Fibroblasts were isolated from diseased (n = 8) and non-diseased (n = 4) lungs. Global gene expression analysis was carried out at the initial point of isolation and after 3 weeks of culture. We identify several genes that are altered by removal of the fibroblast from the IPF environment. Comparison of this subset of genes to four previously published whole lung analyses refined our list to a small subset of key fibroblast specific genes important in IPF. Application of STRING database analysis and confirmation via in-vitro and histological assay highlights the CXCL14/CXCR4 chemokine axis with a possible role in the progression and/or activation of fibroblasts within the IPF lung. Our findings, present a possible therapeutic target for IPF and a model for the study and discovery of novel protein and processes in this terrible disease.Idiopathic Pulmonary Fibrosis (IPF) is a progressive disorder that is marked by an over accumulation of activated fibroblast populations. Despite the improved understanding of many mechanisms within this disease, global gene expression analysis has few focused studies on the fibroblast, the central effector cell of progressive fibrosis. We present a unique analysis of IPF pulmonary fibroblasts as they transition through cell culture and identify  The overabundance of these cells and their excessive extracellular matrix (ECM) deposition aids in the progressive fibrosis observed. This results in tissue distortion, impaired gas exchange, and ultimately organ failure6. The exact source of the IPF fibroblasts population remains unclear, however there are four potential reservoirs, including resident lung fibroblasts, circulating fibrocytes, epithelial cells that have undergone epithelial-mesenchymal-transition (EMT), and pleural mesenchymal cells9. As the fibroblast is so integral to the pathogenesis of IPF, much effort has been directed toward characterizing the specific processes within this cell. However, a comprehensive global understanding of the IPF fibroblast remains elusive.Idiopathic pulmonary fibrosis (IPF) is a lethal form of interstitial lung disease of unknown etiology. IPF has been defined as a disease of unregulated wound repair. This is likely driven by an abnormal epithelium and propagated by a dysregulated overabundant, heterogeneous, fibroblast population in various states of activationin vitro fibroblasts as a model system has revealed many important IPF mechanisms and molecules including transforming growth factor beta (TGF-beta1), tumor necrosis factor, and mediators of the Wnt pathway15. Despite this improved understanding of possible pathogenic mechanisms of IPF, only two therapeutic options have emerged each of which serve to slow disease progression. Global gene expression analysis is a powerful method for investigating the transcriptome and cellular diseased states, however analysis of isolated IPF and normal fibroblasts has shown no discernible genomic differences between populations18. Some data exists on the global genomic phenotype of IPF whole lung tissue in comparison to hypersensitivity pneumonitis, non-specific interstitial pneumonitis (NSIP) and emphysema. In addition, differential gene expression in familial and sporadic interstitial pneumonia has also been described20. However, the use of whole lung tissue in these experiments does not allow for characterization of the specific cellular contributions to disease.Investigation of this disease using in vitro models of disease; specifically the observation that once primary cells such as fibroblasts, chondrocytes, melanocytes, and hepatocytes are placed in tissue culture they undergo phenotypic changes affecting biological processes, cell motility, cytokine production, and cytoskeletal arrangements28. These changes often result in the primary cells bearing little resemblance to their cells of origin.The pace of progress is hampered in part by the imperfections of 17 at the time of isolation. Here we observed and analyzed these cells after three passages (P3) in culture. We expanded our analysis to include our initial list of 1700 differentially expressed genes and four previously published lists that identified differences in genomic expression between IPF and normal whole lung isolates32. These data revealed that through culture the genomic profiles of these cells change in vitro and we hypothesized that characterization of the adaptive processes evident in the transition to in vitro culture may enable the identification of novel IPF related mechanisms or proteins that may be suitable for therapeutic intervention.We have previously reported the development of a method for the isolation of IPF and normal fibroblasts  directly from lung tissue without the confounding effects of tissue culture. This method revealed over 1700 significantly differential expressed genes between normal-F and IPF-F17. However, in this investigation we found that once these cells transition to the in vitro state, this differential profile is lost. Specifically, gene expression analysis, using Statistical Analysis of Microarray ) software, of cultured IPF-F (n = 8) and normal-F (n = 4) demonstrated no statistically significant differential gene expression between these two populations.We have previously demonstrated that IPF-F and normal-F have significantly different global gene expression patterns when freshly isolatedin vitro, revealed 2889 and 1118 genes to be significantly altered, respectively  and normal-F (n = 4) from initial isolation (non- cultured) to 3 passages in vitro (P3) fibroblasts for both normal  and principle component analysis demonstrated clear clustering and separation between the non-cultured fibroblasts (P0) and the mal Fig.  and IPF mal Fig. . This hiAnalysis of the fold change in genes associated with myofibroblast differentiation and subpopulations reveals that the IPF-F and normal-F adapt to tissue culture by the promotion of markers typically associated with fibroblast activation such as ACTA2, CTGF, FGFR1 34. This analysis revealed enrichment for several well characterized processes . A result of particular interest is the CXCR4/CXCL12/CXCR14 axis as our string analysis indicates a possible role of these genes as part of a linker axis for the WNT pathway, VEGF angiogenetic pathway and the HIF1 regulated oxidative stress response. To demonstrate the value of refining the whole lung data sets with our derived IPF list, we performed overlap analysis solely with the data from the public lists. This analysis resulted in 704 genes found in at least 3 of the 4 public data sets  for 24 hours to simulate the oxidative stress present in the IPF lung. We then examined the expression of six genes with close connections to the CXCR4/CXCL12/CXCL14 axis found in the string pathway  regardless of the in-vivo origin, IPF or normal. This analysis also highlights the many genes whose transcriptional control is directly influenced by the IPF environment and are separate from the intrinsic propensity of the fibroblast itself.Analysis of whole lung tissue in IPF has resulted in several important findings identifying many of the key underlying processes in IPF; however, identifying disease specific processes that are primarily related to and/or regulated by the fibroblast is difficult in the whole tissue background. Our hypothesis is predicated on the idea that some fibroblast genomic adaptation to the disease environment is lost through the process of isolation and tissue culture. To address this issue, we documented the differential expression profile of IPF and normal fibroblasts from isolation and during culture. This unique approach demonstrated that there are a number of cellular pathways that are altered by the process of To demonstrate the utility of this approach we first compared four broad IPF whole lung tissue profiles to find overlapping processes within these lists. As expected, there is substantial concordance within these publically available data lists resulting in more than 700 genes overlapping Table . RefinemBy comparing our fibroblast specific list to the same four broad IPF whole lung tissue profiles, we identified a narrow subset of 59 IPF specific genes that we believe are indicative of the environmental impact on the fibroblast phenotype in disease. Many of these genes correspond to signaling peptides indicative of a dynamic crosstalk between the IPF environment and the IPF fibroblast. Using a smaller focused set of genes, we generated a string pathway which resulted in significant enrichment for biological pathways that control cell migration and developmental processes Fig. . As comp39  and migratory associated processes42  that respond to the IPF environment. Through our STRING analysis  and the receptor CXCR4L12 Figs  and 8 inion Figs  and 7 ou49. Prostate tumors develop rapidly and display increased angiogenesis in the presence of CXCL14 overexpressing cancer associated fibroblasts50. We speculate that in the IPF lung the CXCL14/CXCR4 interaction may be a driving force for the recruitment or migration of fibroblasts to the sites of fibrosis and that once the foci form, there is decreased need for this axis and therefore a decrease in expression. This hypothesis is further supported by recent studies into both CXCL12 and CXCL14. Like CXCL12, CXCL14 is over expressed in whole lung tissue of IPF patients51 and also found at higher levels in the serum of IPF patients52 strengthening its potential role as a recruitment mediator. In-vivo studies targeting CXCL12 in lung fibrosis models have overall been inconclusive, however they have demonstrated some success in the slowing of disease progression54. We theorize that inclusion of CXCL14 may reveal a complementary role for this ligand in the axis that when addressed may effectively target this pathway. Further studies into the significance of the co-localization of CXCL14/CXCR4/ACTA2 within the IPF lung needs to be performed, however, we speculate that it may be possible to slow or inhibit fibroblast migration and activation by pharmacological intervention directed at the CXCR4/CXCL14 complex.The role of CXCL14 in fibroblast biology is poorly understood. However CXCL14 has been identified in cancer associated stromal fibroblasts as a promoter of migration and ERK-dependent proliferation55. There is general agreement that therapeutic intervention in fibrotic disease is complicated by the heterogeneity of both the IPF lung and the disease as a whole. Our study highlights an added layer of complexity which further can cloud or even misdirect our understanding of fibroblast biology. Our unique approach highlights the transcriptomic changes observed through the first weeks of tissue culture that has enabled us to identify a subset of genes not previously reported as significant in IPF.IPF is a progressive disease that presents in acute, progressive, and stable variations based on many yet unknown environmental and genetic factorsin-vitro fibroblast culture model both before and after challenge. Finally, we identified co-expression of our most interesting gene, CXCL14, with ACTA2 in vivo in areas of fibrosis. Identification of CXCL14 as a gene of interest in IPF has been recently been reported56, however, to our knowledge, this is the first time that CXCL14/CXCR4/ACTA2 has been demonstrated together in the IPF lung and attributed to disease. This observation has not been previously described, which we hypothesize is due to the environmental limitation inherent to in-vitro tissue culture. We therefore suggest that our data set and model system can be applied to other IPF associated pathways to help identify potential therapeutic targets.A potential limitation to this study may emanate from what we have termed as the initial fibroblast population isolated from the IPF lung. Criticism of this technique, which is widely employed in other fields of study, may call into question the purity of the population at initial isolation. We addressed this concern in three ways: first, we used previously published data sets and string pathway analysis to refine the large number of genes in the IPF list. Second, we confirmed expression in an in-vitro tissue culture model. In doing so we identify a potential role for CXCL14 in IPF within the CXCL12/CXCL14/CXCR4 axis that is in accordance with our data and four published data sets. As a soluble ligand that is upregulated in IPF, CXCL14 serves as a candidate for further study that may also lead to a therapeutic intervention. Finally, we propose that further application of this data set may result in the identification of novel processes of interest in the IPF lung.In conclusion, we have taken a wide range of novel and published data and focused it through a model system that reduces the confounding effects of the traditional IPF lung tissue was obtained through Inova Fairfax Hospital (VA). All normal control lungs were obtained through the Washington Regional Transplant Community (WRTC). Appropriate written informed consent was obtained for each patient and donor by Inova Fairfax hospital and the WRTC. This study was approved by the Inova Fairfax Hospital Internal Review Board (IRB #06.083) and the George Mason University Human Subject Review Board (Exemption #5022). All experiments were performed in accordance with relevant guidelines and regulations. Additionally, no organs/tissues were procured from prisoners.57. Briefly, samples were dissected into 1–2 mm2 pieces and subjected to enzymatic digestion in 0.4% collagenase P  complete media  containing 10% fetal bovine serum (FBS), penicillin (100 I.U/ml), streptomycin (100 MCH/ml), amphotericin B (0.25 M.C.G./ml P/S/A) and 0.1% DNase1, at 37 °C and 5% CO2 for 2 hours. The resulting material was passed through sterile cell filters  to remove undigested tissue and remaining cells were pelleted by centrifugation at 1000 g for 5 min. The pelleted cells were then suspended in complete media and seeded onto tissue culture treated plastic at 37 °C and 5% CO2 for 45 minutes. The attached fibroblast population was vigorously washed with PBS to remove any unattached cells. P0 cell populations were extracted for RNA immediately. P3 populations were continued in culture and maintained in complete media at 37 °C and 5% CO2. All demographic information for the patients is listed below  and 4 normal  controls as previously describedRNA was extracted from IPF-F and Normal-F fibroblasts at P0 and P3 using a Qiagen RNeasy kit  and DNase treated using DNase free . The quality and quantity of the RNA was determined using RNA 6000 nanochips and an Agilent Bioanalyzer. For analysis, 1 µg of RNA was amplified and amino-allylated using the MessageAmp II aRNA kit (Ambion). All microarrays were carried out by the Duke Institute for Genome Sciences and Policy, Microarray Facility using HO36K human chip representing 33,791 transcripts from the Ensembl Human Build (BI-35C) including 22,169 unique genes (Operon Human Oligo set V4). Using two color amino-allylated amplified RNAs: cy5 corresponding to patient and normal samples, and cy3 for Stratagene human reference RNA. Microarray results were confirmed by QPCR analysis using a random selection of 11 genes  data was analyzed using statistical analysis of microarray (SAM)34. Any resulting transcript without a corresponding Unigene ID was not considered in this analysis. Additionally, some unigene IDs did not have a corresponding ID in the Database for Annotation, Visualization, and Integrated Discovery as noted in the discrepancy between Unigene and David IDs below. Table The resulting genepixs files were analyzed using the TM4 software suite  and normalized by locally weighted linear regression (lowess)34. Functional categories were clustered using the Functional Annotation Clustering tool, and clusters with a score of greater than 3.0 were selected for discussion. Protein-protein interactions were obtained using STRING  database. Interactions were parsed in three steps. Initial input of 59 proteins corresponding to differentially expressed genes were entered into STRING. All genes with predicted associations were curated manually and any closely linked non-associated proteins were also selected. The second step was simply the redefined string network that only included the manually curated proteins. Finally, predicted functional partner network linker nodes (string score > 0.995) were added to complete the network.Ontological analysis and classification of the genes identified in each analysis was carried out using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) as previously describedFibroblast populations isolated as described above were seeded in 6-well falcon tissue culture plates for 24-hours in complete media. After this attachment cells were placed in serum free DMEM for 24-hours and then challenged with serum free DMEM containing 250 µM hydrogen peroxide. Cells were then harvested for RNA analysis as described above. Assays were carried out on cells in passages 4–7. Two of the four IPF cell lines selected for this assay were derived from the pool of cells isolated for the microarray analysis and another two cell lines were derived from IPF donors not included in the microarray data set. All non-disease cell lines challenged by hydrogen peroxide were derived from normal donors in the microarray dataset.59  and Quantifast SYBR Green PCR Kit (Qiagen). QPCR was carried out in triplicate and normalized to 18 S expression levels using the delta delta CT methodFormalin fixed paraffin-embedded IPF and normal tissue sections (3–5 µm) were deparaffinized by serial washings in xylene, 100, and 90% ethanol prior to rehydration in nuclease free water. Antigen retrieval was performed by boiling in sodium citrate buffer (pH 6). Slides were blocked for 1 hour in 1% BSA prior to overnight incubation with primary antibodies: anti-actin smooth muscle (Thermo #PA5–16697), EpCAM , CXCR4 (Santa Cruz #sc-53534), CXCL12 (Santa Cruz#P-159X), CXCL14 (Thermo# PA5-528820). Primary antibody incubation was followed by 1 hour secondary incubation in Alexa Fluro 594 or 488 conjugate . Counter staining with DAPI was performed prior to imaging using Life Technologies EVOS FL. All antibodies were tested for cross reactivity prior to use and secondary antibodies were added in the absence of the primary antibody to confirm lack of non-specific binding. No cross reactivity was identified and minimal background florescence was detected at very high light levels which were not used during imaging. All images were taken from three different IPF and three different normal lung sections derived from the lower peripheral lobes. Select images are representative of whole section observations.https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE87175.The microarray data sets used in this analysis are available in the Gene Expression Omnibus repository, Supplmentary Information


Specific corticostriatal structures and circuits are important for flexibly shifting between goal-oriented versus habitual behaviors. For example, the orbitofrontal cortex and dorsomedial striatum are critical for goal-directed action, while the dorsolateral striatum supports habits. To determine the role of neurotrophin signaling, we overexpressed a truncated, inactive form of tropomyosin receptor kinase B [also called tyrosine receptor kinase B (TrkB)], the high-affinity receptor for Brain-derived Neurotrophic Factor, in the orbitofrontal cortex, dorsomedial striatum and dorsolateral striatum. Overexpression of truncated TrkB interfered with phosphorylation of full-length TrkB and ERK42/44, as expected. In the orbitofrontal cortex and dorsomedial striatum, truncated trkB overexpression also occluded the ability of mice to select actions based on the likelihood that they would be reinforced. Meanwhile, in the dorsolateral striatum, truncated trkB blocked the development of habits. Thus, corticostriatal TrkB-mediated plasticity appears necessary for balancing actions and habits. Nevertheless, the mechanisms by which the brain balances actions and habits are still being identified.Flexible action requires shifting between familiar and novel behavioral strategies. Extensive response training and exposure to stressors and certain drugs of abuse can lead to a bias towards habit-based behaviors that are by contrast inflexible. Maladaptive habits may contribute to illnesses characterized by impulse control deficits, such as addiction and obsessive-compulsive disorder5. The posterior dorsomedial striatum (DMS) and orbitofrontal prefrontal cortex (oPFC) are necessary for goal-directed actions, while the dorsolateral striatum (DLS) controls habits8.During the initial acquisition of an instrumental behavior, organisms are typically sensitive to the predictive relationship between actions and their outcomes, and goal-directed action selection strategies dominate. After continued training, reward-related stimuli can gain control over behavior, and behavioral response strategies become automated, or “habitual,” and insensitive to action-outcome associationsBdnf knockdown causes failures in action-outcome decision making and a deferral to habit-based behaviors9. Where, precisely, stimulation of the high-affinity BDNF receptor tyrosine receptor kinase B (TrkB) is important remains unclear, however, given that BDNF is subject to anterograde transport. For example, oPFC-selective Bdnf knockdown reduces BDNF protein in the dorsal striatum7, suggesting that TrkB activation in the oPFC, DMS, or both could support goal-directed action. Resolving these possibilities is important because upon BDNF binding, the intracellular domain of TrkB auto-phosphorylates, creating docking sites for effector proteins that initiate intracellular signaling cascades, e.g., the ERK42/44 and Akt pathways. TrkB impacts a diverse array of neuronal functions including cell survival and differentiation, axonal and dendritic growth and arborization and synapse formation and plasticity.The primary neurotrophin Brain-derived Neurotrophic Factor (BDNF) appears to be a key cortical substrate coordinating goal-directed action selection, given that oPFC-selective Here, mice were trained to generate two food-reinforced behaviors in operant conditioning chambers, then tested for sensitivity to action-outcome associations using a contingency degradation procedure. In this task, food pellets associated with one familiar behavior are delivered non-contingently (“for free”), regardless of the animal’s actions, while the other response remains reinforced Fig. . Mice thTrkB.t1, which lacks an intracellular domain and therefore cannot initiate intracellular signaling pathways (from10). We infused lenti-TrkB.t1 with an HA tag, lenti-Green Fluorescent Protein , or a half-and-half mixture of the two in order to generate multiple lenti-TrkB.t1 “doses”      Fig. , as expe13) Fig. , as alsoure Fig. . Specifi64) Fig. .TrkB.t1 mice were simply unable to energize a response that was reinforced . We feel that this is unlikely, however, given that across all groups, response rates during the reinforced phase of testing were higher than those generated on the last day of training   t5 = 4.385, p = 0.007), even though responding was not reinforced. By contrast, the other groups did not (GFP control 1-sample t-test against no change (0) t4 = −2.414, p = 0.073; Half TrkB.t1 1-sample t-test against no change (0) t5 = −1.233, p = 0.272).Given low response rates during instrumental response training Fig. , it is cns) Fig. . Groups ns) Fig. . Again, TrkB.t1 overexpression in the oPFC block, or instead delay, action-outcome learning and memory? To answer this question, we exposed mice to non-contingent pellet delivery for 2 additional sessions. Ultimately , full-titer TrkB.t1 mice inhibited nonreinforced responding in a goal-directed manner, indicating that TrkB.t1 overexpression delayed, but did not fully occlude, action-outcome-based decision making   Fig. .TrkB.t1 in the oPFC. We euthanized them 3 weeks following viral vector infusion and extracted the oPFC using a tissue punch. In this case, tissue samples would be expected to contain both infected and uninfected cells. Nevertheless, Trkb.T1 protein levels were detectably elevated in mice bearing the lenti-TrkB.t1 virus, as would be expected     , while total ERK42/44 was unchanged     Fig. . Phospho01) Fig. . Meanwhi58) Fig. . Consist26) Fig. . Similar61) Fig.  and no c34) Fig. .Figure 217. To address the possibility that TrkB.t1 overexpression led to an accumulation of cortical BDNF , or alternatively, diminished local BDNF levels, we also quantified BDNF. Neither mature nor pro-BDNF were significantly affected, despite large group sizes     Fig. . To addr27) Fig. . Our fin57) Fig.  further TrkB.t1 overexpression here7. Interfering with oPFC-striatal interactions also impedes an organism’s ability to modify instrumental behaviors when reward value changes8. The striatum contains very little Bdnf mRNA18, but abundant BDNF protein anterogradely transported from cortical sources19. We thus next examined whether TrkB in the dorsal striatum is similarly important for flexible action selection. In this case, we overexpressed TrkB.t1 selectively in the DMS or DLS    Fig. . TrkB.t117) Fig. . Thus, Tinduced habit behavior using a random interval schedule of reinforcement   Fig. .TrkB.t1 overexpression in these striatal sub-regions causes response patterns that bear remarkable resemblance to those following inactivation of each respective structure21. Although TrkB is expressed in both the DMS and DLS22, these patterns were nevertheless somewhat unexpected, given that systemic administration of a putative TrkB agonist blocks habits induced by extensive response training9 and excess glucocorticoids16, rather than facilitating this DLS-dependent behavior. TrkB stabilizes dendritic spine densities and morphologies throughout multiple brain regions23 and is essential for corticostriatal long-term potentiation24. The switch from goal-directed action to habits is thought to reflect a transition in the coordinated control of response strategies by multiple cortico-striatal regions to a predominantly DLS-controlled output . Thus, broad-spread TrkB stimulation  may energize goal-directed action by stimulating multiple cortico-striatal structures 12 competing with the DLS for control over behavior. Further understanding the molecular mechanisms mediating the balance between actions and habits could shed light onto treating disorders characterized by impairments in flexible action and decision making, such as obsessive-compulsive disorder and addiction2.To summarize, TrkB appears to be essential to the functions of both the DMS  and DLS (supporting habits). Indeed, ad libitum access to water and food, except during instrumental conditioning when body weights were maintained at ~90% of baseline. Procedures were approved by the Emory University Institutional Animal Care and Use Committee and were performed in concordance with The Guide for the Care and Use of Laboratory Animals.Experiments used adult male wild-type C57BL/6 mice  . Mice were housed 2–5 per cage and maintained on a 12-hour light cycle (on at 0800) and were experimentally naïve. Mice had TrkB.t1 and an HA tag or GFP under a CMV promotor were generated by the Emory University Viral Vector Core and have been described in detail previously10. Viral vectors were infused at a rate of 0.1 μL/minute, with a total volume of 0.5 μL and the microsyringe left in place for 5 minutes following infusion. In experiments targeting the oPFC, viral vectors were infused at +2.6 mm anteroposterior (AP), −2.85 mm dorsoventral (DV) and +/−1.2 mm mediolateral (ML). Viral vectors targeting the DMS were delivered to +0.74 mm AP, −3.0 mm DV and +/−2.2 mm ML. DLS coordinates were +0.5 mm AP, −3.5 mm DV and +/−2.7 mm ML.Mice were anaesthetized with ketamine/dexdomitor and then mounted onto a digital stereotaxic apparatus . Lentiviral vectors expressing Mice were trained to nose poke for food pellet reinforcers  in Med-Associates  operant conditioning chambers. Mice were trained to nose poke on 2 available apertures using a fixed ratio 1 (FR1) schedule of reinforcement for 5 sessions. Next, mice were trained for 2 additional days using a random interval 30 second (RI30) schedule of reinforcement. Sessions lasted for 70 minutes or until the maximum 60 pellets (30 per nose poke) had been delivered.26. Briefly, during the ‘non-degraded’ session, one nose poke aperture was occluded and responding on the other nose poke aperture was reinforced using a variable ratio 2 (VR2) schedule of reinforcement. The next day, during the ‘degraded’ session, pellets were delivered non-contingently at a rate yoked to the reinforcement rate from the previous session. Responses were recorded, but had no programmed consequences. The location of the ‘degraded’ aperture was counterbalanced across subjects. Mice that decrease their response rates during the ‘degraded’ session are considered goal-directed. Equivalent response rates during the ‘non-degraded’ and ‘degraded’ sessions are thought to reflect habitual responding4.Next, mice were tested for sensitivity to action-outcome contingencies using a modified version of classical action-outcome contingency degradation, the details of which are further discussed in refsIn experiments with oPFC infusions, mice were tested in the modified contingency degradation procedure 3 consecutive times. In experiments with striatal infusions, following the first contingency degradation test, mice were trained for an additional 4 days with 2 available nose poke recesses using an RI60-second schedule of reinforcement. Then, mice were again tested for sensitivity to action-outcome contingency degradation, as above.TrkB.t1 virus, or GFP was imaged. To visualize HA, sections were blocked, then incubated with the primary antibody  overnight at 4 °C. The next day, sections were incubated with secondary antibody  and then mounted with Permount  for fluorescence imaging. Mice with mislocalized infusions were excluded from analysis, resulting in the omission of 1 mouse from each of the TrkB.t1 groups and 2 mice from the GFP control groups in the oPFC infusion experiment and 2 mice from each group in the dorsal striatal infusion experiment.Mice were anesthetized by isoflurane and euthanized by rapid decapitation. Brains were stored for 48 hours in 4% paraformaldehyde and then transferred to a 30% w/v sucrose solution. Brains were then sectioned at 50 μM. To verify infusion sites, sections were immunostained for the HA tag on the Sections were immunostained for the HA tag (as above). Sections were imaged on a Nikon 4550 s SMZ18 stereo microscope . All images were collected in the same session with settings held constant. A sampling area was drawn around the infusion site and the mean integrated intensity was quantified in NIS Elements (Nikon Instruments).TrkB.t1 or GFP as above. Approximately 3 weeks following infusion, matching the onset of behavioral studies, mice were rapidly decapitated and brains were stored at −80 °C, then later sectioned into 1-mm thick sections. The oPFC was dissected using a 1 mm tissue core. Tissue was homogenized in lysis buffer  and protein concentrations were determined by a Pierce BCA Protein Assay kit (Thermo Fisher Scientific). 15 μg of each sample was separated by SDS-page on a 7.5% gradient Tris-glycine gel . Next, samples were transferred to a PVDF membrane (Bio-Rad) and blocked with 5% nonfat dry milk for 1 hour. The membrane was incubated overnight at 4 °C in primary antibodies. Primary antibodies were TrkB , phospho-Trk (Y706/Y707) , Akt , phospho-Akt (T308) , ERK42/44 , phospho-ERK42/44 (T202/Y204) , BDNF , GFAP , PSD-95  and HSP70 . Following 1 hour of incubation in secondary antibodies [goat anti-mouse and anti-rabbit peroxidase labeled IgG ], immunoreactivity was assessed using a chemiluminescence substrate (Thermo Fisher Scientific) and a ChemiDoc MP Imaging System (Bio-Rad). Immunoblot comparisons were generated at least twice.Behaviorally-naïve mice received oPFC-targeted infusions of full-titer lenti-e.g., refs9). Behavioral response rates were compared by 2-factor mixed-design ANOVA and Bonferroni post-hoc comparisons in case of significant interactions. In an additional analysis, response rates during the instrumental contingency degradation testing phases were normalized to response rates associated with the same nose poke port generated on the final day of training. Fold-change values were compared by 2-factor ANOVA, as well as 1-sample t-tests against no change (0).All mice were randomly assigned to condition, and sample sizes were in line with prior reports using the same approaches  in the same lane and then to the control sample mean on the same gel to accommodate fluorescence variance Throughout, normality was confirmed using the Shapiro-Wilk test. Values >2 standard deviations above or below the mean were considered outliers and excluded, resulting in the omission of 1 mouse each from the fold-change calculations in Fig. Behavioral experiments were not performed blind to the condition, but response rates were collected via automated photobeam-based systems, minimizing bias. Similarly, equivalent amounts of protein were loaded in western blotting experiments, also minimizing bias.Data can be made available upon reasonable request.


The deviation of the ratio of reactants from stoichiometric ratio benefits the production of graphene with higher concentration of defects. It is indicated that the temperature of the heat treatment has remarkable influences on the structure of graphene, Raman-sensitive defects can be recovered partly by heat treatment while IR-sensitive defects are closely related with the oxidation and decomposition of the oxygen-containing groups at elevated temperature. This work indicates that SHS is a promising method to produce graphene with special magnetism, and the heat treatment is an effective way to further adjust the magnetism of graphene. This work sheds light on the study to develop carbon materials with controlled ferromagnetism.Magnetism in graphene has stimulated extensive studies to search for novel metal-free magnetic device. In this paper, we use a synthesis method far from equilibrium state named self-propagating high temperature synthesis (SHS) to produce few-layer graphene with different defect contents and then use a heat treatment process  to further control the defects in graphene. We find that the type and content of defects in graphene can be controlled by adjusting the mole ratio of reactants (Mg: CaCO Compared with other properties, magnetism in graphene8 has stimulated extensive studies to search for novel metal-free magnetic device. The emergence of magnetism in versatile natured graphene and the ability to control its properties can lead graphene to be an excellent material for spintronics and other memory based device applications which promise information storing, processing and communicating at faster speed with lower energy consumption. Research on the origin of magnetism in graphene oxide9, graphene nanoflakes12, hydrogenated graphene13 and graphene nanoribbons14 suggested that the magnetic behavior of graphene based materials is to a large part governed by their structures. Although the mechanism of graphene magnetism is complicated, extensive theoretical and experimental studies indicated that defects15, disordering4, covalent-adsorption16 and magnetic edge state in graphene nanoribbons17 and partially hydrogenated epitaxial graphene13 are the potential carriers for the magnetism in graphene.Graphene has generated a lot of activity in the area of material science due to its exceptional electronic and mechanical properties13. Recently, we have developed a facile and cost-effective method named as self-propagating high temperature synthesis (SHS) to produce few-layer graphene18. The SHS process utilizes the heat generated by the exothermic reaction of Mg and CaCO3 to sustain itself in the form of a combustion wave after external ignition. The process is of high reaction temperature, fast heating and cooling speed and far from equilibrium state, so the defect in graphene made by this method is special. We have found that few-layer graphene samples both non-doped and doped with nitrogen produced by SHS method exhibit ferromagnetic properties and have high Curie temperatures (>600 K), and the saturation magnetization and coercive field increase with the increasing of nitrogen contents in the samples19. Taking advantage of the far-from-equilibrium-state SHS process, people are expected to produce graphene with different kinds and contents of defects, which helps further clarify the relationship between defects and the ferromagnetic properties of graphene. However, few works have been done on these issues.Ferromagnetism has also been observed in graphene materials prepared by different methods like thermal exfoliation of graphitic oxide, conversion of nano diamonds, arc evaporation of graphite in hydrogen and graphene oxide partially reduced by hydrazine and further completely reduced by thermal annealing, since graphene obtained by different methods has different types and quantity of defects3) in SHS process. Secondly, in order to further improve the magnetic property of SHS graphene, we proposed a heat treatment method , which is heating the sample in vacuum environment at a certain high temperature and then cooling down to room temperature in atmospheric environment. Our works indicated that the deviation of stoichiometric ratio of the reactants under far from equilibrium state is a promising method to produce graphene with special magnetism, and that the designed heat treatment is an effective way to further adjust the ferromagnetism of graphene.In the present study, firstly, we explored the method to produce few-layer graphene with different defect concentrations by changing the ratio of reactants ; these materials were purchased from Sinopharm Chemical Reagent Co., Ltd.Here, we used the SHS method to synthesize graphene with different content of defects by changing the ratio of reactants: magnesium, (99.5% purity) and calcium carbonate  is a stoichiometric ratio according to the reaction: 2Mg + CaCO3 = 2MgO + CaO + C (graphene), while the ratio (4:1) was designed to deviate from the stoichiometric ratio. The products were expressed as M2C1 and M4C1, respectively, according to the ratios of Mg and CaCO3. 16 grams of Mg for M2C1 and 32 grams for M4C1 were added to 33.3 grams of calcium carbonate and then milled in a mortar for 20 minutes, respectively. Each sample was ignited by an electric ignition device composed by a direct current (DC) power source and a resistance-based wire heater. The ignition current was 22 A. The coarse product was placed in dilute hydrochloric acid (10 v/v %) containing ethanol (20 wt %) and sonicated for 1 h, then washed with deionized water and absolute ethanol in that order. The obtained sample was dried in a vacuum oven at 120 °C for 24 h.The SHS experiments were conducted in a stainless-steel combustion chamber under an atmosphere of carbon dioxide (99.9%)−1 and kept for 5 min in vacuum (10−4 Pa), then cooled down to room temperature within 5 minutes in air by opening the valve.Every graphene sample (M2C1 or M4C1) was divided into 4 parts and three of them were heated at 500 K, 650 K and 800 K, and named as M2C1-500 or M4C1-500, M2C1-650 or M4C1-650 and M2C1-800 or M4C1-800, respectively. As a contrast, the initial M2C1 and M4C1 sample without heat treatment was named as M2C1-G and M4C1-G (G stands for the generated graphene). The heating rate from room temperature to the desired temperature was 5 K·min−1 in air atmosphere form 300 K to 1200 K. The nitrogen adsorption/desorption measurements were carried out on Belsorp mini II (Japan) at 77 K to obtain the specific surface area of M2C1-G and M4C1-G. Before adsorption/desorption tests, the samples were degassed at 150 °C for 4 hours with vacuum pumping.The phase composition of the as-prepared powders was analyzed by powder X-ray diffraction (XRD) analyses (Philips X’Pert diffractometer) with CuKα radiation. Environmental scanning electron microscopy  and high-resolution transmission electron microscopy (HRTEM JEM-2100) were used to observe the morphology of the graphene sheets. The TEM specimens were prepared by dropping ethanol/water (38 v/v %) solution containing 1 wt % graphene onto a copper grid and drying at 100 °C. Raman spectra was obtained using a Raman Station  with a 532 nm wavelength laser corresponding to 2.34 eV. X-ray photoelectron spectroscopy  was utilized to determine the bonding characteristics of the samples. All XPS peaks were calibrated according to the C 1 s peak (284.6 eV). The magnetic properties were measured using a Quantum Design MPMS magnetometer based on a superconducting quantum interference device (SQUID). Thermogravimetric analysis (TGA) was performed on a Netzsch STA 449 F3 under a heating rate of 10 K·min2 g−1 according to the BET method. Then we can evaluate the layer of M2C1-G is about 7 through the SSA of monolayer graphene is 2630 m2 g−1. According to the above analysis and the Raman analyses in the following section, we conclude that the products synthesized by SHS method are few-layers graphene.Figure 3) for SHS reaction. The stoichiometric mole ratio of the reaction between Mg and CaCO3 is 2:1, which is just the ratio for M2C1-G, while the ratio for M4C1-G is 4:1, much higher than the stoichiometric ratio. The deviation of the stoichiometric ratio for M4C1 means that Mg is excessive for the reaction, and the excessive Mg may play multiple roles in the SHS reaction. Firstly, the excessive Mg may melt at 648 °C and volatilize at 1107 °C, which can absorb large amount of heat produced by the exothermic SHS reaction (ΔH = −632 kJ/mol) and decrease the maximum temperature of the reaction. Secondly, the gaseous Mg in the enclosed space of reaction container may affect the growth process of graphene since they may decrease the collision probability of the reactive carbon atoms produced during the SHS reaction process. As a result, we can deduce that the reaction temperature for M4C1-G is lower than that of M2C1-G which benefits the production of smaller and thinner sheets for M4C1-G as shown in Fig. The difference of the morphology between M2C1-G and M4C1-G can be understood by considering the mole ratios of reactants ; the peaks at 1141 cm−1, 1717 cm−1, 2850–2920 cm−1 and 3200–3600 cm−1 are attributed to the C-O-C, C=O, C-H and O-H vibration, respectively. On the one hand, from Fig. 2O (1624 cm−1) and O-H vibration (3200–3600 cm−1) with the increase of heat treatment temperature, suggesting the remove of hydroxyl and water on graphene; the peaks corresponding to oxygen-containing groups are not clear for M2C1-G, suggesting that M2C1-G has good chemical stability. On the other hand, it is interesting to see that the FTIR spectra of M4C1-G is quite different from that of M2C1-G. The peaks corresponding to epoxy, hydroxyl, carbonyl and carboxyl groups can be both found for M4C1-G and M4C1-800; however, the relative intensities of peaks corresponding to epoxy, carbonyl and carboxyl groups for M4C1-800 increase obviously, while the peaks corresponding to O-H vibration almost disappear, compared with those for M4C1-G. Consequently, it can be concluded that M2C1-G has less oxygen-containing groups and is more stable for the heat treatment than M4C1-G and that the oxidization of graphene happens for M4C1-G heat-treated at high temperature.Figure −1 and the result has been added in Fig. 2. From the results of DSC and TG analysis, we conclude that the SHS graphene had two types of structure. One is easily oxidized at low temperature, corresponding to the oxygen-containing groups and carbon defects; the other is more thermally stable, oxidized at higher temperature, assigning to the defect-free parts in SHS graphene20. But the ratio of the two exothermic peaks in the curves of DSC for the two samples is different. The relatively intensity of the first peak to the second peak in M4C1-G is much higher than that in M2C1-G, indicating that M4C1-G contained more oxygen-containing groups and its thermal stability is lower than that of M2C1-G, which are consistent with the results from FTIR and XPS.To better study this behavior, we performed the Thermogravimetric Analysis (TGA) and the differential scanning calorimetry (DSC) of M2C1-G and M4C1-G in air environment at the heating rate of 10 K·min21. Gaussian decomposition and Lorentz decomposition are employed in this fitting. The C1s component can be deconvolved into six components: sp2 C=C (284.4 ± 0.1 eV), sp3 C-C (285.4 ± 0.1 eV), C-O (286.4 ± 0.1 eV), C=O (287.5 ± 0.2 eV), O=C-O (288.6 ± 0.2 eV) and π-π* satellite peak (290.5 ± 0.1 eV)24. In order to obtain more detailed information, the contents of components in C 1 s of M2C1 and M4C1 treated at different temperatures are analyzed according to the fitting and the results are shown in the Fig. XPS characterizations are further performed to analyze the elemental composition and C/O configuration in the samples. The XPS survey spectra of the samples in Fig. 2 and sp3 C in graphene, the well opposite trend suggests that the oxidized sp2 carbons are mostly changed to sp3 carbons and vice versa. Similar trend can also be found in M4C1 sample.Figure XPS results in Fig. 28. The Raman spectra of the M2C1 and M4C1 samples treated at different temperatures are shown in Fig. −1 represents the in-plane bond-stretching motion of the pairs of sp2 hybridized C atoms (the E2g phonons); the D band at 1341 cm−1 corresponds to breathing mode of rings or K-point phonons of A1g symmetry; and the second-order D (2D) band at 2678 cm−1 originates from a two phonon double resonance process29. The 2D peaks of the M2C1 and M4C1 samples around 2678 cm−1,which shift greatly to lower wavenumber compared with that of graphite (2714 cm−1), can identify the samples as few-layer graphene31.Raman spectroscopy is considered to be an effective tool for characterization of mono-, few-, or multil-layer grapheneID) to the G peak (IG) in graphene is directly proportional to the level of defects in the sample33. Here, the intensity ratios (ID/IG) is used to demonstrate the relatively change of ID to IG with the heat treatment temperatures as shown in Fig. −2) of graphene can be investigated and defined as nD = 5.9 * 1014 * EL−4 * (ID/IG)−1 29, where the laser energy EL = 2.34 eV (λ = 532 nm) and the calculated defect density is shown in Fig. 34 and two orders higher than highly ordered pyrolytic graphite irradiated by 140 eV Ar+ ions reported by Ugeda35.The relative intensity of the D peak (ID/IG of the M4C1 sample is much higher than that of the M2C1 sample treated at the same temperature. This result indicates that the mole ratio of the reactants (Mg: CaCO3) plays an important role on the defect density in the SHS products, the deviation of the ratio of reactants (Mg: CaCO3) from stoichiometric one in the case of M4C1 benefits the production of graphene with higher defect density. The ratios of ID/IG of both M4C1 and M2C1 decrease with the increase of heat treatment temperature, while the decline for M4C1 is more obvious than that of M2C1. But the defect concentration in M4C1 is still higher than that in M2C1 overall even after heat-treated at 800 K. The reduction of defect density with the increase of the heat treatment temperature suggests the repair of the Raman-sensitive defects is the main process during the heat-treatment process. Finally, we can conclude that the ratio of reactants is the main factor for the formation of defects in SHS graphene and that the heat treatment can repair part of the defects characterized by Raman spectra, especially for M4C1.From Fig. I2D/IG has been used for the identification of the number of graphene layers. The value of I2D/IG obtained from Fig. I2D/IG value is about 0.6 for M2C1 and about 0.4 for M4C1, which are larger than that of graphite (about 0.3)36. These results also indicate that M2C1 and M4C1 are few layer graphene. In addition, comparing the ratios of the samples treated by different temperatures, we can find that the ratios of I2D/IG for M2C1 and M4C1 at any treated temperatures do not change significantly, which indicates that the heating treatment has no obvious effect on the number of graphene layers. At last, we summarize the full width at half-maximum (FWHM) of the 2D band in the spectra for all samples in Fig. The ratio of 37, the FWHM of the 2D band in graphene could be a quantitative guide to distinguish the layer number (single- to five-layers) of few- layer graphene. However, it is based on graphene produced by mechanical exfoliation which has less defects, and the research about the relationship between defective graphene and FWHM of 2D peak has not yet been reported. In addition, the size of laser light spot for Raman spectra we used is about 100 μm and laser light may penetrate many graphene sheets in its light path, so the 2D peak we got reflects the information of many graphene sheets, which is composed of many overlaid 2D peaks. So the FWHM of 2D peak may not provide us with exact information about the number of SHS graphene layer.As mentioned in the research of J. T. L. ThongPowder X-ray diffraction is used to analyze the phases in M2C1 and M4C1 samples as shown in Fig. Ms) and heat treatment temperatures obtained from Fig. 19, the total ferromagnetic impurities  in SHS graphene are less than 15 ppm, indicating the ferromagnetic contribution of impurities could be neglectable. Therefore, the results represent that the ferromagnetism of SHS graphene is due to the structure defects in it. For comparison, we further summarized the Ms values of several carbon materials at room temperature reported in the references as shown in Fig. To investigate the magnetic properties of the SHS samples, the magnetization behaviors versus magnetic field curves for M2C1-G and M4C1-G and the heat-treated samples by 500 K, 650 K and 800 K are measured at room temperature (300 K) in the magnetic field range from −5000 Oe to 5000 Oe, as shown in Fig. Ms changing tendencies of M2C1 and M4C1 heat-treated at different temperatures are quite different as shown in Fig. Ms for M2C1 and M4C1 are affected by the changes of the defects in them. The changing tendency of defect concentration for M2C1 in Fig. Ms for M2C1, however, it is quite different for M4C1. It may indicate that Raman-sensitive defects must not be the only representation for the ferromagnetism in graphene.Surprisingly, it can be also found that the It is well known that Raman measurement is sensitive to symmetric structures while FTIR spectra is sensitive to asymmetric structures. We divide the defects of the SHS graphene into Raman-sensitive and FTIR-sensitive defects. Edges (zigzag and armchair), vacancies  and disordering are the defects originating from the broken of the C-C bonds which make graphene sheets distorted. These defects can be measured by Raman spectrum and mentioned as Raman-sensitive. The defects corresponding to the oxygen-containing groups include carboxyl group, carbonyl group and hydroxyl group, etc., which are connected to the graphene layers by covalent bonds and also introduce various edges and defect sites. They are FTIR sensitive and mentioned as FTIR-sensitive.Ms tendency between M2C1 and M4C1 by considering the changes of both Raman- and FTIR-sensitive defects. On the one hand, the Raman-sensitive defects in graphene have been repaired in a certain extent as shown in Fig. Ms of the SHS graphene. On the other hand, the content of carboxyl group increases with the increase of heat treatment temperature, which could increase the Ms. M2C1 is more stable at elevated temperature and has less content of carboxyl and other oxygen-containing groups, so the Raman-sensitive defects play a more important role on the ferromagnetism than FTIR-sensitive one, so the Ms for M2C1 has a similar trend with the change of Raman-sensitive defects. Since M4C1 is more thermal sensitive and easy to be oxidized at higher temperature as above mentioned the effect of FTIR-sensitive defects on the ferromagnetism may overcome that of Raman-sensitive defects, as a result the Ms for M4C1 has a similar trend with the FTIR-sensitive defects. In addition, according to XPS results in Fig. Ms for M4C1 or M2C1, the carboxyl group must be the origin of the ferromagnetism for SHS graphene. Based on the analyses above-mentioned, we obtain a more comprehensive scene of the magnetism properties of the SHS graphene and the effect of heat treatment on them. Both Raman-sensitive and FTIR-sensitive defects could contribute to the ferromagnetic properties of graphene. The heat treatment plays an important role on elucidating the effect of different factors for the ferromagnetism of graphene.We could explain the difference of the 3) from stoichiometric ratio benefits the production of few-layer graphene with smaller and more plicated sheets, and also benefits the production of both Raman-sensitive and FTIR-sensitive defects. Secondly, the heat treatment method could adjust the contents and types of defects by using the competitive relationship of the repair and oxidation processes. Thirdly, there are two origins of the ferromagnetism of the SHS graphene, associated with the Raman-sensitive and IR-sensitive defects respectively. The Ms trends could be explained by considering the changes of both Raman-sensitive and FTIR-sensitive defects. As a result, this work sheds light on the study to develop carbon materials with controlled ferromagnetism.In this study, we obtain a more comprehensive scene of the ferromagnetic properties of the SHS graphene and the methods to tune them. Firstly, the deviation of the mole ratio of SHS reactants (Mg: CaCOSupplementary Information


Important characteristics of this low-temperature response are a pronounced selectivity to acid- and base-forming gases and a large disparity of response and recovery time constants which often leads to an integrator-type of gas response. We show that this kind of sensor performance is related to the trend of semiconductor gas sensors to adsorb water vapor in multi-layer form and that this ability is sensitively influenced by the surface morphology. In particular we show that surface roughness in the nanometer range enhances desorption of water from multi-layer adsorbates, enabling them to respond more swiftly to changes in the ambient humidity. Further experiments reveal that reactive gases, such as NO Gases that could be detected under such conditions are NO00 °C or alcohols ,5,6,7. Ialcohols ,12,13. Aalcohols ,17,18,19alcohols ,21,22. Talcohols ,26,27,28alcohols ,31,32,33alcohols ,38,39,40alcohols . In esseaterials . The intaterials ,44 whereaterials ,45.In the following, we shed more light on the ability of semiconductor substrates of acquiring multi-layer water adsorbates and of exhibiting an integrator-type gas response with regard to acid- and base-forming gases. Below, we will demonstrate that Brunauer–Emmett–Teller (BET) water adsorbates ,47 can r2 layers with distinctly different and well-controllable surface morphologies. Typical morphologies obtained with both methods are shown in 2 at room temperature and high-temperature annealing in air. As shown in the surface SEM of 2 grains. Almost all of these grains have a visible sub-structure with feature sizes in the range of a few nanometers only. In addition, a larger number of such smaller grains can be seen in the empty spaces between the dominating larger grains. These latter films were produced by a modified RGTO  technique, starting with a room-temperature evaporation of metallic tin and a high-temperature annealing step in air to allow the molten tin to become oxidized and to be transformed into SnO2. A more detailed description of the preparation processes and the resulting material properties is presented in In order to test the effect of the surface morphology on the room temperature gas response, we have adopted two different methods of preparing SnO2, NH3, H2, C2H4, CO, and ethyl alcohol were performed. All gases were diluted to the desired concentrations in dry synthetic air. The sensor operation temperature could be controlled with the help of a substrate heater, allowing measurements between room temperature and about 100 °C. In order to minimize parasitic condensation on the chamber walls, the walls were slightly heated (~40 °C) during all gas sensing tests. The actual humidity concentration inside the test chamber was independently controlled using a humidity sensor.In order to assess the gas sensing properties of the above layers, the films were fitted with contact pads and gas sensing tests with humidity, NO2, both in its flat-surface and nano-morphology versions, does respond to water vapor with the expected resistivity decrease. The magnitude and the dynamic behavior of the humidity responses, however, clearly depend on the surface morphology. The morphologies of the tested films are those shown in Before starting gas sensing tests, both kinds of sensing materials were heated and stored in a flow of dry synthetic air to remove any humidity that might have existed on the sensor surface. Thereafter the films were cooled to nominally room temperature and subjected to pulses of humidified synthetic air with the level of humidity increasing in each subsequent exposure pulse. Between each exposure pulse the initially dry flow of synthetic air was restored. The data displayed in 2 film with a smooth surface exhibits an almost ideal kind of integrator response with the humidity response increasing linearly during each exposure pulse and with the response staying constant in between exposure pulses. From the fact that there is no visible drop in the sensor response over periods ranging up to 40 min, it is evident that the time constants for desorption from room temperature substrates is in the order of several hours at least. Due to this extremely slow desorption, the flat-surface films accumulate increasing amounts of water as the number of humidity exposure pulses is increased. In the nano-morphology case a different behavior is observed: the humidity response also rises linearly during each exposure pulse. After each exposure pulse, however, the humidity response drops much more rapidly than in the flat-surface case. Desorption time constants in this case were in the order of one hour only.The data in 2 surfaces and that the degree of sticking depends on the surface morphology. Since besides morphology, temperature is an important parameter controlling the adsorption/desorption kinetics, these experiments were repeated at increasingly higher sensor operation temperatures, as reported in These first experiments show that adsorbed water vapor tends to stick to SnO2 and NH3 exposure steps to investigate their response towards water-soluble gases. In order to elucidate the effects of background humidity this experiment was repeated four times, each time increasing the humidity level of the synthetic air carrier gas from almost zero up to about 90% relative humidity. 2 films to these exposure sequences. For better comparison, the sensor resistances of the two versions of SnO2 have been normalized to their starting values at the beginning of these sensing tests.Both kinds of sensing materials were subjected to a series of NO2 do respond to these acid- and base-forming gases. Considering the absolute magnitude of the gas response it is seen that SnO2 layers with nano-crystal morphology exhibit a larger response than their flat-surface counterparts when the background humidity level is low. In the limit of high-humidity levels the magnitude of the gas response becomes more or less similar and independent of the surface morphology. Turning to the kinetics of the gas response, it is seen that both morphologies exhibit a more or less pronounced integrator-type response as long as the background humidity is low and a faster and more reversible response when the background humidity is high. This latter result demonstrates that the presence of water vapor does have an accelerating effect on the desorption behavior of NO2 and NH3 molecules from the adsorbed water layers, independent of the kind of surface morphology. Comparing NO2 and NH3, it is seen that the adsorption and desorption rates of NO2 are relatively high, whereas they are very low in the case of NH3. In this latter case the accelerating effect of the background humidity on the adsorption and desorption processes is much more important than in the case of NO2. In order to reveal any interaction of the dissolved NO2 and NH3 molecules inside the water adsorbate layers, control experiments were performed in which the sequence of the NO2 and NH3 exposure steps was inverted. As these latter experiments yielded similar results, it appeared that the adsorbates do not measurably react inside the adsorbed water layers and that therefore only their relative concentrations do determine the actual level of gas response.As a key result one can observe that both kinds of SnO2 layers. In addition the response to this latter group of gases also did not depend on the level of background humidity. Again, and consistent with the results in 2 response than those with a granular surface.In order to gain further insight into the gas sensing mechanisms operative on both kinds of materials, similar gas sensing tests were performed with the samples being exposed to increasing concentrations of non-acid or non-base-forming gases. These latter data, displayed in 2 layers are very similar to those observed on hydrogenated diamond samples [BETb(T) standing for the so-called BET constant: Overall, the observed gas response characteristics of the SnO samples ,38,39,40 samples  can sati samples ,47,48. I2O molecules to the adsorbent surface is stronger than the binding between water molecules in liquid water The two energy parameters in this latter equation, olecule) . A numerThis latter fact is also illustrated in The considerations in 2 surfaces are reasonably hydrophilic and that therefore BET multilayer adsorbates do indeed form. Supporting evidence into this direction is provided by the fact that the contact angle of water is small on the investigated sensor surfaces  of SnO2 powders. Electron beam evaporation onto room temperature substrates and under standard vacuum conditions yielded amorphous material with an excess amount of tin. In order to arrive at fully stoichiometric, crystalline SnO2 layers, the evaporated films were annealed in laboratory air at 600 °C for 20 h [2. Scanning electron microscopy further revealed that these films have a comparatively flat surface, are compact and do not exhibit any significant porosity. This latter fact is demonstrated in 2 layer on top. In order to obtain metallic Sn layers with a flat surface morphology, the substrates were not heated during deposition. Oxidation of the evaporated tin layers was performed afterwards by annealing at 600 °C in laboratory air. Upon annealing, the evaporated tin layers melt  .x grains as shown in x grains scaled with the thickness of the Sn precursor films. As shown in the example of x layer formed from a 40 nm Sn precursor film consisted of an irregular array of SnOx grains with an average diameter around 40 nm. In addition, one can observe a large number of very small grains interspersed in between the larger ones. Such films did not electrically conduct as the disconnected grains failed to form percolation paths that could connect the contact pads at the periphery of the SnOx samples. In order to obtain electrically conducting layers, a second precursor layer of Sn was evaporated on top of the first layer of SnOx grains and annealed in air as before. These second and third layers of SnOx grains formed bridges between the initially disconnected grains and thus allowed electrical currents to flow over macroscopic distances. A typical example of such an interconnected film is shown in Oxidation of very thin Sn layers . The resulting pH changes in the BET layers are then communicated to the sub-surface electronic system inside the MOx semiconductors, thus forming an electrically detectable output signal. A huge variety of combustible gases , which yield large signals on heated MOx gas sensors, are either completely rejected by the BET water layers  or fail to electrolytically dissociate  and thus go undetected. BET adsorbates thereby considerably contribute to gas selectivity. Concerning the kinetics of those gases that actively contribute to the sensor response, it is found that the desorption of H2O, NO2 and NH3 is much more sensitively influenced by the effects of surface morphology, ambient humidity and sensor operation temperature than their adsorption. While rough surface morphologies, high ambient humidity and elevated sensor operation temperatures enhance desorption, desorption becomes increasingly unlikely in the limit of flat surface morphologies, low ambient humidity and room-temperature operation. In this latter case MOx gas sensors tend to function as gas dosimeters. As has been shown in recent papers [The room-temperature response of SnOt papers ,32,33, a


PGOD materials are natural chemical extensions of graphene that promise to have an enhanced range of properties and applications. Experimentally realised molecules based on two PGOD nodes exhibit a bi-stable closed-shell/multi-radical character that can be understood through competing Lewis resonance forms. Here, following the same rationale, we predict that similar states should be accessible in PGOD materials, which we confirm using accurate density functional theory calculations. Although for graphene the semimetallic state is always dominant, for PGOD materials this state becomes marginally meta-stable relative to open-shell multi-radical and/or closed-shell states that are stabilised through symmetry breaking, in line with analogous molecular systems. These latter states are semiconducting, increasing the potential use of PGOD materials as highly tuneable platforms for future organic nano-electronics and spintronics.Post-graphene organic Dirac (PGOD) materials are ordered two-dimensional networks of triply bonded  The lack of band gap controllability in graphene severely restricts its use in nanoelectronics. Here, the authors predict that post-graphene organic Dirac materials should allow for exceptional electronic tunability between graphene-like semimetallicity and multi-radical and/or closed-shell semiconducting states. Organic nanosystems based on sp2 carbon networks are seen by many as an essential basis for many future technologies1, arguably provoking a paradigm shift in materials science. Graphene, the simplest two-dimensional (2D) hexagonal network of sp2 hybridised carbon, plays an especially prominent role in this respect5. Since its isolation4, researchers have become fascinated with its many exceptional properties, which are progressively being more fully understood and exploited in many fields of science and technology. One particularly appealing characteristic of graphene is the existence of Dirac cones in its electronic band structure5. This feature, due to specific bands crossing at the Fermi level, EF, gives rise to semimetallicity and ultra-high electron mobilities, and has promoted graphene as a potential candidate to replace silicon in the next generation of nanoelectronic devices5. However, the intrinsic connection of these features with the extended high symmetry of its electronic state limits its use in applications requiring nanostructuring .The triply bonded 9 and carbon-based11 2D materials ). An important class of PGOD materials are the so-called graphynes13 and graphdiynes13, which present sp2 carbon atoms linked via alkyne groups in a variety of ways14. Although some graphdiynes have been synthesised15, those predicted to be Dirac materials are still awaiting experimental realisation. Recently, a series of 2D conjugated networks were predicted to present Dirac cones in their bandstructures10. These networks present a common basic skeleton: a hexagonal array of triply bonded sp2 carbon atoms  connected by π-conjugated linkers . Herein, we further propose graphphenyl (GP), obtained when using phenyl ring as linker  or buta-1,3-diyn-1,4-diyl (-C≡C-C≡C-) alkyne linkers, respectively. Finally, graphene is the densest hexagonal array of sp2 carbon nodes directly linked by bonding based on π–π orbital overlap. This family of 2D materials is summarised in Fig. The discovery of graphene has triggered a global search for other 2D materials with similar characteristics, but with higher electronic tunability and greater resilience to nanostructuring. In this direction, ab initio computational modelling has predicted that Dirac cones should be in the band structures of a number of non-carbon-basedsee Fig. . Replaci16, one of the oldest bi-radical compounds ever synthesised. The triply bonded methyl radical carbon atoms in Tschitschibabin’s hydrocarbon bear the two unpaired electrons of the bi-radical molecule. Due to the connectivity linking these two radical sites, the molecule possesses two possible Lewis resonance forms: 1) the open-shell anti-ferromagnetic (AFM) one with two unpaired localised electrons , it is chemically reasonable to expect that these extended resonance forms should lead to the accessibility of similar electronic states in all cases.Focusing on GDP  cutting it into nanoribbons22, (iii) covalent grafting of molecules23 or atoms25, (iv) inclusion of defects26, or (v) applying external strains28. Analogously, computational modelling has predicted similar states in α-graphyne (GY) upon application of strain29 or by forming α-GY nanoribbons31. These studies lend support to our chemically motivated proposal that extended resonance forms may lead to open-shell multi-radical and/or closed-shell solutions being stabilised in all the materials depicted in Fig. As far as we are aware, none of the family of materials in Fig. 35 and that of PGOD materials. In all cases, these solutions are found to be energetically accessible and to significantly affect the electronic properties of the host materials, highlighting the potential of such systems for future organic electronics/spintronics applications.In this work, we present a systematic computational modelling study employing accurate density functional theory (DFT) based calculations to assess the existence and stability of low lying localised electronic states in all the 2D PGOD materials shown in Fig. 36, especially for capturing the most subtle electronic features displayed by many systems . The molecular computational chemistry community has a long tradition of using hybrid functionals where, especially for electronic structure, they are known to generally outperform GGA functionals41. A particular case is that of bi-radical/quinoidal compounds (such as Tschitschibabin’s hydrocarbon), which have attracted much attention in recent years for its use in organic electronics33 and organic magnetism42. When these compounds are reduced or doped by other elements , charge transfer phenomena between the two sp2 centres takes place44. The computational assessment of the electronic ground state of such molecular systems is thus quite challenging38. DFT calculations employing GGA functionals over-delocalise valence electrons, and cannot stabilise the localised states found in experimental observations. Calculations using hybrid functionals, on the other hand, are found to be able to capture the quite complex and sensitive electronic structure of such π-conjugated mixed valence compounds45.Largely due to their computational efficiency and relative accuracy, DFT based approaches have become the dominant method of choice when simulating the electronic structures of periodic materials. For numerous cases  the standard generalised gradient approximation (GGA) produces reliable results. However, hybrid functionals ) are now being widely adopted by material’s modellers46, or local density approximation (LDA)47 functionals, and seldom utilise hybrid functionals27. A reason for this might be that the high tendency of GGA to delocalise electrons ensures the success in finding the known, and highly studied, semimetallic solutions. However, knowing how critical the proportion of HFE is for obtaining the correct localised solutions in π-conjugated molecular systems45, it is reasonable to think that possible localised solutions in Dirac cone materials might have remained elusive due to this bias. In this work, we find that PBE048 is a functional providing a good balance between localisation and delocalisation . Therefore, the main discussion is based on PBE0 results. For comparison with previous works, results obtained by the PBE GGA functional49 are included in Supplementary Figs. Most of the periodic modelling studies assessing the electronic structure of Dirac materials are performed using pure GGAsp2 carbon atoms siting at the nodes of the hexagonal network, due to the inclusion of (-C≡C-)n linkers (where n = 0–2), between them. In these cases Dirac cones appear at the high-symmetry reciprocal space K point, as previously found29. Although the nodal sp2 centres in GP and GDP lay in a plane, these networks cannot be regarded as one-atom planar materials, due to the fact their phenyl rings are partially twisted by ~35o due to inter-ring steric hindrance . These materials also present a zero band gap feature at K. This feature can be understood as resulting from the high symmetry of the hexagonal lattice, which is preserved in all cases.In Fig. see Fig. , x views6 m/s for graphene to 3.04·106 m/s for GDP. These values are in line with those of previous studies50 and indicate that the increasingly separated π-conjugated sp2 carbon centres when going from graphene to GDP has only a modest effect on the ballistic transport.Overall, it is possible to see a clear reduction in the energy scale of the band structures through the series from graphene to GDP, accompanied with a corresponding flattening of the bands. As reported in  Supplementary Fig. sp2 triply bonded carbon atoms  may be possible for larger cells than used herein.[53, covalently grafting of atoms and molecules54 or the application of external strains55. Such approaches, however, tend to significantly affect other fundamental and technological interesting properties of graphene . Conversely, GP and GDP display a sizeable band gap around the Fermi level  arises from sp2 triply bonded carbon atoms, in full agreement with the open-shell character of such centres (see above).Finally, in bi-radical/quinoidal compounds it is known that a ferromagnetic (FM) alignment of unpaired electrons (a triplet state) can be induced by thermal excitationsK-vector reversal symmetry  resulting in a lifting of the degeneracy of spin-up and spin-down bands. One effect of this spin polarisation is to induce a small (~0.3 eV) energy gap at the previously spin degenerate crossing at K resulting in two crossings between bands of the same spin above and below the Fermi level. Additionally, two further crossings between bands of different spin appear at the Fermi level at K + k1 and K−k2. These two crossings close to K are reminiscent of Weyl points, however, as they are constituted by bands of different spin, they indicate the possibility of spin helical Dirac states as found on the edges of graphene nanoribbons (1D)59 and within surfaces of toplogical insulators (2D)60. Applying spin-orbit coupling (SOC) in our calculations reveals that these crossings are maintained and not gapped out  by SOC. The relative energies of these spin-polarised solutions with respect to the purely semimetallic solutions  and chemistry  which, until now, have not had significant overlap. Bi-radical/quinoidal molecules are finite systems and, consequently, their electronic character tends to vary between two localised electronic solutions: namely the bi-radical AFM and the closed-shell quinoidal, as represented by the bottom dotted line in Fig. Electronically, the behaviour of PGOD materials lies between that of bi-radical/quinoidal molecules and graphene ; here using 6 × 6 × 1 and 18 × 18 × 1 meshes for PBE and PBE0, respectively  to obtain reasonable band structures. All DFT based calculations were performed employing the all-electron FHI-AIMS code63, using a light/Tier1 numerical atom-centred orbital (NAO) basis set64. This basis set approximately provides results of a similar or higher quality to those obtained with valence triple-zeta plus polarisation Gaussian type orbitals65. For the periodic band structure calculations the energy zero (Fermi level) is set by the G = 0 component of the long-range electrostatic potential. In addition, the entire set of PBE calculations was also performed using the Vienna ab initio simulation package (VASP)66, using a plane-wave basis set with a 415 eV kinetic energy cut-off. Due to the very-high computational cost required for hybrid calculations using VASP, only single-point PBE0 calculations using the PBE0-optimised structures from FHI-AIMS were used in our comparisons. We find that in all cases the corresponding PBE and PBE0 results from VASP and FHI-AIMS are fully consistent, thus negating any significant influence of the DFT implementation  in our results.Optimizations of the atomic and electronic structures were performed using different initial electronic guesses to induce different electronic solutions . This set of optimizations was separately performed using the GGA functional: PBE45. For such systems, even widely used hybrid functionals such as PBE048 (25% HFE) or B3LYP67 (20% HFE) still tend to over-delocalise electrons due to the inherent self-interaction error in GGA68. However, use of functionals having a relatively large percentage of HFE, although useful for stabilising localised states, can prevent one finding fully delocalised metallic solutions – essential for extended materials such as graphene69. As a consequence, we find that the PBE0 functional provides a good balance between electronic localisation and delocalisation for the 2D materials we consider. We note that the optimised structures obtained at using the PBE functional  are made openly available in Supplementary Data Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1


Immune checkpoint inhibitors targeting the PD-1 pathway have greatly changed clinical management of metastatic urothelial carcinoma and metastatic renal cell carcinoma. However, response rates are low, and biomarkers are needed to predict for treatment response. Immunohistochemical quantification of PD-L1 was developed as a promising biomarker in early clinical trials, but many shortcomings of the four different assays  have limited its clinical utility. Further limitations include the use of archival specimens to measure this dynamic biomarker. Indeed, until PD-L1 testing is standardized and can consistently predict treatment outcome, the currently available PD-L1 assays are not clinically useful in urothelial and renal cell carcinoma. Other more promising biomarkers include tumor mutational burden, profiles of tumor infiltrating lymphocytes, molecular subtypes, and PD-L2. Potentially, a composite biomarker may be best but will need prospective testing to validate such a biomarker. Over the past few years, immune checkpoint inhibitors (CPIs) blocking the programmed cell death receptor 1 (PD-1) pathway have emerged as treatments capable of improving the overall survival of patients with metastatic renal cell carcinoma (mRCC) and metastatic urothelial carcinoma (mUC) . Based oPD-L1 is one of several ligands for the PD-1 receptor, and early trials in advanced mUC suggested that immunohistochemistry (IHC) staining for PD-L1 on paraffin-embedded tumor specimens would serve as a predictive biomarker , 6. BaseThe phase I study of atezolizumab in mUC was initially designed to include PD-L1-positive enriched cohorts, with a dose expansion cohort for all mUC patients regardless of PD-L1 status . The Venp = 0.41) [p = 0.038) [IMvigor 210 (Cohort 2) and KEYNOTE-045 explored the use of atezolizumab and pembrolizumab, respectively, in the post-platinum mUC population. IMvigor 210 enrolled patients with locally advanced or mUC refractory to cisplatin-based chemotherapy. The SP142 rabbit antibody IHC assay was used to assess PD-L1 status in archival specimens as discussed above; PD-L1 positivity was defined as ≥10% PD-L1 positive immune cells in the tumor microenvironment (defined as 3+ in the phase I study of atezolizumab). While the objective response rate (ORR) of the entire cohort was 15%, the ORR was 26% (26/100) in PD-L1 positive patients, compared with only 9% (19/210) in PD-L1 negative patients (Table  = 0.41) . Interes= 0.038) . It is sPD-L1 status did not predict for response in KEYNOTE-045 . More imWhile patients with a positive Ventana SP142 PD-L1 test were almost three times more likely to respond to atezolizumab than those who were PD-L1 negative in IMvigor 210, the absolute difference in ORR between PD-L1 positive versus PD-L1 negative patients was not as large as seen in the phase 1 atezolizumab study. Furthermore, Dako 22C3 PD-L1 expression did not appear to be associated with any treatment response for pembrolizumab in KEYNOTE-045. Assuming that atezolizumab and pembrolizumab have similar clinical activity, it is worth exploring why, PD-L1 “positive” patients are not the same, and why these biomarkers may not have sufficient discriminatory power to predict response to a CPI. As discussed above, these IHC assays used completely different antibodies, and moreover, the assay characteristics define PD-L1 positivity based on staining different cellular populations (tumor cells versus immune cells versus tumor and immune cells). Yet another discrepancy includes the percentage of positive cells for the threshold of positivity within the assay.Three additional CPIs have now been studied in and received accelerated FDA approval for the platinum-refractory population of mUC – nivolumab, durvalumab, and avelumab , 9, 10. For example, in the durvalumab trial, 51% of patients were defined as PD-L1 positive, compared with only 16% of patients in the avelumab trial , 10. ThiDue to a variety of factors, including renal or hearing impairment, poor performance status, and neuropathies, 30% to 50% of patients with chemotherapy-naïve advanced UC are not candidates for platinum-based chemotherapy . Cohort Based on the data presented above, in which patients with mUC may respond to PD-1/PD-L1 blockade even if their archival tumor lacks PD-L1 expression, we do not recommend PD-L1 clinical testing in UC patients. While in some studies PD-L1 positivity may identify patients more likely to have an objective response, and combined tumor/microenvironment testing may further enrich for responders, in others studies this biomarker has no discriminatory power, and given the conflicting results treatment with a CPI should not be withheld based on PD-L1 status in mUC. Prospective studies of PD-L1 as a predictive biomarker are needed, with consideration for contemporary/recent biopsies, tumor heterogeneity assessments, and expression in tumor vs normal immune cells.p = 0.002) [Nivolumab received FDA approval for the treatment of patients with advanced RCC refractory to first-line vascular endothelial growth factor (VEGF) inhibitors, demonstrating improved OS compared to everolimus  . Tumor P= 0.002) . Nivolum= 0.002) . Therefo= 0.002) , 17. TheAtezolizumab has also been investigated in mRCC. The expansion cohort of a phase Ia trial enrolled 70 patients with treatment refractory mRCC; all patients were treated with atezolizumab . Enrollmp = 0.095) and 1.03  for the atezolizumab alone vs sunitinib arm. Early phase trials combining immune checkpoint inhibitors with small molecule VEGF receptor inhibitors inpatients enrolled regardless of PD-L1 status have shown promising results with ORRs ranging from 67% to 100%. These studies demonstrate a signal for potentially improved overall response rates for patients treated with combination therapy. Several phase III studies are currently underway investigating checkpoint inhibitors in combination with VEGF-targeted therapy for patients with mRCC [Atezolizumab has also been investigated in the frontline setting in combination with bevacizumab, a VEGF inhibitor . Bevacizith mRCC –24.p < 0.0001) and ORR  in patients with intermediate-high risk disease [p = 0.98), whereas in PD-L1 positive population, there was a large difference in PFS between these two groups  [p = 0.0002) [Immunotherapy CPI combinations have proven effective in melanoma, and CheckMate-214 was the first in mRCC to use combination of CTLA-4 and PD-1 inhibitors. CHECKMATE 214 was a phase III trial which randomized 1040 patients with metastatic clear cell RCC to treatment with either combination nivolumab-ipilimumab or sunitinib. Co-primary endpoints included ORR, progression free survival (PFS), and OS, specifically in the IMDC intermediate or high risk population. Secondary endpoints included PFS and OS for the intention to treat population (including favorable risk). Nivolumab-ipilimumab improved both median OS (not reached (NR) vs 26.0 months, HR 0.63,  disease . PD-L1 p disease . In the  0.0003) . However 0.0002) . FurtherFurther correlative work has also emphasized the ineffectiveness of PD-L1 as a predictive biomarker in mRCC . PrimaryThe clinical trials for mUC and mRCC mentioned above highlight the numerous challenges facing PD-L1 as a biomarker. First, there is no standardized format to assess PD-L1 immunohistochemically. Several pharmaceutical companies have established partnerships with diagnostic companies to design companion PD-L1 assays. These assays are in a sense more different than they are similar. The only similarity between the assays is that they are all IHC tissue-based assays to detect membrane expression of PD-L1. Critical differences include which components are included for the purposes of scoring (tumor cells vs. immune cells), as well as staining thresholds to define PD-L1 positivity . Thus, iSecond, because these clinical trials have used different companion diagnostic assays, we are unable to indirectly compare the effectiveness of one drug in a “PD-L1 positive” population compared to another drug in a “PD-L1 positive” population. The first two concerns have been ameliorated in the diagnostic testing for non-small cell lung cancer (NSCLC). Ratcliffe et al. studied the concordance between three commercially available PD-L1 IHC assays for NSCLC patients including Ventana SP263 , Dako 22C3 (pembrolizumab), and Dako 28–8 (nivolumab) and found >90% concordance at several levels  of PD-L1 expression . The sepLastly, there are both biological and technical challenges which further complicate PD-L1 testing. This has been well reviewed in the literature , 33. BioThere are several other predictive biomarkers under examination for mUC and mRCC, including tumor mutational burden (TMB), mismatch repair status, gene expression profiles (GEP), TCGA (The Cancer Genome Atlas) profiling, tumor infiltration lymphocytes (TILs), and PD-L2.It has been observed that tumors with a higher mutational burden may have a better response to immunotherapy . Higher p = 0.0041) [p = 0.002) and median PFS (3.02 months vs. 1.87 months) [ERBB2 (HER2) and ERBB3 (HER3) mutations [In mUC, data from IMvigor 210 further suggest that high TMB may not be only prognostic of survival, but potentially predictive of response , 13. Pat months) . Recentlutations . These dNot surprisingly, patients with the highest mutational burden often harbor specific DNA damage response defects, such as microsatellite instability (MSI-H) or are mismatch repair deficient (dMMR) . Thus, cGene expression profiling has demonstrated utility as a predictive biomarker to chemotherapy and immunotherapy –47. The p < 0.0001). GEP score could correctly predict 70 of the 81 responders, compared to the PD-L1 CPS cutoff, which predicted only 41 of the 81 responders. While this is hypothesis generating, the utility of GEP still needs to be validated in large prospective clinical trials.Ayers et al. evaluated 680 tumor and immune related genes using a NanoString nCounter platform and found that the genes most able to separate responders from non-responders of PD-1 therapy were genes linked to interferon gamma signaling . The inip < 0.0001) and increased PD-L1 immune cell prevalence . However, the increase of PD-L1 expression in the basal subtype did not correlate with an objective response. Response to atezolizumab in this post-platinum population was much higher in the luminal cluster II subtype compared with all other subtypes. This relationship was again observed in the front line platinum-ineligible population with the luminal II samples having a higher response rate compared to the other molecular subtypes – however, the sample size was not sufficient to reach statistical significance [Consensus clustering based on gene expression data from TCGA has distinguished two major intrinsic subtypes of high grade bladder cancer – luminal and basal . Rosenbeificance . FurtherPD-L2 is another ligand capable of binding to PD-1. While its structure is quite similar to PD-L1, the exact function of PD-L2 is controversial . PD-L2 pIn RCC, while PD-L1 expression was observed in only 9% of cases in a series of 425 patients, PD-L2 expression was observed in 50% of cases . PD-L1 aCPIs are known to enhance the activity of the adaptive immune system by recruiting CD8 positive cytolytic T cells into the tumor microenvironment . CD8 posp = 0.0265) [High T cell infiltration and clonality in advanced UC has also correlated with treatment response . Increas 0.0265) . These tUltimately, a single biomarker may not adequately predict response to PD-1 immune checkpoint therapies, and a combination of factors may need to be taken into account to predict responses. Based on the level of CD8A and PD-L1 expression, Ock et al. described four distinct tumor microenvironment immune types (TMIT) . Tumors B. fragilis, the mice were able to recover an anticancer response to CTLA-4 blockade. This finding is similar to that reported by Sivan et al., which found that the best responses to PD-L1 blockade required specific Bacteroides - mice which were given oral Bifidobacterium improved tumor control to the same degree as those given an anti-PD-L1 agent [It is well established that the gut microbiota plays a critical role in both innate and adaptive immune homeostasis . Thus, iL1 agent . With reL1 agent . These sThe FDA approval of several checkpoint inhibitors for the treatment of mUC and mRCC represents a major landmark for patients and oncologists. However, it is critical that we choose the right patients, given that these drugs have the potential for not only physical toxicity but also financial toxicity, with costs of up to $20,000 per month . Selecti