While we observed evidence suggesting that L1 expression has a weak positive effect on A3B and A3G expression and A3B promoter activity, neither efficient siRNA-mediated knockdown nor overexpression of functional L1 elements affected catalytic activity of A3 proteins consistently. However, L1 knockdown diminished proliferation of a UC cell line exhibiting robust endogenous L1 expression, but had little impact on a cell line with low L1 expression levels. Our results indicate that UC cells express A3B at levels exceeding A3A levels by far, making A3B the prime candidate for causing genomic mutations. Our data provide evidence that L1 activation constitutes only a minor and negligible factor involved in induction or upregulation of endogenous A3 expression in UC.The most common mutational signature in urothelial carcinoma (UC), the most common type of urinary bladder cancer is assumed to be caused by the misdirected activity of APOBEC3 (A3) cytidine deaminases, especially A3A or A3B, which are known to normally restrict the propagation of exogenous viruses and endogenous retroelements such as LINE-1 (L1). The involvement of A3 proteins in urothelial carcinogenesis is unexpected because, to date, UC is thought to be caused by chemical carcinogens rather than viral activity. Therefore, we explored the relationship between A3 expression and L1 activity, which is generally upregulated in UC. We found that UC cell lines highly express A3B and in some cases A3G, but not A3A, and exhibit corresponding cytidine deamination activity  They re viruses . Importa viruses  and inhi viruses . A3 prot viruses . Recentl viruses . Many tunfection . Indeed,nfection . Additionfection . In contnfection  in urothnfection .A3 locus in primates , and non-LTR retrotransposons such as long interspersed nuclear element-1  and short interspersed nuclear elements (SINEs), have been the original targets of A3 activity and have provided the evolutionary pressure necessary for the continuous expansion of the primates . Mobilizprimates . For insprimates . Importaprimates   used in this study  were cultured in DMEM GlutaMax , supplemented with 10% fetal calf serum . BC61 ceTo minimize DNA contamination, total RNA was extracted by acid phenol extraction followed by column purification. Synthesis of complementary DNA was performed using the QuantiTect Reverse Transcription Kit , according to the manufacturer’s instructions, including an extra DNA removal step by DNase as recommended by the supplier. Briefly, 1 μg of total RNA was subjected to genomic DNA elimination reaction in a 14 μl volume, comprised of 2 μl of a 7x gDNA-Wipeout-Buffer, RNA, and water. The reaction mixture was incubated at 42°C for 2 min and then kept on ice. One microliter of the reaction mixture was taken and mixed with 14.38 μl of water in a new tube , which served as mock RT template for RT-qPCR assay. With the remaining 13 μl reaction mixture, cDNA synthesis was performed , 1 μl RT primer mix, 1 μl water, and 13 μl DNAse treated RNA) by incubating the RT reaction components for 30 min at 42°C and then inactivating the RT enzyme by boiling for 95°C for 3 min (according to the manufacturer’s instruction). In order to minimize potential inhibitory effects of the RT buffer system on qPCR, a 1:10 dilution of the cDNA product was generated prior to the PCR reaction quantifying FL-L1 transcripts. The final nucleic acids concentration of the RNA suspension (used for the Mock RT-qPCR) and the cDNA suspension were both adjusted to 4.64 ng/μl prior to qPCR.Supplementary Table 1. Ct values obtained from mock-RT experiments were found to be comparable with those obtained from blank control (water). The qPCR conditions were as follows: initial denaturation step at 95°C for 15 min, followed by 40 amplification cycles consisting of denaturation at 95°C for 15 s, annealing at 55°C for 20 s and extension at 72°C for 30 s, using the primers presented in the following method section and Supplementary Table 2.The efficiency of DNAse treatment was assessed by qPCR on RNA samples that were not incubated with any Reverse Transcriptase before. Data  obtained from cell lines 5637, VM-CUB1, 639-V, SD, BC61, RT4 are provided in Supplementary Table 2) were employed using the following PCR conditions: initial denaturation at 95°C for 15 min, followed by 40 amplification cycles consisting of denaturation at 95°C for 15 s, annealing at the appropriate temperature for 20 s and extension at 72°C for 30 s. Assay specificity was controlled for by using UCSC In-Silico PCR and melting curve profiles. All measurements were performed at least in duplicates; assay variance was <10%. Relative expression was calculated by the modified ΔΔCt method using TATA-box binding protein (TBP) mRNA levels as a reference gene transcript , UMUC3 , PC3 , 5637 (A3G), and VM-CUB1 (FL-L1), respectively.RT-qPCR was performed on a 7500 Fast Real-Time PCR System  or Roche LightCycler 96  using the QuantiTect SYBR Green PCR Kit  with cDNA (1:10 diluted) from DNAse-treated RNA samples  as described previously . To quananscript . To asceSupplementary Table 2). RT-qPCRs with these primers were performed as previously described ], washed in 1xPBS-T, and incubated overnight with the respective primary antibody at 4°C.Twenty micrograms of each protein lysate were boiled in 3x SDS sample buffer , loaded on 4–12% Bis/Tris gels (Invitrogen), subjected to SDS-PAGE, and electroblotted onto nitrocellulose membranes. After protein transfer, membranes were blocked for 2 h at room temperature in 10% non-fat milk powder in 1xPBS-T . Lysates were clarified by centrifugation for 20 min at 14,800 rpm at 4°C. For immunoblot analyses, samples were boiled at 95°C for 5 min in Roti load reducing buffer , separated on an SDS-PAGE gel followed by transfer to a PVDF membrane. Membranes were blocked in TBST containing 5% skimmed milk powder and probed with the respective primary antibody. A rabbit anti-A3G antibody  that is cross-reactive against A3A and A3B was used to detect A3B and A3G proteins (HEK293T cells were transfected with plasmids expressing hemagglutinin (HA)-tagged A3B or A3G using Lipofectamine LTX (Thermo Fisher Scientific). 5637, UMUC3 and VM-CUB1 UCCs were transfected with the pJM101/L1proteins . Mouse aIn vitro deamination reactions were performed as described , respectively, using a Wallac 1420 VICTOR2TM plate reader . Cell proliferation was measured by an EdU incorporation assay , according to the manufacturer’s instructions. The SA-β-galactosidase assay was performed as described .A3H haplotype I encodes an active cytosine deaminase described to reside in the nucleus and linked to cancer mutagenesis  the unpaired Student’s t-test with Graphpad Prism . Data were represented as the mean ± standard deviation (SD). Significant differences (p < 0.05) are marked by asterisks. Correlation coefficients and significance were calculated by non-parametric Spearman’s rank correlation (Spearman’s rho) and were corrected for multiple testing using the Bonferroni method.A3A, A3B, A3C, A3D, A3F, A3G, and A3H) in 19 UCCs and five independent primary cultures of normal urothelial cells (UPs) by RT-qPCR. A3A was only detectable in the urothelial cell culture UP86, the UCC BFTC905  and in activated PBMCs serving as positive control for A3A expression (data not shown). A3A expression was not detectable in all remaining UCCs and UPs. In contrast, A3B expression was barely detectable in all analyzed urothelial cell cultures, but was high in all analyzed UCCs except for the 647-V line harboring no detectable A3B mRNA at all . A3C, D, and F transcripts were detectable at moderate levels in all tested UPs and the majority of UCCs, but a few UCCs expressed A3C and A3D at extremely low or undetectable levels . A3G expression was robust in UPs but low in most UCCs, especially in those originating from muscle-invasive urothelial cancers . A3H transcripts were below detection level in the majority of muscle-invasive UCCs and in two UPs, while the remaining cell lines and UP cultures displayed moderate or robust transcript levels. In the papillary UCC BC61, A3H expression was exceptionally upregulated .As a first step to investigate if the expression of specific members of the A3 protein family of cytidine deaminases is a response to the upregulation of endogenous L1 expression, we profiled mRNA levels of all seven members of the A3 protein family . If L1 upregulation contributed to activation of A3 enzymes, mRNA expression of L1 and A3 would be expected to correlate. We therefore calculated whether any correlation between FL-L1 mRNA expression levels and mRNA levels of the various A3 genes could be detected in UCCs using Spearmen’s rho correlation , which was lost after Bonferroni correction for multiple testing.Quantification of FL-L1 transcripts revealed moderate L1 expression in all UPs and robust transcriptional L1 upregulation in almost all analyzed UCCs with the exception of the UCCs 253J and 5637 , a specific allele of A3H, has been implicated in breast and lung carcinogenesis . Approximately two thirds of the tested UCCs harbor the G105/K121 allele associated with the A3H-I haplotype in a homozygous (6/18) or heterozygous (7/18) manner. However, expression of A3H was not detectable in selected UCCs irrespective of the A3H genotype . This finding implies A3H as a possible but unlikely source of A3 mutations in several but not all UCCs.Because ogenesis , we addiFigure 1) which is based on the L1.3 reference sequence , elevated amounts of L1 ORF1p were detected in the UCC lines BFTC905, RT-112, VM-CUB1, and SD . More moderate but still detectable L1 ORF1p levels were present in J82, SW-I710, UMUC6, 253J, 5637, 639-V, 647-V, HT-1376, T24, and UMUC3 cells . The relatively modest amounts of L1 ORF1p in BC61 and RT4 cells do not seem consistent with the transcriptional L1 upregulation in these cells , but could be explained by the fact that a subset of FL-L1 elements transcribed in these cell lines does not encode intact L1 proteins. Of note, L1 ORF1p expression was not detectable in the normal urothelial cell culture UP239 and the TERT-NHUC immortalized normal urothelial cell line .Consistent with their previously assessed relative FL-L1 mRNA levels   or high (VM-CUB1 and SD) L1 mRNA and ORF1p expression levels, respectively. Expression of full-length L1Hs elements was downregulated in the four UCCs by transfecting either siRNA#1  or siRNAFigures 2B,D), 5637 , and SD UCCs. L1 mRNA and ORF1p expression was barely detectable in 639-V and remained unchanged after L1 siRNA treatment . Interestingly, RT-qPCR using primer combinations specific for the 5′end of the L1 5′-UTR  revealed that overall FL-L1 transcript levels were reduced only by at most 50% . Since the partial mRNA knockdown observed in these cell lines nevertheless resulted in a highly efficient L1 ORF1p depletion , this observation indicates that the siRNAs target most if not all intact protein-coding L1 elements harboring an intact ORF1 efficiently. In contrast, non-functional FL-L1 elements with mutations in ORF1 that differ from the L1.3 reference sequence were targeted less efficiently.Transfection of each of the two L1-specific siRNAs efficiently knocked down L1 ORF1p expression in VM-CUB1 . A3H expression was only detectable in SD cells among these four UCCs  and not altered by the knockdown of L1 expression in the examined UCCs , whereas L1 knockdown exerted variable effects on the expression of A3B to A3G in the tested UCCs.Following transfection with L1-specific siRNAs, Figures 2E–H), and was associated with a drop of A3B expression by 25% (5637) to 55% (VM-CUB1). A3G expression was diminished by 14, 47 and 36% in VM-CUB1, 5637, and SD cells, respectively, and remained unaffected in 639-V cells. However, only minor, negligible effects on A3C, A3D and A3F transcript levels were observed in VM-CUB1, 5637, and SD cells. In 639-V cells, A3C expression was unchanged, whereas A3D and A3F transcript levels were increased by 56% and 38%, respectively.Transfection of L1_siRNA#1 diminished overall FL-L1 transcript levels in the analyzed UCC lines by 8% (639-V) to 42% (5637) . The knockdown of functional L1 elements was associated with an increase of A3F transcript levels in 639-V cells by 44%, but had no meaningful effect on A3F expression in the remaining cell lines. L1_siRNA#2-mediated knockdown of ORF1p-expressing endogenous L1 elements was associated with a strong increase of A3G transcript levels in three UCCs ranging from 50% in the SD line to fourfold in VM-CUB1 cells but had no consequences for A3F expression in 5637 cells, and no noteworthy effect on 5637 cells .Following transfection with L1_siRNA#2, A3C, D, F, and G transcript levels in three out of four analyzed UCCs, while L1_siRNA#1-mediated knockdown was associated with a comparable major increase in A3C, A3D, and A3F transcript levels only in 639-V cells and a minor increase in VM-CUB1 cells. A decrease of A3 transcript levels was observed only after L1_siRNA#1-mediated knockdown in 5637 and SD UCCs and was minor, ranging from 10 to 50% among the A3 genes. Taken together, no consistent correlation between the knockdown of functional endogenous L1 elements and expression of A3 genes could be observed, but knockdown of intact L1s was more often associated with increases rather than decreases of A3 gene expression. In sum, these findings are not consistent with the hypothesis that expression of A3 genes is upregulated in UCCs as a consequence of functional L1s activation. Furthermore, it is currently unclear why the effects of functional L1 knockdown on A3 gene expression differed between L1_siRNA#1- and L1_siRNA#2-mediated knockdowns.In summary, knockdown of functional L1 elements with L1_siRNA#2 resulted in a general increase of A3B and A3G transcript levels in UCC lines, we next transfected either of the L1 expression plasmids pJM101/L1RP and pAJG101/L1RP  into the cell lines VM-CUB1 and 5637, which are characterized by relatively high and low endogenous L1 transcript levels, respectively . Following transfection, FL-L1 RNA levels increased 2.5- to 3-fold in VM-CUB1 cells and by 23% to 46% in 5637 cells, as demonstrated by the L1_5′-UTR-specific RT-qPCR assay . To analyze whether ectopic L1 overexpression affects endogenous A3 expression, we quantified A3A, A3B, and A3G mRNA levels in the transfected UCCs. We found that expression of A3B and A3G was slightly increased in VM-CUB1 UCC after transfection with pAJG101/L1RP, but only A3B expression changes reached the level of significance . In 5637 UCC, A3B and A3G expression was not altered significantly. Of note, A3A expression remained undetectable in both cell lines (data not shown).To investigate the consequences of ectopic overexpression of retrotransposition-competent L1 elements on endogenous A3B promoter activity, we co-transfected VM-CUB1 and 5637 cells with either of the two A3B promoter luciferase reporter constructs pA3B-120 or pA3B-1200  together with the L1 expression plasmids pJM101/L1RP and pAJG101/L1RP or the empty pCEP4 vector as negative control. Activity of the A3B promoter encoded by pA3B-1200 increased by ∼36% – 42% and 64% – 80%, respectively, after co-transfection of the luciferase reporter construct with pJM101/L1RP or pAJG101/L1RP into VM-CUB1 and 5637 cells relative to the effect of the control vector . This increase in promoter activity is consistent with the increase in endogenous A3B mRNA levels by ∼27% after transfection of plasmid pAJG101/L1RP in VM-CUB1 cells . Taken together, induction of L1 activity has only minor effects on A3B expression as well as promoter activity and significant effects are limited to VM-CUB1 UCC with high L1 expression.To study whether ectopic expression of functional L1 elements can induce A3 mRNA expression levels. We chose specifically (i) the UCC 5637 exhibiting major transcriptional upregulation of A3B and A3G, (ii) UMUC3 showing robust transcript levels of most A3 genes (with A3B expression being the highest), and (iii) VM-CUB1 with very low transcript levels of most A3 family members except for A3B . Distinguishing A3B from A3G by standard immunoblotting techniques is challenging due to the high amino-acid sequence homology between both proteins and their comparable molecular masses . Similarly, A3G expression levels dropped after transfection with A3G-specific siRNA to below 10% in all three UCCs . Simultaneous treatment with A3B- and A3G-specific siRNAs resulted in diminished A3B and A3G mRNA levels in all examined UCCs comparable to those observed after single siRNA treatment .We investigated expression and deaminase activity of A3 enzymes suspected to cause mutations during bladder carcinogenesis in selected UCCs with different r masses . TherefoFigure 4B) and their mRNA expression pattern in 5637 cells . The intensity of the 45 kDa band was slightly diminished after transfection of the cells with A3B-specific siRNA, but the band disappeared almost entirely after transfection with A3G-specific siRNA or a combination of both siRNAs . Expression of the 45-kDa protein was not affected by transfection of control siRNA.Immunoblot analyses of cell extracts isolated from the differently transfected 5637 cells with an anti-A3G antibody (ApoC17)  reportedA3G-specific siRNA strongly depleted the amounts of the 45-kDa protein in both UMUC3 and VM-CUB1 cells, whereas the A3B-specific siRNA had only a minor effect on the 45-kDa protein levels in UMUC3 cells and did not affect its expression at all in VM-CUB1 cells . These findings suggest that the majority of the 45-kDa proteins detected with the anti-A3G antibody represents A3G. A note of caution on the detection of endogenous A3B: Since a more specific antibody capable of selectively detecting endogenous A3B enzyme in UC cell lines is currently not available, we cannot formally exclude that A3B protein is not depleted by A3B-specific siRNA, despite downregulation of A3B mRNA. Of note, A3A expression was not considered because there was no evidence for the presence of A3A mRNA in the analyzed UCC lines , and consistently, immunoblot analysis with anti-A3A antibodies did not provide any evidence for the presence of A3A proteins (data not shown).Transfection of A3B- and A3G-siRNAs as controls . To measure deaminase activity, we applied a qualitative PCR-based in vitro DNA deamination assay to identify C→U conversion in an 80-nt single-stranded DNA substrate harboring the isozyme-specific motif TTCA or CCCA, specifically recognized by A3B or A3G, respectively . Of note, whereas the substrate TTCA (YTCA) was reported as a statistically favorable target for A3A over A3B in cancer tissues . A3G deaminase activity was robust in 5637 cells, but lower in UMUC3 cells and barely detectable in VM-CUB1 cells. Importantly, as expected from the CCCA substrate specificity of A3G . Using the CCCA substrate, A3B downregulation slightly reduced product formation, whereas simultaneous knockdown of A3B and A3G abolished detectable deaminase activity. Conversely, using the TTCA substrate, A3B knockdown, but not A3G knockdown resulted in complete loss of detectable deaminase activity . Taken together, these data confirm that A3G favors the CCCA sequence motif and A3B prefers the TTCA motif, but also indicate that A3B might mutate CCCA sequences on ssDNA substrates with a low frequency. More importantly, combining the deamination assay data  with the A3 expression data presented in Figures 1 and 4A leads to the conclusion that in vitro A3B is the predominantly active member of the A3 family in the tested UCCs, whereas A3G-specific deaminase activity is comparably low.In order to investigate if A3B and/or A3G are enzymatically active in UCC lines, DNA deamination activity assays were performed using cells lysates from the different UCCs transfected with ectively . Catalyt tissues , TTC wasc assays . The assy of A3G , siRNA-mRP in UCCs. Lysates from transfected and untransfected UCCs were then either treated with RNase A to eliminate potential inhibitory RNA molecules, or left untreated, before they were assayed for A3B- or A3G-specific deaminase activity .Next, we investigated the effect of the expression of functional L1 elements on the deamination activity of A3 proteins by ectopically overexpressing transfected functional L1 elements encoded by pAJG101/L1activity . EctopicFigure 5A). The number of viable 5637 UCCs was less severely depleted to 68% and 46% after treatment with L1_siRNA#1 and L1_siRNA#2, respectively . Caspase 3/7 activity, measured as an indicator of apoptosis, decreased to 43% and 8% in VM-CUB1 cells after L1_siRNA#1 and L1_siRNA#2 treatment, respectively . In 5637 UCCs, caspase activity was increased after treatment with L1_siRNA#1, but not with L1_siRNA#2 . According to flow cytometry data, the fraction of VM-CUB1 cells in G2/M phase was increased by roughly 8% in cells treated with either L1 siRNA . In VM-CUB1 cells treated with L1_siRNA#2, the fraction of subG1 cells also decreased. S-phase fraction was unchanged  and accordingly, incorporation of EdU was only slightly diminished, especially following L1_siRNA#1 treatment . Only minor changes in cell cycle distribution and, accordingly, EdU incorporation were seen in 5637 UCCs . Thus, the decrease in viable cells following L1 knockdown does not reflect apoptosis.While our results do not indicate that L1 expression affects A3B (or other A3) expression consistently, L1 silencing by siRNA impaired cell proliferation. In VM-CUB1 cells expressing L1 more strongly, the number of viable cells decreased to 47% and 7% after L1 knockdown using L1_siRNA#1 and L1_siRNA#2, respectively, compared to control siRNA-transfected cells . Moreover, some VM-CUB1 cells showed morphological changes typical of senescent cells and stained positive for senescence-associated (SA)-β-galactosidase after treatment with either L1 siRNA, but very rarely after treatment with control siRNA . Unexpectedly, in 5637 cells, clonogenicity reproducibly increased after L1 knockdown using L1_siRNA#2, whereas L1_siRNA#1 exerted no significant effects on clonogenicity . Accordingly, no indications of senescence were detected in 5637 cells after treatment with L1 siRNAs or control siRNA (data not shown). Thus, L1 knockdown affected VM-CUB1 UCCs with high L1 expression levels more severely than 5637 cells with low L1 expression.In keeping with its effects on cell viability in short-term experiments, FL-L1 knockdown strongly diminished the clonogenicity of VM-CUB1 cells , whereas A3A was essentially undetectable in almost all (16/17) analyzed UCCs. Obviously, expression and enzymatic activity of A3 family members may vary during urothelial carcinogenesis and may not be fully reflected in the pattern seen in UCCs. However, A3 mutational activity was shown to be involved in both early and late mutation events that occurred during urothelial carcinogenesis arguing rather for continuous A3 mutational activity   in UCCs 5637, UMUC3 and VM-CUB1, this is very likely to be the case for A3B too. Moreover, cytidine deamination assays coupled with knockdown experiments convincingly revealed the expected substrate-specific activity levels for both A3B and A3G. Of note, the general DNA motif reported to be recognized by APOBEC proteins to introduce somatic mutations in cancer is “TC”  . Further(NMIBCs) , or may CC lines . However is “TC”  . In addition, endogenous FL-L1 and A3 expression levels did not correlate with each other across the tested panel of cell lines. Here, future investigations are required to unambiguously elucidate any role of L1 expression and/or retrotransposition activity in the activation of A3 proteins in tumor cells.To address the general question of what triggers A3 activation in urothelial cancer cells, we pursued the hypothesis that A3 activation may be elicited by endogenous retroelement activity rather than the presence of exogenous viruses. Expression of functional endogenous L1 elements seems a plausible cause for A3 activation, because in urothelial cancer cells, L1 promoter sequences are frequently hypomethylated, and FL-L1 expression is increased even more than in other cancer types . In comp in UCCs . HoweverORFeus-Hs . This could be explained by the fact that the L1 5′UTR-specific primers used for the RT-qPCR assay also detect transcripts from FL-L1 elements with non-functional ORF1 sequences, which are not or less efficiently targeted by the siRNAs.Knocking down the expression of endogenous FL-L1 elements with two different siRNAs targeting the intact ORF1 coding region resulted in the efficient depletion of endogenous L1 ORF1p. This observation indicates that the majority of transcripts from active L1Hs elements harboring intact ORF1 sequences were removed from the tested cell lines. However, these siRNAs did not decrease the overall FL-L1 transcript levels as measured by RT-qPCR to the same degree , but had less effect on UCCs exhibiting lower L1 expression levels (such as 5637 cells). These results are in good agreement with previous reports that L1 knockdown causes a loss of proliferative ability in tumor cells independent from apoptosis , ultimatFinally, if there is no evidence that A3 activation in UC is elicited by either exogenous virus infection or endogenous L1 retrotransposon activation, what causes it? Several alternative hypotheses deserve investigation. For instance, A3B is induced by several cytokines in normal liver  and throMost advanced muscle-invasive UCs contain mutations inactivating p53, which are rare in non-muscle invasive UC . The p53Moreover, recent results suggest that A3B may target ssDNA accumulating as a result of replication stress  or transThus, we conclude that several factors may cooperate to activate A3 in urothelial carcinogenesis. This work largely excludes the pervasive activation of L1 retroelements as one potential factor. Moreover, in line with some, but not other previous reports, detailed analysis of UCCs suggests A3B rather than A3A as the predominantly active enzyme.The major limitations of our study concern the detection of A3 proteins and the high L1 copy number in the human genome. With respect to A3 proteins, we could not obtain antibodies that are sufficiently sensitive and specific to detect endogenous expression of each isoenzyme. A reliable array of such antibodies would be very helpful to characterize the expression pattern of A3s in UC cell lines and tissues more precisely. Also the highly repetitive character of endogenous L1 retroelements causes major complications for our studies. To fully understand the impact of L1 activity in UC cells and tissues, a complete characterization of the repertoire of transcripts from retrotransposition-competent L1 elements and, ideally, from non-functional L1 elements will be required. Third generation techniques currently under development will hopefully enable this investigation.RP, and participated in drafting the manuscript. UK and AK performed some experiments. WG, WS, AAJV, DH, and CM analyzed the data. WS, GS, and CM contributed reagents and tools. WG, WS, AAJV, GS, and CM wrote the paper.WG, WS, AAJV, and CM conceived and designed the experiments. AV and WG performed most of the experiments. GS performed immunoblot analyses, generated the L1 reporter plasmid pAJG101/L1The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


As a new carbon-based nanomaterial, graphene has exhibited unique advantages in significantly improving the combination properties of traditional polymer hydrogels. The specific properties of graphene, such as high electrical conductivity, high thermal conductivity and excellent mechanical properties, have made graphene not only a gelator to self-assemble into the graphene-based hydrogels (GBH) with extraordinary electromechanical performance, but also a filler to blend with small molecules and macromolecules for the preparation of multifunctional GBH. It fully exploits the practical applications of traditional hydrogels. This review summarizes the preparation methods, properties, and the applications of GBH. Further developments and challenges of GBH are also prospected. Graphene is a new nanomaterial with strict two-dimensional layers structure Geim, . With exThis review aims to demystify the preparation methods, performance aspects, and the applications of GBH. Further developments and challenges of GBH are also prospected.Self-assembly method means that the basic structure of graphene oxide (GO) is spontaneously translated into a stable 3D graphene structure under the interaction of non-covalent π-π bond. It is considered an effective approach for fabricating GBH. Various self-assembly methods such as hydrothermal process  was greater several orders than the traditional hydrogels. The 3D multifunctional GBH, which was self-assembled by combining DNA and GO sheets, possessed good mechanical property, big dye-adsorption capacity, and excellent self-cure capability  / poly (PVA) hydrogel, which was made by SG and PVA mixed solution, showed good mechanical property and intelligent adsorption property for cationic dyes compared to ordinary pure PVA hydrogel  composite hydrogel was synthesized via cross-linking reaction of PAA at low temperature , poly (PDMAA), etc.), GBH are attracted much attention in tissue engineering. Jing et al. studied the mussel-inspired GO/CS composite hydrogel, which was prepared by incorporating protein polydopamine (PDA)  after 42 h. However, only 51% vitamin B12 molecules can be released in the acidic medium (pH = 1.7) at the same time. Li et al. successfully fabricated an NIR-responsive GBH as an active cell scaffold by pNIPAAm derivative solution and arginine-glycine-aspartic acid (RGD). The NIR-responsive GBH could efficiently capture cells, but also could release the cells on the stimulus of NIR light  (pNIPAAm), etc.) has shown attractive prospects in drug controlled release system on account of the huge specific area of GO or graphene. The graphene/Ag (mass ratio of 1:5) composite hydrogel, which was made through the cross-linking reaction of graphene with acrylic acid and methylene bisacrylamide, exhibited good biocompatibility, and high swelling ratio , good cycling performance and high rate capability  hydrogel exhibited excellent mechanical property, high Photo-Fenton activity and big adsorption capacity. The hydrogel was achieved a 90% degradation of Rhodamine B  and a 72.7% degradation of chemical oxygen demand  in wastewater for 1 h under visible light  and L-ascorbic acid (LA). The hydrogel possessed high selectivity to oil-water separating by pre-soaking (Hu et al., The specific properties of graphene, such as high electrical conductivity, high thermal conductivity, and excellent mechanical properties, have made graphene not only a gelator to self-assemble into the GBH with extraordinary electromechanical performance, but also a filler to blend with small molecules and macromolecules for the preparation of multifunctional GBH. It fully exploits the practical applications of traditional hydrogels. In view of the developing trend of hydrogel in recent years, theoretical researches are relatively high. Researchers are very interested in the application prospect of hydrogel for biomedical, tissue engineering, supercapacitor, water treatment, dye absorption, catalyst carrier, and intelligent response for microfluidic system. However compared to the practical applications, the actual operation research is very weak. The formation mechanism of graphene/GO hydrogel in the aqueous solution and the influencing function are not apparent yet. Design and fabrication of GBH with new structures and functions will be a significant challenge in the future.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The lubrication performance of graphene used as additive in 4-n-pentyl-4′-cyanobiphyl liquid crystal (5CB) for steel/steel contacts was studied on a ball-on-plate tribotester. The friction test results show that when the graphene content in the 5CB was 0.15 wt.%, and the lubricant and friction pairs were heated to 44–46 °C before friction tests, the lubrication performance of the 5CB was most improved. Compared with pure 5CB, 5CB+0.15 wt.% graphene suspension reduced the friction coefficient and wear scar diameter by up to 70.6% and 41.3%, respectively. The lubrication mechanisms have been tentatively proposed according to the test results. We speculate that the excellent lubrication performance of graphene/5CB suspensions may be attributed to the low shear resistance adsorption layer formed by graphene and 5CB molecules on the sliding surfaces. As the protective layer, it not only prevents direct contact between the rough sliding surfaces but also is easy to slide. Friction and wear between mechanical parts widely exist in mechanical systems. Friction consumes a lot of energy, and wear will lead to premature failure of parts . In ordeLiquid crystals exhibit mesophase between liquid and crystal phase, which have both crystal anisotropy and liquid fluidity. Liquid crystals can be classified in two categories: (1) monomer liquid crystals, irrespective of whether they are able to polymerize, and (2) polymer liquid crystals . 5CB belRecently, nanoparticles have been introduced into industry as a new lubricant additive ,8,9. The2 stripping of graphite. The antifriction and antiwear properties of the multilayer graphene as an additive of polyalphaolefin-2 (PAO2) oil were studied by using four-ball test method. They pointed out that the PAO2 with 0.05 wt.% multilayer graphene showed the best lubrication performance. By analyzing the worn surfaces, it was found that the excellent lubrication performance of the multilayer graphene additives can be attributed to its small sheet diameter and layered structure, which make the multilayer graphene easily enter the friction interfaces and form a protective layer, thus preventing the friction surfaces from directly contacting.Multilayer graphene can be well dispersed in some specific lubricants, and shows its antifriction and antiwear properties. For instance, Fan et al.  reportedBesides multilayer graphene, few-layer graphene also has been reported as lubricant additive. For instance, Eswaraiah et al.  evaluateTo exert the lubricating properties of graphene in a lubricant, the dispersibility of graphene in the lubricant must be considered. The dispersity of graphene in most lubricants is very poor . ChemicaIn addition to using chemical modification to improve the dispersion of graphene, it has also been reported to improve dispersion by changing the shape of graphene to prevent adsorption and agglomeration between sheets. Inspired by the fact that crumpled paper sheets are not easily adhered to each other, Dou et al.  used capThe dispersion effect of graphene in base oil will directly affect its lubrication performance. However, using chemical modification to improve the dispersion of graphene will affect its chemical properties. At the same time, changing the morphology of graphene through external force to improve its dispersion will destroy the physical structure of graphene.In order to achieve the good dispersion of graphene in lubricating oil without changing its physical and chemical properties, we chose 5CB as the basic lubricant in this study. Since the rod-shaped molecules of 5CB tend to be arranged along the surface of the nanostructure that graphene provides ,31,32, tIn this study, the adsorption between the 5CB molecules and graphene was fully utilized. Considering the fact that the higher the temperature, the more intense the thermal movement of the liquid molecules and the more fierce the Brownian movement, we have achieved good dispersion of graphene in 5CB by heating the graphene/5CB suspension. The lubrication properties of the suspension were tested on a tribometer with a ball-on-plate configuration. Transmission electron microscopy (TEM), optical microscopy, scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS) and Raman spectroscopy were used to characterize graphene and study the wear surfaces. Based on the above tests and analysis results, we deduced the possible antifriction and antiwear mechanism of graphene as additive in 5CB.The 5CB was supplied by the Shijiazhuang Huarui Scientific and Technological Co. . This liquid crystal exhibits a nematic phase at 22–35 °C . The graThe preparation procedures of Graphene/5CB suspension are as follows: Firstly, 5 mg of graphene was added in 10 ml of anhydrous ethanol with ultrasonic dispersion for 1 h to ensure that graphene was uniformly dispersed the solution. Secondly, 10 g of 5CB was added to graphene/anhydrous ethanol solution in 40–50 °C water bath with ultrasonication for 1 h to obtain a homogeneous suspension. After that, the suspension was put in vacuum at 50 °C for 12 h to completely evaporate the anhydrous ethanol. Finally the suspension of 0.05 wt.% graphene in 5CB (5CB+0.05 wt.% G suspension) was obtained. According to the above method, 5CB+0.15 wt.% G suspension, 5CB+0.25 wt.% G suspension and 5CB+0.35 wt.% G suspension were prepared respectively. No additional dispersion or surfactant was used to improve the dispersion of graphene in 5CB.The friction tests were carried out on a high temperature tribometer  with a ball-on-plate configuration, in indoor air conditions . The tribometer is shown in −1. The normal load applied to the steel ball was 3 N. The duration for each sliding friction test was 1 h.Before conducting the friction tests, the steel balls and steel plates were ultrasonically washed with acetone, washed with anhydrous ethanol and dried. During the tests, the steel plate rotated at a speed of 200 rpm and the friction track radius was 18 mm, which was equivalent to sliding at a speed of 0.38 msIt is well known that the concentration of additives in basic lubricants plays an important role in reducing friction and wear . In ordeFor the influence of temperature on lubrication performance of the graphene/5CB suspension, three different temperature conditions were investigated. The first temperature condition: the steel plate was heated to 44–46 °C while the graphene/5CB suspension lubricant was stirred at 44–46 °C for 120 s, to achieve good dispersion of the graphene in the 5CB, then the graphene/5CB suspension lubricant was applied to the plate and immediately stopping heating at the beginning of the friction test. The heating was stopped at the beginning of the friction test to make the 5CB return to the nematic phase, thus restoring the lubrication properties of the 5CB as a liquid crystal lubricant. After the friction test started, the shear force caused the graphene to be uniformly distributed on the surface of the friction region. Therefore, it is not necessary to rely on continuous heating to maintain a good dispersion state of graphene. The second temperature condition was at room temperature (24–26 °C). The third temperature condition was at 44–46 °C.Before the friction tests, the lubricant was dripped onto the steel plate with a dropper. Then the wear track surface was fully covered with a lubricating layer formed by the lubricant droplets. Each test condition was repeated at least three times, and the final results of friction coefficient and wear scar diameter were the average values.Before analyzing the friction surfaces, the steel balls and steel plates were washed with anhydrous ethanol to remove contaminants. An optical microscope  was used to observe wear profile and measure diameter of wear scar. Scanning electron microscopy  was used to further analyze the surface textures of steel ball wear scar. Energy dispersive spectroscopy (EDS) was used to analyze the content of various elements on the wear surfaces. Raman microscope  was used to study Raman spectra of the graphene and wear scar. The transmission electron microscope  was used to analyze the structure and morphology of graphene before and after the friction tests.In order to achieve good dispersion of graphene in the 5CB and make sure the 5CB exhibits a nematic phase during the rubbing process, the friction tests were performed in the first temperature condition. The friction coefficient and the wear scar diameter at different graphene concentrations were studied.The relationship between the concentration of graphene in 5CB and the average friction coefficient is shown in The relationship between the friction coefficient and the time is shown in When the addition amount of graphene is less than 0.15 wt.%, the coefficient of friction of the lubricant is relatively stable, this may be attributed to the absence of excess graphene on the friction surfaces and all graphene is adsorbed on the friction surfaces to fill surface pits or grooves to make the friction surfaces smooth ,37. WhenGraphene, as a sheet-like nano material, is easy to enter the friction region . In addiWhen addition amount of graphene is less than 0.15 wt.%, the average diameter of steel ball wear scar decreases with the increase of concentration. This phenomenon further proves that when the concentration is less than 0.15 wt.%, there is not enough graphene to form an antifriction and antiwear protective layer covering the wear surface . The conHowever, when the graphene concentration is higher than 0.15 wt.%, the average wear scar diameter starts to increase. This phenomenon further proves that graphene tends to agglomerate into large bodies at higher concentrations, and it is not easy to form a continuous and uniform antifriction layer on the friction region ,42. In aThe friction coefficient and wear scar diameter of the 5CB+0.15 wt.% G suspension were tested under the three temperature conditions set. The variation of the friction coefficient with the friction test time is shown in The lubrication mechanism of 5CB+0.15 wt.% G suspension were emphatically analyzed with 5CB as the benchmark. All the test data and images analyzed were obtained under the first temperature condition.To determine how graphene influences lubrication performance, the topography of wear surfaces after tests were investigated. As shown in In order to prove that graphene exists on the friction contact surfaces and plays an important role in the friction tests process, the wear surfaces of the balls lubricated by 5CB and 5CB+0.15 wt.% G suspension were analyzed by SEM and EDS, respectively. The test results are shown in Obviously, the friction surface of the ball for 5CB had irregular and deep furrows, and the severe scratch was attributed to the relatively poor antiwear property of 5CB. The corresponding EDS analysis showed that the content of C element on the worn surface was only 1.44 wt.%, which may be due to the basal component of the steel ball and carbonizations of the 5CB, as shown in By contrast, some very regular and shallow furrows appeared on the wear surface of the ball for 5CB+0.15 wt.% G suspension. Many literatures show that friction coefficient is closely related to surface morphology ,48. In a−1), D peak (1350 cm−1) and 2D peak (2712 cm−1) which are the typical Raman signals of graphene [Because Raman spectroscopy is a most suitable characterization technique for graphene materials , the Ramgraphene . This di2D/IG increased after the friction tests, which indicates that the thickness of the graphene is reduced [D/IG ratio to 0.89, as compared with 0.75 in the pristine graphene powder. This indicates that the defect density of graphene increases after friction tests [Comparing  reduced ,50,51. Ion tests ,53. ThisGraphene protects the friction surfaces and reduces the friction coefficient by forming a protective layer on the friction surfaces during the friction process. Graphene has strong mechanical strength, so the protective layer can greatly protect the friction surfaces from abrasion and corrosion. In addition, graphene sheets with weak van der Waals force are easy to slide between each other, which helps to reduce shear resistance during friction ,24. In oIn this work, the lubrication performances of graphene as additive of 5CB for steel/steel contacts were studied by using a tribometer with a ball-on-plate configuration. Based on the analysis of the test results, the conclusions are as follows.The concentration of graphene with the best antifriction and antiwear properties in the graphene/5CB suspension was 0.15 wt.%. Compared with pure 5CB, 5CB+0.15 wt.% G suspension reduced the friction coefficient and wear scar diameter by up to 70.6% and 41.3%, respectively. This outstanding lubricity is due to the low shear resistance and chemically stable protective layer formed by graphene and 5CB on the friction surfaces. Graphene with a sheet structure easily enters the friction interfaces and forms a protective layer on the friction surfaces, which makes the friction surfaces inert and prevents the two friction surfaces from direct contact, thereby reducing wear. In addition, graphene sheets are easy to slide, which is beneficial to reducing frictional resistance. Therefore, the addition of appropriate amount of graphene into 5CB can significantly reduce the friction and wear.


Tumor expression of immune co-inhibitory ligands, such as PD-L1 and Galectin-9, have potential prognostic value in Hepatocellular Carcinoma (HCC). Circulating levels of these molecules, however, have hardly been studied. This study aims to assess the prognostic significance of circulating PD-L1 and circulating Galectin-9 in patients with resected HCC, and to compare their prognostic significance to the intra-tumoral expression of these same molecules. Archived tissues and stored peripheral blood samples from 81 patients who underwent HCC resection or liver transplantation, with curative intent, were used. Immunohistochemistry was performed to determine intra-tumoral expression of PD-L1 and Galectin-9, while ELISA was used to quantify their respective circulating levels. High circulating PD-L1  and high circulating Galectin-9  levels were both associated with improved HCC-specific survival. Surprisingly, there was no correlation between circulating levels of PD-L1 and Galectin-9 and their intra-tumoral expression levels. In fact, circulating levels of PD-L1 and Galectin-9 were predictive of HCC-specific survival independently of intra-tumoral levels and baseline clinicopathologic characteristics. Combined analysis of circulating levels and intra-tumoral expression of PD-L1  and Galectin-9  resulted in more confident prediction of survival. In conclusion, circulating PD-L1 and Galectin-9 levels prognostically differentiate resected HCC patients, independently of their intra-tumoral expression. Combining circulating and intra-tumoral expression levels of PD-L1 or Galectin-9 further improves the prognostic values of these immune biomarkers. Only 20% of patients with HCC are diagnosed early enough to be candidates for curative treatments such as resection, local ablation or liver transplantation2. Once advanced disease is diagnosed, HCC is incurable and overall survival can be only modestly extended with sorafenib3.Worldwide over half a million people die from HCC4 have brought forth new therapeutic strategies against cancer5. Currently, the most successful immunotherapeutic strategies are those that are designed to overcome immune resistance mechanisms by using antibodies that abrogate co-inhibitory receptor-ligand interactions, the so-called negative immune checkpoint inhibitors6. Recent approval of anti-CTLA-4 and anti-PD-1 antibodies for the treatment of advanced melanoma, non-small cell lung cancer, renal cell carcinoma and bladder cancer has made it clear that immunotherapy is the new wave of anti-cancer treatments. Immunotherapy clinical trials are now ongoing in many cancers, including HCC7, and it is likely that these therapies will be approved in the future in other cancer types as well. In view of the high costs and occasional severe toxicity of these novel therapies, immune specific biomarkers that can predict which patients will benefit are urgently needed. Two such recent promising immune biomarkers in HCC are PD-L1 and Galectin-9 .Developments in our understanding of tumor immunologyin vitro disruption of this interaction restores the function of tumor-derived effector T-cells8. PD-L1 is known to be expressed by HCC cells13. We have previously shown that tumor PD-L1 protein expression is a promising prognostic biomarker in HCC14. In addition, tumor PD-L1 protein expression has shown promise as a predictive biomarker to identify cancer patients that respond to anti-PD1 immunotherapy16.PD-L1 is a ligand that binds PD-1, a co-inhibitory receptor expressed on activated T cells. Binding of the ligand PD-L1 to its receptor PD-1 transduces a negative signal into T-cells inhibiting their activation. In HCC the PD-L1/PD-1 interaction impairs effector T-cell function and 17. Gal-9 causes T-cell inhibition and apoptosis through its binding to the co-inhibitory receptor TIM-3 and blockade of the interaction between Gal-9 and TIM-3 reinvigorates ex vivo responses of T-cells of HCC and melanoma patients to tumor antigens18. Humanized antagonistic antibodies against TIM-3 are currently in preclinical development19. In addition, binding of Gal-9 to CD44 enhances the differentiation of immunosuppressive T regulatory cells20. A direct anti-metastatic role for Gal-9 has also been described22. We and others have demonstrated that Gal-9 is also expressed by HCC tumor cells and that Gal-9 protein expression is a potential prognostic biomarker in HCC24.Gal-9 is a glycan-binding protein and an important modulator of T-cell function25, lung cancer26, gastric cancer27, pancreatic cancer28 and HCC29. Elevated levels of circulating Gal-9 have been observed in metastatic colon cancer30 and in benign inflammatory liver diseases such as chronic hepatitis-C and active chronic hepatitis-B infection32. However, no study has investigated the relationship of circulating Gal-9 to cancer survival before.However, in addition to cell-bound expression, soluble forms of PD-L1 and Gal-9 exist in the circulation. These circulating forms of PD-L1 and Gal-9 have been poorly studied in cancer patients. Circulating levels of PD-L1 have been examined in renal-cell cancerOne may hypothesize that circulating forms of PD-L1 or Gal-9 would correlate with their tumor tissue expression status, since release from tumor cells, or from the tumor microenvironment, may be the source of these molecules in the circulation. In that case, circulating PD-L1 and Gal-9 may act as preferred biomarkers given the easier accessibility of peripheral blood when compared to tumor tissue. On the other hand, these physiologic molecules may have functions in the circulation that are independent of the immune interactions ongoing in the tumor microenvironment. Thus, the potential for the circulating levels of PD-L1 and Gal-9 to act as independent prognostic biomarkers, or independent predictors of immunotherapy treatment efficacy, exists. Thus, the aims of our study were to examine how circulating levels of PD-L1 and Gal-9 compare to tissue expression of these molecules and whether circulating levels have the potential to replace tissue expression, or add to tissue expression, as potential immune biomarkers in HCC patients undergoing surgical resection.Archival blood samples (59 serum samples and 22 plasma samples) and formalin fixed paraffin-embedded tissue samples from 81 patients who underwent hepatic resection or liver transplantation for HCC at Erasmus MC-University Medical Center between January 2007 and March 2013, were used for this study. All patients had undergone procedures with curative intent.Medical ethical approval for this study was obtained from the Medical Ethics Committee of Erasmus MC (MEC-2015-237). All patients gave informed consent for the use of their blood and tissue in medical research. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.33 in the laboratory of Dr. Haidong Dong . The dynamic range of this ELISA is: 86–3670 pg/ml. Limit of detection: 84 pg/ml. Recovery from heparin plasma: 84–105%. Soluble Gal-9 was performed using an ELISA kit according to the manufacturer’s protocol . This ELISA kit has been used in several published studies35. The dynamic range of this ELISA is 7.8–500 pg/ml. Limit of detection 3.3 pg/ml. Recovery from serum: 91–104%. Recovery from heparin plasma: 87–102%. All samples were tested in duplicate and mean values were used for analysis.Sera or plasma samples were drawn pre-operatively  and were stored at −80 °C. Soluble PD-L1 was measured using a validated ELISA14. In brief, three 0.6 mm cores were taken from the tumorous areas and two 0.6 mm cores were taken from the surrounding tumor free liver tissue of each tissue block. The tumorous areas with vital tissue were marked by an experienced pathologist using archived H&E glass slides. The TMAs were made using an automated tissue-arrayer ATA-27 .TMAs were constructed as previously described14. In brief 4 µm thick sections were mounted on Superfrost PlusTM slides. The sections were deparaffinized and rehydrated. Endogenous peroxidase activity was blocked with 0.3% H2O2 for 15 minutes. Antigen retrieval was performed in a microwave for 10 minutes using the appropriate antigen retrieval buffer. After serum block, primary antibodies were applied at 4 °C overnight. The primary antibodies were PD-L1 clone 405.9A1136 , and Gal-9 goat polyclonal31 (R&D systems). HRP-conjugated anti-mouse or anti-goat IgG polymer secondary antibody  was then applied for 1 hour, followed by diaminobenzadine (DAB) as the chromogen detection method. The slides were stained with haematoxylin followed by dehydration. Negative controls consisted of omission of the primary antibody and appropriate positive control tissues included in the TMAs were used to evaluate specificity of all antibodies.Immunohistochemistry and scoring was performed as previously describedScoring was performed by 2 independent investigators blinded to clinical outcome and differences resolved by mutual agreement. Only cytoplasmatic staining was observed for both antibodies. Intensity of tumor cell and hepatocyte staining was scored in a scale from zero to three. Intra-core heterogeneity of staining intensity of tumor cells or hepatocytes was rarely observed, thus percentages of positive tumor cells were not analyzed. Average values of the scores of the different cores were used for analysis. Patients with any evaluable PD-L1 or Gal-9 staining on their tumor cells were considered to have high expression, while patients with complete absence of staining were considered to have low expression.2 tests or the T-test as appropriate. For the correlations between the circulating and intra-tumoral immune biomarkers the Spearman Correlation test was performed. Sensitivity analysis was performed in relation to sample source (serums versus plasma), type of surgery (resection versus liver transplantation) and the use of the TNM classification system. The statistical analysis was performed using the SPSS© 21 software.All analyses were performed in duplicate. Survival curves were estimated by the Kaplan-Meier method. Survival was calculated from the date of surgery to the date of event (recurrence or death), or date of last follow up. The patients who died from causes other than HCC were censored at their time of death. The log-rank test was used to assess differences between survival curves of different groups, while for biomarkers with three linearly associated levels the linear trend for factor levels was used. Optimal high vs low values were established by examining a grid of cutoffs and choosing the cutoff with the lowest −2 log likelihood. For multivariate analysis, the Cox proportional Hazard regression analysis was used. The associations between clinicopathologic parameters with immune biomarkers were examined using the χ−1  showed a non-significant trend toward predicting HCC-specific mortality. Number of lesions, tumor differentiation, vascular invasion and cirrhosis did not predict HCC-specific mortality in our cohort. Tumor size >3 cm also predicted HCC recurrence . Circulating PD-L1 and Gal-9 were not associated with any of the above clinicopathologic characteristics with the exception of a positive association between circulating PD-L1  and cirrhosis (p = 0.045).Pre-operative circulating PD-L1 and Gal-9 levels were studied in 81 HCC patients who underwent hepatic resection or liver transplantation. Plot diagrams of circulating PD-L1 and Gal-9 levels can be seen in Supplementary Fig. −1 (p = 0.060) showed non-significant trends to association with HCC-specific survival.Median pre-operative circulating PD-L1 concentration was 383 pg/ml (intra-quartile range (IQR) 206–774 pg/ml), and median pre-operative circulating Gal-9 concentration was 21 pg/ml (IQR 3–44 pg/ml). With an optimal cutoff of 700 pg/ml, high circulating PD-L1 was associated with improved HCC-specific survival , and with a cutoff of 42 pg/ml high circulating Gal-9 was also associated with improved HCC-specific survival . The respected Kaplan-Meier curves are shown in Fig. In the majority of patients PD-L1 and Gal-9 were measured in sera, but from 22 patients only plasma samples were available. When the patients with plasma samples were excluded from the analysis, the Kaplan-Meier survival curves for the remaining 59 patients are similar to Fig. 14. Specifically, 58 patients from the previous cohort of 154 resected HCC patients had available stored peripheral blood, and were thus included in the current study. An additional 23 patients, unique in the present study, had available stored peripheral blood and thus had their corresponding tumor tissue immunohistochemically stained for PD-L1 and Gal-9. Examples of PD-L1 and Gal-9 immunohistochemical staining of tumor tissues are shown in Supplementary Fig. 14. The respective relationships of intra-tumoral PD-L1 and Gal-9 with HCC recurrence can be seen in Fig. −1 (p = 0.004) were independent predictors of HCC-specific survival.The intra-tumoral expression of PD-L1 and Gal-9, in combination with other immune inhibitory molecules, has been studied by our group before, in an overlapping cohort−1 (p = 0.050). In the case of PD-L1, both intra-tumoral PD-L1 (p = 0.056) and circulating PD-L1 (p = 0.067) showed a non-significant trend towards independently predicting HCC-specific survival, together with AFP > 100 µgl−1 (p = 0.039). In addition, there was no correlation between circulating levels and expression levels of PD-L1 or Gal-9 on hepatocytes in the surrounding tumor free liver tissue.Next we examined if pre-operative circulating levels of PD-L1 and Gal-9 reflected intra-tumoral expression. There was no correlation between intra-tumoral PD-L1 and circulating PD-L1  or between intra-tumoral Gal-9 and circulating Gal-9 . Box-and-whisker plot graphs supporting the above lack of association can be seen in Supplementary Fig. −1 and cirrhosis, are independent predictors of HCC-specific survival  or liver transplantation (n = 23) with curative intent, while Finkelmeier et al., included patients from all stages of HCC of which the majority were treated by local ablation or palliative therapy with sorafenib. Only 21% of their patients were treated with resection (n = 26) or liver transplantation (n = 19). It is entirely possible that circulating PD-L1 may have different roles in a setting of a small amount of resectable disease versus an advanced palliative setting with a large tumor burden. In the early curative setting, high levels of circulating PD-L1 may represent adaptive immune resistance of the tumor against successful immune attack, resulting in increased expression and release of PD-L1 by cells in the tumor in response to pro-inflammatory cytokines secreted by infiltrating immune cells. In this setting high levels of circulating PD-L1 would be associated with good prognosis. In the advanced metastatic setting the anti-tumor immune system may be overburdened and no longer able to suppress tumor growth. In this setting high levels circulating PD-L1 may reflect increased tumor load and therefore be associated with worse prognosis. Indeed, in the Finkelmeier study circulating PD-L1 levels were positively associated with more advanced disease, while this was not the case in our cohort . Similarly, in advanced renal cell carcinoma and advanced lung cancer high circulating PD-L1 levels were positively correlated with disease stage and with worse prognosis26. On the other hand, no correlation between circulating PD-L1 levels and outcome was found in advanced pancreatic cancer28. Such contrasting observations may not be surprising since such differences have been similarly observed in the setting of intra-tumoral PD-L1 expression where the prognostic significance of PD-L1 differs depending on the cancer type and stage. An additional difference with the Finkelmeier et al. study is the fact that in our study 54% of patients did not have cirrhosis and 25% of patients had no identifiable underlying liver disease. In contrast, in the Finkelmeier et al. study, all patients had an identifiable underlying liver disease and all patients were assigned a Child-Pugh score, indicating that all patients had cirrhosis. Thus, our opposite results may also be due to the inclusion of many HCC patients without underlying liver disease in our study. Finally, the use of different ELISA assays could be another explanation for the differing results although, despite using different assays, both studies observed similar median circulating PD-L1 levels  and used similar cutoff values (700 vs 800 pg/ml).No study has yet examined the prognostic significance of circulating Gal-9 in any cancer. On the other hand, the prognostic role of circulating PD-L1 has been examined in a few cancer types before25, suggesting that expression of soluble and membrane-bound PD-L1 are differentially regulated. Preliminary data have shown involvement of matrix metalloproteinases in shedding of membrane-bound PD-L138. More recently it has been shown that tumor cells can produce soluble PD-L1 by alternative splicing39. Moreover, PD-L1 can be released from tumor associated macrophages and dendritic cells. It has been shown by us and by other research groups that tumor associated macrophages and dendritic cells express PD-L1 in the tumor microenvironment in HCC40. Myeloid cells, especially mature dendritic cells, have been shown to release soluble PD-L1 in vitro41 and it has been recently shown that circulating activated macrophages in ovarian cancer patients also express high levels of PD-L143. Finally, another possible source of soluble PD-L may be circulating tumor cells, which have been shown to express PD-L1 in breast cancer patients44. Whatever the source of circulating PD-L1 may be, the circulating ligand is known to retain its PD-1 binding domain and immunosuppressive properties39. Regarding the form of soluble PD-L1, recent studies have shown that PD-L1 can be released from tumor cells in the form of exosomes. Interestingly, pre-treatment levels of circulating exosomal PD-L1 were higher in melanoma patients that failed to respond to pembrolizumab therapy than in non-responders, but this difference was not observed for total circulating PD-L145. In head and neck cancer, levels of PD-L1 carried by exosomes correlated with disease activity but total plasma PD-L1 levels did not46. Therefore, the form of circulating PD-L1, either expressed in circulating exosomes or in non-bound form, may additionally explain why high circulating PD-L1 levels are associated with worse or better prognosis depending on the cancer setting. Another theoretical possibility for differences between studies is the use of differing ELISA systems. It is important to note however that Frigola et al.25, found that high circulating PD-L1 is associated with worse patient survival in Renal Cell Carcinoma, results opposite to ours, using the same ELISA and the same laboratory where our assays were performed. Thus, it is likely that biologic factors, rather than technical factors, account for the differences in results between our study and that of Frigola et al., as we discuss above.An additional consideration that may influence the prognostic role of PD-L1 has to do with the source and the particular form of the circulating ligand. One hypothesis is that PD-L1can be actively shed from membrane-bound PD-L1 expressing tumor cells and/or hepatocytes. Soluble PD-L1 has indeed been shown to be released into cell culture supernatants by several, but not all, membrane PD-L1 expressing tumor cell lines23. Gal-9 does not have a signal peptide, and its secretion must involve a non-classical pathway. Cleavage from the cell surface by matrix-metalloproteinases has been suggested as a secretion pathway47, while other studies showed that cancer cells can secrete Gal-9 via exosomes48. Therefore, circulating levels of these molecules are probably not passive reflections of tissue expression. In addition, while their expression in tumor tissues probably determines their local effects, their circulating counterparts may exert systemic effects, e.g. by inhibiting systemic immunity or, in case of Gal-9, prevent formation of distant metastasis by hampering extravasation of tumor cells into other tissues22. This means that future studies on the role of PD-L1 and Gal-9 as prognostic or predictive markers should take both intra-tumoral and circulating levels into consideration to maximize the potential for biomarker optimization.Like PD-L1, Gal-9 is not only expressed on tumor cells, but also at high levels on tumor-associated macrophages in HCC24. We have previously studied the prognostic value of intra-tumoral expression of PD-L1 and Gal-914. Our current results regarding intra-tumoral expression of PD-L1 and Gal-9  indicating that sample size may be another explanation for the difference, and that perhaps our results are not that discrepant with the results of the Fujita et al., study. In addition, it is important to keep in mind that in multivariate analysis the association of circulating PD-L1 and Gal-9 with survival was independent of the presence or absence of cirrhosis. Thus cirrhosis was not a confounder of PD-L1 or Gal-9 levels in regards to patient survival.We found an association between circulating PD-L1 and the presence of cirrhosis. A similar relationship was suggested by Finkelmeier Our study has several strengths. It is the first study to examine the role of circulating Gal-9 in HCC and one of only two studies, to date, to examine the role of circulating PD-L1 in HCC. In addition, few studies have studied circulating levels of these molecules in other cancers, thus this is a fairly unexplored field of research. Most importantly both the intra-tumoral expression and the circulating levels of these molecules were compared. The observation that intra-tumoral and circulating levels independently contribute to prognostication, is novel. Testing for the expression of these molecules in the surrounding tumor free liver tissue also allowed us to show that it is not the expression on hepatocytes in the diseased liver that determines circulating levels of PD-L1 and Gal-9. Our study has also limitations. One limitation of our study is the use of both serum and plasma samples for our analysis. While we show in Supplementary Fig. In summary, pre-operative circulating levels of PD-L1 and Gal-9 do not correlate to their expression in tumor tissue, but have prognostic value in resected HCC patients independently of their expression in tumor tissue. Combined circulating levels and intra-tumoral expression of PD-L1 and Gal-9 provide more confident prognostic immune biomarker profiles compared to each separately.SUPPLEMENTARY INFO


For the first time, we estimated perspectives for using a new 2D carbon nanotube (CNT)–graphene hybrid nanocomposite as a base element of a new generation o optical nanodevices. The 2D CNT–graphene hybrid nanocomposite was modelled by two graphene monolayers between which single-walled CNTs with different diameters were regularly arranged at different distances from each other. Spectra of the real and imaginary parts of the diagonal elements of the surface conductivity tensor for four topological models of the hybrid nanocomposite have been obtained. The absorption coefficient for p-polarized and s-polarized radiation was calculated for different topological models of the hybrid nanocomposite. It was found that the characteristic peaks with high intensity appear in the UV region at wavelengths from 150 to 350 nm (related to graphene) and in the optical range from 380 to 740 nm irrespective of the diameter of the tubes and the distance between them. For waves corresponding to the most intense peaks, the absorption coefficient as a function of the angle of incidence was calculated. It was shown that the optical properties of the hybrid nanocomposite were approximately equal for both metallic and semiconductor nanotubes. The spin degeneracy is already taken into account in the above equations by the factor 2, η is a phenomenological parameter characterizing electron scattering processes.where , the known substitution P(k)→ was used, where  is the Hamiltonian. The detailed description for the calculation of the matrix elements of the momentum operator is given in [xx is similar to the spectrum of graphene, while the spectrum of σyy has complex and multiple peaks. As previously shown [To calculate the elements of the impulse matrix given in . The Hamly shown , a complly shown ).Special attention should be paid to the peak of great intensity observed in the wavelength range of 800–830 nm for models with tube  . One canA) for four types of the tubes with an intertube distance of 13 hexagons, and The calculation results of the absorption of electromagnetic waves of the CNT–graphene hybrid nanocomposite are presented in The analysis of the diagrams in XZ-plane; 2) the wave vector lies in the YZ-plane. YZ-plane, and at angles of 85–90° for the irradiation in the XZ-plane. The absorption reaches values of 45–50% at these angles of incidence. Thus, one can say that the optical properties of the composite do not explicitly depend on the type of the tubes.For wavelengths corresponding to the most intense peaks, a diagram of the dependence of absorption coefficient  on the aIn summary, it can be concluded that the new 2D CNT–graphene hybrid nanocomposite is very promising for optoelectronic devices. In particular, the established regularities of change in absorbance as a function of the angle of incidence of the electromagnetic wave allows us to suggest the possibility of using the CNT–graphene film as a polarizer for electro-optical and magneto-optical thin film modulators. The advantages of such polarizers are a wide spectral range and low loss.


AbstractPorcellionidespruinosus, a terrestrial isopod, was chosen as a non-target species to investigate such possible effects. Two different experiments were conducted to study two different neem-derived plant protection products, i.e., NeemAzal T/S (1% azadirachtin) which is a commercial product registered in the EU, and neem leaf extract from dried neem leaves (1%).The latter simulates the plant protection product, is domestically produced, and widely used by farmers in India and other tropical and subtropical countries. Findings are consistent with previous results obtained with other non-target organisms, i.e., neither of the tested neem products have adverse effects on the mortality of P.pruinosus. However, further research on a wider range of soil organisms is needed to prove the safety of neem-based products as biological control agents and to be part of integrated pest management.Neem-based products have gained major attention over the last few years due to their wide range of applications in pest management, and have been in the focus of biological plant protection research in the past decade. Yet, there is limited information available to understand the side effects of these neem-derived pesticides on non-target species in soil. Therefore,  Azadirachtaindica)-based biological insecticides requires more information about their possible side effects on non-target organisms. Such organisms are the woodlice species, important decomposers of organic material in agricultural areas. It was demonstrated that neither a commercial product of neem nor a domestic neem leaf extract had any adverse effect on a ubiquitous woodlice species, Porcellionidespruinosus.The safe use of neem (Azadirachtaindica A. Juss) which has been known in the Indian sub-continent for more than 4000 years  (Lepidoptera), one of the most important cabbage pests in agriculture. Extensive research is being conducted with neem and its derived products against target pests. Aphisgossypii (Glover) and found that the aqueous neem seed extract is efficient in nymph mortality and reducing the survival period and fecundity of the cotton aphid.Neem extracts and products have been used against different orders of insects such as Daphniamagna  and Hyalellaazteca . Nicrophorusvespilloides (Herbst 1783), a common burying beetle, feeding on a variety of animal carcasses. Nicrophorusvespilloides were fed with cockchafers which were previously fed with neem-treated leaves. They found that it had no negative impact on the reproduction nor they found any impacts on the morphology of N.vespilloides.The effect of neem-derived products on non-target organisms has also been studied. However, information on the effect of neem-based pesticides and formulations on non-target organisms of soil biota are still limited. Isopoda, Oniscidae) are ubiquitous saprophagous members of the soil fauna  and Enchytraeusalbidus  an annelid, commonly known as white worm, using avoidance as test parameter. For both the species, patterns of antagonism were found when exposed to dimethoate and atrazine, synergism for lindane, dimethoate, and atrazine, synergism for lindane and dimethoate exposures and concentration addition for cadmium and zinc occurred, while the exposure to cadmium and dimethoate showed dissimilar patterns   . ES can  society . Soil fu society . If the  society , or heav society  that esp society .A small-scale terrestrial ecosystem containing soil collected from an agricultural field in Central Portugal was used to evaluate the effects of the combination of the herbicide glyphosate and the insecticide dimethoate in another study. The application of dimethoate led to a decrease in feeding activity in all concentrations tested. The mortality of isopods exposed to dimethoate in single and binary exposures was high. Exposure to dimethoate decreased the acetylcholinesterase activity of isopods .Porcellionidespruinosus. We selected P.pruinosus as a non-target organism, because it is ubiquitous and it occurs in anthropogenic environments, where pest control is applied. They play a vital role in the fragmentation and decomposition process of leaf litter, thereby causing changes in soil quality and soil services  (t (1833) .Preparation of neem leaf extract: For neem leaf extract, air-dried neem leaves were obtained from local growers in India, Maharashtra, Konkan Division, Mumbai Suburban area. A stock concentration of 1% was prepared by soaking 1g of crushed dried neem leaves in 100 ml distilled water overnight and then filtered using a non-sterile filter paper. Different working concentrations  of neem leaf extract were prepared from 1% stock solution using distilled water in the laboratory and were used on the same day. Generally the applied dosage used by local growers in India has a maximum concentration of 1%. In this experiment, we tried to model the concentration used by the local growers in the field conditions.Preparation of azadirachtin: NeemAzal T/S (Trifolio-M GmBH), a commercial product containing 1% azadirachtin, registered in the EU, was used. A stock concentration of 1% azadirachtin was prepared  by diluting 1 ml NeemAzal T/S in 100 ml of distilled water which equals to 0.01% azadirachtin. It was further diluted to get the 0.0005, 0.001, 0.0025, 0.005, 0.0075 and 0.01% azadirachtin concentrations respectively and was used on the same day. The registered dosage of azadirachtin ranges from 0.0025 to 0.005%, depending on the plant culture in the EU.A control with only distilled water was used for both experiments. The working concentrations and distilled water were sprayed using a hand sprayer under laboratory conditions.Experimentaldesign: Five adults of Porcellionidespruinosus were placed in glass Petri dishes (13 cm in diameter), with 1 g of commercial horticultural soil (pH = 7.0) and approximately 1 g of fresh potato as a food source. Two milliliters of different working concentrations of neem leaf extract and azadirachtin were sprayed using a hand sprayer. After spraying, the Petri dishes were kept in the dark, checked after time periods of 1, 24, 48, 72, 96, and 120 hours post-application of neem leaf extract and azadirachtin respectively, and mortality data was recorded. The mortality data obtained after 120 hrs was subjected to statistical analysis using R software (R Core Team 2017). Logistic regressions were fitted  to check the effect of the two different products on isopod mortality. To test whether the concentrations have significant effect on mortality, chi-squared tests were performed on model deviances. Prior to running the tests model diagnostic plots were investigated to assess homoscedasticity and residual normality  as studied by Nicrophorusvespilloides, which co-occurs with the forest cockchafer Melolonthahippocastani  and also feeds on the carcasses of M.hippocastani. In their study, they fed N.vespilloides with dead M.hippocastani which were previously fed with neem-treated leaves. They neither observed any impact on the mean larval weights nor on the morphology of N.vespilloides. Still, they authors did not dismiss the possibility of azadirachtin passing through the food chain and affecting other non-target organisms.While there are numerous literature references available on the effect of neem and neem-derived products on target organisms, some of the studies reported data on non-target organisms as well. For instance, Philosciamuscorum  and did not find any negative effects on P.muscorum. The results of our experiments were found to be similar and this experiment for the first time investigated the effects of two different neem products on this non-target isopod species, i.e., Porcellionidespruinosus.P.pruinosus.From our results it can be concluded that neither NeemAzal T/S nor neem leaf extracts pose any risk to the terrestrial isopod species studied in the tested concentrations. However, further research is needed to test the possible effect of various neem products on the members of the soil fauna. Also, it can be concluded that NeemAzal T/S and domestic neem leaf extract do not differ in respect to their mortality effects on PD and AMP designed and conducted the experiments. MS analyzed the data. PD wrote the manuscript. AMP, FT, GT, and MS gave their feedback. All authors read and approved the manuscript.


Complexation of cis-Pd(tmeda)(NO3)2 with L1 at a 1:1 or 3:2 ratio produced [Pd(tmeda)(L1)](NO3)2 (1a). The reaction mixture obtained at 3:2 ratio upon prolonged heating, produced a small amount of [Pd3(tmeda)3(L1)2](NO3)6 (2a). Complexation of Pd(NO3)2 with L1 at a 1:2 or 3:4 ratios afforded [Pd(L1)2](NO3)2 (3a) and [(NO3)2@Pd3(L1)4](NO3)4 (4a), respectively. The encapsulated NO3– ions of 4a undergo anion exchange with halides  to form [(X)2@Pd3(L1)4](NO3)45a–7a. The coordination behaviour of ligand L1 and some dynamic properties of these complexes are compared with a set of known complexes prepared using the regioisomeric ligand bis(pyridin-3-ylmethyl)pyridine-3,5-dicarboxylate (L2). Importantly, a ligand isomerism phenomenon is claimed by considering complexes prepared from L1 and L2.The complexation study of  N-donor ligands) is particularly advantageous for the construction of a variety of metallocages (NO3)2  variation was also carried out to successfully prepare a series of complexes [Pd(tmeda)(L1)](BF4)2 (1b); [Pd(tmeda)(L1)](ClO4)2 (1c); [Pd(tmeda)(L1)](OTf)2 (1d). The complexes 1a–d were prepared by mixing the corresponding metal component Pd(tmeda)(Y)2 with the ligand L1 where Y = NO3−, BF4−, ClO4− and OTf−. The metal components were prepared in situ by reacting Pd(tmeda)(Cl)2 with AgY in DMSO-d6 followed by separation of the precipitated AgCl.The addition of one equivalent of 1a was characterized by various NMR techniques  as compared to the free ligand L1. The peak positions of Hf and Hg remained unchanged which indicated that the central pyridine ring is not involved in the complexation. The 1H NMR spectra of compounds 1b, 1c and 1d are very much comparable to that of 1a (NO3)2 to a clear solution of two equivalents of ligand L1 in DMSO-d6 produced a turbid mixture. However, a clear yellow solution was obtained upon stirring the mixture at 90 °C for 5 min. The progress of the complexation reaction was monitored by 1H NMR spectroscopy. We targeted a Pd3L’3L2-type complex [3(tmeda)3(L1)2](NO3)6 ](NO3)2 (1a) and uncomplexed cis-Pd(tmeda)2+. The reaction was allowed to continue where upon the integration ratios of the peaks corresponding to Hf and Hg were lower than expected and that of Hd was higher than expected. In addition, a new peak was observed at around 9.15 ppm 2+ remaining in solution. The peaks assigned to Hf and Hg of 2a are shifted downfield compared to those of Hf and Hg in 1a whereas other signals of 2a merged with the respective signals of 1a. The observed downfield shift of the Hf signal in 2a is complexation-induced and found to have a unique position. The downfield shift of the Hg signal in 2a could not be induced by complexation and is best described by considering an anisotropy effect of the nearby carbonyl groups the ligand strand. The Hg signal of 2a, however, merged with the Hd signal of 1a.The addition of three equivalents of  complex , i.e., (NO3)2  variation was also carried out to successfully prepare a series of complexes [Pd(L1)2](BF4)2 (3b), [Pd(L1)2](ClO4)2 (3c), and [Pd(L1)2](OTf)2 (3d). These complexes were prepared by complexation of Pd(Y)2 with the ligand L1 where Y = BF4−, ClO4−, and OTf−. It is important to note that Pd(Y)2 solutions were prepared by reacting PdI2 with AgY and the precipitated AgI was removed by filtration. Following this procedure, the presence of iodide as impurity could not be ruled out but its presence was found to not influence the formation of the targeted complex. In contrast, the presence of chloride remaining as impurity when PdCl2 was reacted with AgY to prepare Pd(Y)2, contaminated Pd(Y)2 and produced upon complexation with L1 complexes 3a–d along with some other products. The choice of PdI2 is on the basis of our previous experience from related cages (NO3)2 (3a) and [(NO3)2@Pd3(L1)4](NO3)4 (4a) in the mixture  with the ligand L1 at a 3:4 ratio resulted in the formation of only mononuclear complexes 3b–d (depicted in a later Scheme) and the unutilized proportion of Pd(Y)2 remained in solution. Thus, the counter ion nitrate has a determining role by acting as template for the cavities in the formation of the trinuclear complex 4a. It is proposed that the anion is essential to avoid charge repulsion between the metal centers in the ensuing cavities. However, the anions should be of fitting sizes only to get accommodated in the cavities so that discrete architectures are formed. Larger anions such as BF4−, ClO4− and OTf− are not accommodated in the cavities and not helpful as templates. The Pd(NO3)2 sample prepared from PdCl2 and AgNO3, contained chloride as impurity and resulted in a mixture of products along with the targeted 4a (3)@Pd3(L1)4](NO3)4 (6a’) and [(Cl)2@Pd3(L1)4](NO3)4 (6a) and their proportion was found to depend on the amount of chloride as impurity. The influence of chloride on the product composition is discussed in a later section.A solution containing three equivalents of commercially acquired Pd as compared to the free ligand L1. The signal of Hf also got downfield-shifted indicating that the central pyridine ring is involved in the complexation.The complex of peaks  featured4a confirmed the formation of a trinuclear complex ]2+, [Pd3(tmeda)3(L1)2]6+, [Pd(L1)2]2+, and [(NO3)2@Pd3(L1)4]4+ are shown in 3(tmeda)3(L1)2]6+ could not be prepared exclusively, we looked into the energetics of the system. The overall Gibbs free energies (∆G) and the enthalpies (∆H) for the formation of the trinuclear complex [Pd3(tmeda)3(L1)2]6+ considering its formation from 1 equivalent of [Pd(tmeda)(NO3)2] and 2 equivalents of [Pd(tmeda)(L1)]2+ were found to be not feasible  and endothermic , respectively (L1)2](NO3)2 (3a) was found to be stable at room temperature for days in DMSO-d6  got reduced to palladium(0). In another experiment, Pd(NO3)2 was added to a solution of 3a at a 2:1 ratio where upon complex-to-complex conversion was observed at room temperature or upon heating to afford 4a (3)2 a complete formation of 4a was observed within 20 min at rt or 5 min at 90 °C  to match the stoichiometric requirement for the formation of 3a. Although the formation of 3a was observed, it remained only as a minor product, and the added ligand L1 was partially consumed. Thus, the unbound ligand remained in its free state along with 4a. No further change was observed after 30 min 2@Pd3(L1)4](NO3)4 (4a) was prepared by mixing Pd(NO3)2 with ligand L1 at a 3:4 ratio (4)2, Pd(ClO4)2 or Pd(OTf)2 with L1 at a 3:4 ratio did not afford the analogous trinuclear complexes; rather the corresponding mononuclear complexes were formed and the uncomplexed palladium(II) remained in solution   in four separate experiments using DMSO-d6 as the solvent. The anion exchange processes were monitored by 1H NMR spectroscopy of the samples (NO3)@Pd3(L1)4](NO3)4 (6a’) and [(Cl)2@Pd3(L1)4](NO3)4 (6a). With further addition of TBACl, the proportion of 6a increased at the cost of 4a and 6a’ to finally yield compound 6a as the only product. Similarly, the addition of TBABr initially produced a mixture of 4a, [(Br)(NO3)@Pd3(L1)4](NO3)4 (7a’) and [(Br)2@Pd3(L1)4](NO3)4 (7a) with complex 7a as the exclusive final product. In the case of TBAF, initially there was no change observable except a slight broadening of the signals for Hb and Hc. Further addition of TBAF led to the formation of [(F)2@Pd3(L1)4](NO3)4 (5a) along with minor impurities. However, the addition of TBAI to 4a showed no changes and hence iodide encapsulation did not happen. The 1H NMR spectra for the mixtures of products formed at intermediate and final stages are provided in 4a, 5a, 6a and 7a are shown in 1H NMR spectra of these anion-encapsulated complexes are influenced by coordination of ligand L1 with palladium(II) and interaction of the encapsulated anion with the endohedrally oriented hydrogens of the cages.Each of the two cavities of cage  earlier –23. Hali4−, ClO4− or OTf− could not support the formation of the double-decker cage even though the required amount of palladium(II) was available. The addition of TBAI to any of these solutions containing Pd(Y)2 and 3b, 3c or 3d, respectively, did not lead to double-decker cages indicates that I− is not suited for the cavity. However, addition of TBANO3, TBAF, TBACl and TBABr produced the corresponding anion encapsulated double-decker cages as shown in steps (iv), (v) and (vi) of 1H NMR spectra for the conversion of 3b to corresponding products 4b, 5b, 6b and 7b are shown in Is iodide not capable of replacing the preexisting nitrate in a competition or iodide is not suited at all for the cavity irrespective of any competition? The following argument might answer this question. The complexation reaction shown in steps (ii) of 1H NMR spectral analysis of the two nitrate anions incorporating compound 4a discussed in an earlier section revealed downfield shifts of particular signals as compared to the free ligand L1 and the Δδ values were 1.42, 0.82 and 1.02 ppm for the signals of Ha, Hb, and Hf, respectively. A similar comparison for the (i) two F− encapsulated compound 5a: Δδ = 1.75, 0.99 and 1.78 ppm for Ha, Hb, and Hf, respectively), (ii) the two Cl− encapsulated compound 6a: , and (iii) the two Br− encapsulated compound 7a:  are in line with the expectation. Although fluoride (F–) could replace NO3– in 4a to afford complex 5a, the complex 5a was found to be unstable and it decomposed within a few hours. Thus the 1H NMR and ESIMS spectrum of 5a were recorded from freshly prepared samples. The complexes 4a, 6a and 7a are quite stable and no decomposition was observed. Detailed characterization data of 4a–7a form a variety of NMR techniques are provided in The 6a and 7a were detected. Also, one of the mixed halide–nitrate encapsulated complexes, i.e., 7a’ was also detected.The molecular compositions of the halide encapsulated complexes were confirmed by recording ESIMS data for the systems. The double halide encapsulated complexes 6a ](NO3)2 (1a), [Pd(L1)2](NO3)2 (3a), [(NO3)2@Pd3(L1)4](NO3)4 (4a), and [(X)2@Pd3(L1)4](NO3)45a–7a fulfill the definition of ligand isomerism when compared with the reported complexes [Pd(tmeda)(L2)](NO3)2 (8a), [Pd(L2)2](NO3)2 (10a), [(NO3)2@Pd3(L2)4](NO3)4 (11a), and [(X)2@Pd3(L2)4](NO3)412a–14a, respectively. We have demonstrated ligand isomerism in Pd2L4-type cages [3L4-type double-decker coordination cages for the first time.As discussed in the introduction section “The definition of ligand isomerism includes metal complexes (at least two) having the same molecular formula but are composed of different structural isomers of the ligand.” The complexes prepared in this work namely [Pd(tmeda)(NO3)2 with L1 produced the trinuclear complex [Pd3(tmeda)3(L1)2](NO3)6 (2a), the ligand L2 did not afford the targeted [Pd3(tmeda)3(L1)2](NO3)6 (9a). The complex [Pd(L1)2](NO3)2 (3a) was unstable when heated in DMSO medium whereas the corresponding complex [Pd(L2)2](NO3)2 (10a) was stable under comparable conditions. The addition of two equivalents of L1 to a solution of [(NO3)2@Pd3(L1)4](NO3)4 (4a) produced only a small amount of [Pd(L1)2](NO3)2 (3a) and the added ligand remained in solution. On the other hand, addition of the required amount of L2 to a solution of [(NO3)2@Pd3(L2)4](NO3)4 (11a) resulted in complete transformation to complex Pd(L2)2](NO3)2 (10a). The F− encapsulated complex [(F)2@Pd3(L1)4](NO3)4 (5a) decomposed within a few hours whereas [(F)2@Pd3(L2)4](NO3)4 (12a) was stable for a few hours. These differences are ascribed to the positional exchanged functionalities in the ligands L1 and L2. Probably, the coordination ability of the central pyridine ring is better than that of the terminal pyridine rings in case of L1. However, in the mononuclear complexes of L1 the central pyridine remained uncomplexed, which may be due to the formation of metallomacrocyclic rings. This behavior is not observed in the case of mononuclear complexes of L2. Thus, the mononuclear complexes of L1 are reluctant to form  and prone to decomposition. As far as trinuclear complex formation is concerned the central pyridine ring of L1 is in a relatively favorable situation, thus the complex 4a could form and 3a was a kinetic product.The complexation behavior of cis-protected palladium(II) and bare palladium(II) components with the new tridentate ligand L1. A variety of counter anions were employed to broaden the scope of the choice of metal components. Mononuclear complexes with PdL’L composition could be prepared easily, however, Pd3L’3L2-type trinuclear complexes were obtained in only small amounts. Also, mononuclear complexes of PdL2 and trinuclear complexes of Pd3L4-type compositions were prepared easily. The choice of the counter anion did not influence the formation of mononuclear complexes whereas the counter anion displayed a template role for the formation of trinuclear complexes, especially for Pd3L4-type complexes. The anions helped to screen the charge repulsion between the palladium(II) ions. The complexation behavior of palladium(II) components with the ligand L2 have been reported earlier +.[Pd(tmeda)(L1)](NO3)2 (1a): To a solution of cis-Pd(tmeda)(NO3)2 , in 3 mL of DMSO ligand L1  was added. The reaction mixture was stirred at room temperature for 10 min to obtain a clear yellow solution. The product was precipitated by the addition of ethyl acetate (10 mL), separated by centrifugation, washed with acetone (4 mL) and dried under vacuum to afford complex 1a . 1H NMR  δ 9.90 , 9.35 , 8.67 , 8.54–8.52 , 8.18 , 7.85 , 5.91–5.29 ; 13C NMR  δ 164.50, 154.60, 151.91, 147.21, 140.92, 133.03, 132.24, 129.41, 127.31, 63.41, 62.31.[Pd(L1)2](NO3)2 (3a): To a solution of ligand L1  in 3 mL of DMSO, Pd(NO3)2  was added and the reaction mixture was stirred for 10 min at room temperature to give a clear yellow solution. The product was precipitated by the addition of 10 mL of ethyl acetate. The pale yellow precipitate was separated by centrifugation, washed with acetone and dried under vacuum to afford complex 3a . 1H NMR  δ 9.92 , 9.30 , 8.75 , 8.55–8.53 , 8.31 , 7.82 , 5.63 ; 13C NMR  δ 162.43, 154.57, 151.59, 147.83, 141.30, 133.97, 132.31 129.57, 127.38, 63.86; ESIMS (m/z): 866.10 [3a − 1NO3]+; 402.05 [3a − 2NO3]2+.[(NO3)2@Pd3(L1)4](NO3)4 (4a): To a solution of ligand L1  in 2 mL of DMSO, Pd(NO3)2  in 1 mL of DMSO was added and the reaction mixture was stirred for 5 min at 90 °C to give a clear yellow solution. The product was precipitated by the addition of 10 mL of ethyl acetate. The pale yellow precipitate was separated by centrifugation, washed with acetone and dried under vacuum to afford the complex 4a . 1H NMR  δ 10.54 , 9.74 , 9.66 , 8.58 , 8.26 , 7.95–7.93 , 5.45 ; 13C NMR  δ 162.40, 155.05, 153.19, 149.91, 141.88, 138.39, 133.83, 128.66, 127.68, 64.84; ESIMS (m/z): 982.04 [4a − 2NO3]2+; 634.03 [4a − 3NO3]3+; 460.03 [4a − 4NO3]4+.File 1Experimental procedures, NMR, ESIMS data, and theoretical study.