The demand for transparent conductors is expected to grow rapidly as electronic devices, such as touch screens, displays, solid state lighting and photovoltaics become ubiquitous in our lives. Doped metal oxides, especially indium tin oxide, are the commonly used materials for transparent conductors. As there are some drawbacks to this class of materials, exploration of alternative materials has been conducted. There is an interest in films of carbon nanomaterials such as, carbon nanotubes and graphene as they exhibit outstanding properties. This article reviews the synthesis and assembly of these films and their post-treatment. These processes determine the film performance and understanding of this platform will be useful for future work to improve the film performance. As electronic devices, such as touch screens, displays, solid state lighting and photovoltaic devices become more prevalent in our lives, the demand for transparent conductors increases. Doped metal oxides, especially indium tin oxide (ITO) ,2 are ofConducting polymers have exhibited good electrical, optical and mechanical properties . HoweverCarbon nanotube (CNT), a one-dimensional material, is extensively studied by various groups due to its attractive properties. CNT exhibits low electrical resistivity ,25 and hVarious techniques have been developed to synthesize CNTs. Some common techniques include arc discharge , laser aCarbon nanotubes (CNTs) can be dispersed in solution via three main ways: dispersion in organic solvents , dispersCarbon nanotubes (CNTs) are often dispersed in solution by ultrasonication. This step is essential as it breaks down the CNT aggregates to disperse them in the solvent. As the process is very harsh, defects are introduced, which can degrade the electrical conductivity of the CNT . BesidesCarbon nanotube (CNT) films formed directly via chemical vapor deposition (CVD) ,42 generAnother important factor that determines the carbon nanotube (CNT) film property is the characteristics of individual CNTs. Synthesized CNTs are not uniform in conductivity type, length and diameter. Longer and larger diameter metallic CNTs are more desirable for transparent conductor applications. The lower resistivity of metallic carbon nanotubes (CNTs) relative to semiconducting CNTs is attributed to the longer mean free path in metallic CNTs. Ballistic conduction in metallic CNTs can span up to micrometer range but semiconducting CNTs have a series of barriers to conduction along their lengths ,44. ResiBesides the lower sheet resistivity of metallic CNTs, the improvement in electrical conductivity of a film based on metallic CNTs only is attributed to the lower junction resistance between two metallic CNTs compared to junction resistance between one metallic CNT and one semiconducting CNT . The junHence, sorting of carbon nanotubes (CNTs) or selective synthesis of metallic CNTs is very crucial. Various groups have explored different means of sorting or selective synthesis, such as selective chemical functionalization ,51, seleLarger diameter CNTs are preferred for transparent conductors because they carry more current than smaller diameter CNTs ,57. In fav1.46 [Another important factor that influences the conductivity of the CNT film is the tube length of the CNTs ,63. The av1.46   and transmittance (T) of carbon nanotube network are related byDC and σOp are the electrical and optical conductivities respectively [DC/σOp is desired for a low sheet resistance and high transparency network. The electrical conductivity, σDC, is found to correlate with the network morphology. The mean diameter of the CNT bundles (bundled due to agglomeration of solution processed CNTs) has an inverse relationship with σDC. This is because the smaller the bundle, the higher the junction density and number of electrical pathways in the network. Hence, debundling of CNTs is desirable [Sheet resistance  grown CNTs has proven to be an effective method to transfer CVD grown CNTs. The method is deterministic and the CNTs can be completely transferred from a donor substrate to a receiving substrate . It is eAfter the assembly of the CNT film, post deposition treatment is often carried out to improve the electrical conductivity by improving the junction resistance between two CNTs ,71, remoThe junction resistance between two CNTs is expected to decrease when they are joined. Two CNTs can be joined by Joule heating . By cont3 [The electrical conductivity of CNT network can also be improved by removing surfactants often used to disperse CNTs in solution. Some surfactant residues are deposited together with CNTs during CNT film assembly. As the surfactant is insulating, the electrical conductivity of the CNT film is compromised. Hence, after CNT film assembly, removal of the surfactant improves the electrical conductivity. Surfactant can be removed by washing with water, followed by acetone  or by ac3 .2 shifts the Fermi level closer to the valence band and conductivity improves [3, SOCl2 or I2 also prove effective [Another commonly used method to improve the electrical conductivity of CNT network is by doping. Various types of dopants have been studied and found to improve the electrical conductivity by different extents. Doping of CNTs by vapor phase reactions with bromine (electron acceptor) or potassium (electron donor) has yielded decreased resistivity by a factor of 30 . Doping improves . P dopinffective ,77. Anotffective ,79. HoweGraphene, the two-dimensional allotrope of carbon, has generated much interest because of its high mobility , transpaSolution processed graphene films have been studied extensively because their production via roll-to-roll processing can potentially be scaled up for commercial applications. There are two main forms of precursors to graphene in solution: graphitic precursors ,84,85,86et al. [i.e., the width and length) tend to be <400 nm, which will increase the number of inter-flake interfaces when a large surface area film is formed from such dispersions. The large number of inter-flake interfaces present will decrease the electrical conductivity of the film formed. Thin films can be formed from graphite dispersions via various techniques like vacuum filtration [As graphite is hydrophobic, surfactants are often used to assist the graphite to disperse in organic solvents. Lotya et al.  demonstrltration ,85, spraltration ,85 or Laltration . Another group  reportedAnother form of precursor to graphene in solution is graphite oxide precursors. By oxidizing graphite via the Hummers method  or a modet al. compared the sheet resistance and transparency of films formed via various reduction treatments [Becerril eatments . Three teatments . et al. also reported that incomplete reduction has detrimental effects on the electrical properties of reduced graphene films [3 bonds with the carbon atoms in the basal plane. The sp3 bonds disrupt the transport of charge carriers delocalized in a sp2 network. Hence, the electrical conductivity is lower in a partially reduced film relative to a completely reduced film. Mattevi ne films . The reset al. [Besides complete reduction of graphene oxide films, large graphene sheets are desired as they decrease the inter-flake interfaces. Large-scale graphene sheets with an area up to 20 × 40 µm have been demonstrated by Tung et al.  as shownThe main advantage of solution processed films is their compatibility with large scale and low cost roll-to-roll processing. The key disadvantages are the incomplete reduction of graphene oxide and the small dimensions of the graphene sheets formed. Development of methods to overcome these issues will result in films with better electrical conductivity, which is an important figure of merit for transparent conductors.Graphene films can also be formed by chemical vapor deposition (CVD) growth. The process usually involves breaking down a gaseous carbon feedstock  in hydrogen gas at high temperature on a metal catalyst to form a graphene film. Graphene film growth via CVD is very appealing because large scale growth is possible and the electrical conductivity of CVD grown graphene films is generally better than that of solution processed graphene films. et al. [et al. has also demonstrated very outstanding sheet resistance of 30 Ω/sq and 90% transmittance. High quality graphene films with dimensions up to 30 in have been demonstrated by Bae et al. . This isDespite the impressive progress in the field, some challenges remain to be overcome. CVD grown graphene films that demonstrate very low sheet resistance are often doped chemically. However, chemical doping is unstable and the sheet resistance of graphene films increases with time. This will be discussed in greater detail in a subsequent Grain boundaries in graphene films have been found to decrease the local work function, leading to potential barriers that scatter charge carriers by both backscattering and intervalley carrier scattering . The caret al. reported an approach that achieves grain size up to tens of micrometers by pre-patterning seed crystals to control the graphene nucleation [Yu cleation . In the et al. found that high temperature, low partial pressure and methane (carbon feedstock for graphene growth) flow rate during CVD growth result in fewer nucleation sites. Hence, they proposed a two-step CVD process [Other groups have also adopted a similar strategy of decreasing the nucleation sites to increase the grain size and decrease the grain boundaries by various clever methods. Li  process . The firMillimeter-sized grains have been demonstrated by various groups ,107,108 Besides grain boundaries, wrinkles in graphene films can impede electrical transport across the folds . WrinkleA graphene film grown via chemical vapor deposition (CVD) is often grown on a metal foil or metal film on substrate. Graphene film, which is used for transparent conductor applications has to be transfer printed from the growth substrate to a transparent substrate. Polymeric substrates are often used as they are flexible and transparent. It is not feasible to grow graphene film directly on a polymeric substrate via CVD because the polymeric substrate degrades under the high temperature employed during CVD. In this section, we discuss the various techniques developed to transfer print graphene films and relate the transfer printing process to the quality of the film transferred. The transfer printing process ,119,120 et al. reported dry and wet transfer techniques using PMMA, as illustrated in Various types of supporting substrates have been investigated. They include poly(methyl methacrylate) PMMA ,115, polIn the dry transfer process, PMMA is spin-coated onto graphene film grown on copper foil. A PDMS frame was used to support the PMMA/graphene film sandwich while etching the copper foil using ammonium persulfate. This copper etchant is preferred over iron(III) nitrate, because it does not leave behind contamination residues like iron oxide. After the copper foil is completely etched, the graphene film is transferred to a receiving substrate. Heat treatment above the glass transition temperature of PMMA is performed after the graphene transfer to improve the contact between the graphene film and the receiving substrate. This is achieved by softening the PMMA so that the gap between the graphene film and the receiving substrate is reduced. The improved adhesion of the graphene film to the receiving substrate prevents cracks and tears from forming when the PMMA is removed. The wet transfer process is similar, as illustrated in et al. [Liang et al.  also repi.e., ~275 Ω/sq), but subsequent transfers quickly decrease the sheet resistance. Hence, it was postulated that the adhesion between the first layer of film transferred directly onto the receiving substrate and the receiving substrate is poor, which results in mechanical damage of the film when the thermal release tape is removed. Hence, the sheet resistance is poor. Subsequent transferred layers do not interact directly with the substrate, so the sheet resistance is lower.The importance of adhesion between the transferred graphene film and receiving substrate is emphasized in . When trIt is evident that the transfer printing process of graphene films to an arbitrary receiving substrate strongly influences the quality of the transferred film. This is because the transfer process can introduce residual contaminants and mechanical damage to the transferred film . Hence, 3 in nitromethane, HNO3 in nitromethane and HCl. These strong oxidizing agents withdraw electrons from the graphene film and increase the doping density, leading to a decrease in sheet resistance [3 in nitromethane, the doping effect is transient. The sheet resistance increases by ~100% after 80 days in ambient condition at room temperature [x, also suffers sheet resistance degradation with time [i.e., only 8% decrease in sheet resistance). Typical sheet resistance of a transferred (via PMMA) graphene film is ~125 Ω/sq. This sheet resistance is low enough for certain applications, such as touch screens. However, when integrated into a solar cell or large area display, it will not be suitable as the series resistance is too high. Wet doping agents can be used to enhance the electrical conductivity of the graphene films ,122,123,perature . Anotherith time . A self-et al. reported a novel method of using nonvolatile ferroelectric polymer poly(vinylidenefluoride-co-trifluoroethylene) (P(VDF-TrFE)) gating to decrease the sheet resistance of the graphene film [Ni ene film . The advAlthough single layer of chemical vapor deposition (CVD) grown graphene film has relatively low sheet resistance <500 Ω/sq, the search for a scalable, non-volatile and highly effective method to dope graphene continues so that graphene films can be used in more commercial applications . The scaVarious groups have also explored hybrid films ,133,134.As demand for transparent conductors in flexible devices is expected to be high in future, alternative materials to substitute metal oxides are sought. Films of carbon nanomaterials have been identified as one potential class of substitute materials. More work remains as the sheet resistance of carbon nanomaterial films is still unable to reach <10 Ω/sq at 90% transparency refer to  below, wNonetheless, the films, which have been demonstrated may be suitable for less demanding applications, such as touch panels. Further exploration in their stability in devices over time is necessary. The scalability, reproducibility and cost effectiveness of integrating them into practical devices also have to be carefully evaluated. Finally, the success of practical application of carbon nanomaterial films in transparent conductors is also dependent on the development of competing alternative materials, such as thin metal films, metal nanowire films, conducting polymers and various other forms of hybrid films.


The U3 small nucleolar RNA (snoRNA) is an essential player in the initial steps of ribosomal RNA biogenesis which is ubiquitously present in Eukarya. It is exceptional among the small nucleolar RNAs in its size, the presence of multiple conserved sequence boxes, a highly conserved secondary structure core, its biogenesis as an independent gene transcribed by polymerase III, and its involvement in pre-rRNA cleavage rather than chemical modification. Fungal U3 snoRNAs share many features with their sisters from other eukaryotic kingdoms but differ from them in particular in their 5’ regions, which in fungi has a distinctive consensus structure and often harbours introns. Here we report on a comprehensive homology search and detailed analysis of the evolution of sequence and secondary structure features covering the entire kingdom Fungi. The U3 small nucleolar RNA (snoRNA) is a box C/D snoRNA with an exceptional structure. Like many other snoRNAs it is involved in pre-rRNA processing. However, in contrast to typical box C/D snoRNAs ,2 it doepansions . In addipansions .Saccharomyces cerevisiae), while the hinge between the first and the second hairpin binds to the 5S. cerevisiae U3 genes, which are excised by the canonical pre-mRNA splicing machinery [Neurospora crassa [Fungal U3 snoRNAs deviate from the eukaryote consensus in particular close to their 5a crassa . In conta crassa . So far,a crassa ,12,13 arhttp://www.bioinf.uni-leipzig.de/publications/supplements/16-018). The 54 fungal U3 snoRNAs from 52 distinct organisms reported in [muscle [infernal v1.1 [muscle alignments comprising both previously published U3 sequences and the homologs detected in the initial infernal search. Novel sequences found by these CMs were furthermore added to the alignments.We surveyed a total of 147 fungal genomes from Microsporidia, Mucoromycotina, Blastocladiomycota, Basidiomycota, and Ascomycota; a complete list is supplied in the Supplemental Material  database.Structural models were computed and iteratively improved using mLocARNA , RNAz [1NA [RNAz , as well package  as well RNAduplex a component of the ViennaRNA package [RNAduplex does not handle complex internal structures of the interaction partners. As a control we used random sequences with the same length and nucleotide background distribution as the 18S rRNA fragments.Base pairing between U3 and potential target sites were computed with  package  designed package . Homologmeme [Introns were identified using meme  to retri species  reports Schizosaccharomyces japonicus and Allomyces macrogynus with 11 and 10, resp., nearly identical paralogs. Duplications are evolutionarily recent and appear to be species- or genus-specific. There is no evidence for diverged families of paralogs. Instead, paralogous copies are presumably subject to concerted evolution [U3 snoRNA genes were identified in all 147 genomes included in this study, supporting the indispensable nature of U3 in rRNA biogenesis. We found a total of 310 genes, i.e., the U3 snoRNAs, similar to snRNAs, tend to appear in multiple genomic copies. Almost all investigated genomes contain three or fewer copies. Notable exceptions are volution  similar The secondary structures of U3 snoRNAs vary substantially between kingdoms [Rhodotorula minuta has a 50 nt insert in the loop of stem M6, the yeast Kluyveromyces lactis shows an even larger 80 nt insert in M8, while another yeast, Candida glabrata has an extra stem of about 50 nt between M9 and M10.The characteristic sequence motifs offer no surprises in the fungal kingdom. Motif ATACTY is nearly perfectly conserved, while A = GYATCW is more variable. Both C and CRTGATGA typical for box C/D snoRNAs. As in other box C/D snoRNAs, box CWYGATGA. The pattern for box B is quite variable at both ends but conserves the core motif AGYGA. Box D = CTGA is very stringently conserved and matches the pattern perfectly in almost all of the 310 sequences. Not unexpectedly, box DThe guiding potential of the predicted U3 snoRNA genes can be quantified by the number of base pairs and the interaction energy between the 5Kluyveromyces genus [Baudoinia compniacensis, Cucurbitaria berberidis, and both U3 genes in Rhodotorula graminis). In eight genomes we observed multiple copies of the U3 gene of which at least one is intronless and another one contains an intron, e.g., Coprinopsis cinerea, a basidiomycete, carries two U3 transcripts where one is intron interrupted. The remaining seven species are spread over Dothideomycetes , Leotiomycetes , and Sordariomycetes .Of the 310 genes from 147 organisms 138 from 78 genomes are interrupted by at least one intron. Intron-bearing genes are not found in early branching lineages such as Microsporidia, Mucoromycotina, Chytridiomycota, or Blastocladiomycota. They are broadly distributed across the Basidiomycota but not present in Taphrinomycotina. In Pezizomycotina they are found in the vast majority of analyzed organisms. Among the Saccharomycotina, only Saccharomycetaceae have introns. U3 genes both with and without introns are present in the es genus . A more GT-AG3GC-AG3Pyrenophora tritici-repentis and 5GC-GG3Nadsonia fulvescens. Notably, GC-AG junctions have been observed a common exception also in large surveys of fungal introns [GTRWGT, where the underlined nucleotides are absolutely conserved. Almost all of the remaining donor have a mismatch in exactly one of the other three positions. All except one acceptor sites share YAG with a strong preference of CAG over TAG (129 versus 8 introns). The patterns conform very well to the consensus motifs derived from large sets of introns [AYRCTR [TRACRC in 129/138 cases. The distance between branch and acceptor sites is strongly constrained, varying between 6 to 36 nt in fungal intron [S. cerevisiae and N. crassa [The U3 introns have an average length of 92 nt, approximately matching the typical intron length in most fungi . Almost  introns ,25. Near introns ,26. The  [RCTRAY . The prel intron ,27. The Baudoinia compniacensis, which is inserted in stem M3 upstream of box CThe introns are inserted almost exclusively in the most-5 motifs  . 133 of Penicillium or Saccharomyces genera, for example exceed 75%. Between Aspergillus and Penicillium we still observe between 40% and 60%. On the other hand there is also evidence for intron sliding [The tight clustering of intron locations in the U3 snoRNA sequence suggests that at least large groups of the introns are homologous, dating back to the same insertion event. Due to the rapid rate of sequence evolution in introns direct evidence for a common origin can be obtained from sequence similarity however only for closely related species. Introns with the  sliding . The intS. cerevisiae strains, which found that typically effects of individual mutations are small but may have very large epistatic effects acting in particular to maintain base pairing patterns [The U3 snoRNA is much better conserved in fungi and in eukaryotes in general than all other box C/D snoRNAs. Despite the presence of several important, well-conserved sequence motifs and well conserved core secondary structure elements, it shows extensive variability and clade-specific peculiarities. Among its well-preserved key features is also the propensity of its 5patterns .Fungal U3 genes are the only snoRNAs that are frequently interrupted by introns. These share canonical pre-mRNA splice sites and are clearly processed by the spliceosomal machinery. Introns have been inserted almost exclusively in the 5U3 introns appear only in the crown group fungal lineages. Introns may have been inserted only a single time in fungal history . If thisAlthough the 5


In addition, survival analysis showed that DLBCL patients who co-expressed PD-L1 and p-AKT had significantly poorer outcome than patients with single positive or both negative expression (P<0.05). In vitro, total PD-L1 and membrane PD-L1 (mPD-L1) proteins were overexpressed in five DLBCL cell lines by western blot and flow cytometry. We observed that AKT/mTOR pathway was activated in DLBCL cells after stimulated with human recombination PD-1/Fc. Taken together, these results suggested that the combination of PD-1/PD-L1 antibodies and AKT/mTOR inhibitor might be a promising and novel therapeutic approach for DLBCL in the future.Programmed death-1 (PD-1) /programmed death-ligand 1 (PD-L1) engagement usually leads to diminished antitumor T-cell responses, which mediates the immune escape of tumor cells. However, little is known whether PD-1/PD-L1 could directly activates intracellular oncogenic signaling pathways in tumor cells. The purpose of this study is to investigate whether intracellular AKT/mTOR signaling could be directly activated by PD-1/PD-L1 during the malignant progression in diffuse large B-cell lymphoma (DLBCL). Detection of the expression of PD-L1 and p-AKT by immunohistochemistry (IHC) showed that both proteins were overexpressed in 54% and 48% DLBCL cases, respectively. Spearman test showed that PD-L1 expression was correlated with p-AKT expression (R=0.244, χ Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma (NHL) accounting for approximately 30-40% of newly diagnosed NHL cases . It is aProgrammed death ligand 1 , a member of the B7 family, is an important ligand of programmed death 1 , which is an immune inhibitory receptor expressed on the surface of T cells, B cells and monocyte upon activation . RecentlInteraction between PD-1 and PD-L1 leads to the suppression of T-cell function and further facilitates immune escape of tumor cells. However, apart from evading host immunity, little is known about the intracellular signal transduction in tumor cell after PD-L1 binding to PD-1. PD-L1 is a type I transmembrane protein with 290 amino acids, consisting of immunoglobulin V-like and C like domains, a hydrophobic transmembrane domain, and a cytoplasmic tail domain . SeveralIn this retrospective study, due to lack of tumor tissue or clinical data of some cases, only 100 DLBCL patients between Jan 2008 and Dec 2011 in our hospital were evaluable. Clinical characteristics of the patients were summarized in Table Positive PD-L1 expression was found in 54 DLBCL cases (54%) by IHC. Representative IHC staining patterns of negative and positive PD-L1 expression were given in Figure 2=5.260; P=0.026, Table 2=5.702; P=0.027, Table 2=5.962; P=0.017, Table We also found that positive PD-L1 expression was significantly associated with pathological subtype  and 5 years OS (P=0.007) compared to patients with both negative expression of PD-L1 and p-AKT (Table Median follow-up duration was 52.4 (ranging from 1.5 to 89.1) months. All of the 100 patients were available for the 3-year overall survival analysis, however only 72 patients could be done for the 5-year overall survival analysis. The results revealed that 3-year and 5-year overall survival rates were 73% and 59.2%, respectively. Clinically, some DLBCL patients were only treated with CHOP/CHOPE. But other DLBCL patients were treated with standard R-CHOP. Considering the influence of different therapy schemes on DLBCL patient prognosis, we separately analyzed 61 patients who adopted CHOP/CHOPE regiment and 39 patients who adopted R-CHOP regiment. Kaplan-Meier survival curves of 61 patients who adopted CHOP/CHOPE regiment showed that DLBCL patients with either positive PD-L1 or p-AKT expression corresponded with a significantly shorter 3 years OS  and 5 years OS (P=0.014) compared to patients with both negative expression of PD-L1 and p-AKT , pathological pattern , clinical stages , IPI scores , chemotherapy regimens , PD-L1 expression , p-AKT expression , co-expression of PD-L1 and p-AKT  were associated with poor prognosis in DLBCL patients. To determine the prognostic value of PD-L1 and p-AKT expression, multivariate cox regression models for comparison with prognostic factors were applied. We found that clinical stages , chemotherapy regimens , PD-L1 expression  and p-AKT expression  were the independent prognostic factors negatively impacting survival.Subgroup analyses were performed according to gender, age, pathological pattern, clinical stages, IPI scores and chemotherapy regimens, PD-L1 expression, p-AKT expression and co-expression of PD-L1 and p-AKT. The results of univariate and multivariate cox analysis were summarized in Table In short, PD-L1 expression was correlated with p-AKT, and the co-expression of PD-L1 and p-AKT would result in a poorer prognosis compared with single positive or both negative expression of PD-L1 and p-AKT in DLBCL. Even PD-L1 and p-AKT were the two independent prognostic factors negatively impacting survival.We selected five human DLBCL cell lines to determine the total PD-L1 expression by western blot and mPD-L1 expression by flow cytometry. The results indicated that all of the five DLBCL cell lines expressed PD-L1 Figure  and mPD-To further determine whether PD-1/PD-L1 binding could directly activate the intracellular AKT/mTOR signaling in tumor cells, DLBCL cell lines were treated with human recombinant PD1/Fc for 24h and 48h. Total and phosphorylated AKT expressions were evaluated by western blot. Results revealed that p-AKT level was significantly up-regulated in three DLBLC cell lines including DHL-16, U2932 and Ly-3, suggesting that the intracellular AKT/mTOR pathway in DLBCL cells was activated Figure . Taken tP =0.0323) [P<0.05). Therefore, PD-1 or PD-L1 blockage might be a novel therapeutic approach for DLBCL and other tumors. Several clinical trials carried out in the past two years had revealed that both anti-PD-1 antibodies  and anti PD-L1 antibodies (such as MPDL3280A) had shown significant antitumor effects in patients with metastatic melanoma [With the advent of rituximab, the OS of DLBCL patients has been significantly improved. However, still 30~40% DLBCL patients inevitably develop into relapsed/refractory disease. So further studies are needed to investigate the underlying mechanism and develop novel therapeutic approaches for DLBCL. Recent studies have highlighted the role of PD-1/PD-L1 pathway in negatively regulating host antitumor response. As the major ligand of PD-1, PD-L1 is widely expressed and correlated with poor prognosis in many human cancers, including melanoma , lung ca=0.0323) , which wmelanoma , bladdermelanoma , and lunmelanoma .P=0.033, P=0.028), and R-CHOP for 5-years OS (P=0.023), but not R-CHOP for 3-years OS (P=0.051).DLBCL tumors have several important oncogenic signaling pathways which make DLBCL to be a heterogeneous disease and impact overall survival of DLBCL patients. The AKT/mTOR signaling pathway has been demonstrated to be constitutively activated in DLBCL , and AKT2=5.962; P=0.017). In addition, we found that the DLBCL patients who co-expressed PD-L1 and p-AKT had the worst clinical outcome compared with the patients who were single positive or both negative expression of PD-L1 and p-AKT for 3-years and 5-years OS (P<0.05). So we hypothesized that PD-1/PD-L1 binding might directly activate the intracellular AKT/mTOR oncogenic signaling in tumor cells to promote DLBCL progression. To confirm this hypothesis, we selected five DLBCL cell lines, which overexpressed PD-L1 protein and mPD-L1, to be stimulated with human recombinant PD-1/Fc protein for 24h and 48h. Interestingly, we found that level of p-AKT protein was significantly up-regulated with a time-dependent manner, suggesting that PD-1/PD-L1 binding could directly activate the intracellular AKT/mTOR oncogenic signaling in DLBCL tumor cells. Otherwise, Parsa et al found that expression of the gene encoding PD-L1 increased after loss of PTEN gene and activation of PI3K pathway in human glioma [In our study, spearman test showed that PD-L1 expression was correlated with p-AKT expression in DLBCL tissues in the Tianjin Medical University Cancer Institute and Hospital  from Jan 2008 and Dec 2011 were studied. Archived FFPE tumor tissues were obtained from our Department of Pathology and these cases were reclassified according to the 2008 WHO classification and Hans algorithm by experienced hematopathologists. In addition, 10 specimens of normal lymph gland tissue obtained from patients with reactive hyperplasia of lymph node were used as normal controls. All clinical information was obtained by reviewing the patients’ medical charts. The study and all protocols below were approved by the Ethics Committee of TMUCTH.IHC staining for PD-L1 and p-AKT proteins were performed using the streptavidin–peroxidase method (SP method). Briefly, the paraffin-fixed slides were dewaxed in xylene and rehydrated through graded alcohols. Antigen retrieval was respectively carried out using EDTA buffer (pH 8.0) for anti-PD-L1 and citric acid buffer (pH 6.0) for anti-phospho-AKT (Ser473) in 120°C for 2 minutes and 30 seconds. Endogenous peroxidase activity was blocked using 0.3% hydrogen peroxide for 10 minutes at room temperature in dark place. Nonspecific binding of the primary antibody was blocked by incubating the slides with 10% normal horse serum for 30 minutes at 37°C. Then they were incubated with the primary antibodies including rabbit anti-PD-L1 polyclonal antibody  and rabbit anti-phospho-AKT (Ser473) polyclonal antibody  at 4°C overnight. And then they were incubated with secondary anti-rabbit IgG/HRP at 37°C for 30 minutes. Subsequently, for visualisation of the antigen, the sections were performed with the chromagen 3, 3′-diaminobenzidine. The slides were counterstained with hematoxylin and mounted under coverslips.Percentages of PD-L1 positive tumor cells and staining intensity were evaluated for each slide. Staining for PD-L1 was considered high expression, if ≥5% of the tumor cell population showed 2+ or 3+ membrane staining. In addition, if ≥20% of the total tissue cellularity showed 2+ or 3+ membrane or cytoplasmic staining in malignant and/or nonmalignant cells, it was considered to have a microenvironment positive for PD-L1 . p-AKT eThe DLBCL cell lines: TMD8, U2932, SUDHL-16 were cultured in RPMI-1640 medium  containing 10% fetal bovine serum . OCI-LY3 (LY3) and OCI-LY7 (LY7) were cultured in IMDM medium  supplemented with 15% fetal bovine serum . All medium were supplemented with 1% penicillin-streptomycin and all cells were incubated at 37°C in 5% CO2.For flow cytometry, DLBCL cell lines were harvested, washed, and then labeled with fluorochrome-conjugated PE anti-Human CD274   for 30 minutes at 4°C in dark place. Then the mPD-L1in all samples was measured by flow cytometry . Results were analyzed with FlowJo software (version 7.6).All five cell lines were cultured in complete medium containing 100ug/ml human recombinant PD1/Fc  for 24h and 48h. And then total proteins were isolated and run using western Blot to detect the total AKT and p-AKT expression.Cells were harvested and suspended in RIPA lysis buffer containing 1 mM phenylmethylsulfonyl fluoride (PMSF). 20-50μg total protein (depending on different proteins) were separated by sodiumdodecyl sulfate–polyacrylamide (SDS–PAGE) gel electrophoresis and then transferred to polyvinylidene difluoride membranes , which were then blocked in 5% non-fat milk in Tris-buffer saline-Tween (TBST) for 1 h. The immunoblotting was performed by incubation with the primary antibodies including PD-L1 , p-AKT and total-AKT , and β-actin , which was used as an endogenous protein for normalization at 4°C overnight. Blots were then washed and incubated with a 1:3000 dilution of Goat anti-Rabbit IgG H&L (HRP)-conjugated secondary . Signals were detected by enhanced chemiluminescence Plus reagents  and formed images by Chemiluminescence Imaging System. Signal quantification was obtained using Quantity One software  and normalized to β-actin.Clinical, pathological, and chromosomal charac- teristics of the patients were compared using chi-square tests or Fisher's exact tests. The Kaplan-Meier method was used to estimate the over survival (OS) distributions, and the log-rank test was performed to compare the survival difference. Univariate analysis were performed with log-rank test. Multivariate-adjusted Cox regression models were used to evaluate independent prognostic factors. Difference was considered significant when the P value was <0.05 and all reported P values were two sided. These statistical analyses were performed with SPSS 17.0 statistical software .


In this work, the relative dielectric permittivity of graphene oxide (GO), both its real and imaginary parts, have been measured under various humidity conditions at GHz. It is demonstrated that the relative dielectric permittivity increases with increasing humidity due to water uptake. This finding is very different to that at a couple of MHz or lower frequency, where the relative dielectric permittivity increases with decreasing humidity. This GO electrical property was used to create a battery-free wireless radio-frequency identification (RFID) humidity sensor by coating printed graphene antenna with the GO layer. The resonance frequency as well as the backscattering phase of such GO/graphene antenna become sensitive to the surrounding humidity and can be detected by the RFID reader. This enables battery-free wireless monitoring of the local humidity with digital identification attached to any location or item and paves the way for low-cost efficient sensors for Internet of Things (IoTs) applications. It was established that oxygen functional groups in GO drive intercalation of water molecules between individual GO sheets, which results in an increase in the inter-layer spacing in GO thin films5. The presence of inter-layer water in a GO film can be crucial for a number of its properties, such as electrical conductivity7, molecular permeation9, mechanical10 and dielectric properties11.Graphene oxide (GO) is a chemical derivative of graphene functionalised with hydroxyl and epoxy groups. GO is a hydrophilic material and is capable to absorb a significant amount of water. Its water uptake depends strongly on the humidity of the environment and was previously studied by X-ray and neutron diffraction and 12. Of particular interest would be the relative dielectric properties of multi-layered GO as a function of water uptake, considering the low intrinsic relative permittivity εr of GO and the high 12. While the equivalent circuit technique works well at low frequency, it is not suitable for high frequency, such as at GHz, due to parasitic effects. In this work the GO relative dielectric permittivity was obtained based on the measured transmission and reflection parameters (S-parameters) at GHz. There is no need of equivalent circuit model for the GO.Multi-layered GO electrical and dielectric properties under various humidity conditions have been studied at low frequency6. We experimentally determine both the real and imaginary parts of the GO relative dielectric permittivity at GHz. The findings in this work are very different to those reported in12 – both the real and imaginary parts of the GO relative dielectric permittivity decrease with decreasing humidity, from ∼17 at 100% RH to 12 at 10% RH  and from 6 at 100% RH to 2 at 10% RH (the imaginary part of the relative permittivity). In particular, the imaginary part  method, free space method, resonator cavity, etc.14. However, all these methods do not suit permittivity measurement for small and thin piece of GO under different humidity environments.The electrical property of GO can be completely characterized by its relative dielectric permittivity, Here, to measure the relative permittivity of the GO layer under various humidity conditions, a resonator circuit was designed Fig. , with GO16. The saturated salt solutions used were LiCl (RH-11%), K2CO3 (RH-43%), Mg (NO3)2 (RH-55%), NaCl (RH-75%) and K2SO4 (RH-98%) aqueous solutions prepared by dissolving excess amount of salts in deionised water. Before each measurement with each particular salt, the humidity was set to be stabilised for at least 48 hours. All measurements are done at 24 °C. When the electrical property of GO (such as its permittivity) changes with humidity, it alters the loading of the resonator and results in a shift of the resonance frequency as well as change of the backscattering phase.Both the GO and the calibration circuits were placed in a hermetic container  for the samples with and without printed GO layer are displayed in Fig. The measured transmission coefficients S1 for theFrom the measurement results, it becomes clear that the sample with GO layer has responded to the humidity change, whereas the sample without GO hasn’t. The different responses of these two resonators can only be caused by the change of GO electrical properties due to water uptake. For the resonator with GO layer, it can be observed that the resonance shifts to lower frequency and its fractional bandwidth increases as the humidity rises. This reveals that both the real  antenna. Instead of using GO capacitor to sense the humidity12, the phase shift of the backscattering signal due to the humidity change was detected by the RFID reader. The GO sensor is battery-free, wireless and fully printable. Battery-free wireless sensing is in the heart of IoTs technology19, allowing collection of information about the immediate state of the object without the need of batteries. Here we demonstrate a battery-free RFID humidity sensor by combining printable graphene RFID antenna with GO coating.It’s well known that GO is sensitive to humidityRFID antennas are sensitive to changes in the environment due to proximity effects. When a layer of GO is printed on top of an RFID antenna – the resonance frequency of the latter becomes sensitive to the permittivity of the GO layer (and thus to humidity) as it alters the antenna impedance. Other parameters, such as the phase of the backscattered signal, change as well, which can be easily detected by the RFID reader. The operating principle and use of the phase of the backscattered signal to measure the relative humidity (RH) with the GO-coated printed graphene RFID antenna, shown in Fig. 21.When a RFID reader transmits an electromagnetic wave signal  to an RFID antenna, the antenna draws energy from this forward signal and activates the RFID chip on the antenna. The backscattered signal is both amplitude and phase modulated by the RFID chip through varying the chip’s input impedance. Modulation occurs as the RFID chip rapidly switches between two discrete impedance statesThe operating principle and equivalent circuit for the antenna’s amplitude and phase modulation are schematically shown in Fig. Va on the antenna in Fig. 22PAnt is the power available at the antenna port, Za is the antenna impedance. The switching between the two input impedance states ZC1 and ZC2 generates two different currents at the antenna port, which can be calculated as:22Different to a normal antenna, impedance of a RFID antenna is typically designed to conjugately match to the higher impedance state of the chip in order to maximize the collected power. The equivalent open source voltage 23). The resistance of the GO coating is still several orders of magnitude higher than the resistance of the printed graphene RFID antenna in this experiment (fractions of Ohm24), so the effects of the change of the GO conductivity can be ignored and only the change in dielectric property are taken into account. The GO dielectric property change alters the antenna impedance Za. As Za changes so do I1and I2, causing the backscattered signal phase varies accordingly. The backscattered signal phase change can be detected by the RFID reader. In this work, the backscattered signal phase was measured using Voyantic Tagformance under various humidity conditions and depicted in Fig. 25.When the humidity changes, the GO layer on the RFID antenna changes its dielectric property. At high humidity, the ionic conductivity due to the intercalated water increases and even pristine GO becomes conductive but only poorly (mega ohms resistance at 100% RH and Giga ohms at 0% RHRH) can be observed, unambiguously demonstrating the effectiveness of wireless printed graphene enabled battery-free RFID GO humidity detection.From Fig. 28. In those reported works, the minimum power-on-tag was measured and the resonance frequency was then extracted from the minimum power-on-tag. This technique requires the reader to scan the whole allocated UHF RFID frequency spectrum and post-measurement data processing to find out the minimum power-on-tag and resonance frequency. In this work, the backscattered signal phase was measured. The advantage of measuring the backscattered signal phase is that there is no need for the reader to scan the whole allocated frequency spectrum in order to find out the resonance frequency. As it can be seen in Fig. It is worth to notice that the technique used here to detect the humidity change is very different to that employed in other reported printed battery-free UHF RFID sensors7) and decreases with decreasing humidity at GHz. Furthermore the dielectric property has been used to design and build a RFID sensing tag which can act as a battery-free wireless humidity sensor, by coating GO layer on top of the printed graphene RFID antenna. Such combination can form bases for future energy harvesting enabled RFID sensors for IoTs applications. Furthermore, backscattered signal phase rather than minimum power-on-tag or resonance frequency has been used to detect the humidity change, which can significantly simplify and speed up the monitoring process.We have experimentally extracted the GO relative dielectric permittivity under various humidity conditions at GHz. The measurement results clearly reveal that the GO dielectric property  changes with the humidity but in a different manner as it does in a couple of MHz or lower frequency. Most distinguishingly, the relative dielectric permittivity does not have large changes , etc. The S-parameters were used to extract the relative dielectric permittivity in this work.30. In brief, 4 grams of graphite was mixed with 2 grams of NaNO3 and 92 mL of H2SO4. KMNO4 was subsequently added in incremental steps in order to achieve a homogeneous solution. The temperature of the reaction was monitored and kept near 100 °C. The mixture was then diluted by 500 mL of deionised water and 3% H2O2. The resulting solution was washed by repeated centrifugation until the pH value of the solution was around 7. The GO was then diluted to the required concentration. Lateral size of GO flakes is about 500 μm × 500 μm.Modified Hummers method was employed to prepare GO. The typical oxygen content for GO produced by this technique is around~ 30–40%31.The water uptake with two different flake sizes (0.5 μm and 10 μm) has been measured by monitoring the weight change of GO exposed to different humidity conditions. The results show that the small and large flakes only have a few percentage decrease in mass uptakes indicating similar hydration behaviour (see Supporting Materials, Fig. 32. The lateral SEM view of the GO coated on printed graphene on paper substrate is shown in Fig. For the purpose of coating GO on a printed graphene RFID antenna, a 10 grams per litre viscous GO solution was used. This allowed direct screen printing of the GO on the antenna, which was left to dry overnight in a fume hood under continuous air flow. The printed graphene RFID antenna is made with screen printing and rolling compressionAll data generated or analysed during this study are included in this published article and its supplementary information file.Supplementary Information


Staphylococcus aureus (S.aureus), and acceptable antibacterial ability against Escherichia coli (E. coli). Meanwhile, antibacterial efficiency closely correlates with coverage area of graphene film, and larger graphene coverage always leads to better antibacterial performance. The underlying mechanism is thought to be the integrative action of phospholipids disturbance and electron extraction at the interface between graphene and biomembrane. Meanwhile, the electron extraction action would further lead to the activation of platelet. This study might provide some new insights into the relationship between antibacterial ability and hemocompatibility based on graphene functionalized biomedical device.Germanium (Ge), as an elemental semiconductor material, has been an attractive candidate for manufacturing semiconductor microelectronic device. In the present investigation, to improve the biocompatibility of Ge-based device, graphene film is directly deposited on the Ge surface with different coverage area by controlling the growth time. Compared to bare Ge, the presence of graphene film entitles Ge with satisfactory antibacterial ability against  The rapid developments of biotechnology, biomaterials and tissue engineering have resulted in advances in medical diagnostics. Of particular significance are the bio-electronic devices . Nevertheless, the biocompatibility is always a crucial issue which limits the feasibility and longevity of the implantable bio-electronic device235Considering the sensibility of physiological environment, biomaterials and biochips remain subjected to the host immune response against foreign bodies342 hybridized carbon atoms packed in two-dimensional honeycomb structure, has been one of the most fascinating nano-materials ascribed to its exceptional physical and chemical performance1314151516171819Graphene, as a single-atom-thick sheet of spOur previous works have proved that relatively perfect monolayer graphene films can be directly deposited on Ge surface by CVD2021−1), the 2D peak (~2700 cm−1) and the defect related D peak (1350 cm−1), is shown in 232D/IG) above 3 is observed in 3-hybridization peak located at 285.1 eV, which comes from natural carbon contamination2-hybridization peak at 284.6 eV as well as a slight decrease in the intensity of the sp3 peak can be observed in 2 hybridization of the C 1 s spectra dominates (The graphene films are deposited on the Ge(110) substrate by atmospheric pressure chemical vapor deposition (APCVD). ominates , suggestCorrosion resistance is one of the significant factors to be considered in course of practical application of biomedical devices. In order to evaluate the impact of the graphene coverage area on the corrosion resistance of the underlying Ge, the dynamic potential curve test was conducted in the physiological saline (0.9 wt% NaCl solution). As depicted in Staphylococcus aureus (S.aureus) and Gram-negative Escherichia coli (E. coli) are used as model bacterium to inoculate on the surface of graphene coated Ge substrate. In order to better visualize the interactive behaviors of bacteria when contacting with graphene covered Ge substrate, Live/Dead Kit is utilized for bacteria fluorescent staining. As shown in S.aureus significantly. Meanwhile, the damaged bacteria display even more remarkable tendency, and observation vision is filled with intense red fluorescence derived from the listless and dead bacteria due to the damaged cell membrane, as depicted in the middle panel. However, the obvious increase in the proportion of the dead bacteria is observed on the Half-Gr where the surface is covered with graphene films, although not completely covered. Furthermore, massive damaged bacteria fill up the whole surface of Full-Gr. However, in case of E. coli, the red spots which refer to the dead bacteria (in web version) on the None-Gr are barely observed, while the surface is occupied by green spots, which refer to the living bacteria (in web version), indicating the inability of None-Gr to inactivate E. coli. The dead bacteria are hard to be observed on Half-Gr either , and only appear on Full-Gr with trace amount. Therefore, compared to E. coli, S.aureus is more susceptible to the presence of graphene coverage on Ge substrate.Gram-positive S.aureus and E. coli which are detached from the surface of the None-Gr are observed on the agar plate. However, when the bacteria contact with the Half-Gr surface, the number of S.aureus colonies reduces significantly, while the number of E. coli colonies remains high. In addition, the number of S.aureus colonies continues to decrease on the Full-Gr. Meanwhile, the number of E. coli colonies which are detached from Full-Gr ones also present a partially decrease compared to that from the Half-Gr ones. The result of reduction of Alamar blue in In addition, the bacterium solution is re-inoculated on the agar plate after detaching from the surface of incubated specimen to further incubation for 16 h, and the typical photographs is depicted in S.aureus on the None-Gr surface basically maintains original spherical appearance whereas those on the Half-Gr surface are disintegrated with severely distorted shape. Furthermore, the glomerate bacteria on the Full-Gr surface exhibit apparently deathlike state due to the crack of membrane. Meanwhile, the evident residue of exudative cytoplasm appears around the bacteria swarm. The similar manner is also observed for E. coli. On the None-Gr surface, the bacteria keep the typical rod-like shape. When the bacteria contact with Half-Gr surface, a minority of bacteria behaves with disrupted shape and loses the integrity of cell membrane while the majority of bacteria still remain the intrinsic shape. Nevertheless, the bacteria on the Full-Gr show irregular appearance characteristics, some bacteria have totally lost the cytoplasm ascribe to the fracture of membrane, which indicates poor living state. And, some robust bacteria with intact membrane still exist as well.To further confirm the influence of the graphene coverage on the bacteria phenotype, SEM is utilized to inspect the membrane integrity and morphology of the two distinct bacteria, as displayed in Infaust interface interaction between red blood cells and biomaterials in the physiological saline has the opportunity to give rise to the leakage of intercellular hemoglobin, which indicates the occurrence of hemolysis. According to the ISO 10993-4, biomaterials with the hemolysis rate less than 5% can be considered as feasible blood-contacting materials. As shown in S.aureus and E. coli at the same conditions, this could be due to the bacteria structure difference where the outer film of E. coli protects itself against extracellular perturbation, whereas S.aureus has no outer filmFor the past few years, the antibacterial mechanism of graphene and its derivates proposed by previous works mainly focus on physical damage caused by piercing through the bacteria membrane due to the sharp edge structure of graphene272920I-V characteristic curve . After heating to the desired temperature (910 °C), methane was introduced to deposit graphene films for various durations ranging from 100 min to 200 min. After deposition, the supply of methane was switched off and the furnace was cooled down to room temperature under the mixture of flowing H2 and Ar gas.The graphene films were deposited on the Ge (110) substrate by atmospheric pressure chemical vapor deposition (APCVD). Ge substrate was cut into small pieces and put in an inner quartz processing tube (50 nm in diameter) located at the center of horizontal quartz tube. Then the quartz tube was evacuated to approximately 0.01 Pa and followed by filling with gas mixture comprised of 200 standard cubic cm per min (sccm) argon  and 50 sccm hydrogen (HThe surface morphology was characterized by atomic force microscope (AFM Bruker Multimode 8 system) in tapping mode and scanning electronic microscopy (SEM Zeiss Supra 55) with an acceleration voltage of 3 keV. Raman spectroscopy (HORIBA Jobin Yvon HR800) was utilized to evaluate the quality and uniformity of the CVD-grown graphene film. The analysis of the X-ray photoelectron spectroscopy (XPS) was carried out by using a Gaussian–Lorentzian peak shape after removing background spectra by Shirley algorithm.A typical system consisting of three electrodes was used to conduct the electrochemical test. The graphite rod, saturated calomel electrode and the test sample were used as a counter electrode, reference electrode and working electrode, respectively. The physiological saline (0.9 wt% NaCl solution) was used as the electrolyte during the electrochemical test. The scanning parameter of the electrochemical workstation (CHI760C) was controlled at 1 mV/s from the cathode to the anode direction.®BaclightTM Bacterial Viability Kit  was used to implement fluorescence staining to reveal the viability of bacteria on the samples, and then the fluorescence staining results were observed by fluorescence microscopy (Olympus GX71). This kit contains a mixture of a SYTO®9 green fluorescence nucleic acid stain and red fluorescence nucleic acid stain, propidium iodide (PI).A LIVE/DEAD7 CFU mL−1 was seed on the sample surface, incubated at 37 °C for 24 h, fixed by 2.5 v% glutaraldehyde solution for night at 4 °C and then dehydrated by graded ethanol  for 10 min each. Subsequently, samples were finally dried by hexamethyldisilazane ethanol solution series followed by coating platinum before SEM observation.For scanning electron microscope (SEM) observation, 60 μL bacterium solution with the concentration of 10To evaluate the hemolysis rate (HR) of the samples, fresh human whole blood was obtained from the healthy donors. The blood collecting procedures were performed in compliance with institutional guidelines approved by Zhongshan Hospital, Shanghai, and the experiments were conducted according to National standard of China GB/T 16886.4-2003 protocol under the condition of donors’ consent. All samples were sterilized by 75 v/v% ethanol solution and blow-dried. Then samples were rinsed with physiological saline and then immersed in physiological saline at 37 °C for 30 min. Meanwhile, untreated physiological saline and ultrapure water were utilized as the negative and positive controls respectively. Subsequently, diluted human whole blood was add to each well and incubated at 37 °C for another 1 h. Afterward, the mixture solution were centrifuged for 5 min at 3000 rpm. Finally, supernatant were measured at 545 nm by Multimode Reader (Biotech Cytation 5), and HR can be acquired by the equation as follows545, N545 and P545. respectively.the absorbance for samples, negative control and positive control were represented by SPlatelet rich plasma (PRP) was obtained by centrifugation of human whole blood for 15 min at 1500 rpm. The samples was sterilized and dried as mentioned above, and then incubated for 30 min. Soon afterward, 50 μL PRP was seeded on each surface of the samples and incubated for another 2 h. After rinsed with PBS , samples were fixed by immersing in 2.5 v% glutaraldehyde solution for night at 4 °C. In order to observe the microstructure morphology, the platelets on the samples were dehydrated by the same processing conditions as mentioned in section of Antibacterial ability assessment.How to cite this article: Geng, H. et al. Antibacterial ability and hemocompatibility of graphene functionalized germanium. Sci. Rep.6, 37474; doi: 10.1038/srep37474 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Trypanosoma cruzi-infected mice had a marked reduction of cardiac inflammation, fibrosis and galectin-3 expression when compared to controls. Serum concentrations of galectin-3, IFNγ and TNFα, as well as cardiac gene expression of inflammatory mediators were reduced after DMS treatment. The gene expression of M1 marker, iNOS, was decreased, while the M2 marker, arginase1, was increased. DMS-treated mice showed an improvement in exercise capacity. Moreover, DMS caused a reduction in parasite load in vivo. DMS inhibited the activation of lymphocytes, and reduced cytokines and NO production in activated macrophage cultures in vitro, while increasing IL-1β production. Analysis by qRT-PCR array showed that DMS treatment modulated inflammasome activation induced by T. cruzi on macrophages. Altogether, our results demonstrate that DMS, through anti-parasitic and immunomodulatory actions, can be beneficial in the treatment of chronic phase of T. cruzi infection and suggest that S1P-activated processes as possible therapeutic targets for the treatment of Chagas disease cardiomyopathy.Chagas disease cardiomyopathy is a parasite-driven inflammatory disease to which there are no effective treatments. Here we evaluated the therapeutic potential of N,N-dimethylsphingosine(DMS), which blocks the production of sphingosine-1-phosphate(S1P), a mediator of cellular events during inflammatory responses, in a model of chronic Chagas disease cardiomyopathy. DMS-treated,  Trypanosoma cruzi infection, both in the cardiac and in the digestive form, are associated with the occurrence of an inflammatory reaction1. Chronic Chagas disease cardiomyopathy (CCC) involves cardiac myocytes undergoing necrosis and cytolysis via various mechanisms, and areas of myocellular hypertrophy and mononuclear cell infiltration occur4. In response to the myocardial damage, fibrotic areas occur and may contribute to the disruption of the cardiac conduction system and appearance of dysrhythmias, as well as to myocardial thinning and cardiac hypertrophy5. Given the lack of an effective specific therapy, CCC is treated similarly to all other heart failure syndromes using therapies to mitigate symptoms6. Therefore, the development of new alternative treatments for CCC is needed.The pathological manifestations of chronic Chagas disease, caused by 7. Among them, sphingosine-1-phosphate (S1P), produced by phosphorylation of sphingosine (Sph) by sphingosine kinases (SphK1 and SphK2) in response to various stimuli, plays important roles in several cellular processes, including cell growth and cell trafficking9. The balance of Sph and S1P determines the progress of many diseases and there is evidence that sphingolipid metabolism and the expression of S1P receptors (S1PR1-5) are altered in inflammatory processes10. S1P drives the differentiation of different immune cell types, inducing changes in their functional phenotypes and regulating production of pro-inflammatory cytokines and eicosanoids. In particular, S1P has emerged as a central regulator of lymphocyte egress12.Sphingolipid metabolites are emerging as important lipid signaling molecules in both health and diseasein vitro assays.Due to the persistent inflammation found in CCC, which is a hallmark of the disease, and the critical role of S1P-activated pathways on the regulation of inflammation, we hypothesized that N,N-dimethylsphingosine (DMS), a pan SphK inhibitor, has a beneficial effect in chronic Chagas disease. Thus, in the present study we investigated the effects of DMS in a murine model of chronic Chagas disease cardiomyopathy, as well as its mechanisms of action on T. cruzi were treated with DMS or vehicle   Fig. . Inflammne) Fig. . Adminisne) Fig. . Gene exent Fig. . Similarent Fig. .Figure 113. To evaluate the effects of DMS on the expression of this important mediator of inflammation and fibrosis, we performed confocal microscopy analysis in the heart tissue. Vehicle-treated, T. cruzi-infected mice had a high expression of galectin-3, while a reduction of galectin-3 expression was seen after DMS treatment  or anti-CD3 plus anti-CD28. A concentration-dependent inhibition of lymphoproliferation was seen when DMS was added to the cultures  to T. cruzi-infected macrophages significantly increased the activation of caspase 1, whereas infection by T. cruzi alone induced a slight increase of caspase 1 activity  during 1 and 24 h and total RNA was extracted for gene expression analysis, compared to uninfected macrophage cultures , whereas 2 genes were found altered only at 24 h time point (48 h of infection)  Fig. . DMS treon) Fig. . When T.k13 Fig. , Il6, Ilp1a Fig. . Additiolls Fig. . Analysiays Fig. .Figure 76. Therefore, the development of therapeutic strategies aiming at modulation of inflammation without affecting parasite control is of great interest. Here we show, using a mouse model of chronic Chagas cardiomyopathy which reproduces the pathological findings observed in human hearts, a potent effect of DMS, causing reduction of heart inflammation and fibrosis, modulation of pro-inflammatory mediators and improvement of exercise capacity. Importantly, the residual parasite load found in mice chronically infected with T. cruzi was reduced by DMS treatment.Persistent inflammation is one of the hallmarks of chronic Chagas disease cardiomyopathy, and leads to a progressive destruction of the myocardium and heart dysfunctionin vitro reinforces that, in addition to a reduction of cell migration, a direct effect of the drug on immune cells may cause the potent immunomodulatory effect of DMS observed in vivo in our model. In line with this idea, it was recently shown that FTY720, a recently approved drug that inhibits the S1P pathway, also modulates the activation of human T lymphocytes and leads to a reduction of IFNγ production16.In our study, a marked reduction of inflammation was seen after DMS treatment, as shown by two different analyses, morphometry and qRT-PCR for quantification of CD45 expression. Moreover, the production of IFNγ and TNFα, two cytokines known to promote the chronic Chagas myocarditis, were significantly reduced in the heart, as well as in the serum, showing a local and a systemic effect of DMS. The fact that DMS modulates the activation of lymphocytes and macrophages 19. The expression of SphK1 is an important factor regulating the proliferation of cardiac fibroblasts20. SphK1-transgenic mice which overproduces endogenous S1P showed 100% occurrence of cardiac fibrosis, involved with activation of the S1P3-Rho family small G protein signaling pathway and increased ROS production21. Additionally, DMS-treated mice had a reduced expression of TGFβ and galectin-3, two pro-fibrogenic factors that stimulate the proliferation and production of extracellular matrix proteins by cardiac fibroblasts23. Here we observed a reduction on fibrotic area in the heart of DMS-treated mice, corroborating the importance of S1P signaling for heart fibrosis.All of the three major S1P receptor (1–3) subtypes are also expressed in cardiac fibroblasts and participate in cardiac remodeling by the activation of signaling pathways through S1P. Moreover, both FTY720 and DMS have been shown to reduce fibrosisin vivo, despite causing a reduction on the production of pro-inflammatory factors, such as IFNγ and TNFα, known to play important roles in resistance to T. cruzi infection25. We showed here that DMS has not only a direct effect on the parasite, causing several cellular alterations and death of trypomastigote forms, but also an indirect effect, by inducing the increase of NO and ROS production in infected macrophages in vitro. These results are in accordance with a previous report showing S1P down regulating iNOS expression in macrophages through the inhibition of NF-κB, AP-1 and/or STAT-1 activation26. This suggests that the inhibition of S1P production by DMS treatment may lead to an increased NO production, also contributing to the in vivo antiparasitic mechanisms of DMS.An important finding was the antiparasitic effect of DMS, leading to a reduction of parasite load 27. The reduction of parasitism by benznidazole, a drug used to treat Chagas disease, reduces cardiac alterations during the chronic phase of infection28. Additionally, the reinforcement of immunological tolerance to myocardial antigens also caused reduction of inflammation in a mouse model of T. cruzi infection29. Thus, the fact that DMS affects both inflammatory cells as well as the parasite suggests that these effects may contribute to the modulation of inflammation seen after DMS treatment.Chagas disease cardiomyopathy may result from multiple pathological mechanisms, including immune responses against the parasite, as well as self-reactive responses against cardiac antigensTrypanosoma brucei SphK causes attenuation of cell division, microtubule elongation at the posterior tip, and altered organelle positioning. SphK inhibitors, such as DMS and safingol, are toxic to T. brucei, both of them having a 10-fold therapeutic index versus human cells, suggesting their potential use against T. brucei infection30. Here we demonstrated that DMS also induces morphological alterations and death in T. cruzi, suggesting that TcSphK may be a candidate target for drug development against T. cruzi. The reduced parasite load on infected macrophages may be also an important mechanism to reduce the dissemination of parasites, since it is likely that T. cruzi-infected macrophages circulate in vivo31.SphK is a highly conserved enzyme in eukaryotes, and while there are two isoforms in mammals, only one is found in trypanosomatids. Depletion of 32. Both Sph analogues are capable of inducing IL-1β production, and our data reinforces the ability of DMS to activate the inflammasome pathway. T. cruzi infection has also been shown to induce inflammasome activation in mice, being a resistance factor to infection by regulating the production of nitric oxide and ROS35. Here we found that DMS induced inflammasome activation in T. cruzi-infected macrophages, as shown by increased caspase-1 activation and IL-1β production. Moreover, DMS increased the production of ROS and NO, which may be contributing to parasite clearance by infected macrophages. Interestingly, the increase on production of nitric oxide was not accompanied by an upregulation of iNOS gene transcription, suggesting that post-transcriptional and post-translational mechanisms are responsible for DMS-induced NO production36. The transcription of genes activated in response to ROS production, however, were increased in DMS-treated macrophages, such as NFEl2l, SOD-1 and catalase 1.A previous study has shown that Sph and its analogues DMS and FTY720 act as a danger-associated molecular patterns (DAMPs), inducing mature IL-1β secretion and promoting inflammasome activation38. Although we did not see interference of PKC and MAPK inhibitors on the effects of DMS in macrophage cultures, further studies are required to demonstrate whether the beneficial effects of DMS in vivo are solely dependent on Sphk inhibition. The fact that DMS may have off-target effects, however, may not be detrimental, since FTY720 and other approved drugs are known to have off-target effects39.In addition to its effects on Sphk, DMS was shown to inhibit the activity of protein kinase C and mitogen-activated protein kinase (MAPK)41. The analysis by PCR array performed in our study indicates that DMS induces the expression of inflammasome genes known to repress NF-κB activation, such as Nlrc5 and Nlrx143, which suggests that inhibition of T. cruzi-induced NF-kB may be linked to the activation of these inflammasome mediators. Both Sph and its analogue FTY720 have already been described as regulators of the NLRP3-inflammasome and IL-1β release from macrophages32. Although NLRP3-inflammasome activation plays a significant role in the activation of IL-1β/ROS and NF-kB signaling of cytokine gene expression for T. cruzi control in human and mouse macrophages, it was observed that NLRP3-mediated IL-1β/NF-kB activation is dispensable since it is compensated by ROS-mediated control of T. cruzi replication and survival in macrophages35. Therefore, our results suggest that, in addition to the NLRP3 inflammasome previously described32, DMS activates other inflammasome pathways.Despite the increased production of IL-1β by DMS-treated macrophages, a reduction of TNFα and IL-6, two proinflammatory cytokines, was found. This may be due to a modulation of NF-κB activation by DMS, as observed in our study and in previous reportsin vitro and in vivo by its antiparasitic and immunomodulatory effects and suggest that inflammasome activation is a promising strategy for the development of anti-Chagas disease treatment. Further studies are required to demonstrate the usefulness of inhibitors of S1P pathway, which are being already used in the clinical setting, as potential candidates for the treatment of Chagas disease cardiomyopathy.In conclusion, our results demonstrate a potent effect of DMS ad libitum. All experiments were carried out in accordance with the recommendations of Ethical Issues Guidelines, and were approved by the local ethics committee for animal use under number 001/15 .Four weeks-old male C57BL/6 mice were used in all experiments. They were raised, maintained in the animal facility of the Center for Biotechnology and Cell Therapy, Hospital São Rafael , and provided with rodent diet and water T. cruzi strain were obtained from culture supernatants of infected LLC-MK2 cells. Infection was performed by intraperitoneal (i.p.) injection of 1000 T. cruzi trypomastigotes in saline and parasitemia was monitored during infection. Groups of chronically infected mice were treated i.p. with N,N-dimethylsphingosine  based on a previous report by Lai and col. (2008)44, 3×/week during two months  was used to exercise the animals. The speed of the treadmill and the intensity of the shock (mA) were controlled by a potentiometer . Total running distance and time of exercise were recorded.45.Electrocardiography was performed using the Bio Amp PowerLab System , recording the bipolar lead I. All data were acquired for computer analysis using Chart 5 for Windows (PowerLab). The EKG analysis included heart rate, PR interval, P wave duration, QT interval, QTc, and arrhythmias. The QTc was calculated as the ratio of QT interval by square roots of RR interval (Bazett’s formula)46.The hearts of all mice were removed and half of each heart was fixed in buffered 10% formalin. Sections of paraffin-embedded tissue were stained by the standard hematoxylin-eosin and Sirius red staining methods for evaluation of inflammation and fibrosis, respectively, by optical microscopy. Images were digitized using a color digital video camera  adapted to a BX41 microscope . Morphometric analyses were performed using the software Image Pro Plus v.7.0 , as described before46. Sections were incubated overnight with anti-galectin-3, diluted 1:50  followed by incubation, for 1 h, with Alexa fluor 633 (1:200)  Nuclei were stained with 4,6-diamidino-2-phenylindole . The presence of fluorescent cells was determined by observation on a FluoView 1000 confocal microscope (Olympus).Sections of formalin-fixed paraffin embedded hearts were used for detection of galectin-3 expression by immunofluorescence as described before47, and the quantification of parasite load was performed as described previously46. To calculate the number of parasites per milligram of tissue, each plate contained an 8-log standard curve of DNA extracted from trypomastigotes of the Colombian T. cruzi strain in duplicate. Data were analyzed using 7500 software 2.0.1 (Applied Biosystems).For DNA extraction, spleen fragments were submitted to DNA extraction using the NucleoSpin Tissue Kit , as recommended by the manufacturer. Primers were designed based on the literature5 cells/mL in a 24 well-plate with rounded coverslips on the bottom in RPMI-1640 medium  supplemented with 10% fetal bovine serum  and 50 µg/mL of gentamycin  and incubated for 24 h. Cells were then infected with trypomastigotes (1:10) for 2 h. Free trypomastigotes were removed by successive washes using saline solution. Cultures were incubated for 24 h to allow full internalization and differentiation of trypomastigotes into amastigotes. Cultures were then incubated in complete medium alone or with test inhibitors for 72 h. Cells were fixed, then stained with hematoxylin and eosin, and submitted to manual counting using an optical microscope (Olympus).Peritoneal exudate macrophages obtained from C57BL/6 mice, four days after thioglicollate injection, were seeded at a cell density of 2 × 10Ptprc, Lgals3, Tnf, Ifnγ, Il10, Tgfβ, Nos2, Chi3l3, Il1β, Arg1, Nfe2l2, Cat and Sod1. All reactions were run in triplicate on an ABI 7500 Real Time PCR System (Applied Biosystems) under standard thermal cycling conditions. A non-template control (NTC) and non-reverse transcription controls (No-RT) were also included. The samples were normalized with 18S and Hprt. The threshold cycle (2-ΔΔCt) method of comparative PCR was used to analyse the results48.RNA was extracted of the heart samples and macrophage cultures using TRIzol . cDNA was synthetized using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The qPCR was prepared with TaqMan® Universal PCR Master Mix (Applied Biosystems). qRT-PCR assays were performed to detect the expression levels of 6 cells/mL) were incubated in 24 well-plates with supplemented RPMI for 24 h. After washing with saline solution to discard non-adherent cells, infection was performed with trypomastigotes (1:10), for 24 h. Free trypomastigotes were removed by successive washes. Cultures were then incubated with complete medium alone or with DMS (5 µM) in triplicate for 24 h. RNA was extracted using the Rneasy Plus Mini Kit . Quantification of RNA and degree of purity were performed in a spectrophotometer . Sample integrity was observed using a 1% agarose gel. cDNA synthesis was performed using the RT2 First Strand kit (Qiagen). For target gene expression analysis, we used RT2 Profiler PCR Arrays Mouse Inflammasome (Qiagen), the SYBR®Green system and 96-well plates. The 7500 Real Time PCR was used (Applied Biosystems). All experiments were performed in DNase/RNase free conditions. The analysis was performed by Threshold Cycle Method48, obtained by calculating 2-ΔΔCt. The QIAGEN’s qPCR analysis web portal date, available on http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php was used to assist in the analysis, and analysis of differentially expressed genes and pathways prediction were done through free online bioinformatic sites BioVenn (www.cmbi.ru.nl/cdd/biovenn) and Enrichr (amp.pharm.mssm.edu/Enrichr).Peritoneal exudate macrophages  only or with anti-CD3 and anti-CD28 (Themo Fisher Scientific), in the absence or presence of DMS at different concentrations . After 48 h, plates were pulsed with 1 μCi of methyl-3H-thymidine  for 18 h. The plates were harvested and the 3H-thymidine uptake was determined using a β-plate counter . Dexamethasone (Sigma-Aldrich) was used as positive control.Spleen cell suspensions from naive C57BL/6 mice were prepared in DMEM medium  supplemented with 10% FBS and 50 μg/mL of gentamycin. Splenocytes were cultured in 96-well plates at 1 × 10in vivo study were used for galectin-3, TNFα and IFNγ and TGFβ determination. Quantification of cytokines was performed by ELISA, using specific antibody kits , according to manufacturer’s instructions. To estimate the amount of nitric oxide (NO) produced, macrophage culture supernatants were used for nitrite determination by the Griess reaction, as previously described49.Serum samples from the 6) were obtained and infected with T. cruzi. Cultures were then incubated with complete medium alone or with DMS for 30 min. After incubation, macrophages were removed from each well using 0.01% trypsin and labeled with 10 μM of 2′,7′-dichlorofluorescin diacetate (Sigma-Aldrich) for 30 minutes at 37 °C. Cells were then washed and analyzed using a cell analyzer  with FlowJo software .Thioglicollate-elicited peritoneal exudate macrophages (1 × 106) were obtained and infected with T. cruzi as described above. Cultures were then incubated with reagents for 2 h and after that, caspase-1 activity was measured using caspase-Glo® 1 inflammasome assay , according to the manufacturer’s instructions. The luminescence of each sample was measured in a Glomax 20/20 luminometer (Promega).Thioglicollate-elicited peritoneal exudate macrophages (1 × 10T. cruzi trypomastigotes (5x107) were treated with DMS  and incubated for 24 h at 37 °C. After incubation, parasites were fixed for 1 h at room temperature with 2% formaldehyde and 2.5% glutaraldehyde  in sodium cacodylate buffer  for 1 h at room temperature. After fixation, parasites were processed for transmission electron microscopy as previously described50. Images were obtained in a JEOL TEM-1230 transmission electron microscope.P < 0.05.All continuous variables are presented as means ± SEM. Data were analyzed using 1-way ANOVA, followed by Newman-Keuls multiple-comparison test with Prism 5.0 . All differences were considered significant at values of Supplementary materialSupplementary Table S1Supplementary Table S2Supplementary Table S3Supplementary Table S4Supplementary Table S5Supplementary Table S6Supplementary Table S7