability is augmented by capsules of the talocalcaneal and the TCNJ.On the lateral aspect, the calcaneofibular ligament is the most important stabilizer of the STJ in inversion. The anterior, lateral, and posterior talocalcaneal ligaments are small, inconsistent, and offer little support by themselves Figure . On the 5The talus has a complex blood supply and receives contributions from the posterior tibial, anterior tibial, and the peroneal arteries. The deltoid branch of the posterior tibial artery supplies most of the talar body and medial part of the talar neck. The perforating branches of the dorsalis pedis artery enter the talar neck through its dorsal surface and supply the talar neck and the talar head. The peroneal artery has the least contribution, and supplies, the lateral part of the talar neck.4 From a functional and biomechanical perspective, the STJ is considered a uniaxial joint with triplanar motion. As described by Manter et al,6 the STJ axis is directed medially, anteriorly, and superiorly. Movements of the STJ can be described in the frontal, sagittal, and transverse  planes. Frontal plane movements are varus and valgus and describe the movement of the calcaneus relative to the talus in the medial and lateral direction, respectively. Sagittal plane movements are dorsiflexion and plantar flexion and represent the upward and downward movement of the anterior end of the calcaneus, respectively. Transverse plane movements are adduction and abduction and describe the movement of the forefoot and midfoot in the medial and lateral directions relative to the hindfoot. Supination and pronation are essentially movements of the forefoot. Supination describes the position of the foot where the medial border is lifted off the ground, and pronation occurs when the lateral border of the foot is off the ground. Mosca7 compares the STJ motion to the act of wringing a towel and describes the subtalar motion in terms of movement of the acetabulum pedis around the head of the talus. Inversion is a combination of plantar flexion and varus of the heel along with adduction and supination of the forefoot. It may be thought of simply as the down and in position. In contrast, eversion is a combination of valgus and dorsiflexion of the heel, along with abduction and pronation of the forefoot; this may be thought of as the up and out position.7Although the TCJ and the TCNJ are anatomically distinct joints with separate capsules, they function as a single unit.4 This unlocks the foot and allows it to adapt to uneven surfaces in the stance phase. On the contrary, the axes of the subtalar and midfoot joints converge during propulsion. This motion locks the forefoot and makes it a rigid lever, allowing the triceps surae to generate adequate power for push-off.7The complex movements of the STJ contribute to the midtarsal joint locking mechanism, which is an essential component of the gait cycle and helps maintain the foot's flexibility. During the stance phase, the axes of the subtalar and midfoot joints become parallel, increasing the sagittal motion of the forefoot.8 showed that the contact pressures in the TCJ (posterior facet) were highest in inversion. However, contact pressures in the TCNJ were not found to be affected by the foot position. Reeck et al9 demonstrated that the contact area and the contact pressure in the TCJ were higher in the toe-off position compared with the foot-flat position.Joint kinetics of the STJ has been studied in detail. Wagner et alIn summary, the STJ inverts during the toe-off phase, decreasing foot motion while increasing the TCJ contact pressures. It everts during the midstance phase in sharp contrast, allowing for greater flexibility and lowering TCJ contact pressures.10 These are rare injuries and are thought to account for <1% of all dislocations, although accurate epidemiologic data lack.12 In a literature review,11 the etiology was traffic accidents in 43.7% of all cases, falls in 32.9%, sports injuries in 13.9%, sprains in 5.3%, and other causes in 4.2%. Males were more commonly affected than females, and the dislocations occurred more frequently on the right side.11Judey and Dufaurest were the first to describe subtalar dislocation in 1811.13 classified subtalar dislocations as medial, lateral, and posterior, depending on the position of the dislocated foot with respect to the talus. An anterior type was added later by Malgaigne and Henke.14 Medial subtalar dislocations account for approximately 75% of all cases and are characterized by medial dislocation of the foot and the heel in relation to the talus. Lateral dislocations account for 17% to 26% of all cases; the heel and foot dislocate laterally with respect to the talus.16 Anterior and posterior dislocations are rare injuries characterized by anterior and posterior dislocation of the foot and heel in relation to the talus, respectively.17181920In 1853, Broca16Medial subtalar dislocations result from forced inversion of the plantarflexed foot; these generally result from low-energy trauma. In this position, the talar neck hinges and rotates around the sustentaculum tali, resulting in rupture of the lateral talonavicular joint capsule and ligaments, followed by the subtalar ligaments. The inversion force may also result in fracture of the posterior process of the talus, which is a common occurrence with medial subtalar dislocations. The talar neck may break off the sustentaculum tali in rare cases as it swivels around this pivot.21 Posterior subtalar dislocations are caused by forced plantar flexion of the foot, whereas anterior subtalar dislocations result from anterior traction to the foot, with the leg in a fixed position.17181920In contrast, lateral subtalar dislocations are produced by high-energy trauma and are the result of forced eversion of a dorsiflexed foot. In this position, the talar head pivots around the anterior process of calcaneus, resulting in rupture of the talonavicular and subtalar ligaments and joint capsules. The same force may also result in partial or complete rupture of the deltoid ligament. An impaction fracture of the talar head or a push-off fracture of the anterior process of the calcaneus may occur as the soft talar head rotates around the anterior process of the calcaneus. Because of the high-energy mechanism of trauma, lateral subtalar dislocations often present with associated foot injuries, such as fractures of the ankle, talus, cuboid, cuneiforms, navicular, and metatarsals.17 think that a subtalar dislocation represents the first stage of a total talar dislocation, wherein the talus dislocates from all its articulations. In a medial total talar dislocation, forced plantar flexion causes the foot to dislocate from the ankle joint. Next, adduction and internal rotation forces cause the foot to rotate 90° along its vertical and longitudinal axis, dislocating the talus from the talocalcaneal and talonavicular joints. Finally, the displacing foot returns to its position leaving the talus dislocated laterally. Similarly, eversion forces may lead to a lateral total talar dislocation. Both of these are high-energy mechanisms.22Leitner et al21Medial subtalar dislocations result in an obvious foot deformity, likened to an acquired clubfoot. The foot is dislocated beneath the talus, the heel is displaced medially, and the forefoot is adducted and plantarflexed. The talar head is often palpable laterally Figure . In cont23The diagnosis of a subtalar dislocation can be made on plain radiographs in most cases. The AP projection of the ankle will show medial or lateral dislocation of the foot beneath the talus. The lateral projection of the ankle will reveal loss of the normal talonavicular articulation. In a normal foot, the convexity of the talar head matches the concavity of the proximal articular surface of the navicular, the so-called head within the cup appearance Figure . This re23 If closed reduction can be performed, this is done first. A postreduction CT helps assess the congruency of the subtalar and talonavicular joints and any associated injuries that might require surgical intervention  and partial weight bearing may be needed for unstable high-energy dislocations that have associated foot injuries. Physical therapy focuses on regaining the complete range of motion at the ankle and STJs.22 Irreducibility by closed means is more common in lateral subtalar dislocations. In medial subtalar dislocations, irreducibility is generally the result of buttonholing of the talar head through the capsule of the talonavicular joint or entrapment within the extensor retinaculum, extensor tendons, or the extensor digitorum brevis muscle. Rarely, interposition of the deep peroneal nerve and dorsalis pedis artery has also been demonstrated as potential blocks to the closed reduction of medial subtalar dislocations.26 Another irreducible variety is the locked dislocation wherein the navicular is impacted into the talar neck.16Closed reduction may be unsuccessful in up to 14% of all cases; several anatomic structures could contribute to the irreducibility.25 Interposed osteochondral fragments can also result in irreducibility and should be sought if there are no soft-tissue obstacles to reduction.25Irreducibility in lateral subtalar dislocations is usually attributable to entrapment of the tibialis posterior tendon or the flexor hallucis longus tendon around the talar head.When indicated, open reduction may be accomplished through an incision centered over the palpable dislocated talar head. After obstacles to reduction are identified and addressed, reduction is achieved easily Figure . The sub11; these are managed on the lines of other open injuries. Early institution of broad-spectrum antibiotics and meticulous wound débridement and lavage are the cornerstones of management. Temporary ankle spanning external fixation may be needed in the presence of large wounds to facilitate wound inspection and change of dressings.Approximately 20% to 25% of all subtalar dislocations are open27; however, this view has been challenged.28 The final outcome depends on several factors, including the severity of the trauma, articular cartilage damage, need for open reduction, prolonged immobilization, and associated foot injuries. Isolated, low-energy dislocations that are stable after closed reduction and are mobilized early can be expected to have an excellent prognosis29 (Figure 24 Posttraumatic arthritis is perhaps the most common complication of subtalar dislocations and invariably involves the STJ, the reported rates varying from 40% to 89%.30 Midfoot arthritis is much less common. Patients with radiographic evidence of subtalar arthritis may not necessarily be symptomatic. In those who have troublesome subtalar arthritis, subtalar fusion may be needed. Chronic instability of the STJ is rare and attributable to inadequate immobilization. Mild to moderate instability may be managed with orthoses and muscle strengthening or ligamentoplasty. Gross instability with painful arthrosis of the STJ would necessitate subtalar arthrodesis.16It was traditionally thought that lateral subtalar dislocations have poor outcomes and medial dislocations have excellent outcomes9 Figure . HoweverSubtalar dislocations can occur due to low- or high-energy injury mechanisms. Up to 60% of these injuries have associated fractures. Hence, a CT scan is indicated to avoid the sequelae of missed injuries. Low-energy, isolated medial subtalar dislocations that can be reduced by closed means have the best prognosis. On the other hand, high-energy and open dislocations that have associated foot injuries fare worse. Subtalar arthrosis is the commonest complication; however, all patients with subtalar arthrosis do not require surgical intervention.


Moreover, fresh tumor samples from 34 patients with HNSCC were examined by flow cytometry and immunohistochemistry to further investigate their properties and cytotoxic capabilities and their correlation with regulatory T cells (Tregs) in the tumor immune microenvironment (TIME). mIHC and flow cytometry analysis showed that TCF1−Texterm represented a greater proportion of CD8+PD1+Tex than TCF1+Texprog in most patients. TCF1+Texprog produced abundant TNFα, while TCF1−Texterm expressed higher levels of CD103, TIM-3, CTLA-4, and TIGIT. TCF1−Texterm exhibited a polyfunctional TNFα+GZMB+IFNγ+ phenotype; and were associated with better overall survival and recurrence-free survival. The results also indicated that larger proportions of TCF1−Texterm were accompanied by an increase in the proportion of Tregs. Therefore, it was concluded that TCF1−Texterm was the major CD8+PD1+Tex subset in the HNSCC TIME and that these cells favor patient survival. A high proportion of TCF1−Texterm was associated with greater Treg abundance.The heterogeneity of exhausted T cells (Tex) is a critical determinant of immune checkpoint blockade therapy efficacy. However, few studies have explored exhausted T cell subpopulations in human cancers. In the present study, we examined samples from two cohorts of 175 patients with head and neck squamous cell cancer (HNSCC) by multiplex immunohistochemistry (mIHC) to investigate two subsets of Tex, CD8 The programmed death-1 (PD-1)-blocking antibody nivolumab was the first immune checkpoint inhibitor approved by the United States Food and Drug Administration (FDA) for metastatic head and neck squamous cell cancer (HNSCC), receiving approval in 2016 on the basis of the results of the CheckMate 141 trial.6 The characteristics of exhausted T cells are a high expression of inhibitory receptors, reduced cytotoxic activity, and reduced proliferation potential.7 Recently, researchers have made progress in understanding exhausted CD8+ T cells (Tex). Two subpopulations of Tex were found in the TIME, progenitor exhausted T cells (Texprog) and terminally exhausted T cells (Texterm), and these subpopulations are characterized by distinct epigenetic and transcriptional states.11 They are distinguished by the expression of TCF1, which is encoded by TCF7 and plays a fundamental role in T cell development, stemness, and memory formation.14 TCF1 was highly expressed in Texprog cells, but not in Texterm, thus TCF1 expression could unambiguously identify Texprog cells.13 CD8+PD1+TCF1+ Texprog (TCF1+Texprog) possessed stem-like properties and could be differentiated into Texterm but exhibited relatively lower cytotoxicity. CD8+PD1+TCF1− Texterm (TCF1−Texterm) expressed more cytokines and exerted strong antitumor activity. In particular, TCF1+ Texprog were discovered within the tumor region and lymph node and responded preferentially to checkpoint blockade in melanoma.16The term “T cell exhaustion”, which was originally derived from a mouse chronic virus infection model, is now commonly used to define a dysfunctional status of T cells stimulated by continuous antigen burden.+ T cells and suggested that the abundance of infiltrating CD8+ T cells was associated with better overall survival (OS).19 Further, a subset of CD8+CD103+ tissue-resident memory T cells (TRM) are critical for protective immunity in HNSCC.21 However, previous studies have shown that the prognostic value of Tex is controversial. In hepatocellular carcinoma, PD1hiCD8+ Tex was associated with poor outcomes, whereas in triple-negative breast cancer, they were associated with a better prognosis.25 A possible explanation might be that the TIME consists of distinct Tex subsets that exhibit distinct cytokine production and phenotypic characteristics. Therefore, to further understand the heterogeneity and prognostic value of PD1+CD8+Tex, we aimed to explore the profiles of TCF1+Texprog and TCF1−Texterm in HNSCC.Previous studies on HNSCC focused mostly on whole tumor-infiltrating CD8+Texprog and TCF1−Texterm in HNSCC, including their spatial distribution, prognostic value, cytokine secretion, and correlation with regulatory T cells (Tregs) in the TIME. We found that CD8+PD1+TCF1+ Texprog and CD8+PD1+TCF1− Texterm coexisted in the HNSCC TIME and that the latter was the major subset. The density of tumor-infiltrating TCF1−Texterm could serve as an independent prognostic indicator in HNSCC. In addition, the level of infiltrating Tregs increased as the proportion of TCF1−Texterm became higher. We propose that these findings could provide new strategies and indications for optimal immunotherapeutic regimens for HNSCC patients.In this study, we applied multiplex immunohistochemistry (mIHC) and flow cytometry to investigate the comprehensive profile of TCF1+Texprog and TCF1−Texterm. In this scheme, CD8+PD-1+TCF1+ cells represented TCF1+Texprog, and CD8+PD-1+TCF1− cells were TCF1−Texterm.8 Interestingly, these two subtypes were both observed in formalin-fixed, paraffin-embedded (FFPE) samples from HNSCC patients , anti-CD8 (red), anti-PD-1 (green), anti-TCF1 (yellow), and DAPI (blue) was developed to comprehensively profile the spatial distribution of TCF1nts Fig. . Then, wion Fig. . To furtion Fig. . The resion Fig. . In addi+CD8+Tex in 34 fresh HNSCC tumor samples and 10 paired draining lymph nodes (dLNs). The clinicopathologic characteristics of HNSCC patients are shown in Table +PD-1+TCF1− Texterm than CD8+PD-1+TCF1+ Texprog in HNSCC tumor samples which were consistent with the mIHC results  cohort with 102 patients , tumor necrosis factor-α (TNFα), and granzyme B (GZMB) was analyzed. TNF-α, IFN-γ, and GZMB are important cytokines that play a critical role in antitumor response. However, there are differences in their functions. TNF-α could promote T cell proliferation and survival and GZMB acts as exocytosis of granule components in the direction of the target cell, leading to a lethal hit of the target cell. We found that GZMB production was significantly higher in CD8+PD-1+TCF1− Texterm than in CD8+PD-1+TCF1+ Texprog , cytotoxic T lymphocyte antigen 4 (CTLA-4), lymphocyte activation gene-3 (LAG-3), and T cell immunoglobulin and ITIM domain (TIGIT) in these two subsets by flow cytometry. CD103 is a marker of Trog Fig. . We also27 Tregs promote intratumoral T cell exhaustion by modulating the expression of several coinhibitors and the exhaustion-associated transcriptomic signature of CD8+ T cells.28 We, therefore, used flow cytometry to investigate the association between the percentages of Tregs and the ratios of TCF1+Texprog to TCF1−Texterm which represented the status of PD1+CD8+ Tex proportion. We found that, although not statistically significant, the proportion of CD4+Foxp3+ Tregs increased as the ratio of TCF1+Texprog/TCF1−Texterm decreased  to induce a higher proportion of CD8+PD-1+TCF1+ Texprog, resulting in a superior antitumor response in combination with a PD-L1 blockade.33 In addition, Ptpn2 deletion in CD8+ T cells enhanced the generation and cytotoxicity of TCF1−Texterm, promoted tumor control, and improved PD-1 checkpoint blockade responses to B16 tumors.34Survival analysis revealed that a high abundance of TCF1+Foxp3+ Tregs were elevated as TCF1+Texprog/TCF1−Texterm ratio decreased. Previous studies reported that high levels of tumor-infiltrating Foxp3+ Tregs were associated with better prognosis and locoregional control in HNSCC.37 However, Foxp3 is a crucial regulator of Tregs that is responsible for a substantial amount of their suppressive potential.38 This result indicates that the good prognosticator, Foxp3+ Tregs, might be accompanied by high levels of TCF1-Texterm. Foxp3 expression correlated with decreased TCF1 and reduced accessibility of TCF1-bound chromatin regions.39 Treg-derived IL-10 and IL-35 can also directly induce the expression of multiple coinhibitors and drive T cell dysfunction.28 This result suggested that Tregs might affect the formation of TCF1+Texprog through transcriptional regulation, resulting in an imbalance in Tex subtypes. In addition, we found that TCF1−Texterm expressed higher levels of TIM-3, CTLA-4, and TIGIT, which are markers of a dysfunctional subset of tumor-infiltrating CD8+PD1+ T cells in cancer. In a transgenic HNSCC mouse model, anti-TIM-3 monoclonal antibody injection induced a reduction in Tregs and increased IFN-γ production on CD8+ T cells, suggesting that the antitumor immune response was enhanced by TIM-3 blockade through relieved suppression of negative immune factors.40 Therefore, increased Tregs and upregulated inhibitory receptors on TCF1−Texterm might construct an immunosuppressive microenvironment in the TIME, resulting in a barrier to the efficacy of immunotherapy. These findings indicated that ICB therapy in combination with Treg deletion might serve as a new strategy to achieve a stronger T cell immune response in the TIME when designing immunotherapy regimens for patients with HNSCC, especially for those with TCF1+Texprog/TCF1−Texterm ratios lower than 0.6.Even though progenitor and terminally exhausted T cells played important roles in the antitumor response, we noticed that CD4−Texterm as the major subtype in the TIME and then showed that this subtype is indicative of a favorable prognosis in HNSCC. The TCF1−Texterm in the tumor environment was correlated with that in dLNs, which showed a higher proportion of Texprog. Moreover, we found that a high proportion of TCF1−Texterm was associated with more tumor-infiltrating Tregs. Overall, our study might provide a theoretical basis for optimal ICB immunotherapy in HNSCC.In summary, the present study first identified TCF1N = 102; The Stomatologic Hospital, School of Medicine, Zhejiang University, ZJU cohort, N = 73) and 34 HNSCC patients’ fresh tumor tissues and 10 paired dLNs during surgery between Jan. 2020 and Jun. 2021 were obtained from the Hospital of Stomatology, Guanghua School of Stomatology, Sun Yan-Sen University. All the samples were HPV negative. All the samples used in this study were approved by the ethics committee of the Hospital of Stomatology, Guanghua School of Stomatology, Sun Yat-sen University. Their clinical data were kept anonymous.A total of 175 consecutive patients’ FFPE specimens with histologically verified primary HNSCC from January 2006 to 2010 came from two clinical centers . Next, the slides were incubated with Foxp3  primary antibodies at 4 °C overnight. After being treated with HRP goat anti-rabbit secondary antibody, the slides were stained 3,3-diaminobenzidine (DAB). Then Aperio AT2 scanner (Leica) was applied to scan the stained slides. At least three random interested areas  of every section were chosen for quantification. Mean Foxp3 H-scores were quantified with background subtraction using Aperio eSlide Manager Quantification software.43IHC staining was performed on FFPE samples as previously described.+Texprog and TCF1−Texterm phenotypes were identified and quantitated using the tissue cytometry of StrataQuest software .44 Threshold cut-offs depicted by horizontal and/or vertical lines were determined manually by backgating to the greyscale and multiplexed tissue images for each biomarker .45Opal 7-color fluorescent IHC kit  was used to stain the multi-parametric immunofluorescence panel. First, an initial deparaffinization procedure was performed on all slides, followed by fixation in 10% formalin fixation and Tris-EDTA (pH = 9.0) antigen retrieval. Afterward, the slides were incubated with primary antibody, secondary-HRP antibody, and Opal TSA dyes. Subsequently by the other cycles of staining consisted of antigen retrieval, blocking, primary antibody and secondary-HRP antibody incubation, and Opal TSA dye staining. Finally, the slides were mounted with Antifade Reagent . DAPI was used for the nuclear counterstain. Images of the whole tissue specimens were acquired by the TissueFAXS platform (TissueGnostics). TCF1The Opal detection fluorophores for exhausted T cells analysis panel was: Opal 690-CD8 , Opal 650-PD-1 , Opal 570-TCF1 , Opal 540-PanCK .23 Tumor tissues were cut into several pieces. One-piece was fixed in 10% formalin for IHC or mIHC verification and the other pieces were incubated with 1 mg·mL−1 Collagenase IV, 0.5 mg/mL Collagenase I (Gibco) and 0.1 mg·mL−1 DNase I (Biofroxx) in complete RPMI 1640 (Gibco) medium with 10% FBS for 30 min at 37 °C. Digested tissues were filtered using a 70 μm cell strainer. Lymph nodes were ground and filtered in the complete medium by a 40 μm cell strainer. Cells were then washed and treated with 1× red blood cell lysis buffer (BD Biosciences) for 2 min at room temperature. After washing with PBS (Gibco), the cell suspensions were then used for flow cytometry analysis.Tissue dissociation was performed as the previous study.For intra-nuclear staining, the samples were first stained with Viability Ghost Dye (Tonbo Biosciences) for 15 min at room temperature, then incubated with the cell surface markers: CD45 , CD20 , CD3 , CD4 , CD8 , PD-1 , CD103 , TIM-3 , TIGIT , LAG-3  for 30 min at 4 °C. Afterward, Foxp3/Transcription Factor Staining Buffer Set (Tonbo Biosciences) was applied according to the manufacturer’s instructions for the next nuclear molecules staining. TCF1  was stained for 45 min at room temperature. Lastly, Alexa Fluor 488 donkey anti-rabbit secondary antibody , CTLA-4 , and Foxp3  were stained.2. Then the samples were washed with PBS and stained with Viability Ghost Dye. Next incubated with the membrane molecules: CD3, CD4, CD8, and PD-1. Followed by Foxp3/Transcription Factor Staining Buffer Set application and TCF1 staining. Lastly, the cell suspensions were incubated with donkey anti-rabbit secondary antibody, TNFα , GZMB , IFNγ  overnight. Stained cells were washed and resuspended in PBS. All the samples were acquired on BD LSRFortessa (BD Biosciences). Flow cytometry data were analyzed by FlowJo software (Tree Star).For cytokine detection, the samples were stimulated for 5 h with a cell stimulation cocktail (Tonbo Biosciences) at 37 °C, 5% COt test. To determine the optimal cutoff point for the high- and low-density groups in our cohort and validation cohort, we used the maximally selected rank statistics from the “maxstat” R package.47 The cutoff point for RFS was set to the same as the value for OS analysis. Survival analysis was performed and presented by using the Kaplan–Meier method. The difference in survival curves was tested by the log-rank test. The correlation analysis between groups was determined by Spearman’s correlation test. P < 0.05 was considered statistically significant .All results were summarized as mean ± SEM, and statistical analysis was performed with Prism 7.0 (Graphpad Software). Differences between groups were evaluated by paired or unpaired two-tailed Student’s Supplementary materials


The high flexural strength and modulus of the friction composite, i.e., 61 MPa and 6.4 GPa, respectively, were obtained. The friction composite also showed high thermal stability, such as 410 °C for degradation temperature and 215 °C for glass transition temperature. The results indicated that the obtained UFNBRPs-modified polybenzoxazine friction composite meets the industrial standard of brake linings and pads for automobiles; therefore, the UFNBRPs-modified polybenzoxazine friction composite can effectively be used as a replacement for asbestos-based friction materials.Asbestos-free friction composite based on ultrafine full-vulcanized acrylonitrile butadiene rubber particles (UFNBRPs)-modified polybenzoxazine was successfully developed. The UFNBRPs-modified polybenzoxazine friction composite was characterized for chemical, tribological, and mechanical properties as well as thermal stability. The UFNBRPs not only act as a filler to reduce noise in the friction composites due to their suitable viscoelastic behaviors but also play a key role in friction modifiers to enhance friction coefficient and wear resistance in the polybenzoxazine composites. The chemical bonding formation between UFNBRPs and polybenzoxazine can significantly improve friction, mechanical, and thermal properties of the friction composite. The outstanding tribological performance of the friction composite under 100–350 °C, i.e., friction coefficients and wear rates in a range of 0.36–0.43 and 0.13 × 10 Polymer composite materials composed of different reinforcements and fillers into several engineering polymers to replace metallic and ceramics are currently used in structural applications such as automotive, construction, spacecraft, marine, etc. ,2,3. In Phenolic resins are traditionally used as a binder for friction polymer materials such as brake pads due to their low cost and a suitable combination of mechanical properties and suitable wetting capability with most ingredients ,6,7,8,9.Benzoxazine resin (BA-a) was based on bisphenol A supported by PTT Phenol Co., Ltd. , formaldehyde purchased from Merck Co., Ltd. , and aniline obtained from Panreac Quimica S.A. . Aramid short fiber was provided by DuPont . All chemicals were used as received.Bisphenol A/aniline-based benzoxazine resin (BA-a) was synthesized from bisphenol A, formaldehyde, and aniline according to a patented solventless method . The obtUltrafine full-vulcanized acrylonitrile butadiene rubber particles (UFNBRPs)-modified polybenzoxazine at UFNBRPs contents in a range of 0–15 wt% were prepared. The molding compounds were mixed in an internal mixer at 110 °C for 30 min and thermally cured at 200 °C for 2 h under a hydraulic pressure of 10 MPa by a compression molder. The samples were then air-cooled to room temperature before characterizations.UFNBRPs-modified polybenzoxazine friction composite was produced from the ingredients listed in Differential scanning calorimetry  was performed under a heating rate of 10 °C/min from 30 to 300 °C, nitrogen purging at 50 mL/min. A small amount of samples ranging from 5 to 10 mg was placed on the aluminum pan with lids, and three samples of each compound were evaluated.−1 and a spectral range of 4000–400 cm−1. Three samples of each poly(BA-a), UFNBRPs, and poly(BA-a)/UFNBRPs composite were measured.FTIR measurement was conducted at room temperature on a Spectrum GX FTIR spectrometer from Perkin Elmer. The samples were sufficiently thin to be within a range where the Beere–Lambert law is obeyed. All spectra were taken with 64 scans at a resolution of 4 cmThermogravimetric analysis (TGA) from Perkin Elmer (Diamond TG/DTA) was performed under nitrogen purging with a constant flow of 100 mL/min. A sample mass of 10 mg was heated at a linear heating rate of 20 °C/min from room temperature to 900 °C. Three samples of each composite were evaluated.Dynamic mechanical analyzer (DMA) from Netzsch Inc., model DMA 242 C in the three-point bending mode and a support span of 40 mm was used to examine loss tangent (tanΔ) of the samples. Three samples of each composite with dimensions of 10 × 50 × 3 mm were tested. The tests were performed in a temperature sweep mode with a fixed frequency of 1 Hz. Each sample was tested using a heating rate of 2 °C/min from 30 to 250 °C.Flexural properties were conducted by a Universal Testing Machine, Model Instron 5567. The measurement was carried out in a three-point bending mode, with a support span of 48 mm and at a crosshead speed of 1.2 mm/min. Five samples with a dimension of 25 × 60 × 3 mm were tested, and the average values for the samples were determined.A tribological test under dry sliding at a temperature of 25 °C was conducted on a pin-on-disk tribometer having a maximum temperature of 950 °C from CSM Instrument Ltd., Switzerland. The test condition is 10 N for normal load and 0.366 m/s for sliding speed for 1000 m distance. Three samples of each composite with a dimension of 25 × 25 × 7 mm were tested, and the average values for the samples were determined. At a temperature range of 100–350 °C, the sliding speed of the frictional surface of the disk against the test sample shall be a constant speed given within 6 to 8 m/s, and the sliding test under a pressure of 1 MPa according to TIS97-2557 and JIS D 4411 standards. Two samples of each friction composite with a dimension of 25 × 25 × 7 mm were tested, and the average values for the samples were reported.The worn surface of the samples was evaluated with a JSM5410LV scanning electron microscopy (SEM) from JEOL Ltd. using an acceleration voltage of 15 kV. All samples were coated with a thin film of gold to make the surfaces conductive.Curing behavior of benzoxazine resin (BA-a) filled with 0, 2, 5, 10, and 15 wt% of ultrafine full-vulcanized acrylonitrile butadiene rubber particles (UFNBRPs) was studied to be able to control reactivities and optimize processing times of UFNBRPs-filled polybenzoxazine composites. In In addition, ΔH of BA-a part in each of BA-a/UFNBRP molding compounds is also calculated and listed in −1, and 3320 cm−1 due to a tetra-substituted benzene ring mode and the phenolic hydroxyl (–OH) group formation, respectively [−1, while the bands at 2848 and 2925 cm−1 were attributed to the C-H stretching vibration of CH2 groups in the rubber backbone. The characteristic bands at 969 and 892 cm−1, which are the trans-1,4 and 1,2-vinyl characteristic frequencies of the butadiene moieties due to out-of-plane deformation of the =C−H, were also observed. Moreover, the absorption band with a frequency of 1630 cm−1 and 1449 cm−1 indicated CH2 bending vibration of a methylene group, and >C=C< unsaturated sites of the UFNBRPs, respectively [−1 due to the –OH group tended to decrease when compared with that of the poly(BA-a), implying to the consumption of the –OH group in the poly(BA-a). While the new band at 1107 cm−1 assigned to ether linkages (C−O−C stretching) formed between the –OH group in the poly(BA-a) and the −CH=CH2 groups of the UFNBRPs [−1 typical of oscillations of the C=N group also appears in the spectrum, while the intensity of the band at 1630 and 2233 cm–1 typical of oscillations of the >C=C< and C≡N groups, respectively, disappeared. Therefore, all appearance bands of the poly(BA-a) filled with UFNBRPs are clear evidence of the chemical bonding formed between the poly(BA-a) and the UFNBRPs.To further confirm the chemical reaction between UFNBRPs and poly(BA-a), the IR spectra of poly(BA-a), UFNBRPs, and poly(BA-a)/UFNBRPs composite are shown in ectively ,27,28. Fg) calculated by loss modulus of the poly(BA-a)/UFNBRPs composites at various UFNBRPs contents are summarized in gs of the composites was enhanced by the addition of UFNBRPs due to chemical bonding formation between poly(BA-a) and UFNBRPs as discussed in FTIR analysis, resulting in crosslink density improvement. The increased Tg with the addition of UFNBRPs of poly(BA-a)/UFNRPs composites would lead to substantial enhancement of friction force and specific wear of poly(BA-a)/UFNBRPs friction composite since the relatively higher Tg (or higher glass-rubber transition temperature), resulting in better ability to stabilize the tribological property, i.e., friction coefficient and wear rate, under relatively higher braking temperature [Thermomechanical property, i.e., storage modulus at 30 °C (E′) and glass transition temperature (Tperature . It was d5 and Td10) and char yield of the poly(BA-a)/UFNBRPs composites tended to be quite stable with an addition of UFNBRPs up to 5 wt% followed by a sharp decrease with the incorporation of UFNBRP more than 5 wt% UFNBRPs. The obtained degradation temperature and char yield of the composites, i.e., poly(BA-a)/UFNBRPs (2 wt%) and poly(BA-a)/UFNBRPs (5 wt%) was still similar, indicating that the composites should have a high degree of crosslinking, which requires more energy for degradation. Moreover, the formation of char is an effective method of increasing the fire resistance of polymers as it can also be confirmed by the limiting oxygen index (LOI), representing the minimum concentration of oxygen, expressed as a percentage, that will support ignition of a polymer. LOI was determined with Van-Krevelen and Hoftyzer equation [From equation  when CR LOI values of the poly(BA-a)/UFNBRPs composites at various UFNBRPs contents were in the range 24.9–27.6, as the results are summarized in avg.) and wear rate at 25 °C under dry sliding friction using pin-on-disk of poly(BA-a)/UFNBRPs composites at various UFNBRPs were measured and depicted in avg. of the composites was sharply increased from 0.50 to 0.60 in a range of UFNBRP contents from 0 to 5 wt%. This behavior can be suggested that the development of friction force at the sliding surface for the poly(BA-a) and the poly(BA-a)/UFNBRPs composites pursues different mechanisms. UFNBRPs can be deformed on the composite’s contact surface to generate friction force by an exchange with thermal energy after applied load and velocity. As a consequence, the μavg. increased with an increase in the actual contact area and the surface roughness. The addition of greater than 5 wt% UFNBRPs revealed a slight increase in the μavg. of the composites. It is possible that an agglomerate formation of UFNBRPs changed in the surface roughness, which resulted in a significant reduction in the actual contact area between rigid asperity and the composite surface. Furthermore, it was also found that the addition of UFNBRPs up to 5 wt% significantly improved the wear resistance of the poly(BA-a) as the wear rate was decreased from 3.35 × 10−4 mm3/Nm for poly(BA-a) to 1.83 × 10−4 mm3/Nm for poly(BA-a)/UFNBRPs (5 wt%) composite. The addition of UFNBRPs greater than 5 wt% resulted in an increase in the wear rate of the composites. It seems like that the 5 wt% UFNBRPs, having uniform dispersion in the poly(BA-a) is optimum content for the poly(BA-a) to be a binder for poly(BA-a)/UFNBRPs friction composite.Average friction coefficient  of the poly(BA-a)/UFNBRPs (5 wt%) composite, which may have an effect on adhesion ability of the poly(BA-a) and UFNBRPs.The influence of temperature should be paid special attention to since the different temperatures will cause a great change in the friction coefficient. To investigate the effect of temperature on the tribological property of the poly(BA-a) modified with UFNBRPs, the poly(BA-a)/UFNBRPs (5 wt%) composite was selected to measure μIn terms of the wear rate of the composite, it can be seen that the wear rate was very different, and they generally became more pronounced with temperature. The wear rate slowly increased with the temperature up to 200 °C, and then rapidly increased as the temperatures increased above 200 °C. This characteristic was due to the lower thermal stability of the composite at a temperature of above 200 °C. Moreover, it is possible that a portion of friction energy was converted to heat, and the remainder resulted in plastic deformation, micro-cracks, and a change in surface roughness.Scanning electron microscope (SEM) photographs of the worn surface of poly(BA-a)/UFNBRPs (5 wt%) composite at different temperatures are illustrated in avg. and wear rate values of both friction polymer composites at various temperatures are plotted in avg., i.e., 0.40–0.43 of poly(BA-a)/UFNBRPs friction composite as the temperature increased from 100 to 300 °C was obtained, and the μavg. of the friction composite was then reduced to 0.36 at 350 °C. It showed the same behavior observed for poly(BA-a)/UFNBRPs (5 wt%) composite that the μavg was clearly decreased at elevated temperature. It is possible that the temperature of 350 °C is higher than the Td5 of the poly(BA-a), i.e., 325 °C; in consequence, the wear debris was formed, resulting in a decrement of friction coefficient of the poly(BA-a)/UFNBRPs friction composite. However, the obtained μavg. values of the friction composite still meet the required standard for brake pads, i.e., μavg. at 100–350° is in a range of 0.2–0.7 [avg. at the various temperatures of the poly(BA-a)/UFNBRPs friction composite showed higher than that of the phenolic one, which was in a range of 0.36–0.41. This characteristic suggested that the adhesion between the poly(BA-a)/UFNBRPs friction composite and the brake disc showed more effectiveness than that between the phenolic type and the brake disc. Moreover, in comparison, the friction coefficient of the poly(BA-a)/UFNBRPs friction composite with that of commercial asbestos-based friction material. It was found that the poly(BA-a)/UFNBRPs friction composite showed a more stable friction coefficient against the different temperatures than the asbestos-based friction because of the inherent thermal stability of the poly(BA-a)/UFNBRPs matrix.As all results above mentioned was analyzed, it was observed that the amount of UFNBRPs content for poly(BA-a)/UFNBRPs composite should not be more than 5 wt%, showing the balanced properties between tribological and thermal properties, and also base on the rubber content in friction composites designed by Akebono Brake Industry Co Ltd. for friction composite materials [ 0.2–0.7 ,34. In a−4 mm3/Nm at 100 °C and less than 3.5 × 10−4 mm3/Nm at 350° [The wear performance of the poly(BA-a)/UFNBRPs friction composite was measured in terms of wear rate and also depicted in  at 350° ,35. Moref) and flexural modulus (Ef), are important for friction polymer composites used for brake friction materials. From f = 33 ± 2 MPa and Ef = 3.3 ± 0.2 GPa. This characteristic can be confirmed that the UFNBRPs-modified poly(BA-a) has higher interfacial adhesion to the ingredients than that of the phenolic one. In addition, it is possible that the flexural strength of the poly(BA-a), i.e., 126 MPa [Mechanical properties, i.e., flexural strength  friction composite was investigated, as also listed in d5 and Tg, i.e., 410 and 215 °C, respectively, of the poly(BA-a)/UFNBRPs friction composite was found to be similar to those of the phenolic one, i.e., Td5 = 424 °C and Tg = 210 °C. The values can be related to the thermal degradation of the organic binder resin of the friction composite. It can be implied that the friction composites may show substantial brake fade resistance. It describes the status of friction stability under severe temperature conditions, and it refers to the loss of brake effectiveness due to excessive frictional heat from the extended brake applications [In general, the ideal brake friction material should have a constant friction coefficient under various operating conditions such as temperature and applied loads. In this work, the thermal properties such as Tications . In addiThe evaluation of friction polymer composites based on ultrafine full-vulcanized acrylonitrile butadiene rubber particles (UFNBRPs)-modified polybenzoxazine can be summarized as follows. The UFNBRPs-modified polybenzoxazine friction composites showed an outstanding friction coefficient and wear rate. The significant mechanical properties, i.e., flexural strength and modulus, were obtained due to suitable interfacial adhesion between the UFNBRPs-modified polybenzoxazine and all ingredients. The high thermal stability, resulting in a better ability to stabilize the friction coefficient and wear rate under relatively higher temperatures, can help better design friction composites. In addition, the ether bonds formed between the polybenzoxazine and the UFNBRPs are attributed to the significant improvement in tribological, mechanical, and thermal properties of the obtained UFNBRPs-modified polybenzoxazine friction composites.