Accumulating evidence suggests that exogenous cellular stress induces PD-L1 upregulation in cancer. A DNA double-strand break (DSB) is the most critical type of genotoxic stress, but the involvement of DSB repair in PD-L1 expression has not been investigated. Here we show that PD-L1 expression in cancer cells is upregulated in response to DSBs. This upregulation requires ATM/ATR/Chk1 kinases. Using an siRNA library targeting DSB repair genes, we discover that BRCA2 depletion enhances Chk1-dependent PD-L1 upregulation after X-rays or PARP inhibition. In addition, we show that Ku70/80 depletion substantially enhances PD-L1 upregulation after X-rays. The upregulation by Ku80 depletion requires Chk1 activation following DNA end-resection by Exonuclease 1. DSBs activate STAT1 and STAT3 signalling, and IRF1 is required for DSB-dependent PD-L1 upregulation. Thus, our findings reveal the involvement of DSB repair in PD-L1 expression and provide mechanistic insight into how PD-L1 expression is regulated after DSBs. PD-L1 is upregulated in many cancers due to exogenous cellular stress. Here the authors show that PD-L1 is upregulated in response to DNA double strand breaks via STAT and IRF1 signalling. PD-1 has a role to inhibit T-cell proliferation and interferon-gamma (IFNγ) production in T cells2. Programmed death-ligand 1 (PD-L1) is identified as the ligand of PD-13. The major role of PD-1/PD-L1 interaction is to regulate autoimmune response in the peripheral tissue; PD-1-deficient mice show hyperactivation of the immune system2. Further, mounting evidence showed that PD-1 or PD-L1 deficient mice developed different autoimmune diseases in each genetic background2. Alternatively, the downregulation of PD-1/PD-L1 interaction can be a cause of human autoimmune disease. The association was reported between single-nucleotide polymorphisms (SNPs) on human PD-1 gene or augmented PD-1 expression and human autoimmune diseases4. Thus, the interaction between PD-1 and PD-L1 is critical to control a balanced global immune response in the human body. In addition, PD-1 has roles in cellular response; for example, PD-1 is involved in the immune reaction against HIV infection5.Programmed cell death-1 (PD-1) is an immune receptor, which is expressed on activated CD4+ T cells and CD8+ T cells as well as on B cells in the periphery7. Notably, PD-1 therapy provides significant clinical benefits for patients with an advanced stage cancer11. Despite this therapy having a substantial effect in advanced cancers in many cases, 20–40% of patients still show progressive disease. A recent clinical report described that, in advanced PD-L1-positive non-small-cell lung cancers, anti-PD-1 therapy as a first-line treatment is associated with significantly longer progression-free survival and improved overall survival compared with the standard platinum-based chemotherapy12. In addition, high responders frequently show elevated PD-L1 expression due to gene amplification and upregulation of an ectopic promoter by translocation14. Thus, the evidence suggests that PD-L1 level in tumours is an important factor influencing therapeutic efficacy for responders17, although the correlation is not perfect and the mechanisms underlying this correlation in non-responders have not been precisely revealed18. Recent studies have suggested that cancers with mutations of genes contributing to genomic stability could be targets for anti-PD-1 therapy. Significantly high rates of responses in cancer with microsatellite instability (MSI), which is a hallmark of genome instability, to anti-PD-1 therapy have been reported in colon cancer20. Accumulating studies suggested that MSI-positive tumours presenting neoantigens promote the release of IFNγ from tumour-infiltrating lymphocytes (TILs), and the released IFNγ upregulates PD-L1 expression in immune cells and tumours24. Therefore, PD-1 therapy is considered to be useful for tumours exhibiting high MSI. Consistent with this notion, patients with mismatch-repair (MMR)-deficient cancer showed a higher rate of progression-free survival following anti-PD-1 therapy, demonstrating that MMR status is a potent predictive marker25. High expression of PD-1/PD-L1 in TILs was also observed in DNA polymerase ε-mutated and MSI cancers26. Furthermore, the possible involvement of homologous recombination (HR) repair has been suggested recently; specifically, enrichment of mutations in BRCA2, an HR factor, has been identified in melanomas responsive to anti-PD-1 therapy27. HR deficient tumours were also shown to exhibit greater neoantigen loads, TILs and PD-1/PD-L1 expression in immune cells23. Thus, the evidence suggests that genomic instability causes high levels of mutations and neoantigen loads in tumours, resulting in greater PD-1/PD-L1 expression in cells surrounding tumour microenvironment.Since decades ago, several distinct strategies for cancer immune therapy have been proposed. Among them, anti-PD-1 antibody has been newly developed as a next-generation immunotherapy agent that blocks the immune checkpoint pathway29. Importantly, PD-L1 expression in cancer cells was found to be transiently upregulated following IR, that is, for several days after irradiation, and when the PD-1/PD-L1 interaction was blocked during this upregulation, the anti-PD-1 antibody treatment rescued T-cell activity and delayed tumour growth after irradiation in an immunocompetent mouse model31. Anti-PD-1 therapy was also found to enhance the efficacy in patients treated with carboplatin, suggesting its beneficial effect when used in combination with chemotherapy32. DNA double-strand breaks (DSBs) are the most critical type of DNA damage induced by radio/chemotherapy and activate signal transduction by phosphoinositol-3-kinase-related kinases, ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3-related protein (ATR) and DNA-PKcs. Such signalling was previously shown to promote DNA repair and arrest the cell cycle, or to trigger apoptosis. In addition to these functions, previous studies have shown that induction of DNA damage response upregulates the expression of cell-surface molecules, such as NKG2D ligand33. However, so far, the relationship between DSB-dependent damage signal and PD-L1 expression in cancer cells has not been elucidated. In this study, we showed for the first time that DSBs upregulate PD-L1 expression in an ATM/ATR/Chk1-dependent manner. Depletion of BRCA2 enhances PD-L1 upregulation after DSBs. PD-L1 expression in Ku-depleted cells requires Chk1 after Exonuclease1 (EXO1)-dependent resection. Finally, we showed that DSBs activate STAT1/STAT3 phosphorylation and IRF1, and IRF1 is required for DSB-dependent PD-L1 upregulation, i.e. through the canonical pathway. This new understanding of the molecular mechanism underlying PD-L1 expression in cancer cells should aid the development of strategies to predict prognosis and may contribute to improving the efficacy of therapies in DNA-repair-defective cancer cells, particularly when anti-PD-1 therapy and radio/chemotherapy are combined.In cancer treatment, radiotherapy and chemotherapy  are applied to induce lethal DNA damage in cancer cells. Recent studies have shown that ionising radiation (IR) synergistically promotes antitumor immunity when applied in combination with immune checkpoint inhibitorsTo test the responsiveness of PD-L1 expression in cancer cells after DNA damage, we examined PD-L1 protein levels by immunoblot analysis. PD-L1 expression in osteosarcoma (U2OS), lung cancer  and prostate cancer (DU145) cell lines was upregulated after IR, which induces DSBs, in a time- and dose-dependent manner Fig. . The uprU2OS cells were mainly used in this study because they have been widely utilised in the analysis of DNA repair and signalling due to their intact DNA damage response compared with that of other cancer cell lines. Thus, these findings suggest that the upregulation of PD-L1 occurs in living cancer cells after IR. In contrast, we did not observe an obvious PD-L1 upregulation in primary fibroblast cells (48BR) after DNA damage in the time range of our analysis . Strikingly, IR-, CPT-, Etp- or APH-dependent induction of PD-L1 expression was significantly suppressed by ATM inhibition, suggesting that PD-L1 expression is upregulated in response to DSB-dependent damage signalling . Notably, IR-induced PD-L1 upregulation was substantially suppressed by ATR or Chk1 inhibition  also induce DSBs but indirectly via DNA replication. ATM, which is the most important signal transducer, serves as a sensor of DSBsing Fig. . The MRNSBs Fig. . After Iion Fig. . The PD-ion Fig. . Furtherion Fig. . The uprion Fig. . Taken tThe findings above prompted us to identify the factors regulating PD-L1 expression in DSB repair and signalling. To identify DSB repair factors regulating PD-L1 upregulation after IR, we carried out a screen using an siRNA library targeting DSB repair and signalling . Consistent with the data in the first siRNA screen, depletion of BRCA2 by using another siRNA targeting BRCA2 substantially increased PD-L1 expression after IR Fig. . In a cllls Fig. . The enhlls Fig. . Furtherlls Fig. ; similarlls Fig. . In addi38. The Ku70/80 complex has two major roles in DSB repair. One is the recruitment of NHEJ factors to initiate the downstream part of the repair process. The other is to protect DNA ends from unscheduled digestion by DNA nucleases40. Since DSB-dependent PD-L1 expression requires Chk1 activity, which is activated by ATR on single-stranded DNA (ssDNA)-replication protein A (RPA), we hypothesised that the greater PD-L1 upregulation after IR in Ku80-depleted cells may be due to Chk1 overactivation following an increase in DNA end resection. To address this issue, we examined resection activity and Chk1 signal by monitoring the phosphorylation of RPA Ser4/8 (a resection marker) and Chk1 Ser345, respectively. Greater RPA/Chk1 phosphorylation was observed in Ku80-depleted cells after IR, suggesting that Ku80 depletion enhances DNA end resection and Chk1 activation  in U2OS and H1299 cells . We found that PD-L1 in tumours  with BRCA2, PALB2 or Ku70/80 mutations showed higher expression than in tumours without such mutations  showed that there was no significant difference in PD-L1 expression in TNBC between BRCA1 carriers and noncarriers49. In our screen, BRCA1-depleted cells did not show a significant increase in IR-induced PD-L1 expression compared with that in control cells. These paradoxical results can be explained by our model that Chk1 activation via DNA end resection is a key process in DSB-induced PD-L1 expression. Both BRCA1 and BRCA2 are required for HR, however, BRCA1 and BRCA2 have distinct roles in HR. BRCA1 promotes DNA end resection by relieving the barrier posed by 53BP1 in HR. Therefore, BRCA1 deficiency attenuates the DNA damage signal after IR unless the 53BP1 pathway is functioning normally51. In particular, the ATR/Chk1 signal is not effectively activated if the resection is impaired in BRCA1-defective cells. Notably, the loss of 53BP1, or the loss of RIF1 or REV7, rescues DNA end resection and HR in BRCA1-defective cells54. Disruption of the 53BP1 pathway, which rescues BRCA1 defect, was observed in subsets of sporadic TNBC. Notably, ~70% of BRCA-associated TNBC cases were found to revert to HR via an unknown mechanism. Therefore, the BRCA1 gene status alone may not be simply correlated with PD-L1 expression. On the other hand, BRCA2 is dispensable for resection, but is required for RAD51 loading following the formation of RPA on resected DNA ends. The protein switch from RPA to RAD51 is promoted by BRCA2, and the recruitment of BRCA2 on chromatin requires PALB2. Therefore, the defect of BRCA2 causes the stalling of HR repair at the step of RAD51 loading, but sustains activation of Chk1 at unrepaired DSBs. This effect was confirmed when BRCA2-depleted cells were treated with PARPi. PARP inhibition causes replication fork stalling, which requires BRCA2 to resolve and repair DNA. Notably, the addition of a Chk1 inhibitor cancelled the PARPi-dependent PD-L1 upregulation in BRCA2-depleted cells. Hence, we propose that a defect of DSB repair does not simply influence PD-L1 upregulation; however, the Chk1 activity plays a central role in the upregulation of PD-L1 after DSBs.A recent cohort study reported associations of CD3+ TILs and BRCA1/2 mutation status with survival38. In addition to this role, the Ku complex plays a significant role in protecting DSB ends. To remove the Ku complex from DSB ends safely without excessive resection, the initiation of resection is highly regulated by MRE11/CtIP endonucleases44. However, the lack of the Ku complex causes an increase in DSB end resection, which requires EXO1/BLM40. This increase in resection results in Chk1 activation. We found that depletion of the Ku complex caused high level of PD-L1 expression even without DNA damage. The greater endogenous PD-L1 expression (without DNA damage) in Ku80-depleted cells is likely due to the accumulation of replication-associated damage because the Ku complex also binds to replication-associated DSBs55. In addition to the upregulation of endogenous PD-L1 expression, we found a substantial increase in PD-L1 by Ku depletion after IR or Etp. An experiment using a Chk1 inhibitor clearly revealed that DSB-induced upregulation of PD-L1 in Ku-depleted cells is mediated by Chk1 following the excessive resection, supporting the notion that DSB-induced PD-L1 is regulated in resection-Chk1 pathway. Throughout the present study, we noticed that the magnitude of PD-L1 upregulation between mRNA and protein levels was not always perfectly matched. Because PD-L1 is controlled by post-translational modifications, such as ubiquitination56, and also multiple ubiquitin ligases and deubiquitinating enzymes are activated following DSBs57, the PD-L1 levels may be fine-tuned by the modifications depending on the cellular situation after DNA damage. Further studies are required to reveal the post-translational regulation for controlling PD-L1 expression after DNA damage. In an investigation of clinical specimens, reduction of Ku70/80 expression was observed in samples with the progression of melanoma58. In addition, BRCA2 mutations are enriched in melanomas responsive to anti-PD-1. Thus, downregulation of either Ku or BRCA2 or both may affect the efficacy of PD-1 therapy through PD-L1 upregulation by IR, and also via neoantigen pathway.Ku70/80 is a heterodimer protein that binds to DSB ends and promotes the recruitment of NHEJ proteins11. In addition, a meta-analysis reported that PD-L1 expression on cancer cells is one of the promising candidates as a selection marker17. A phase III clinical trial that involved only patients who had non-small-cell lung cancer with PD-L1 expression on at least 50% of tumour cells showed that pembrolizumab was associated with significantly longer progression-free and overall survival than chemotherapy12. These findings suggest that PD-1 therapy is effective in patients showing high PD-L1 expression. In the TCGA analysis, we showed a correlation between PD-L1 expression and mutations in the BRCA2, PALB2 and Ku70/80 genes. This data set analysis may not represent a direct correlation between PD-L1 expression and DSB signalling because the enhancement might largely be due to the high expression of neoantigens. However, because the statistical significance of the enhancement of neoantigens is less than that of the enhancement of PD-L1, we speculate that DNA damage signalling may partly contribute to the increase in PD-L1 expression in tumours when DNA repair and signalling are downregulated, particularly in patients who are treated with radio/chemotherapy.In previous studies, PD-1 therapy was shown to achieve remarkable clinical responses in patients with several types of cancer; however, ~20–40% of patients still remained poorly responsiveTaken together, in the present study, we showed that the depletion of BRCA2 or Ku80 upregulates PD-L1 expression in the DSB signal cascade. Our siRNA screen showed that the downregulation of DSB repair genes is not always associated with PD-L1 upregulation. Notably, we demonstrated that factors regulating Chk1 activity are important. Thus, BRCA2, PALB2 and Ku70/80 identified in this study could be good markers for predicting the efficacy of combined PD-1 and radio/chemotherapy, although a combined therapy may be sufficiently effective regardless of the gene status. In conclusion, we propose that understanding the signalling pathway underlying the response to DSB induction by radio/chemotherapy, which leads to PD-L1 upregulation, is of high importance to develop prognostic molecular markers and PD-1 blockade cancer immunotherapy. The factors could be pharmaceutical targets to influence the therapeutic efficacy when combined PD-1 and radio/chemotherapy is administered. These open questions will be investigated in future work.59. PARP inhibitor (PARPi)  was also added at 10 μM, and the medium was refreshed with 10 μM PARPi every 48 h until the indicated time point.U2OS, H1299 and DU145 cells were obtained from the American Type Culture Collection (ATCC). Cancer cells were cultured in Eagle’s Minimum Essential Medium (EMEM) with 10% foetal calf serum (FCS). X-ray irradiation was performed at 100 kVp and 20 mA with copper (0.5 mm)–aluminium (1.0 mm) filters . Dose rate was set at 0.5 Gy per min. Etp (Sigma-Aldrich), a topoisomerase II inhibitor, was added at 100 or 500 nM, or CPT (Wako), a topoisomerase I inhibitor, was added at 50 nM, and cells were incubated until harvesting. Etp directly causes DSBs, whereas CPT causes replication-dependent DSBs in S phase cells. APH (Wako), a replicative polymerase inhibitor, was added at 500 nM, and cells were incubated until harvesting. In addition, 10 μM ATM inhibitor (ATMi) , 10 μM ATR inhibitor (ATRi) , Chk1 inhibitor  was added at 30 min prior to DNA damage induction. A previous study has shown that 100 nM UCN-01 specifically inhibits Chk1 but not Chk2siRNA transfection was performed using HiPerFect (Qiagen). siRNA was added to suspended cells after trypsinisation. After 24 h, cells were re-transfected with siRNA in suspended cells after trypsinisation. Cells were incubated for 24 h after the second transfection before DNA damage induction. The sequences of the siRNA oligonucleotides used in this part of the study are listed in Supplementary Table 44. The uncropped versions of immunoblotting are shown in Supplementary Fig. i microscope by using NIS-Elements D imaging software with a 40 × objective via a DS-Qi2 camera. The mean signal intensity of PD-L1 in DAPI-negative areas was measured by ImageJ v1.48. Cells positive for PI, representing dead cells, were excluded from this analysis. Similar results were obtained in at least two independent experiments. The antibodies used are listed in Supplementary Table Immunoblotting was performed by using whole cell lysate−ΔΔCt method. qPCR settings were as follows: initial denaturation at 95 °C for 10 min, followed by 45 cycles of denaturation at 95 °C for 15 s, and annealing and extension at 60 °C for 1 min. The primers and probes used for the qPCR are listed below:Total RNA was extracted from cells using NucleoSpin RNA (MACHEREY-NAGEL) at the indicated time points after DNA damage induction. PrimeScript RT Reagent Kit  (TaKaRa) was used to reverse-transcribe cDNA from total RNA, in accordance with the manufacturer’s instructions. Quantitative PCR (qPCR) was performed using StepOnePlus (Life Technologies). Reactions (20 μl each) were prepared in duplicate in MicroAmp Fast Optical 96-Well Reaction Plate (Applied Biosystems). Each reaction contained 0.5 μM of each primer, 0.2 μM probe, 10 μl of Taqman Universal PCR Master Mix (Applied Biosystems) and cDNA as a template. The expression levels were normalised to GAPDH and calculated using the 2PD-L1 forward: 5′-GGAGATTAGATCCTGAGGAAAACCA-3′PD-L1 reverse: 5′-AACGGAAGATGAATGTCAGTGCTA-3′PD-L1 probe: 5′-AGATGGCTCCCAGAATTACCAAGTGAGTCC-3′PD-L2 forward: 5′-GCTTCACCAGATAGCAGCTTTATTC-3′PD-L2 reverse: 5′-CTCCAAGGTTCACATGACTTCCA-3′PD-L2 probe: 5′-CATTCCAGGGTCACATTGCTGCCATGCT-3′GAPDH forward: 5′-CTCCTCTGACTTCAACAGCGA-3′GAPDH reverse: 5′-CCAAATTCGTTGTCATACCAGGA-3′GAPDH probe: 5′-ATGCCAGCCCCAGCGTCAAAGGT-3′Cancer cells were incubated for 48 h after exposure to 10 Gy X-rays or PAPRi with or without inhibitors and then harvested for flow cytometry analysis. Adherent cells were harvested by shake-off in 1 mM EDTA-PBS without trypsinisation. Harvested cells were washed with ice-cold 1 mM EDTA-PBS and then stained with anti-PD-L1 antibodies for 20 min on ice. Dead cells detected by PI (Sigma-Aldrich) were excluded in the analysis. Flow cytometry analysis was performed on an Attune NxT Flow Cytometer (Thermo Fisher Scientific). The MFI (PD-L1–isotype) is calculated as: the MFI (PD-L1) is subtracted by the MFI (isotype control).http://tcia.at/). The studies for analysis were chosen based on the following two conditions: the number of cases with wild-type PD-L1  was more than 300 and the number of cases with mutations in each set of genes was more than 1% of the total N. Samples with or without mutations, including indel and point mutations, in BRCA2, PALB2, Ku70/80 or any of these four genes were compared using the Mann–Whitney U-test. Box plots were created using the BoxPlotR tool60. The samples that had the RPKM value = 0 were omitted from the log plots. The results of analyses of other tumours are shown in Supplementary Table The normalised RNA sequence and mutation status data provided by The Cancer Genome Atlas (TCGA) project were downloaded from the Genomic Data Commons Data Portal. The neoantigen data were obtained from The Cancer Immunome Atlas  upon reasonable request.Supplementary InformationPeer Review File


The spliceosome is a eukaryote-specific complex that is essential for the removal of introns from pre-mRNA. It consists of five small nuclear RNAs (snRNAs) and over a hundred proteins, making it one of the most complex molecular machineries. Most of this complexity has emerged during eukaryogenesis, a period that is characterised by a drastic increase in cellular and genomic complexity. Although not fully resolved, recent findings have started to shed some light on how and why the spliceosome originated.In this paper we review how the spliceosome has evolved and discuss its origin and subsequent evolution in light of different general hypotheses on the evolution of complexity. Comparative analyses have established that the catalytic core of this ribonucleoprotein (RNP) complex, as well as the spliceosomal introns, evolved from self-splicing group II introns. Most snRNAs evolved from intron fragments and the essential Prp8 protein originated from the protein that is encoded by group II introns. Proteins that functioned in other RNA processes were added to this core and extensive duplications of these proteins substantially increased the complexity of the spliceosome prior to the eukaryotic diversification. The splicing machinery became even more complex in animals and plants, yet was simplified in eukaryotes with streamlined genomes. Apparently, the spliceosome did not evolve its complexity gradually, but in rapid bursts, followed by stagnation or even simplification. We argue that although both adaptive and neutral evolution have been involved in the evolution of the spliceosome, especially the latter was responsible for the emergence of an enormously complex eukaryotic splicing machinery from simple self-splicing sequences.This article was reviewed by W. Ford Doolittle, Eugene V. Koonin and Vivek Anantharaman. Eukaryotic genes are in general composed of coding sequences interspersed by non-coding parts, the introns. Only after removal of these introns and splicing of the exons, a functional protein can be synthesised. The splicing reaction requires one of the most complex machines in the eukaryotic cell, the spliceosome, which consists of five snRNA molecules and over a hundred proteins , 2. Two The spliceosome is one of the numerous complex characteristics that emerged during eukaryogenesis. Eukaryotes are considered far more complex than prokaryotes, because of these evolved characteristics such as their larger genomes, cell sizes and intracellular compartmentalisation. However, some complex eukaryote-like features, such as large cells and internal membranes, have been observed in certain prokaryotes and some eukaryotes are less complex in organisation, cautioning for a too eukaryocentric view on complexity . It has The greater complexity of eukaryotes is additionally observed in the complexity of molecular machines, both for machines that are also present in prokaryotes  –11 and eMany steps were needed for the emergence of the complex spliceosome in the last eukaryotic common ancestor (LECA). The aim of this review is to reconstruct these steps and the subsequent changes in the complexity of the spliceosome in the distinct eukaryotic lineages, which is important for understanding why and how the complexity of this machine has evolved. This could additionally provide more insight into the evolution of other complex molecular machines. In this review we will focus on the snRNAs and main proteins of the major spliceosome.To separate the evolution of the spliceosome during eukaryogenesis from its evolution after the eukaryotic diversification, the spliceosome of LECA has to be reconstructed. The presence of spliceosomal components in all major eukaryotic lineages has revealed that LECA already had a complex major spliceosome, with five snRNAs and around eighty proteins . TherefoIt has become clear that this complex spliceosome had to remove numerous introns from LECA’s transcripts. Multiple approaches have been followed for reconstructing the introns in LECA . ThThe probable function of LECA’s spliceosomes can be inferred from experimental research on present-day spliceosomes, most of which has been performed in yeast, animals and plants. The main components of LECA’s major U2-type) spliceosome were the five small nuclear ribonucleoproteins (snRNPs), consisting of snRNAs and dozens of other associated proteins   proteins and heterogeneous nuclear RNPs (hnRNPs), present across eukaryotes , were inAlthough some earlier studies suggested otherwise , 27, addtrans-splicing, in which the SL RNA, which is carried by the SL snRNP, donates the first “exon” to the mRNA. This splicing mechanism is especially prevalent in some protist lineages, where it in some cases may account for all splicing events [trans-splicing can therefore not unambiguously be established.Most snRNPs of the major spliceosome are also involved in another related splicing reaction called spliced-leader (SL) g events . Based og events . Howeverg events  or recurg events –37. WhetLECA likely already possessed two spliceosome types to process two different kinds of introns. These spliceosomes were approximately as complex as the ones typically observed in present-day eukaryotes. This poses the question how the complex spliceosome evolved during eukaryogenesis. Where did the proteins come from, how were they recruited into the spliceosome and what functions did their prokaryotic homologues, if present, execute?The function of the spliceosome is removing introns from pre-mRNA molecules. The question how the spliceosome originated cannot be decoupled from the origin of the introns they remove. Without introns the spliceosome would be functionless and without the spliceosome the introns would cause the production of aberrant proteins. Different hypotheses have been proposed for the origin of spliceosomal introns. These will shortly be discussed before we turn to the origin of the spliceosome. Both the spliceosomal core and the introns themselves are likely derived from the very same origin, namely self-splicing introns.The similarities between spliceosomal introns and group II self-splicing introns have been recognised for a long time. The latter are present in prokaryotes and in eukaryotic organelles. In mitochondria and plastids these introns are bona fide introns that lost their mobility potential, whereas in prokaryotes they are more properly regarded as retroelements , 39. GroThere is an overwhelming amount of evidence supporting the homology between spliceosomal introns and group II self-splicing introns. The splice site recognition, branching mechanism, stereochemical course of the splicing reaction and the presence of similar RNA domain structures and a homologue of the IEP in the spliceosome (see below) demonstrate the similarities between the two intron types , 50, 51.Since group II introns are especially abundant in alphaproteobacteria and present in certain mitochondria, the most accepted view, first suggested by Cavalier-Smith in 1991 , is thatAs demonstrated by relatively recent intron gains, not all spliceosomal introns in present-day eukaryotes are derived from group II introns. These introns have an endogenous origin and different sources have been suggested, such as transposable elements, internal gene duplications and intronisation of translatable sequences . AlthougLactococcus lactis demonstrated the potential for trans-splicing [Numerous studies have noted the striking similarities in function and structure between the snRNAs and the group II intron domains and especially U6 snRNA and domain V look very similar Fig.  39, 40,,40, 51. splicing . These oAs mentioned above a group II intron usually encodes an IEP. A homologous protein of IEP functions in the spliceosome, namely pre-mRNA processing protein 8 (Prp8), which is present in the U5 snRNP. Prp8 is present in the spliceosomal catalytic core and likely functions as an assembly platform , 69, 70.Group II introns can be classified based on RNA structures or phylogenetic groupings of IEP , 79–81. Each snRNA in the spliceosome is accompanied by a heteroheptameric ring consisting of either Sm or Lsm proteins, which are both members of the Sm family of proteins. For U6 snRNA it is an Lsm ring made up of Lsm2–8, whereas the ring surrounding the other snRNAs consists of SmB, −D3, −G, −E, −F, −D2 and –D1 , 82–85. Homologues of Sm and Lsm proteins are present in both bacteria and archaea. The bacterial homologue, Hfq, is encoded by a single-copy gene . Hfq proAlthough an earlier study was unable to confidently infer the deep phylogenetic relationship between the eukaryotic Sm and Lsm genes , a more Presumably, it started with a homoheptameric flexible Lsm-like ring Fig. . A firstThe ATP-dependent RNA helicases in the spliceosome are mainly from three families within the SFII superfamily, which is especially predominant in eukaryotes . One of Another protein in U5 snRNP is the aforementioned GTPase Snu114, which interacts with Brr2 and Prp8 and is located near the catalytic site. Snu114 was already present in LECA  and is hThe SR splicing regulator proteins are characterised by an RNA recognition motif, which is also present in multiple prokaryotes, especially cyanobacteria . A phyloThe evolutionary history of many other spliceosomal proteins has been clarified to a lesser extent. The exact source of each component, i.e. whether it was present in the archaeal host, the bacterial endosymbiont, was acquired later via HGT or was a unique eukaryotic invention, is obscure as well. The aforementioned examples demonstrate that duplicates of proteins active in other RNA processes in the first stages of eukaryogenesis supplemented the group II intron core in the emerging spliceosome. Subsequent expansions of these protein families resulting in many paralogues within the spliceosome contributed to the vast complexity of the machine Fig. .Fig. 3EvSeveral papers put forward a speculative order of events that led to the emergence of the spliceosome. The starting point for these scenarios is the presence of self-splicing group II introns, including their maturases, in the host genome. For example, Anantharaman et al.  proposed2+ concentration in eukaryotes may have posed a barrier to group II introns in eukaryotic genomes, including protein-coding genes [The timing of the decay of self-splicing introns to spliceosomal ones, on the other hand, differs in these scenarios. Since group II introns have not been detected in nuclear genomes, all introns were apparently converted to spliceosomal introns or completely lost at some point before LECA. Complications with the expression of the targeted gene that arise when a group II intron is integrated in a nuclear gene were suggested to have caused their disappearance , 98. Howng genes , it doesng genes , 98. TheEvidently, much research has focused on the many steps leading to the complex nature of the spliceosome in LECA. Nevertheless, the lack of access to intermediate stages poses a challenge to precisely reconstruct the evolution of the spliceosome. The wide diversity of eukaryotic spliceosomes provides a rich source of complementary data that show both further complexification as well as simplification of the spliceosome Fig. . The occIn at least two lineages the spliceosome has become more complex. The most prominent complexification is the expansion of splicing regulator proteins, which are involved in the recognition of exons and introns, in plants and animals. The SR family has expanded in multicellular eukaryotes, especially in plants , 101. AnAlthough the high number of alternative splicing events in animals relative to other eukaryotes could be related to the expansion of the splicing regulator repertoire in these organisms , this isIn many other eukaryotic lineages the evolution of the spliceosome is characterised by simplification. Both the loss of some subunits and the complete loss of the spliceosome have occurred. Based on draft genomes, introns and spliceosomal genes seem to be completely absent in a few microsporidia species , 106 andCaenorhabditis elegans [In contrast with the aforementioned loss of both the major and minor spliceosome and corresponding introns, the loss of only the latter has been more common. The minor spliceosome is present in representatives of all eukaryotic supergroups, but has at least 9 times been lost during eukaryotic evolution , 30. Thi elegans . Losses  elegans .Saccharomyces cerevisiae [Giardia lamblia have characteristics of both major and minor spliceosomal snRNAs and therefore the reduced spliceosome of this organism is suggested to be a hybrid [Cyanidioschyzon merolae [C. merolae seems to perform splicing without a U1 snRNP, as both U1 snRNA and U1 snRNP-specific proteins appear to be missing [In addition to the complete loss of either the minor spliceosome or both types of spliceosomes, reduced spliceosomes have been observed and more thoroughly analysed in at least three lineages. This loss of spliceosomal subunits is associated with a lower number of introns –112. Numrevisiae , 102. Evrevisiae , 90. Thea hybrid , 113. Maa hybrid . A similNumerous scenarios for the evolution of the spliceosome have been suggested. Usually this concerns a description of what happened, but to truly comprehend the evolution of the spliceosome a transition has to be made from a mere description to addressing the evolutionary forces that shaped this complex machine. A number of hypotheses concerning these forces have been proposed, as mentioned in the introduction. They propose that either the addition or loss of each component of a complex is an adaptation or that solely neutral processes are responsible for the shifts in complexity.Since the establishment of the power of natural selection, adaptive explanations for biological observations have been the most prominent and widely accepted. Many biological papers propose an adaptive explanation for their observations, albeit often implicitly. Such explanations can in many cases be criticised as being just-so stories that lack proper evidence . The rolIn the constructive neutral evolution model the increase in complexity can be seen as a ‘drunkard’s walk’ into the more complex possibilities of a system . The con+-ATPase ring and suggested that this could have been the case in other multi-paralogue complexes as well. As the spliceosome comprises multiple paralogues, such as the Sm proteins and helicases [A well-established example of a similar neutral process resulting in increased complexity is subfunctionalisation of paralogues after gene duplication. A combination of constructive neutral evolution and subfunctionalisation may explain the formation of a heteromeric protein complex from a homomeric state. Finnigan et al.  demonstrelicases , 90, 93,It should be noted that it is difficult to classify an increase in complexity as neutral. As pointed out by Lynch , each emtrans-acting RNAs. These primordial snRNAs and an IEP that acts as a general maturase, which does not only assist splicing of its own intron, would have made the RNA domains of the introns and a dedicated IEP redundant. In this way previously deleterious mutations in the introns are now presuppressed by the action of this trans-acting RNP complex, resulting in the loss of self-splicing features. This primordial spliceosome would also allow the spread of inactive group II introns and intronised sequences unrelated to group II introns in the genome. The already established nucleus would have prevented aberrant protein synthesis upon invasion of the introns into protein-coding genes. The emergence of introns in genes would have made the eukaryotic lineage dependent on the spliceosome.In a neutral scenario the spliceosome would have evolved from the addition of new RNAs and proteins that do not improve the efficacy of the splicing reaction to the catalytic core inherited from the group II intron ancestor. Moreover, at some point the structural RNA elements in the group II introns were replaced by fragments of other group II introns that acted as Numerous proteins were added to the spliceosomal core during eukaryogenesis. Many of these are clearly derived from proteins that already had an RNA-binding function , 86, 93.Giardia has resulted in a significant loss of introns and spliceosomal components [The lack of clear direct benefits of a complex splicing machinery in the early eukaryotes is a strong argument against an adaptive scenario for its evolution. The only plausible direct benefits are compensations for maladaptive features. In light of the small effective population size inferred to have been present during eukaryogenesis based on the fixation of introns , 38 or pmponents , 113. ClThe scenario we infer corresponds to a biphasic pattern of evolution, in which a short explosive innovation phase is followed by a much longer reductive phase , 21. MosThe spliceosome is a complex molecular machine that arose during eukaryogenesis and removes introns from pre-mRNAs, which is required to prevent the production of aberrant proteins. The spliceosome consists of five snRNPs, each comprised of an snRNA and proteins, and additional proteins. There is ample evidence that both the spliceosomal core and the spliceosomal introns originated from self-splicing group II introns, which are widely believed to have been transferred from the mitochondrial endosymbionts to the host DNA. The snRNAs, at least U2, U5 and U6, are likely derived from fragmented group II introns and the U5-snRNP-specific protein Prp8 evolved from the IEP of these introns. Sm proteins, helicases and other proteins were at some point recruited to the spliceosomal core. This addition and the extensive expansion of especially Sm proteins and helicases have drastically increased the complexity of the spliceosome during eukaryogenesis. Apparently, all group II introns in the nucleus were either lost or converted to spliceosomal introns before LECA. During eukaryotic evolution a pronounced increase in spliceosomal complexity occurred in plants and animals, which mainly involved the regulatory proteins. In other lineages the spliceosome simplified, with U2 and U5 snRNP proteins being the least affected, and concomitantly the number of introns decreased.trans-splicing complexes from self-splicing group II introns, as they are less complex and have evolved more recently. Splicing facilitated by general maturases and other protein factors in plant organelles [trans-acting RNA and protein factors in the plastids of the green alga Chlamydomonas reinhardtii [Euglena [The spliceosome-like machineries involved in group II intron splicing in some eukaryotic plastids and mitochondria could be an interesting model for the evolution of ganelles  and by anhardtii , and sug[Euglena  are inteThe spliceosome is one of the most complex machines that emerged during eukaryogenesis. Other complex features that originated in the eukaryotic lineage are for example the nuclear pore complex, an elaborate endomembrane system, the RNA interference machinery and the kinetochore –126, to Reviewer comments:There is much detailed information here about the evolutionary history and likely prokaryotic origins of many components of the eukaryotic spliceosome. I assume that it is up-to-date and correct. We seem now to know quite a bit about this, and have little doubt that the last eukaryotic common ancestor (LECA) already had a modern-type splicing apparatus and many introns requiring its services . Nobody now seems to question the notion first mooted by Cavalier-Smith , that introns entered eukaryotic nuclear genomes as transfers of group II introns from the alpha-proteobacterial endosymbionts that became mitochondria, and, as RNAs, broke down into something like the “five easy pieces” described by Sharp (authors’ reference 66). My “introns early” hypothesis , appealing as it might have appeared in the late 1970s, is dead and buried.We thank the reviewer for the references he put forward on the historical embedding of this topic and we have added these, if not present yet, in the main manuscript. Although the alphaproteobacterial ancestry (viathe endosymbiont) of the spliceosomal introns is widely believed, we would like to caution that is based on circumstantial evidence. Alternative scenarios in which group II introns were already present in the Asgard-related host before the endosymbiosis event or were transferred from another donor after this event are also plausible. The inferred HGT events of organellar group II introns among organelles, between different eukaryotic groups and between bacteria and eukaryotes [weaken the link between mitochondrial group II introns and spliceosomal introns, as their presence in LECA’s mitochondria is not evident. This does not preclude the proposed effect of the mitochondrial endosymbionts on the available amount of energy and effective population size, which may have resulted in massive intron proliferation and the origin of the complex spliceosome.Authors’ response: karyotes , 79 weakBut there are many questions still to be answered, I think. For instance, why, if invasion of the nuclear genome by Group II introns from mitochondria happened before LECA, and eukaryotic nuclear genomes provide a wealth of targets (non-protein coding regions) where it could happen again, has it not? Inferred genetic and physiological barriers seem  too weak to explain total absence.This is indeed an important remaining question and we have included some additional sentences on this aspect in the manuscript. We agree that the proposed gene expression problems and lower Mg2+concentration do not provide a satisfying explanation for the exclusion of group II introns from the nuclear genome. Additional experiments, also in other eukaryotes, and a closer inspection of relatively recent transfers of group II introns to the nuclear genome, such as described in [, might elucidate this enigma in the future.Authors’ response: ribed in , might eAnother question worth pondering: it is relatively easy to imagine how selective and neutral processes could have given rise to the remarkable complexity of the spliceosome. Indeed, I particularly like how this paper gives credence to the latter, especially what has been called “Constructive Neutral Evolution” (authors’ references 9 and 15). Much harder to imagine is how a complex spliceosome, once it has become essential to the expression of most of the genes in a genome, could ever be simplified. And yet it has been, several times. This may be no more amazing than other instances in which what seem to be wired-in fundamental processes and structures can be completely dispensed with or radically transformed. Selection might be not nearly so important as we Darwinists want to believe, either in the building or the dismantling of complexity (author’s reference 18).Finally, and to me most interestingly, how can we combine multi-level selection theory with reasoning about introns as adaptations ? It may well be that multicellular eukaryotes of a certain type  have gained considerable evolvability (and consequent diversity) from having alternatively spliceable introns. But clearly, introns were not added to the genome of LECA so that more than a billion years later this advantage could be realized. Authors are  down on such teleological rationalizing, but might we imagine such evolvability to be an adaptation at some much higher level ?We did not mean to neglect or downplay the importance of multi-level selection and evolvability in the evolution of the complex nature of the spliceosome and introns. We have added a short discussion of this aspect at the end of “An interplay between neutral and adaptive evolution explains spliceosomal evolution” when we discuss the biphasic model.Authors’ response: Reviewer comments:In this review article, Vosseberg and Snel discuss the origin of the spliceosome that was initiated by the domestication of bacterial self-splicing introns. It is a subject of obvious importance and interest, and a long-standing, hard problem in evolutionary biology. The difficulty of the problem stems from the appearance of “irreducible” complexity: the most primitive eukaryotes we are aware of already have a (more or less) full-fledged spliceosome, with the implication that such was also the case for the LECA. Actually, as the authors emphasize, LECA most likely possessed both known types of spliceosomes, U12 and U2. The spliceosome is one of the best showcases for the evolution of eukaryotic cellular complexity because there can be no direct ancestors of the spliceosome in prokaryotes given the non-existence of splicing other than that of self-splicing introns. And, indeed, the authors summarize the relevant information and make a compelling case for the origin of both the spliceosome and the spliceosomal introns themselves from Group II self-splicing introns. In the discussion of the subsequent evolution of the splicesomes, the authors make a good case for a constructive neutral evolution scenario. I fully agree that, in the least, constructive neutral evolution is the appropriate null hypothesis for the evolution of the spliceosome and other complex eukaryotic features. The authors make a very interesting point about the relatively simple, spliceosome-like complexes that are involved in splicing of Group II introns in organelles. These are not ancestral but their evolution might recapitulate that of the spliceosome, so analysis of such complexes indeed might illuminate spliceosome evolution. On the whole, this is a very useful, interesting and insightful review, and a good read, too.We thank the reviewer for his excellent summary and appreciate the constructive feedback he has provided in his report.Authors’ response: I do not have particularly serious criticisms of this article. I find the discussion of the potential of phylogenetic analysis of the IEP and Prp8 (lines 230–237) to be rather disingenuous. I agree with the authors that such analysis is unlikely to be particularly informative. However, it seems to me that one should either try and actually do it or drop this line of discussion altogether.We apologize that our initial wording could be seen as disingenuous and we have decided to remove it.Authors’ response: In the discussion of the evolution of Lsm/Sm proteins, the following “A small number of introns are even shared between certain Lsm-Sm pairs. This is not trivial, as it implies that splicing could already take place before the diversification of the Sm family in eukaryotes” (lines 272–274) is indeed a non-trivial observation, and I think additional details are needed for the reader to be able to assess its validity and impact.We have added more details on these findings and discussed these in light of the low number of shared introns for paralogues originating from duplications during eukaryogenesis and the high number of shared introns for orthologues present in LECA. Upon mapping the location of introns onto the alignments of Lsm/Sm proteins in 22 eukaryotic species, Veretnik et al. [made the striking observation that for some pairs, and also between certain pairs, at the same position in the alignment an intron was located in multiple species for each paralogue, suggesting that this intron was already present before the duplication event. These findings have two major implications: 1) before the diversification of these proteins, which are essential for present-day splicing, splicing already took place; 2) these gene duplications likely did not occur early during eukaryogenesis.Authors’ response: k et al.  made theFor a review article, the current manuscript seems to be somewhat insufficiently referenced. In many case, the authors rely on previous reviews where several original references would do better. I will not suggest a full list of references to add and will only mention two: Along with ref. 96, the following should be cited: López-García P, Moreira D. Selective forces for the origin of the eukaryotic nucleus. Bioessays. 2006 May;28(5):525–33. The absence of this reference which presents a comprehensive reconstruction of intron gain and loss in eukaryotic evolution is surprising: Csuros M, Rogozin IB, Koonin EV. A detailed history of intron-rich eukaryotic ancestors inferred from a global survey of 100 complete genomes. PLoS Comput Biol. 2011 Sep;7(9):e1002150.Upon a critical reassessment of the references we have added 28 references to primary papers throughout the paper and we believe that the references are now more balanced. Initially, we did not include a discussion on the number of introns in LECA, but we agree that this was an omission and therefore added a small paragraph about this in the section "LECA’s spliceosome", including the latter reference. We did not discuss ref.96in light of the hypothesis on the origin of the nucleus, but for the scenario they propose for the origin of the spliceosome. In the revised version of this review we shortly mention the origin of the nucleus in the “Adaptive model” section, referring to both papers.Authors’ response: Further, in my view, there are too few figures in the article, and those included are too crude schematics. It would be good to show a better comparison between elements of self-splicing introns and snRNAs, and perhaps, a complete general scenario for the evolution of the spliceosome.Figure was drawn as a schematic figure on purpose, to immediately appreciate the similarities between group II and spliceosomal introns. Excellent figures with more details have been made before . We have added more details in Fig. by depicting also the RNA structures that are not similar and indicating that not the entire proteins are homologous. As suggested by the reviewer, we have also made a new figure  depicting the general scenario we propose with regards to spliceosome evolution pre- and post-LECA.Authors’ response: ig. 5 in ). We havReviewer comments:The authors have written a review of the evolution of the complexity of the spiceosomal machinery. They have touched on the various components of the spliceosome and their evolution. While there are many detailed discussions of this material available, this is a useful update summarizing the various ideas. Hence I find the review satisfactory and worthy of being published as is.The authors have presented a review of the prevalent ideas in the subject satisfactorily. I do not have any major recommendations.In pg 9–10 the authors discuss the loss of RT activity in Prp8 and point to a 2015 article. The inactive Prp8 and its possible link to Group II intron and spliceosomal evolution has been discussed in a much earlier 2012 paper (pmid: 22919680) by our group.The reference to the 2015 paper was actually for the last part of this sentence. We have added references to the suggested paper and another paper when discussing the loss of RT activity.Authors’ response:


We experimentally observed an asymmetric field screening effect relative to the polarization of the applied gate bias into the surface. Surprisingly, such behavior allows selection of the electronic states that screen external fields, and it can be enhanced with increasing of the number of layers of the semiconducting MoS2 rather than the semi-metal. This work not only provides unique insights on the screening properties of a vast amount of heterojunction fabricated so far, but also uncovers the great potential of controlling a fundamental property for device applications.A long-standing challenge facing the combination of two-dimensional crystals into heterojunction is the unknown effect of mixing layer of different electronic properties  on the screening features of the fabricated device platforms including their functionality. Here we use a compelling set of theoretical and experimental techniques to elucidate the intrinsic dielectric screening properties of heterostructures formed by MoS 2/graphene van der Waals heterostructures and identify an asymmetric electric response under different directions of the applied electric field.Charge density reorganization at the interface between 2D materials may lead to electric field screening. Here, the authors investigate the dielectric screening properties of MoS With the properties of the individual layers being characterized since the discovery of graphene and other 2D materials7, the great challenge is how to combine them in order to obtain unusual physical and chemical phenomena not observed on the original sheets1. The continuous development of experimental methods that allow atomically thin materials to be fabricated on-demand and to be placed on desired locations with an unprecedented control and accuracy have opened pioneering avenues to fabricate complex device architectures using regular bottom-up approaches4. The atomic flatness and lack of dangling bonds at the surface of 2D layered materials, such as graphene, boron nitride (BN) and transition metal dichalcogenides (TMDCs), allow them to form non-covalent interactions with a wide range of materials without the condition of lattice matching that normally heterojunctions would have. One of the first realizations of such vdWHs was the fabrication of BN/graphene interfaces and the probing of their electronic properties using gate bias8. Such system remarkably showed that graphene could develop superior electrical properties, achieving levels of performance comparable to those observed with freestanding layers. In this assembly, BN layers work as an underlying substrate screening out graphene from any dangling bonds, corrugations and charge inhomogeneities that are inherent to standards SiO2 surfaces to play a role on its electronic properties9. A direct extension of this system was the complete encapsulation of graphene, TMDCs and the combination of them between BN layers11. This rapidly showed that such device framework displayed improved transport properties with high-mobilities10. It is important to remark that in all these systems electrostatic gating schemes have been one of the main driving force to probe the chemical and physical properties of either isolated 2D materials before assembling, or their vdWHs afterwards. Electric gate bias measurements have become a feasible way to control, induce and manipulate electronic properties of almost any vdW crystals since graphene early days14. This relatively simple setup allows deep insight into the charge density reorganization between different stacking sequences, and on the electric-field screening, which determines most of the exquisite device properties observed in vdWHs. However, no direct measurements of the electrostatic screening features of different combinations of 2D materials have been reported yet, which ramp up further understanding and developments of vdWHs into any technological platform.The van der Waals heterostructures (vdWHs) composed of two-dimensional (2D) crystals and precisely assembled in a deterministic order constitutes a remarkable paradigm for promising electronic and optoelectronic applications with enhanced features and performance2, displays an asymmetrical behavior relative to the polarization of the applied gate bias into the surface allowing an asymmetric screening. Using electric force microscopy (EFM), we observe that graphene/MoS2 heterostructures exhibit distinct electric response to external fields with respect to the stacking sequence and the direction of electric field. A large depolarization field is recorded at the MoS2 side, which is shown to be dependent majority on the number of MoS2 layers into the heterostructure. A multiscale theoretical framework is developed for elucidating such stacking-dependent asymmetric electric screening phenomenon in graphene/MoS2 vdWHs. At the atomistic level, we employed quantum mechanical ab initio simulations based on density functional theory (DFT) using a total energy vdW functional to resolve various electronic properties of the vdWHs, including polarization, charge transfer and band structures. Our results indicate that the asymmetric formation of interfacial dipole moments preferably at the MoS2 facet accounts for the asymmetric response. We further show that such asymmetric behavior can be explained using a classical quantum capacitor model, described by a set of self-consistent electrostatic conservation equations and treating the 2D layers according to their individual electronic genomes . Our theoretical framework has good consistency with the experimental results at both ab initio and classical levels, showing that the combinations of 2D materials with distinct electronic structures can result in vdWHs with rich screening features. Furthermore, our theoretical framework is readily applicable for other vdWHs beyond graphene/MoS2 to explore a wide range of 2D material combinations with programmable electronic screening properties, which may greatly benefit the design of 2D vdWH-based functional devices.Here we show that the dielectric properties of an archetypal vdWH, involving graphene and MoS2 of different thicknesses on Au coated Si wafer were achieved by two rounds of polymethyl-methacrylate (PMMA) transfer15. Figure 2 (90 nm)/Si substrate. Their thickness was confirmed by the Raman spectra (514.5 nm wavelength) as shown in Fig. 16. The absence of Raman D band suggests the high quality of the graphene. Atomically thin MoS2 nanosheets were exfoliated on a SiO2 (270 nm)/Si substrate , which can be described as22:∂F/∂z is the local force gradient, representing the derivative of the electrostatic force felt by the cantilever tip; k is the spring constant of the cantilever; and Qcant is the Q factor of the cantilever. For simplicity, the interaction between the cantilever tip and the sample in EFM is often viewed as an ideal capacitance  due to the mismatch of the work functions between the tip and the sample. The raw data of the EFM spectroscopy from the Au substrate, 4L graphene, bulk MoS2, and three heterostructures, namely 1L MoS2/1L Graphene, 2L MoS2/4L Graphene, and 3L MoS2/4L Graphene are shown in Fig. y-axis as a function of the DC voltage. Considering the quadratic function in Eq. VCPD was fixed for each sample, the formation of the parabolic curves was due to the sweeping V. In other words, V was a dominant parameter in our EFM measurements. The opening-up means attractive capacitive interactions under both positive and negative voltages21. However, the different samples gave rise to slightly different shapes of the parabolas. This was caused by the other parameters, especially C and z. The distance z was inevitably slightly different from sample to sample during the EFM spectroscopy measurements. Although the local capacitance C is very difficult to define, as it depends on many factors, including the shape, size, conductivity, and dielectric property of a sample and cantilever, C was different from sample to sample. Therefore, it is understandable that the different samples resulted in the slightly different parabolic shapes in EFM spectroscopy.We used EFM to measure the electric field screening properties of the MoSres Fig. . Subtle 2/4L Graphene  of the 3L MoS2/4L Graphene heterostructure ; and a classical electrostatic approach using a capacitance model based on charge conservation equation solved variationally (see Methods for details). We first address the quantum mechanical part of the MoS2, respectively, but with a distinct number of graphene layers. Strikingly, only negative bias affected 2 sheets present in the heterostructures. This is in remarkable agreement with the experimental results, where an asymmetrical response was recorded only from graphene and few-layer MoS2 heterostructures , 2L (0.03) and 3L (0.05)), which indicates that thicker MoS2 tends to be more correlated with variations in the number of graphene layers. This follows the behavior observed from EFM measurements, which heterostructures involving thicker MoS2 sheets in contact with graphene gave rise to a more asymmetric EFM phase parabola . For negative fields towards MoS2 layers, the second term on the right-hand side in Eq. Eρ that would overcome the applied external bias. This resulted in smaller electric fields inside the heterojunction  tend to tune their Fermi level almost linearly with the electric field, which is a property intrinsically present at the pristine layers28. As the number of MoS2 sheets increased, ΔEFermi displayed variations at positive fields as large as 0.34 eV for 3L MoS2/3L Graphene but almost negligible for the negative electric field with the formation of a plateau at −0.2 V nm−1 and beyond. Such asymmetric behavior has been observed when MoS2 layers are used in metal-insulator-semiconductor junctions29. Carrier doping induced by the electric field was responsible for the variation of the Fermi level or the work function of MoS2, mainly along one direction, which is directly related to the unbalance of charge density between both sides of the semi-metal and the semiconductor interface. This indicates that the intrinsic character of the electronic structure of each system in vdW heterostructures contributes to the formation of the asymmetric screening observed. This effect has several main implications on the fundamental electronic structure of the MoS2/Graphene interfaces as can be appreciated on the band structure calculated at different magnitudes of gate bias for a sample system (e.g. 3 L MoS2/1L Graphene) in Fig. 2. At 0.0 V nm−1, the Fermi level crossed the Dirac point of graphene, as no charge-imbalance was present between both systems. Several MoS2 states at the conduction band were observed at 247.86 meV relative to the Fermi level, which also corresponds to the Schottky barrier presents at the interface30. At finite fields, those states are observed to shift up (down) with positive (negative) electric bias, with a consequent split as large as ~0.20 eV. This modifies their occupation, as some graphene states can become occupied (positive bias) or unoccupied (negative bias) according to the field polarization. The insets in Fig. 2 states near the Fermi level. This indicates that for electric fields toward the dichalcogenide layer, the states mainly composed of the conduction band of MoS2 with minor contribution from graphene were responsible for the charge-screening effect and vice versa. This suggests the important role of the interface on the electrical properties of the vdW heterostructures, as the polarity of the electric field can select, which states can screen the system against external bias.On the basis of the previous analysis, several implications on the electronic structure of the heterojunction can be foreseen. Figure eet Fig. , which gace Fig. . This choS2 Fig. . Thin sy2/Graphene vdW heterostructures under an external electric field can be well described using a classical electrostatic model, taking the quantum capacitance into consideration31. As the 2D vdWHs are stacked via non-covalent interactions, it is found that the individual properties of 2D materials can still be largely preserved in their stacked layers, which are coupled by the Coulombic interactions32. The idea behind our classical electrostatic model is that each individual layer has its own electronic “genome”  extracted from ab initio calculations, which can be used as building blocks in solving the electrostatic conservation equations of the whole vdWH, under the non-coupling assumption. Figure 2/Graphene vdWH used in the classical model. Due to the fact that there is no electron drift in the vdWH at equilibrium , the Fermi level EFermi aligns throughout the MoS2/Graphene vdWH. For simplicity of the model, we further assume that (i) the density of states (DOS) of individual layer is invariable with the stacking order and the external electric field and (ii) the interlayer distances (di) are not affected by the external electric field. Note that although the transition of band structure is ignored in assumption (i), it has been shown that such classical treatment using Coulombic coupling has relatively high consistency with the ab initio simulations32. The charge and potential distribution in the vdWH is solved by several conservation equations in a self-consistent approach33: (i) the charge of individual 2D layer Qi follows the charge conservation of the vdWH, (ii) Qi is determined by the electric displacement field adjacent to the 2D layer, (iii) Qi determines the work function of layer i, ϕi and (iv) the work function difference between two adjacent layers is determined by the electric displacement field. More details about the mean-field model can be found in Methods. For the simplest case of 1L MoS2/ 1L Graphene, we plot in Fig. ϕi of both materials, as a function of the external electric field strength (Eext) ranging from −8 to +8 V nm−1. Highly n-doped and p-doped MoS2 regimes can be found when Eext <−2.3 V nm−1 or Eext > 5.5 V nm−1, respectively. The fermi level of MoS2 shifts close to its conduction band (CB) or valence band (VB) in both regimes, respectively, which is accounted for the charge accumulation in the vdWH. Note that a noticeable charge accumulation (>1012 e cm−2) occurs even when the Fermi level of MoS2 is still ca 0.1 eV away from the band edges, due to the fact that the low quantum capacitance of graphene near its intrinsic Fermi level. On the other hand, when Eext is between −2.3 and 5.5 V nm−1, the Fermi level of MoS2 lies far away from the band edges, resulting that the vdWH is merely not polarized and the work function of graphene ϕGr shows little change with Eext. We find that the polarization of the MoS2/Graphene vdWH is more enhanced under negative external electric field, which corresponds with the findings in Fig. 2: MoS2 is considered as a n-type semiconductor with its intrinsic Fermi level (−4.5 eV) closer to its CB (−4.0 eV) than its VB (−5.8 eV)36, while graphene has symmetric linear band structure around its Dirac point (−4.6 eV)37, all energy levels are compared with the vacuum level that is set at 0 eV. Due to their close Fermi level values, little charge transfer occurs between graphene and MoS2 under weak electric field, and the degree of charge transfer is mainly determined by the position of the Fermi level with respect to the CB or VB of MoS2. Following the same procedure, we calculated the dipole moment δ , a considerable amount of total dipole moment can still be observed under weak electric field (|Eext| < 0.5 V nm−1), when the charge transfer between graphene and MoS2 is negligible (<1011 e cm−2). This indicates that the electric field is well screened by multilayer graphene under such conditions, since the DOS of graphene is finite around the intrinsic Fermi level. The screening in the MoS2 becomes more important only when the Fermi level reach the band edges, that is, when the DOS increases greatly. To verify such statement, we reconstructed the band diagram of 3L MoS2 /1L Graphene system under electric fields of −2 V nm−1 , that the local capacitance of the vdWHs can be varied under different electric field , here we further propose that such asymmetry can be described by the quantum capacitance, within both theoretical frameworks. The vdWH can be considered as a capacitor, characterized by the quantum capacitance (CQ), which is a function of the DOS at the Fermi level of the whole vdWH: CQ = DOS(EFermi)e2. The total DOS of the vdWH can be obtained quantum mechanically at high accuracy. On the other hand, the apparent quantum capacitance in the classical model can be calculated by the differentiating the charge density by the potential drop across the vacuum level ΔEvac  and DFT calculations (CQDFT) as functions of Eext in various MoS2/Graphene systems in Fig. −2 in 3L MoS2 /3L Graphene configuration, under an electric field of −1 V nm−1. We find the layer dependency of quantum capacitance is very similar to that of the dipole moment and charge transfer: the layer number of MoS2 is dominating the magnitude of the total quantum capacitance under strong electric field. This is reasonable due to the higher quantum capacitance of MoS2 than graphene when Fermi level shifts to the band edges33. Note that the DFT calculations predict a non-zero quantum capacitance of vdWHs with 2L and 3L graphene even without external electric field, as a result of the interlayer coupling, which is not included in the classical model. Despite the minute difference between both theoretical frameworks, it is clear that the quantum capacitance of the MoS2/Graphene vdWH, as a combination of the energy levels and DOS, can describe the asymmetric screening behavior with good precision. Our multiscale theoretical framework is thus readily applicable for a variety of vdWHs beyond graphene/MoS2, by utilizing the electronic “genome”, in particular, the quantum capacitances of individual 2D layers. A full picture of electric screening of 2D vdWHs can be built benefited from the framework proposed in this work, extending the “tip of the iceberg” of the electrostatic nature of two-layer 2D vdWHs revealed by recent studies40.The classical electrostatic model shows sound agreement with the ab initio calculations in predicting the asymmetric screening behavior of the MoS2. Graphene/MoS2 constitutes an archetypal of vdW heterostructure with exciting possibilities for electronic devices based on atomically thin films. We have shown an asymmetric electric field response on the screening properties of MoS2/Graphene stackings via high-resolution EFM spectroscopy and a multiscale theoretical analysis that involve quantum mechanical ab initio density functional theory, including vdW dispersion forces, and a classical electrostatic approach considering the quantum capacitance. Our ab initio calculations are further unified in a quantum capacitance-based model, showing that the difference between the energy levels and band structures between graphene and MoS2 is account for the asymmetric screening behavior. After the transfer of MoS2 on graphene, the screening of either isolated graphene or MoS2 changes accordingly to the sign of the electric bias utilized becoming polarity-dependent. The EFM phase spectrum shows an asymmetry with the tip voltage, as the number of MoS2 layers increases relative to that of the graphene. Electric fields towards MoS2 tend to be better screened than those directed to graphene as an asymmetrical polarization associated with charge transfer at the MoS2/Graphene interface, generate response fields that opposed to external bias. Such charge rearrangement also polarized the interface inducing the appearance of dipole moments and consequently giving a directional character to the underlying electronic structure. In particular, external fields in such vdW heterostructures can select which electronic states can be used to screen the gate bias, which clearly give an external control on the screening properties according to the stacking order and thickness. Our computational-experimental framework paves the way to understand and engineer the electronic and dielectric properties of a broad class of 2D materials assembled in heterojunctions for different technological applications, such as optoelectronics and plasmonics.In summary, our findings reveal fundamental knowledge of the screening properties of van der Waals heterostructures using widely used two-dimensional materials, such as graphene and MoS2 nanosheets were mechanically exfoliated by Scotch tape. Highly oriented pyrolytic graphite (HOPG)  and synthetic MoS2 crystals  were used as received. Si wafers with 90 and 270 nm thick thermal SiO2 were used for graphene and MoS2, respectively. An Olympus optical microscopy (BX51) equipped with a DP71 camera was used to search atomically thin nanosheets, and a Cypher AFM  was employed for topography measurements. The Raman spectra were collected by a Renishaw Raman microscope using 514.5 nm (for MoS2) and 633 nm (for graphene) lasers and an objective lens of ×100. For the fabrication of the heterostructure, the identified MoS2 nanosheets were firstly coated by thin layer of PMMA, then peeled off from the SiO2/Si via etching by NaOH, stacked on graphene on SiO2/Si under the optical microscope. The MoS2/Graphene structure was transferred to Au (100 nm) coated SiO2/Si substrate following a similar procedure. The Au coating was produced by a Leica ACE600 sputter with a crystal balance monitoring the coating thickness in real time. The EFM measurements were conducted on the Cypher AFM. The EFM phase data were collected by a Pt/Ti-coated cantilever with a spring constant of ~2 N/m  dwelling above the heterostructure at a sampling rate of 2 kHz, while a voltage sweeping linearly from +9 to −9 V was applied on the cantilever over a period of 20 s.The graphene and MoS41 and the VASP codes43. Projected augmented wave method (PAW)45 for the latter, and norm-conserving (NC) Troullier-Martins pseudopotentials46 for the former, have been used in the description of the bonding environment for Mo, S and C. The shape of the numerical atomic orbitals (NAOs) was automatically determined by the algorithms described in ref. 41. The generalized gradient approximation47 along with the DRSLL48 functional was used in both methods, together with a double-zeta polarized basis set in Siesta, and a well-converged plane-wave cutoff of 500 eV in VASP. We have explicitly checked the effect of different modifications on the exchange part of the vdW density functional on the interlayer distance between graphene and MoS2 as shown in Supplementary Table −8 eV Å−1. The self-consistent field (SCF) convergence was also set to 1.0 × 10−8 eV. To model the system studied in the experiments, we created large supercells containing up to 394 atoms to simulate the interface between different number of graphene and MoS2 layers. We have optimized the supercell for the MoS2/Graphene interface using a 5 × 5 graphene cell on a 4 × 4 MoS2 cell, where the mismatch between different lattice constants is smaller than ~2.0% (i.e. the systems are commensurate). We have kept the lattice constant of the MoS2 at equilibrium, and stretched the one for graphene by that amount. Negligible variations of the graphene electronic properties are observed with the preservation of the Dirac cone for all systems. To avoid any interactions between supercells in the non-periodic direction, a 20 Å vacuum space was used in all calculations. In addition to this, a cutoff energy of 120 Ry was used to resolve the real-space grid used to calculate the Hartree and exchange correlation contribution to the total energy in SIESTA. The Brillouin zone was sampled with a 9 × 9 × 1 grid under the Monkhorst-Pack scheme49 to perform relaxations with and without van der Waals interactions. Energetics and electronic band structure were calculated using a converged 44 × 44 × 1 k-sampling for the unit cell of Graphene/MoS2. In addition to this we used a Fermi–Dirac distribution with an electronic temperature of kBT = 20 meV to resolve the electronic structure.Calculations were based on ab initio density functional theory using the SIESTAEext=Ez across the vdW heterostructures is simulated using a spatially periodic sawtooh-like potential 2/Graphene heterostructures. Such potential is convenient to analyze the response of finite systems  to electric fields55, while problematic for extended systems . The magnitudes of the spatially varying electrostatic potential P(r) is calculated by the integration of l the thickness of the vdW heterostructure, and c the height of the supercell24.The electric field 2 layers are treated as individual layers, with the the band structures  considered as invariable to external electric field (i.e. the effect interlayer coupling on band structure is neglected). We further consider that the interlayer distance di between the i-1 and i-th layers is fixed, and taken as the interlayer distance in DFT calculations under zero field. The interlayer dielectric constant εi between the i-1 and i-th layer is considered as uniform. For simplicity, we consider that εi is independent of the external field, while the current model can be easily adapted for field-dependent dielectric constant in multilayer 2D materials using ab initio calculation results24. We consider the interlayer electric field Ei to be uniform. The charge density Qi and work function ϕi of each layer can thus be solved through the following conservation equations in a self-consistent way:In the classical model, the graphene and MoSThe charge neutrality of the vdWH:i-th layer by Gauss law:Charge balance of the Qi as a function of the work function ϕi of i-th layer:ϕi0 is the intrinsic work function of the i-th layer.The potential drop between 2 adjacent layers:CiQ,  = DOSi·e2. We simplify the quantum capacitance of graphene using a linear model: CiGraphene, = 26.1 μF cm−2 V−1 Δϕi, while the quantum capacitance of MoS2 is a step function, where no density of states exist within the band gap, while 2 are set at 4.6 and 4.5 V, respectively. The energy levels of CB and VB of MoS2 are taken as −4.0 and −5.8 eV, respectively.Note that for individual layers, The data that support the findings of this study are available from the corresponding author upon reasonable request.Supplementary InformationPeer Review File


Programmed cell death ligand 1 (PD-L1) is an immunosuppressive molecule expressed on tumor cells. By interacting with programmed cell death-1 (PD-1) on T cells, it inhibits immune responses. Because PD-L1 expression on cancer cells increases their chemoresistance, we investigated the correlation between PD-L1 and multidrug resistance 1/ P-glycoprotein (MDR1/P-gp) expression in breast cancer cells. Analysis of breast cancer tissues using tissue microarrays revealed a significant correlation between PD-L1 and MDR1/P-gp protein levels. Increased expression of PD-L1 was associated with lymph node metastasis and histological tumor grade. In addition, interaction of PD-L1 with PD-1 induced phosphorylation of AKT and ERK, resulting in the activation of PI3K/AKT and MAPK/ERK pathways and increased MDR1/P-gp expression in breast cancer cells. The PD-1/PD-L1 interaction also increased survival of breast cancer cells incubated with doxorubicin. These findings suggest that the PD-1/PD-L1 inhibition may increase chemotherapy efficacy by inhibiting the MDR1/P-gp expression in breast cancer cells. Programmed cell death ligand 1(PD-L1), also known as B7 homologue 1 (B7-H1) or CD274, is an immune checkpoint molecule belonging to the B7 family –6. PD-1,Breast cancer is diagnosed in nearly 1.4 million women every year and is a major cause of cancer-related death in women. The expression of PD-L1 is more frequent in basal breast cancers and basal type breast cancer cell lines , 12. HigInterestingly, recent reports indicate that PD-L1 also acts as an anti-apoptotic molecule on cancer cells. Knockdown of PD-L1 leads to an increase in spontaneous apoptosis and doxorubicin-induced apoptosis in breast cancer cells . PD-L1 eMDR1 gene, represents one of the mechanisms of how cancer cells reduce the intracellular concentration of anticancer drugs. P-gp is a 170 kDa plasma membrane glycoprotein that is a member of the ATP-binding cassette (ABC) transporter protein superfamily, and functions as an energy-dependent ATP efflux pump [/P-gp expression in breast cancer cells.Overexpression of P-glycoprotein (P-gp), coded by lux pump . P-gp islux pump . P-gp is/P-gp expression in breast cancer cells, and this up-regulation is mediated by the activation of PI3K/AKT and MAPK/ERK pathways.In this study, we demonstrate that the PD-L1 expression correlates with the MDR1/P-gp expression in breast cancer tissues. In the presence of PD-1, PD-L1 up-regulates the MDR1p < 0.01; Table Since MDR1/P-gp and PD-L1 play an important role in drug resistance, we investigated whether PD-L1 expression correlates with MDR1/P-gp in 150 breast cancer tissues. PD-L1 and MRD/P-gp protein expression was analyzed by immunohistochemistry (IHC) staining of tissue microarrays . The levp = 0.0009), and histological grade of tumors; PD-L1 was increased in grade I/II compared with grade III (p = 0.0022). In addition, Her-2 positive patients exhibited increased PD-L1 levels (p = 0.0043). No correlation was found with patients’ age, tumor node metastases (TNM) classification, and ER or PR status. We also investigated the correlation between PD-L1 expression and clinicopathological features in 59 triple-negative breast cancer (TNBC) patients. Increased expression of PD-L1 was associated with lymph node metastasis (p = 0.0362), suggesting that high levels of PD-L1 may promote lymph node metastasis in TNBC patients. Together, these data show that the PD-L1 expression is associated with lymph node metastasis, histological grade, and Her-2 status  compared with IgG1 Fc control. Exposure of IFN-γ-treated (24 h) T47D cells to PD-1-Fc increased the MDR1/P-gp mRNA level 3.03–fold .To investigate the contribution of PD-1/PD-L1 interaction to drug resistance, MDA-MB-231 cells were incubated with PD-1-Fc for 24 h prior to exposure to doxorubicin, and their survival was analyzed. As shown in Figure In this study, we found that the PD-L1 expression is increased in breast cancer tissues, and correlates with the expression of MDR1/P-gp. The increased expression of PD-L1 also correlates with lymph node metastases in breast cancer patients, histological grade of tumors, and their Her-2 status. Interaction of PD-L1 with PD-1 increases PI3K/AKT and MAPK/ERK signaling in breast cancer cells, resulting in the increased MDR1/P-gp expression.PD-L1 is up-regulated in many types of tumors. Previous studies mainly focused on the function of PD-L1 as a ligand that interacts with PD-1 on T cells. This interaction results in the inhibitory signal delivered to T cells, resulting in apoptosis and exhaustion of T cells –25. SeveMDR1 gene. In this study, we found a correlation between MDR1/ P-gp and PD-L1 expression in breast cancer tissues. We further showed that the PD-L1 expression on breast cancer cells increased the MDR1/P-gp expression in the presence of PD-1. In MDA-MB-231 cells, knockdown of PD-L1 with siRNA inhibited the MDR1/P-gp up-regulation after PD-1-Fc treatment, demonstrating that the up-regulation of MDR1 was due to the reverse signal derived from PD-L1 on breast cancer cells. To our knowledge, the present work is the first demonstration that PD-L1 reverse signal can up-regulate the expression of MDR1/P-gp.Drug resistance refers to the status of poor responsiveness of tumor cells to chemotherapeutic drugs. The main mechanism of drug resistance is overexpression of the ATP-dependent efflux pump, known as P-gp , which iPrevious studies have indicated that PI3K/AKT and MAPK/ERK pathways are involved in the P-gp biosynthesis and degradation , 30. OurOur results have shown that the PD-L1 expression in breast cancer tissues correlates with lymph node metastasis and HER2 expression, which is consistent with previous reports , 34. HowOne of the limitations of this study is that we did not evaluate correlation between PD-L1 and patient prognosis. Other reports have indicated that PD-L1 correlates with poor prognosis in breast cancer patients , 11. AnoIn conclusion, we have shown that PD-L1 binding to PD-1 up-regulates the MDR1/P-gp expression in breast cancer cells, indicating that this may contribute to the resistance to chemotherapy drugs in breast cancer patients. In addition, our results indicate that the PD-L1/PD-1-induced MDR1/P-gp expression is mediated by the activation of PI3K/AKT and MAPK/ERK pathways. Furthermore, we demonstrate that the PD-L1 expression in breast cancer tissues is associated with histological grade of tumors, lymph node metastasis, and HER2 status. Additional studies are needed to elucidate the mechanisms regulating MDR1/P-gp expression in breast cancer cells, and the extent of how MDR1/P-gp contributes to drug resistance. However, our present findings suggest that the PD-1/PD-L1 inhibition may increase chemotherapy efficacy by inhibiting the MDR1/P-gp expression in breast cancer cells.2. MDA-MBA-231 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, 100 IU/ ml Penicillin, and 100 mg/ ml Streptomycin.T47D cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 100 IU/ml Penicillin, and 100 mg/ml Streptomycin at 37°C in 5% COFlow cytometry analysis was used to access the cell surface PD-L1 expression. Cells were harvested and stained with phycoerythrin (PE)-labeled anti-PD-L1 antibody (BD Biosciences) or isotype control IgG for 30 min. The cells were washed with PBS twice and suspended in 500 µl of PBS. The cells were analyzed using BD FACSCaliber flow cytometer (BD Biosciences). FlowJo software (Tree Star Inc.) was used for data analysis.Cells were seeded in 35 mm wells, incubated 24 h with recombinant human IFN-γ  when the monolayer cell density reached ∼70% confluency, and analyzed for PD-L1 expression by flow cytometry. Cells were supplemented with human recombinant PD-1-Fc  or IgG Fc  for 24 h and harvested for further analysis.MDR1 mRNA were assessed using FastStart Universal SYBR Green Master (ROX) (Roche). Real-time PCR was performed on an ABI 7500 Real-Time PCR System. Relative gene expression levels were analyzed using the 2-ΔΔCt method.Total RNA was extracted using Trizol reagent (Sigma), and RNA concentration was determined using NanoDrop 2000c spectrometer (Thermo Scientific). cDNA was prepared with RevertAid Stand cDNA Synthesis Kit (Thermo Scientific); β-actin was used as an internal control. Levels of human Cells were lysed with RIPA lysis buffer (Beyotime Biotechnology). Lysates were cleared by centrifugation at 12,000 g for 5 min at 4 °C and the total protein concentration was determined by BCA Protein Assay Kit (Beyotime Biotechnology). The lysates were separated using SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked in 5% non-fat dry milk in TBST buffer for 1 hour at room temperature. The membranes were incubated with primary antibody followed by HRP-conjugated goat anti-rabbit secondary antibody (ZSGBbio). Bands were visualized using Pro-light HRP Chemiluminescent Kit (Taingen). Primary antibodies against MDR1/P-gp, ERK, p-ERK and p-AKT were purchased from Cell Signaling Technology. β-actin and AKT antibodies were purchased from Bioss.4 cells per well. After 18 to 24 hours, the monolayer cell density reached to the ∼50% confluency and the PD-L1 siRNA or negative control were transfected using GenMute siRNA Transfection Reagent (Signagen) according to the manufacturer’s instructions. Cells were cultured for 48 h and analyzed for membrane expressions of PD-L1.PD-L1 expression was knocked down in MDA-MB-231 cells using a specific siRNA (GenePharma). Non-targeting siRNAs were designed as negative controls (NC). Cells were plated in 24-well plates at 5×102O2 for 30 minutes, followed by serum blocking, and staining with anti-PD-L1 and anti-MDR1/P-gp antibodies overnight at 4°C. Then the slides were incubated with HRP-conjugated secondary anti-rabbit IgG at 37°C for 30 minutes. Slides were visualized with diaminobenzidine (DAB), counterstained with hematoxylin, and mounted. The percentage of stained cells was defined as follows: 0, negative; 1, 1%–10%; 2, 11%–50% and 3, 51%–100% of the cells. Immunostaining intensity was scored as follows: 0, no staining; 1, weak staining; 2, moderate staining; and 3, heavy staining. The immunostaining intensity scores were multiplied by the percent of cells stained to obtain the final staining score. The final staining score less than or equal to 4 was defined as ‘low expression’ and more than 4 was defined as ‘high expression’.Formalin-fixed and paraffin-embedded (FFPE) tissues from breast cancer patients were analyzed by tissue micro arrays (TMAs). The levels of PD-L1 and MDR1/P-gp in breast cancer samples were assessed using IHC with anti-PD-L1 (Proteintech) and anti-MDR1/P-gp antibodies (Proteintech). Antigen retrieval was performed in pH 6.0 citrate buffer  in microwave oven. Slides were incubated with 3% HMDA-MB-231 cells were plated in six-well plates and incubated with PD-1-Fc for 24 h. Then, the cells were incubated with doxorubicin (1 µM) or control DMSO for 1 hr. Cells were then seeded at various densities in triplicates. After 7–14 days, colonies were fixed with methanol, stained with dilute crystal violet, and counted.t test. Correlation coefficients (r) were calculated using the nonparametric Spearman rank correlation test. Differences with p < 0.05 were considered significant. Statistical analyses were performed with SPSS 17.0 statistical software .Quantification of target changes was performed using the two-tailed Student’s


The BER performance as a function of the relative time offset between three signals  is also evaluated showing favorable performance.We propose graphene-assisted multiple-input high-base optical computing. We fabricate a nonlinear optical device based on a fiber pigtail cross-section coated with a single-layer graphene grown by chemical vapor deposition (CVD) method. An approach to implementing modulo 4 operations of three-input hybrid addition and subtraction of quaternary base numbers in the optical domain using multiple non-degenerate four-wave mixing (FWM) processes in graphene coated optical fiber device and  quadrature phase-shift keying ((D)QPSK) signals is presented. We demonstrate 10-Gbaud modulo 4 operations of three-input quaternary hybrid addition and subtraction  in the experiment. The measured optical signal-to-noise ratio (OSNR) penalties for modulo 4 operations of three-input quaternary hybrid addition and subtraction  are measured to be less than 7 dB at a bit-error rate (BER) of 2 × 10 Graphene possesses the properties of tunable Fermi level, saturable photon absorption, and adjustable refractive index45101112et al.(3)). It also indicates that the nonlinear response of graphene is essentially dispersionless over the wavelength. Such nonlinearity of graphene can be utilized to realize various nonlinear functional devices for telecommunications, such as optical switches, wavelength converters, and signal regenerators. Optical bistability, self-induced regenerative oscillations and four-wave mixing (FWM) have been consecutively observed in graphene-silicon hybrid optoelectronic devices182018Graphene2Recently, spectrally efficient advanced modulation formats have been widely used in optical fiber transmission systems232425262729−3.In this paper, we propose an approach to performing modulo 4 operations of three-input optical addition and subtraction of quaternary base numbers using multiple non-degenerate FWM processes based on graphene coated fiber device. By adopting (D)QPSK signals , we demonstrate 10-Gbaud modulo 4 operations of three-input hybrid addition and subtraction of quaternary base numbers . The received optical signal-to-noise ratio (OSNR) penalties for modulo 4 operations of three-input quaternary hybrid addition and subtraction are measured to be less than 7 dB at bit-error rate (BER) of 2 × 10where the subscripts A, B, C, i1, i2, i3 denote input signal A, signal B, signal C, output idler 1, idler 2 and idler 3, respectively. Considering the phase wrap characteristic with a period of 2π, the linear phase relationships in In the experiment, the wavelengths of three-input signals A, B and C are fixed at 1548.52, 1550.12 and 1552.52 nm, respectively. −3 (7% enhanced forward error correction (EFEC) threshold). The increased OSNR penalties might be mainly due to the relatively low conversion efficiency for converted idlers and accumulated distortions transferred from three-input signals . The insets in To characterize the performance of the proposed graphene-assisted modulo 4 functions of three-input high-base optical computing, we further measure the BER curves as a function of the received OSNR for back to back (B-to-B) signals and three converted idlers. For the graphene-assisted modulo 4 functions of three-input high-base optical computing, we also study the performance tolerance to the relative time offset between three input signals. −3 are measured to be less than 7 dB for three converted idlers . The BER performance as a function of the relative time offset between three signals  is also evaluated and a favorable performance tolerance is achieved. With future improvement, graphene coated fiber device might be employed to facilitate more interesting optical signal processing applications.In summary, an innovative scheme to perform graphene-assisted modulo 4 functions of three-input high-base optical computing is presented. By exploiting multiple non-degenerate FWM processes in a single-layer graphene coated fiber device and adopting (D)QPSK signals, we experimentally demonstrate 10-Gbaud modulo 4 operations of three-input quaternary hybrid addition and subtraction of A + B − C, A + C − B, and B + C − A, respectively. The received OSNR penalties at a BER of 2 × 103 = 8 possibilities of the operations of A, B and C. We only demonstrate 3 of them in the experiment, i.e. A + B − C, A + C − B and B + C − A. Actually, the left 5 possibilities of the operations could be also implemented based on the same multiple non-degenerate FWM processes in graphene coated fiber device. For instance, when using –C instead of C as the input , one can easily get A + B + C, A − C − B, B − C − A. As a consequence, when selectively using –A/–B/–C instead of A/B/C at the inputs, it is possible implement all the 8 possibilities of the operations of A, B and C based on the same multiple non-degenerate FWM processes in graphene coated fiber device, i.e. A + B + C, A + B − C, A − B + C, A − B − C, −A + B + C, −A + B − C, −A − B + C, −A − B − C. Note that the conversion from C to –C can be achieved using conjugated degenerate FWM process which was demonstrated in our previouis worksRemarkably, for three-input optical computing, there are, in principle, 2Additionally, in order to verify the the enhancement of graphene coated fiber device, we measure the output spectrum without graphene for reference under the same experimental conditions. Moreover, we repeat the experiment by adding extra 2 m and 5 m single mode fibers in the setup and get almost the same experimental results. 3 solution. The resultant PMMA/graphene film (5 mm × 5 mm) is then washed in deionized water several times and transferred to deionized water solution or Si/SiO2 substrate. Then, the floating PMMA/graphene sheet is mechanically transferred onto the fiber pigtail cross-section and dried in a cabinet. After drying at room temperature for about 24 hours, the carbon atoms could be self-assembled onto the fiber end-facet thanks to the strong viscosity of graphene. The PMMA layer is finally removed by boiling acetone. By connecting this graphene-on-fiber component with another clean and dry FC/PC fiber connector, the nonlinear optical device is thereby constructed for multiple-input high-base optical computing applications based on non-degenerate FWM processes.In order to fabricate the nonlinear optical device based on a single-layer graphene, as illustrated in 2/Si substrate. The grown graphene sheet is also transferred on silicon-on-insulter (SOI) for SEM characterization, as show in −1, respectively. The obtained I2D/IG ratio of 1.65 demonstrates the formation of monolayer graphene2/Si substrate is almost defect-freeIn the experiment, optical image is taken with an optical microscope (Olympus DX51), Raman spectroscopy is performed with a laser micro-Raman spectrometer , and scanning electron microscopy (SEM) images are obtained by Hitachi-S4800. How to cite this article: Hu, X. et al. Graphene-assisted multiple-input high-base optical computing. Sci. Rep.6, 32911; doi: 10.1038/srep32911 (2016).