Cervical cancer elevated the expression of PD-1 and PD-L1 in mRNA level. PD-1 expression in peripheral blood of cervical cancer patients was related with tumor differentiation, lymph node metastasis, and invasiveness. PD-1/PD-L1 pathway inhibited lymphocyte proliferation but enhanced the secretion of IL-10 and TGF-β in vitro. In summary, our findings demonstrate that elevated levels of PD-1/PD-L1, TGF-β, and IL-10 in peripheral blood of cervical cancer patients may negatively regulate immune response against cervical cancer cells and contribute to the progression of cervical cancer. Therefore, PD-1/PD-L1 pathway may become an immunotherapy target in the future.The present study is to measure the expression of programmed death-1 (PD-1) and programmed death ligand-1 (PD-L1), as well as its clinical significance in cervical cancer patients. Our results showed that different T cell subsets in patients with cervical cancer had high expression of PD-1, and DCs had high expression of PD-L1. High expression of PD-1 on Treg cells in cervical cancer patients facilitated the production of TGF- Cervical cancer has the second highest morbidity and mortality rates among all female genital tract malignant tumors . SurgeryProgrammed death-l (PD-1) is a recently discovered costimulatory molecule on the surface of T cells that binds with PD-ligand to conduct inhibitory second signals. Programmed death ligand-1 (PD-L1) is a member of costimulatory signal B7 family and plays an important role in tumor immune responses . PD-L1 iA total of 78 patients  who were first diagnosed with cervical cancer by colposcopy pathological examination at our hospital between January 2010 and October 2014 were included in the present study  . AnotherμL) were mixed with APC-CD4, PECy7-CD25, PE-PD-1, and FITC-CD8 antibodies (20 μL each) and APC-CD4, PECy7-CD25, PE-IgG, and FITC-CD8 antibodies (20 μL each), respectively. Then, the mixtures were shaken before incubation at room temperature for 15 minutes in the dark. Afterwards, the mixtures were washed with phosphate-buffered saline (PBS) twice before adding 400 μL PBS to resuspend the cells. For the detection of PD-L1, one aliquot of PBMCs (20 μL) was mixed with PERCP-CD11c and PE-PD-L1 antibodies (20 μL each), and the other aliquot of PBMCs (20 μL) was mixed with PERCP-CD11c and PE-IgG (20 μL each). After shaking, the mixtures were incubated at room temperature for 15 minutes in the dark before washing with PBS twice. Then, 400 μL PBS was added to resuspend the cells. After the samples were prepared, they were subjected to flow cytometry using FACS Aria II  for the detection of PD-1 expression on CD4+ and CD8+T cell surface and PD-L1 expression on dendritic cell (DC) surface.Peripheral blood mononuclear cells (PBMCs) were separated from 5 mL venous blood using Ficoll-Hypaque density gradient centrifugation. For the detection of PD-1, two aliquots of PBMCs  10, and interferon- (IFN-) γ in serum were determined using ELISA kits  according to the manufacturer's manuals. The optical density at 450 nm was measured using a reader .The contents of transforming growth factor- (TGF-) β-actin (233 bp) were TAGGCGGACTGTTACTGAGC (upstream) and TGCTCCAACCAACTGCTGTC (downstream). The PCR reaction system (20 μL) was made using SYBR-Premix Ex TaqTM II according to the manufacturer's manual . PCR reaction conditions were denaturation at 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. The data were analyzed using iQ520 Standard Edition Optical System Software V2.0 . β-actin was used as internal control. The 2−ΔΔCt method was used to determine the cycle number Ct value corresponding to a specific fluorescence threshold and to quantify the target genes. Experiments were repeated for at least 3 times. The level of mRNA was expressed as the ratio of target gene against β-actin.Peripheral blood (5 mL) was collected from fasting healthy subjects and patients in the morning and plasma was extracted. Total RNA was extracted from PBMC of three groups using TRIzol . Synthesis of cDNA first strand was performed using Fermentas kit  according to the manufacturer's protocols. The sequences of primers for PD-1 (289 bp) were TGCAGCTTCTCCAACACATC (upstream) and CTGCCCTTCTCTCTGTCACC (downstream). The sequences of primers for PD-L1 (101 bp) were CCTGGAGGTTTCGAGATTCA (upstream) and GGCAAAGCCAAGGTACTCC (downstream). The sequences of primers for 6 cells/mL using RPMI-1640 medium. Then, 100 μL cell suspension was added into 96-well culture plates (1 × 105 cells/well) for cultivation of 72 h. To block PD-1/PD-L1 pathway, anti-PD-L1 antibody  was added before incubation for 72 h. The cells were divided into normal control group, control + anti-PD-L1 antibody group, CIN group, CIN + anti-PD-L1 antibody group, cervical cancer group, and cervical cancer + anti-PD-L1 antibody group. Stimulator ConA was added into the medium , followed by incubation at 37°C in 5% CO2 for 72 h. MTT assay was performed to analyze lymphocyte proliferation. Culture supernatants from each group were subjected to CBA assay for the determination of IL-10 and TGF-β levels.Lymphocytes and DCs separated from cervical tissues were labeled with anti-CD3 antibody  and anti-CD11c antibody . Lymphocytes (CD3+) and DCs (CD11c+) were separated using MACS system . The density of cells was adjusted to 1 × 10t-test. Differences between groups were compared using analysis of variance. Comparison among groups was performed using χ2 test.All results were analyzed using SPSS 16.0 statistical software . The data were expressed as means ± standard deviations. Intergroup comparison of ages was performed using P > 0.05)   . In cervical cancer patients, the levels of TGF-β and IL-10 were significantly enhanced, and the level of IFN-γ was significantly reduced  and was negatively correlated with IFN-γ (r = −0.421)  . These rP < 0.05)  . The res+ on CD8+T cells, we studied clinical characteristics such as age, tumor staging, histological types, tumor differentiation, lymph node metastasis, tumor diameter, invasion depth, and tumor metastasis. The data showed that the expression of PD-1 in CD8+T was related with tumor differentiation, lymph node metastasis, and tumor metastasis (P < 0.05), but not age, tumor staging, histological types, tumor diameter, or invasion depth (P > 0.05)  . The resβ, PD-L1 was blocked using anti-PD-L1 and stimulated by ConA. MTT assay showed that lymphocyte proliferation in cervical cancer group and CIN group was significantly lower than control group (P < 0.05). However, blockade of PD-L1 in cervical cancer group significantly enhanced lymphocyte proliferation (P < 0.05) . After addition of anti-PD-L1 antibody, the levels of IL-10 and TGF-β were significantly reduced (P < 0.05) but still significantly higher than those in control (P < 0.05)  . CBA ass Figures . These rβ and IL-10 are elevated, consistent with the results by Yang et al. Further study shows that PD-1 expression on the surface of CD4+T and CD8+T cells, PD-L1 expression on DC surface, and mRNA expression of PD-1 and PD-L1 are significantly enhanced in cervical cancer patients, being higher than that in CIN group. As the development of cervical lesions, the level of PD-1 is elevated, suggesting that PD-1/PD-L1 expression is closely related with the development of cervical lesions. PD-1/PD-L1 signaling pathway may facilitate the occurrence of cervical cancer by inhibiting the immune functions of T cells.PD-1 is an important negative immune regulatory molecule belonging to CD28/CTLA-4 family. The extracellular IgV-like domain of PD-1 contains four important N-linked glycosylation sites that can bind with its ligands PD-L1 or PD-L2. It plays a regulatory role in immune responses by conducting inhibitory signals via immunoreceptor tyrosine-based inhibition motif . Yang etHamanishi et al. report that PD-1 expression and CD8+T infiltration degree in ovarian cancer are negatively correlated with the prognosis of patients . Shi et +T cells and PD-L1+APCs, leading to the development and metastasis of cervical cancer [We discover that the increase of PD-1+CD8+T is correlated with tumor differentiation, lymph node metastasis, and tumor metastasis. High expression of PD-1 in CD8+T cells may inhibit the scavenging effect of CD8+T cells on tumor cells. Therefore, tumor proliferation is promoted and invasion and metastasis occur. Heeren et al. claim that lymph node metastasis of cervical cancer is correlated with the increase of PD-1l cancer .β and IL-10 can also help antigen-presenting cells express PD-L1 via STAT-3 to exert negative regulation effect [β and IL-10 are elevated while that of IFN-γ is reduced in cervical cancer. Correlation analysis shows that PD-1 expression in Treg cells is positively correlated with TGF-β and IL-10 but negatively correlated with IFN-γ, suggesting that PD-1/PD-L1 participates in the inhibition mechanism of Treg cells via TGF-β and IL-10. It is speculated that PD-1/PD-L1 signaling pathway has potential effect in regulating the immune suppression function of Treg cells in cervical cancer patients, leading to the malignancy of the disease.Our previous studies demonstrate that Treg cells are increased as the aggravation of cervical cancer lesions. We speculate that Treg cells play important roles in immune evasion of tumor cells , 18. Anon effect . After bn effect . The preγ. These facts support the speculation that PD-1/PD-L1 pathway blocking therapy can be used to enhance antitumor immunity [β is reduced after blocking of PD-L1. Yang et al. report that PD-1/PD-L1 pathway inhibits cellular immunity, leading to the occurrence and development of CIN [β and IL-10, and results in the proliferation of differentiation of Tregs in cervical cancer microenvironment, finally leading to the escape of tumor cells from immune system. PD-L1 expression in tumor cells enhances the apoptosis of antigen-specific T cells. Therefore, blocking of PD-L1 reduces T cell apoptosis. Recent studies show that depletion of tumor-infiltrating lymphocytes in tumor microenvironment is correlated with PD-L1 secreted by tumor cells or tumor-derived myeloid cells. Blocking of PD-1/PD-L1 signaling pathway enhances the function of effector CD8+T cells, inhibits the functions of Treg cells and myeloid-derived suppressor cells, and increases the antitumor immunity effect [The study also shows that PD-L1 on DC cells is capable of inhibiting T cell activation and lysing tumor cells or sometimes leads to the apoptosis of tumor-specific T cells. Blocking of PD-L1 enhances the efficacy of immune therapy. A study shows that PD-L1 positive P815 tumor cells resist immune therapy, but blocking of PD-L1 restores the effect of anti-CD137 monoclonal antibody therapy . PD-L1 iimmunity , 28. Usit of CIN . Our resy effect –31. Therβ and IL-10, leading to the occurrence of cervical cancer. The present study provides a new idea that blocking of PD-1/PD-L1 signaling pathways using PD-1 or PD-L1 antibodies may provide a therapeutic method in the immune treatment of cervical cancer.In summary, the present study finds that upregulation of PD-1 on T cells and PD-L1 on DCs in cervical cancer may inhibit T cell proliferation and function in tumor microenvironment, leading to immune suppression that facilitates the growth and metastasis of tumors. The results of relevance between changes of PD-1/PD-L1 and Treg cells demonstrate that PD-1/PD-L1 signaling pathway may exert its immune suppression effect via Treg cells and its factors TGF-


A new concept in the formulation of hybrid nanostructured materials combining high quality graphene 3D supported by Nickel foam and polyporphyrins for visible light photocatalytic application is here reported. Our innovative approach involves the development of a freestanding device able to: i) offer a high surface area to bind the photosensitizers by π-π interactions, and ii) enhance stability and photocatalytic efficiency by using cyclic porphyrin polymers. For these purposes, homo- and co-polymerization reactions by using different porphyrin (free or zinc complexed) monomers were performed. The microscopic structures and morphology of graphene polymer nanocomposites were investigated by using Scanning Electron Microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and Atomic Force Microscopy (AFM). Finally, photocatalytic activity under visible light irradiation of the obtained nanocomposites was tested, by using methylene blue (MB) as organic pollutant. The obtained data suggested that hindered cyclic polymeric structures stacked on graphene surface by non-covalent interactions, restrict the formation of non photoactive aggregates and, as a consequence, induce an enhancement of photocatalytic activity. Remarkably, our systems show a degradation efficiency in the visible-light range much higher than other similar devices containing nanoporphyrin units reported in literature. Indeed, the combination of graphene with photoactive materials aims to strongly improve the photocatalytic performance of the formulated systems by boosting the separation and transfer of photo-generated charges, which constitutes a critical step in photocatalytic reactions. As it turns out, the merging of the excellent visible-light absorption abilities of organic photosensitizers with the charge transfer properties of graphene-based materials  represents the cutting-edge on water treatment technology4. Moreover, the low cost, eco-friendliness, flexibility and versatility of the molecular design of these basic organic molecules constitute appealing features, both environmentally and economically.The design of graphene-based hybrid nanomaterials for photocatalytic applications is one of the most promising routes in the field of water remediation5. As thoroughly reported in the literature, the chemical structures of porphyrins, as well as the sizes and morphologies of their self-assembly aggregates, negatively influence their photocatalytic efficiency8, being limited by the fast recombination of photo-induced electron-hole pairs and the low photo-stability of porphyrins themselves. Indeed, the latters tend to undergo photobleaching. In spite of these limitations, the self-assembly of nano-porphyrins have been recently proven to explicate great potential in visible-light photocatalytic applications, promoting a boost of dye degradation efficiencies5. As reported by Guo et al., differences in molecular packing lead to morphology-dependent catalytic performance. In particular, a distinct photocatalytic performance is observed whenever a fibrous zinc-tetra(4-pyridyl)porphyrin nanostructure is used with respect to the spherical one in the degradation of organic pollutants6. Similar findings have been reported for nanostructured meso tetra(4-carboxyphenyl)-porphyrins (TCPP), suggesting that porphyrins molecules existing in a total J-aggregation facilitate the electron transfer process that prolongs the life-time of the excited state and, therefore, the photocatalytic performance9.Among these, porphyrin molecules have shown great potential in visible-light photocatalytic applications because of their large extinction molar coefficient in the visible-light region and the well-defined organic structures with controllable size and morphology17. Some complications arise from the use of graphene-based materials as co-catalysts. In fact, although these materials generate the benefits discussed above, some tricky features have to be taken into account during the photodegradation processes, such as the “shielding effect” as well as the radical scavenger activity. The first phenomenon is induced by the high weight addition of graphene-based materials as co-catalyst. Due to their opacity, a weakening of the light irradiation depth through the graphene-based composites restricts the efficiency of graphene in promoting the photoactivity18. This intrinsic negative effect imposes a weight addition ratio of graphene derivatives into the composite lower than 5%. However, greater amounts of carbon materials would be necessary to inhibit the recombination of electron-hole pairs photogenerated by the photocatalyst18. In covalently graphene oxide (GO) and reduced graphene oxide (rGO) functionalized composites, an increasing number of photoactive sites are clearly related to an improving of photocatalytic efficiencies. To boost the number of such sites, it is indispensable to increase the weight addition ratio or, alternately, the oxidation points onto the graphene surface. The latter indeed introduces significant structural defects that strongly affect the electron mobility and thus the charge transfer and separation processes. Furthermore, a huge adsorption capacity against organic contaminants in water is displayed from oxidised graphene-based materials. This peculiar property, that in the case of GO versus methylene blue (MB) was quantified at 714 mg/g19, makes difficult to discern between adsorption and photodegradation processes during photocatalytic tests. Finally, another crucial point is related to the antioxidant activity of carbon materials versus organic molecules. Multiple experiments have shown that graphene-based materials are an effective scavenger of OH• radicals, avoiding the desired purification catalyzed by photoactive molecules20.Significant efforts in electron transfer process have been made also by anchoring porphyrins onto graphene based materials via covalent and non-covalent interactions  strategies, which permit to bind molecules to the selected substrateTo obtain an efficient device overcoming the aforementioned disadvantages, a freestanding hybrid nanocomposite by using high quality graphene 3D supported by Nickel foam and photoactive macromolecules was realized. The novelty of our approach consists in exploiting extensive non-covalent interactions between the high surface area of graphene co-catalyst and a coating of cyclic porphyrin polymers. The unusual choice of porphyrin polymers (PPrs) as photoactive sites is based on two assumptions: i) their hindering conformational geometries might significantly avoid formation of agglomerates of single porphyrin units, that act the rapid quenching of excited state; ii) if compared to isolate photo-active molecules, polymers are able to much easily coat and protect the co-catalyst surface. Moreover, our structure shows very high avalaible surface thanks to its foam nature, further improving its photocatalytic performances.This new concept of graphene polymer nanocomposites formulation might avoid the shielding effect as well as the radical scavenger activity, maximising the charge transfer process during photocatalysis. The photocatalytic efficiencies of these nanocomposites were tested and compared to graphene/porphyrin monomers by photodegrading MB dye in water under visible-light irradiation. Our results are below reported and discussed.21. Indeed, this growth mechanism is not self-limiting like on copper.The nickel-graphene foam obtained by CVD growth is shown in Fig. 4. Porphyrin systems can act as photocatalytic media as free molecules, supported molecules, nanostructured assemblies and thin films14. Whatever is the strategy adopted, the formation of porphyrin agglomerates triggers the rapid quenching of excited state preventing photocatalysis. We used a simple polymerization process to concatenate higher number of photoactive sites in more stable cyclic chain structures25. In addition, cyclic polymerization of such porphyrins were chosen to restrain the formation of non-photoactive porphyrin aggregates26. In Fig. 27. The synthetic details of polymerization reactions of homo-PPr and co-PPr are reported in the experimental section. The obtained materials were purified and characterized by MALDI-TOF MS, GPC and UV-Vis spectroscopy. MALDI-TOF analysis, performed after samples purification, indicated that zinc derivate (homo-PPr Zn) contained oligomers lower than 3 repeat units, whereas polymerization of porphyrin Zinc free (homo-PPr), displayed a higher number of porphyrin units along the cyclic chains . The Table The heterogeneous catalysis from porphyrins and metal-porphyrins under visible-light irradiation is well reported in the literaturesee Fig.  ESI†. Th2/g, was used as solid support and embedded with the polymeric photosensitizers. The GF pieces of about 5 mm × 20 mm were characterized by Scanning Electron Microscopy (SEM) and Raman spectroscopy  related to the GF/homo-PPr and GF/co-PPr composites are shown in the left panel of Fig. 29 . As widely reported in the literature, the N1s XPS represents a sensitive probe of the charge distribution in the porphyrin macrocycle, being strongly affected by their periphery and intermolecular interactions as well34. In agreement with the literature data related to the free-base porphyrin, the N1s signal consists of a doublet with the two components centered at 400.2 eV and at 398.2 eV, in both GF/Co-PPr and GF/Homo-PPr spectra. Such binding energy (BE) values are diagnostically attributed to the presence of the pyrrolic (−NH−) and to the iminic (−N = ) species, characteristic of the porphyrin ring36. Nonetheless, as seen by comparing Fig. †). The analyses, in fact, further demonstrate the spectral differences between the two porphyrin-based structures, showing the broadening of the N1s XPS bands in the case of homopolymers, compared with the copolymers. Based on our results, we retain that the organic chains used as linker to join the porphyrinic units can modulate the intermolecular π-π stacking of the aromatic rings and, consequently, the final electronic structures of the macromolecular system.In order to inspect the electronic structure of the porphyrin-based compounds, we investigated the high-resolution region related to the N1s and C1s signalsSEM analysis of GF/homo-PPr and GF/co-PPr were also carried out to observe the morphology of polymeric coating onto the GF surface. In Fig. • radicals.However, their morphology shows clear differences, appearing quite smooth in the co-PPr case Fig. , while i†) evidenced a well distributed and extended coating for homo- and co-PPrs films, if compared to porphyrin monomers. Nonetheless, higher magnification AFM analyses of the three samples demonstrated relevant morphological differences.To finely explore the morphologies and appreciate the roughness parameters of porphyrins and cyclic-porphyrin polymers onto the graphene surface, we performed AFM analysis onto 2D reference nickel/graphene substrates. These samples were prepared following the same procedure used for 3D GF/cyclic porphyrin polymers. Lower magnification topographic AFM images Fig.  ESI† eviIn Fig. et al.37, MB could not be an appropriate probe molecule in photocatalytic test under visible light exposure since in this range it can give a contribution in photoabsorption. Thus, it can induce incorrect measurements of MB concentration during catalytic processes. We faced up this problem by measuring the absorption spectrum of aqueous MB solution (without the graphene/Ni foam) under visible light irradiation  and subtracting these absorption contributions to the absorption profile produces by our systems during the exposure time. We underline that the use of MB is still considered an ISO procedure and hence its use allows to obtain an immediate and easy comparison of our results with those recently reported in literature by using similar procedures13.The photocatalytic activity under visible-light irradiation of the nano-hybrid materials was evaluated and compared by degrading MB dye water solution. As argued by Yan see Fig.  ESI† and0 values  of MB as a function of the photo-exposure time during both the dark and visible-light experiments are reported. To dispel any doubts on the efficiency of the GF device in promoting photocatalysis, homo-PPrs were tested also on pristine Nickel foam surface (without graphene). As shown in Fig. 0 values, indicating a very low photocatalytic activity. Similarly, GF with porphyrin monomers, as well as GF/homo-PPr Zn did not give significant catalytic efficiencies , carried out after the third run, shows a sensible damage of polymer coating on graphene surface. This phenomenon could be attributed to the reactive oxygen species (ROS) which have acted a significant erosion. This evidence suggests that the reduction of photocatalytic action was mainly due to degradation of macromolecules on graphene surface. To test if graphene surface was still able to exploit its co-catalytic properties, we embedded again the GF with the co-PPrs polymers solution, washed with DMF and dried for 12 hours obtaining a restored material for a new recyclability experiment. The same procedure was repeated two times. Figure SEM analysis of GF/homo-PPr and GF/co-PPr Fig.  ESI†, ca• radicals, which drastically reduce the photocatalytic efficiencies. On the other hand, the use of sterically hindered cyclic polyporphyrins increases the number of photoactive sites, limiting the formation of agglomerates that hampered the desired charge transfer process onto the GF surface. The photocatalytic degradation tests carried out by using MB in water solution showed higher efficiencies of homo- and co-polyporphyrins compared to porphyrin monomers coating. Additionally we highlighted that only 400 μg of polymers supported on 6 mg of GF explicated an outstanding photocatalytic efficiencies with a good recyclability capabilities. This evidence can be a game changer in the formulation of polyporphyrins as photoactive compounds for such kind of graphene hybrid materials. The co-monomers nature, the MM values, the sequence length as well as the topology of the co-polymers, could significantly influence the photoactivity behaviour of cyclic polyporphyrins materials, reserving new stimulating finding. At the same time, the polymer deposition methods and parameters could be also taken into account to control aggregate formation. As for Ni/GF, interesting future perspectives put forward the use of nickel free graphene 3D as charge transfer material.The combination of organic photosensitizers with graphene-based materials has become a hot topic in water remediation. We demonstrate a novel innovative approach that consists in combining properties of graphene 3D and porphyrin polymers in a freestanding device. This simple assembly method promotes a direct and extensive contact between the graphene and the photosensitizer polymers through the instauration of non-covalent interactions at the materials interface. At the same time, the polymer coating can confine graphene materials to the direct exposure of light, dyes and OHA Waters 515 HPLC pump, connected to four Ultra-Styragel HR columns joined in series , and a Waters R401 differential refractometer, were used for GPC analysis. Polymer solutions  were injected and eluted at a flow rate of 1 ml/min. Calibration curve was obtained using a set of primary polymethylmethacrylate (PMMA) standards.MALDI /mass spectra were recorded in reflector mode using a 4800 MALDI TOF/TOF™ Analyzer , equipped with a Nd:YAG laser (λ = 355 nm) and working in positive-ion mode. External calibration was performed using an Applied Biosystems calibration mixture consisting of polypeptides with different molecular weight values. Mass accuracy was about 50 ppm. The samples were prepared by mixing approximately 0.1 mmol of the monomer or polymer and 40 mmol of trans-3-indoleacrylic acid  on the sampler target, using THF as a solvent. The m/z nominal values reported in the spectra, are referred to molecular ions constituted by the most abundant isotopes of each element present in the molecule.UV-Visible spectra were recorded on a Shimadzu spectrophotometer at room temperature using toluene as a solvent and quartz cuvettes with a path length of 1 cm. The porphyrin content in co-PPr was evaluated by considering the absorption value at 423 nm (λmax for porphyrin group) using Beer’s law and a molar absorption ε of 378692, determined for porphyrin monomer in toluene. The calculated compositions were reported in Table AFM analyses were performed using a Bruker-Innova microscope operating in high-amplitude mode and ultra-sharpened Si tips were used . The Si tips were substituted as soon as a resolution loose was observed during the AFM images acquisition. The AFM images were analyzed by using the SPMLABANALYSES V7.00 software. In order to give representative parameters, statistics on the AFM images were carried out over different areas of the same sample.−9 Torr. The measurements were carried out at 45° photoelectron take-off angle relative to the sample surface with an acceptance angle of ±3°. The analyzer pass energy was set at 23.5 eV for the high-resolution spectra. The binding energy (BE) scale was calibrated by centering the C1s signal of the aliphatic/aromatic component at 285.0 eV). The analyses were performed on 2 mm diameter surface area of the sample, much greater than the lateral size of any carbon island on the nickel foam surface.XPS were performed by a PHI ESCA/SAM 5600 Multy technique spectrometer with the use of an Mg standard X-ray source. During the analyses, the pressure in the chamber was ~10Micro-Raman Stokes spectra were taken in backscattering geometry with a HORIBA Jobin-Yvon system, equipped with Olympus BX41 microscope. He-Ne laser radiation at a wavelength of 632.8 nm is focused to a spot size of about 1 μm by a 100× objective. The laser power on the sample was about 5 mW, and a 550 mm focal length spectrometer with 1800 lines/mm grating was used.The morphology of hybrid structure were analyzed by scanning electron microscopy  using a Zeiss Supra 25 microscope operating at 2.0 kV, both in the Inlens and secondary lens operation mode.All solvents and basic materials were commercial products appropriately purified before use.23 by reaction of 5,10-di[p-(9-methoxytriethylenenoxy)phenyl]−15,20-di[p-hydroxyphenyl]-porphyrin (see ESI† for synthesis method) with CH2Br2. In a 2 mL vial, equipped with magnetic stirrer and placed in a water bath at 70 °C, 10 mg (0.01 mmol) of 5,10-di[p-(9-methoxytriethylenenoxy)phenyl]−15,20-di[p-hydroxyphenyl]-porphyrin were solubilized in 0.5 mL of N-methylpirrolidinone (NMP) together with 3 mg (a large molar excess) of powdered KOH. After 1 hour, 0.3 mL of CH2Br2 (a large excess) were added and the vial closed. After 24 hours, the solution was poured into 5 mL of 5% HCl under vigorous stirring and the obtained suspension was centrifuged to give a purple polymeric residue. The material was solubilized in 2 mL of THF, precipitated in 5 mL of water, centrifuged and dried in vacuum for 24 hours (yield about 80%). The polymer was characterized by MALDI-TOF mass spectrometry, GPC and UV-Vis spectroscopy. Homo-polymer based on zinc-porphyrin monomer was synthesized in a similar way starting from the specific porphyrin derivative.The homopolyformal −15,20-di[p-hydroxyphenyl]-porphyrin (0.011 mmol) and 8.1 mg of di(bisphenoxy-A)eicosane (0.011 mmol) were solubilised in 3 mL of toluene in a 10 mL closed vial and heated at 70 °C. Then, 4 mg of NaOH , solubilised in 1 mL of water together with 7.1 mg of TBAB (0.022 mmol), were added and the solution was stirred at the same temperature for 1 hour before adding 0.5 mL of CH2Br2. After 24 hours, the organic solution was separated from the aqueous one and poured, under stirring, into a solution of 10 mL of EtOH acidified by 0.5 mL of CH3COOH. After separation by centrifugation, the precipitated material was dissolved in THF and precipitated in EtOH a further two times, washed with H2O, then dried under vacuum. 14 mg of a purple fibrous polymeric material was recovered with a yield of about 75%. The copolymer was characterized by MALDI-TOF mass spectrometry, GPC and UV-Vis spectroscopy.Random copolymer [co-PPr in Fig. 2, porosity 95%, 20 pores/cm, purity 95% and 1.6 mm thickness) were used as 3D scaffold templates for the CVD growth of GF. They were cut into pieces of 5 × 10 mm, then immersed in acetic acid to remove Ni native surface oxide layer and finally placed in the chamber of the AIXTRON’s Black Magic Chemical Vapor Deposition (CVD) system. The chamber was firstly pumped down to 4 × 10–3 mbar and then gradually heated both from the top and bottom side of the chamber up to 900 °C and maintained at this temperature for 20 minutes under Ar and H2 flows  to further clean the surface and eliminate the residual thin surface oxide layer. Then, the temperature was raised up to 1000 °C and the CH4 flow was opened for 40 minutes  and then cooled down at 15 °C/min to grow the graphene layers on the Ni foam surface. The working chamber pressure was maintained 25 mbar both in the cleaning and growth steps.Nickel foams  was dissolved in 1 mL of dimethylformamide (DMF). After, a GF piece of 5 mm × 20 mm was immersed in each polymeric solution. Samples were statically impregnated in the mixture overnight, then removed from vials. The resulting hybrid materials were dried under vacuum overnight at 50 °C.2 and equipped with a cut-off filter (λ > 400 nm) was used as the light source. For each experiment, a MB dye aqueous solution (0.015 mM) was prepared. The samples were immersed in 2 ml of the MB solution and left for 12 h in the dark to reach the adsorption-desorption equilibrium. Afterwards, at given time intervals of 1 h, the variation of the concentration of the MB was spectrophotometrically evaluated using a PerkinElmer Lambda 45 UV–Vis spectrophotometer, observing the absorption peaks at 664 nm. The photodegradation ratio was defined as C/C0 where C is the absorption of the dye at a certain time and C0 represents the absorption value of the MB initial concentration. In particular, these curves were previously subtracted by the contributions of MB photoabsorption, that were measured by irradiating MB aqueous solution (without graphene/Ni).The photocatalytic activities of the nanohybrids were evaluated through the degradation of MB dye under visible light irradiation. A xenon lamp operating at 1.5 mW/cmSupplementary information


Drug-specific risk and characteristics of lupus and vasculitis-like events in patients with rheumatoid arthritis treated with TNFi: results from BSRBR-RA. RMD Open 2017;3:e000314.Jani M, Dixon WG, Kersley-Fleet L, The surname of the third author of this paper was spelt incorrectly. The correct surname is “Kearsley-Fleet”.


We can identify single cells with an accuracy >90% for more than half of the communities consisting out of two bacterial populations. In order to assess to what extent an in silico community is representative for its synthetic counterpart, we created so-called abundance gradients, a combination of synthetic  communities containing two bacterial populations in varying abundances. By showing that we are able to retrieve an abundance gradient using a combination of in silico communities and supervised machine learning techniques, we argue that in silico communities form a viable representation for synthetic bacterial communities, opening up new opportunities for the analysis of synthetic communities and bacterial flow cytometry data in general.Bacterial cells can be characterized in terms of their cell properties using flow cytometry. Flow cytometry is able to deliver multiparametric measurements of up to 50,000 cells per second. However, there has not yet been a thorough survey concerning the identification of the population to which bacterial single cells belong based on flow cytometry data. This paper not only aims to assess the quality of flow cytometry data when measuring bacterial populations, but also suggests an alternative approach for analyzing synthetic microbial communities. We created so-called  Microbial communities are primary contributors in most biogeochemical processes on Earth . As suchTo cope with these bottlenecks, synthetic microbial communities, assembled through the selection of individual microbial populations, and studied under controlled environmental conditions, have recently been suggested as promising intermediary platforms –5. AdvanWith the number of synthetic ecology studies ever increasing, the analysis of low-complexity community compositions, i.e. quantifying the abundance of each constituent taxon, remains the most significant challenge. A study by Saleem et al. used traditional plate counting, which entailed the cultivation of all individual members on agar plates followed by subsequent enumeration of the colony forming units (CFU) . Their cEscherichia coli mutants, the analysis of complex synthetic communities that consist of diverse taxa  faces considerable bias due to taxon-dependent nucleic acid extraction efficiencies, varying amplification efficiency and also primer selectivity. In extremis, this has limited studies with complex synthetic communities to relate their temporal observations only to the initial community composition [Other studies, such as the one by Mee et al., applied quantitative PCR (qPCR) . Yet, whposition . Overallbioinformatics, is growing accordingly [Flow cytometry (FCM) offers a multiparametric description of individual cells, which can be applied to study microbial communities , 14. As ordingly .in silico communities. These are communities created by an aggregation of FCM data coming from axenic cultures which are being measured separately through FCM. The great advantage of using this approach is that we know which cell stems from which bacterial population. This enables us to apply a supervised machine learning approach, which has shown previous success in the recognition of leukemia [The main goal of this paper is to explore in a systematic way the possibilities of using FCM data to identify bacterial single cells, in order to be able to characterize the composition of synthetic bacterial communities. We will do this by introducing the concept of leukemia  or to fileukemia . More spleukemia , 19. Appleukemia  or to anleukemia . HoweverIn the first part of the paper we analyze to what extent data coming from FCM can be used in order to separate microbial populations at the single-cell level. To do so we have cultivated twenty axenic cultures and characterized them by FCM. We performed single-cell predictions using Linear Discriminant Analysis (LDA), an established method for performing multivariate analyses in microbial ecology , and a Rabundance gradients, a combination of in vitro communities which consist out of two populations in varying abundances. We will show that we are able to retrieve these relative abundances, using a classifier trained on an in silico community; this result enables researchers to perform a supervised analysis of synthetic microbial communities.In the second part we show to which degree an in silico community is able to identify a synthetic bacterial community. This is not a foregone conclusion due to the heterogeneous character of bacterial populations, which is reflected in FCM data . In ordemulticlass setting. To do so we created and evaluated in silico communities containing more than two populations. The results show that our approach is valid for communities of lower complexities, furthermore FCM gives rise to data that should be feasible for higher complexities as well. A schematic overview of the proposed method can be found in In the third part of the paper we estimate to what extent bacterial communities can be analyzed for higher population complexities, i.e., in a S = 2, S denoting the number of populations making up a community, i.e., the population richness. This results in 190 in silico communities, where the same amount of cells was sampled for each population, thus creating evenly distributed in silico communities. We calculated the mean for the area under the ROC curve (AUC) and the accuracy (acc), accompanied with their standard deviations and the percentage of communities which reported a score higher than 0.90; results are reported in The performances using LDA and a Random Forest classifier were calculated for all possible pairwise combinations considering twenty populations for We conclude that for a majority of in silico communities we are able to perform single-cell predictions up to high performances, especially when using Random Forests; in this case more than half of our communities report results higher than 0.90 for both AUC and the accuracy. Our highest performances top off at an AUC of 0.999 and an accuracy of 0.996. To further illustrate our findings we have visualized the AUC and the accuracy for all in silico communities, where performances have been ranked in descending order according to the results of applying a Random Forest classifier .On average we see that a Random Forest classifier performs better than LDA. However, it is not always necessary to use a ‘black-box’ non-linear classifier such as a Random Forest. For some of the in silico communities we see that the performance of LDA is similar to the performance of Random Forests; 45% of the in silico communities report an increase in AUC of less than 0.03, 17% report an increase in accuracy less than 0.03. Moreover, note that pairwise combinations of populations give rise to performance accuracies ranging from 99% to near random guessing predictions. Hence, our dataset is highly representative, that is, we were not biased towards highly discriminative populations.An abundance gradient consists out of a set of bacterial communities containing two populations in varying abundances. We constructed these gradients for three combinations of bacterial populations, combinations for which we initially reported a low (Comb. 1), medium (Comb. 2) and high performance (Comb. 3) respectively. We created these gradients in vitro, but, because we measured the bacterial cultures separately beforehand through FCM, we were also able to construct these gradients in silico. In order to explore to what extent in silico communities can be used to identify synthetic bacterial communities, we have predicted the relative abundances of both in silico and in vitro abundance gradients, using LDA and a Random Forest classifier trained on a full evenly distributed in silico community. Ideally, a classifier which is able to achieve a high AUC and accuracy on a held-out test set of this in silico community gives rise to a well-predicted abundance gradient, both in silico and in vitro. The predicted abundance gradients are visualized in S. oneidensis population.As expected, the predicted abundance gradients for Comb. 2 and 3 match the target abundance gradients , whereasSecond, we note that for Comb. 2 the predicted gradients highly overlap; this means that an analysis using LDA and a Random Forest gives rise to very similar results. This is however not the case for Comb. 3, where the use of Random Forests results in a gradient that lies closer to the target gradient, which is reflected for both the in silico and the in vitro analysis. These observations are reflected in the root mean squared error (RMSE), which is calculated between the predicted gradients and the known target abundance gradients . The RMSShannon diversity, noted as D1 [evenness of a community . To quantify our results we calculated the mean accuracy for every S; results are displayed in In order to explore to what extent single-cell predictions can be made when we increase the population richness, we created in silico communities in a S our approach is able to make single-cell predictions significantly better than random guessing. As S increases, both the mean accuracy and the size of the confidence interval decreases. As the richness increases, the degree in overlap between populations in the multiparametric ‘FCM-space’ starts growing accordingly. Therefore it is harder for classifiers to make a distinction between populations, which results in performances that are lower and more centered.For all values of S increases. This means that for communities with a low richness  LDA might provide a sufficient method to make single-cell predictions, but as S increases Random Forest will be a better option for most communities. This also implies that although for low S a linear combination of variables already discriminates populations quite well, predictions can be improved by resorting to classifiers which have the possibility of detecting non-linear relations between variables.The difference in performance between the two classifiers increases as Using the concept of in silico communities, we are able to use supervised machine learning techniques to taxonomically identify bacterial cells up to high accuracies based on FCM data. We note that this approach has not yet been adopted to analyze the composition of synthetic bacterial communities. A possible reason for this is the lack of incorporating these methods in standard FCM software .one-versus-one (OVO) or one-versus-all (OVA) approach [Using a full combination of fluorescence and scatter signals, we demonstrated that using ‘off-the-shelf’ classifiers without further data manipulation already results in acceptable to high performances for low population richness. Compared to previous research, we note that Rajwa et al. were not able to use LDA in order to make proper single-cell predictions . While tapproach .technical variability and biological variability. It has been shown that both sources give rise to heterogeneity in FCM data when studying bacterial axenic cultures [Although it has been briefly mentioned in literature that an in silico community can be representative for its in vitro counterpart , there icultures , althougcultures .S = 2, A & B, we see that for a significant number of combinations the difference in classifier performance is noteworthy, with a mean difference of 2% and a standard deviation of 11%. For clarity, we added the Random Forest performances  for all in silico communities in Technical variability has been suggested to arise from the time-dependent bleaching and leaking of fluorochrome molecules . Its effBiological variability is another and perhaps more important factor to take into account when analyzing microbial communities with FCM. Vives-Rego et al. hypothesize that biological variability in FCM stems from cell size diversity and cell cycle variations . In thisTo do so, a more comprehensive protocol is required to make our in silico approach fully operational, a need which has been pointed out before . This prFor now, in silico communities can already be exploited for various purposes. For environments where limited physiological variation in the axenic populations is expected, or where the in silico populations have been defined for all possible physiological states, our approach can be used to retrieve the community composition for low-complexity microbial communities. Furthermore, by using evaluation tools for classifiers such as the accuracy or the AUC, one can quantify which populations are distinguishable and which are not. One intuitive tool which is extensible to a multiclass setting, is the use of a confusion or misidentification matrix. This allows one to inspect which populations are likely to overlap and which are not; an example is given in Secondly, as we have shown that in silico communities form a viable representation of their in vitro counterparts, we are allowed to extrapolate properties of in silico communities to in vitro microbial communities. This means that in silico communities can be used as a stand-in for in vitro communities, enabling us to use them to develop new data-driven techniques, which will ultimately lead to novel applications for microbial FCM.S) of an in silico community varies from S = 2, …, 20.An in silico community consists out of an aggregation of data coming from axenic cultures, which are being measured separately through FCM. As we have twenty axenic cultures at our disposal, the population richness , in order to keep it computationally feasible. For every increment of S we calculated the mean accuracy, averaging the accuracies for all 150 randomly chosen in silico communities.For abundance gradient to prove that properties of in silico communities can be used for the identification of their in vitro counterparts. An abundance gradient consists out of a set of microbial communities where populations have been mixed in varying abundances. We created an abundance gradient both in silico and in vitro for three combinations of two populations, with abundances ranging from 1% to 99% for the one population and vice versa for the other. This was possible as we measured the axenic cultures separately through FCM beforehand. We chose three different combinations of two populations to create abundance gradients, combinations which initially reported a low, medium and high performance respectively, based on the performance of the Random Forest classifier for S = 2. For every community in an abundance gradient we sampled 10,000 cells (both in silico and in vitro), except for one in vitro community of Comb. 3, for which we were not able to register enough cells (see further on).We used the concept of an Nax = 5,000 cells per bacterial population to create the in silico community upon which we trained our classifier; as the abundance gradient acts as our test set, we trained our classifier on the full in silico community. Note that we have cultivated and measured new axenic cultures in order to create both the in silico and in vitro abundance gradients.We trained a classifier on an evenly sampled in silico community to predict the label of individual cells for all communities in an abundance gradient. We sampled www.bccm.belspo.be), Leibniz Institute DSMZ (www.dsmz.de) and Helmholtz Centre for Environmental Research (www.ufz.de/index.php?en=13354), respectively. For cultivation, all bacteria were grown on rich, solid lysogeny broth medium . A single colony was picked and cultivated for 48h in liquid LB medium. Finally, fresh LB medium was inoculated with 10% (v/v) inoculum and incubated for 24h; all samples were measured in duplicate. A bivariate scatterplot (FL1-H vs. FL3-H) after denoising can be found for every culture . >10,000 cells were registered for each measurement, except for Comb. 3 where we registered 3.084 cells for 1% of S. oneidensis.To create abundance gradients, we chose three combinations (Comb.) of two bacterial populations based on Random Forest performances calculated during the in silico analysis of dataset 1 . We measAs we measured the populations separately beforehand through FCM, we were also able to construct an abundance gradient in silico, by sampling communities according to the same relative abundances as described above.6 cells mL-1 in 0.2 μm filtered buffer solution was reached  and stained with a final concentration of 1% (v/v) nucleic acid stain SYBR® Green I (100x concentrate in 0.2 μm filtered dimethyl sulfoxide). Samples were incubated for 20 minutes in the dark at 37°C. FCM measurements were performed on a C6 Accuri flow cytometer  equipped with four fluorescence detectors , two scatter detectors and a 20mW 488nm laser. This results in a multiparametric description of each cell consisting out of twelve variables .Samples were diluted until an approximate cell density of 10x) transformed FL1-FL3 bivariate space, following the guidelines by Hammes et al. [write.FCS function. The cell densities for each individually measured taxon were calculated by the filter function; both functions are available from the flowCore package (v.1.38.1). As axenic cultures and synthetic communities have been measured in a number of replicates, we pooled these replicates together first, before subsampling cells to create our in silico and in vitro communities we use for analysis.The FCM data were denoised from (in)organic and instrument noise by means of a reproducible digital gating strategy in the arcsinh is a linear classifier which tries to find the optimal linear combination of features in order to separate objects or classes. It assumes the data are distributed according to a Gaussian distribution. It has no hyperparameters to tune and is able to handle problems in the multiclass setting in a natural way. For more information, see  or chaptensemble method, a method in which various classifiers are trained and in which a majority vote is taken to predict the outcome of an unknown sample. In this case the ensemble consists out of decorrelated unpruned trees grown on bootstrap samples. The trees are decorrelated because at every split only a random subset of the total number of K variables is available (K = 12). This results in a decrease in variance for only a slight increase in bias, hence lowering the overall classification error. For more information see [A Random Forest classifier is an example of an tion see  or chapttion see .S = 2, …, 19 using the preset We grew 200 trees when training a Random Forest and chose the gini criterion when making a split. We note that there is no need to tune the number of features that are available to choose from when making a split. We applied the preset nce with . This haaccuracy and the area under the receiving operating characteristic curve (AUC). The in vitro analysis is expressed in terms of the root mean squared error (RMSE).We used various performance metrics in order to evaluate our methodology. We evaluated the in silico analysis in terms of the N denotes the total number of elements to predict, y the true label and 1 the indicator function, which returns the value of 1 when its argument is true and 0 otherwise. It can also be expressed in terms of the true positives (tp), the number of correctly predicted elements belonging to a certain class j, true negatives (tn) the number of correctly predicted elements not belonging to class j, false positives (fp), the number of incorrectly predicted elements belonging to class j and false negatives (fn), the number incorrectly predicted elements not belonging to class j. In this setting, the accuracy can be written as:S denotes the total number of classes.The accuracy can be defined in the following way:receiving operating characteristic (ROC) curve and can be used as a performance measurement for a binary classifier [The AUC measures the area under the assifier , 46. TheCalculating this area results in a number between 0 and 1; the higher this number, the better the performance of a classifier. The AUC can be interpreted as the probability that a classifier will rank a randomly chosen positive higher than a randomly chosen negative. Using the AUC has a number of favorable properties. Most notable are the fact that it gives an indication of how well separated the positive and negative class are and that it is insensitive to prior skewness concerning class distributions.p, opposed to the predicted n being the total number of bacterial communities constituting an abundance gradient. Therefore when the set of predictions are close to the ground truth, the RMSE lies close to zero.Expressing the known relative abundance as A confusion matrix is a tool which helps to describe the performance of a classifier. It reports the tp, tn, fp and fn, and is naturally extendable to a multiclass setting. In this way one can inspect to what extent a classifier ‘confuses’ certain labels of classes. An example for the binary setting is given in Applied to the use of in silico communities, one is able to inspect which populations are easily separated by a classifier and which populations have a similar FCM fingerprint.https://github.com/prubbens/InSilicoFlow.Our code has been made available on github: .fcs format on the FlowRepository database [Our data has been made freely available in database , and canAxenic cultures: FR-FCM-ZZSH.Abundance gradients: FR-FCM-ZZSG.flowCore, a package of computational Tools written in R for the analysis of FCM data [The data has been preprocessed and exported using FCM data .Scikit-learn is an open-source library of various machine learning methods, which can be used in Python [n Python . We usedD1 [pi denotes the relative abundance of population i:Knowing the relative abundance of a population, we can determine the Hill number of order one, i.e., the first alpha diversity metric, denoted as D1 . This inD1 is that we can combine uncertainties for the relative abundances. As it has been established that the instrumental variation ϵI for FCM is less than 5% considering cell counts [p can be calculated from a division of two cell counts, pi (as pi can be determined independently) to determine the variance for D1 in the following way: An additional advantage using a diversity index such as l counts , we can S = 2 (as p2 = 1−p1), this formula can be expressed in terms of the relative abundance of the first population p1 as follows, combining the two formulas above:D1 for every community constituting the abundance gradient, and additionally, we gained a confidence interval within which our in vitro constructed D1 should lie, setting ϵI = 0.05; this information is visualized in For S1 File(XLSX)Click here for additional data file.S1 FigS = 5. Every element of the matrix mij gives the fraction of the population i that is predicted as population j.Example of a confusion matrix calculated on a 30% held-out test set for an in silico community with (TIF)Click here for additional data file.S2 FigFlow cytometric characterization of 20 bacterial taxa. Each point represents one single cell characterized by two fluorescence parameters (FL1-H and FL3-H). The data were denoised from (in)organic noise based on a reproducible digital gating strategy (explained above) and was adjusted for each taxon.(TIF)Click here for additional data file.S3 FigA Comb. 1: P. putida—P. fluorescens; B Comb. 2: A. rhizogenes—Janthinobacterium sp. B3; C Comb. 3: S. oneidensis—M. luteus. Data filtering strategy for the FCM data for each abundance gradient based on a reproducible digital gating strategy (explained above); data points outside the filter represent (in)organic noise. Examples are given for the 40%-60% abundance files.(TIF)Click here for additional data file.S4 FigAB Comb. 1: P. putida—P. fluorescens; CD Comb. 2:A. rhizogenes—Janthinobacterium sp. B3; EF Comb. 3: S. oneidensis—M. luteus. Both predicted and target D1 is plotted against the relative abundance of the first population for every combination, both for the in silico (left panel) and in vitro (right panel) abundance gradients; the 68%-CI and 95%-CI for the in vitro gradients are also visualized, determined as described in Appendix: Alpha diversity analysis.(TIF)Click here for additional data file.


Multiple clinical studies have revealed a link between genomic instability and response to anti-PD-1/PD-L1 therapy in cancer management. A recent study has revealed an important role for the ATR/Chk1 DNA damage checkpoint in regulating PD-L1 expression, raising important clinical and translational questions for therapy selection and study design. A recent report by Sato et al. begins to shed light on the molecular underpinnings of signal transduction from DNA damage to the immune checkpoint, and identifies a key role for the DNA damage sensor kinases ATM, ATR, and Chk1 in this process  was recently approved by the FDA for treatment of patients with advanced MMR-deficient tumours.The association between repair deficiency and improved response to PD-1/PD-L1 blockade has now been demonstrated for several DNA repair pathways. Tumours with mismatch repair (MMR) pathway deficiency exhibit a microsatellite instability (MSI) phenotype, and are characterised by a high mutation burden, an activated immune microenvironment, and increased PD-1/PD-L1 expression on tumour and immune/stromal cells. As such, MSI tumours exhibit high response rates to anti-PD1/PD-L1 therapy,BRCA1, BRCA2, and PALB2) have increased mutation burden, predicted neoantigen load, and PD-1/PD-L1 expression. This raises the possibility that HR-deficient tumours may be sensitive to PD-1/PD-L1 inhibitors; however, their response to these agents appears less robust compared with MMR-deficient or other hypermutated cancers, such as melanomas and non-small cell lung cancers.5 In addition, tumours with mutations in the exonuclease (proofreading) domain of polymerase epsilon (POLE) or polymerase delta (POLD1) have among the highest mutation burdens identified to date, and POLE/POLD1-mutated tumours exhibit high levels of immune activation and PD-1/PD-L1 expression, and are sensitive to immune checkpoint blockade.7Associations between alterations in other DNA repair pathways and tumour immune features have also been reported. Tumours with modifications in the homologous recombination (HR) pathway ] resulted in the largest increase in radiation-induced PD-L1 expression. Again, they observed that pharmacologic inhibition of ATM, ATR, or Chk1 was sufficient to abrogate PD-L1 upregulation. Interestingly, PD-L1 upregulation in Ku80-depleted cells could also be abrogated by depletion of either EXO1 (exonuclease) or BLM (helicase); two genes that are required for DSB processing and end resection. Similarly, in their screen, loss of BRCA1 did not show a significant increase in ionizing radiation-induced PD-L1 expression, compared with control cells. This highlights the importance of Chk1 activation via end resection as a key process in DSB-induced PD-L1 expression. Although both BRCA1 and BRCA2 are required for HR, they have distinct roles in this process; BRCA1 promotes DNA end resection by relieving the barrier posed by 53BP1 in HR, a process where BRCA2 is not involved. Therefore, according to their model, DNA end resection is impaired in BRCA1-defective cells, meaning ATR/Chk1 signalling is not effectively activated and PD-L1 activation does not occur. This is in stark contrast to BRCA2-defective cells where DNA end resection is not impaired as BRCA2 is not involved in this process. Finally, the authors showed that damage-induced ATM/ATR/Chk1-mediated PD-L1 upregulation is dependent on IRF1 signalling through phosphorylated STAT1/3.Despite these robust clinical associations, the mechanisms linking DNA damage to increased PD-L1 expression and immunotherapy response are incompletely understood. To functionally characterise the impact of DNA damage on PD-L1 expression, Sato et al. subjected several DNA repair-proficient cancer cell lines to ionizing radiation, double strand break (DSB)-inducing drugs such as etoposide, or a poly(ADP)-ribose polymerase inhibitor (PARPi). Treatment resulted in a time-dependent increase in PD-L1 expression at both the transcriptional and protein levels. To investigate the role of the DNA damage response in this process, they added specific inhibitors of ATM, ATR, or Chk1, and demonstrated that inhibition of any of these DNA damage checkpoint kinases could suppress PD-L1 upregulation. Next, the authors screened an siRNA library targeting DNA repair genes; they found that depletion of The work by Sato et al. provides important mechanistic insights linking DNA damage with immune activation, by identifying a critical role for the DNA damage checkpoint in regulating PD-L1 expression. Although these data have provided a window into one aspect of the signalling network that links genomic instability with immune signalling, the molecular details of much of the network remain to be elucidated. Particularly, the events downstream of ATR activation, including transcriptional changes and/or direct activation (or repression) of other signalling factors, may uncover additional mechanisms through which the immune response can be modulated by DNA damage.BRCA2-/- cells) are unclear. In addition, many of these experiments will ultimately have to be conducted in immune-competent model systems in order to fully understand the breadth of the immune response to DNA damage.Temporal aspects of DNA damage (or repair deficiency) may also impact the immune response: in the experiments by Sato et al., changes in PD-L1 levels were measured following acute damage exposure or siRNA-mediated depletion of a repair factor, and the observed PD-L1 increase did not persist beyond 14 days. Therefore, the dynamics of PD-L1 expression following chronic damage exposure or in a constitutively DNA repair-deficient background , radiation therapy, or DSB-inducing chemotherapy agents.1 The data by Sato et al. raise the possibility that, at least in the setting of exposure to DSB-inducing agents, DNA damage checkpoint inhibitors  may decrease tumour response to immune checkpoint inhibition by suppressing PD-L1 expression, thereby arguing against a triplet of DSB-inducing therapy/ATR-Chk1 blockade/PD-1-PD-L1 blockade. Nonetheless, the optimal sequence of conventional or targeted agents with anti-PD1/PD-L1 therapy may be highly context-dependent, and this work highlights the important role for pre-clinical studies in identifying potential mechanisms of synergy or antagonism.This work also has several important clinical implications. The optimal timing and combinations of DNA damaging agents with immunotherapy are not known. Furthermore, both immune stimulating and suppressive effects of combined regimens have been noted.BRCA1-mutated tumours, resistance to PARPi may occur due to a rescue of DNA end resection ability via loss of 53BP1, REV7, or Ku70/80, which increase HR capacity.11 In this setting, re-establishing end resection may promote PD-L1 upregulation via EXO1/BLM (and ATM/ ATR/ Chk1) activity, and thus sensitise tumours to PD-1/PD-L1 blockade with or without DSB-inducing agents. In BRCA2-mutated tumours, resistance to PARPi may occur via protection of the replication fork,13 which is dependent on ATR activity.14 This ATR activity may lead to upregulation of PD-L1 and promote response to PD-1/PD-L1 blockade. In both scenarios of PARPi resistance in BRCA1/2-mutated tumours, the study by Sato et al. suggests that PD-1/PD-L1 inhibitors may be a useful therapeutic strategy (with or without concurrent DNA damaging agents) for tumours that have progressed on PARPi.Another clinical implication of this work is the selection of anticancer therapy after PARPi progression. PARPi are synthetically lethal with HR deficiency and are now FDA approved for clinical use in ovarian cancer, while also being evaluated in several other HR-deficient tumour settings. However, a substantial fraction of patients eventually develop resistance to PARP inhibition and several mechanisms of PARPi resistance are now emerging. In The work by Sato et al. highlights an important role for the DNA damage ATR/Chk1 checkpoint in regulating PD-L1 expression, thus linking DNA DSB signalling with regulation of the immune response. These observations have important clinical implications for therapy selection, particularly following progression on PARPi and other DNA damaging agents. They also have translational implications for the design of appropriate correlative studies in ongoing and future clinical trials of DNA damaging agents in combination with immunotherapy.


We propose a broadband terahertz absorber consisting of nonstructured graphene loaded with arrays of elliptic dielectric cylinders. The relative bandwidth for the absorption above 90% reaches about 65%. The working mechanism of broad bandwidth mainly comes from two aspects. One is that the nonstructured graphene loaded with elliptic dielectric cylinders provides multiple discrete graphene plasmon resonances with large relative frequency interval. The other is that, for each discrete resonance, there exists a set of continuous plasmon resonances because the width of the dielectric structure varies continuously and gradiently. The broadband terahertz absorber we demonstrate here, based on geometrically gradient dielectric structures and nonstructured graphene, avoids the graphene processing, which shows great potential applications in related devices. Many applications of THz devices have been proposed so far, such as communicating, spectroscopy, sensing and imaging4. Among these attempts, the absorber plays an important role in many devices working in THz range, including detectors5, sensors6, modulators7, thermal emitters8, camouflage devices9, etc. Traditionally, metallic metamaterials are the key components of many THz absorbers, structures of various geometries have been designed to realize different absorption functions such as dual-band11, multi-band13 and broadband16. Recently, graphene, a single-layered carbon atom arranged in a honeycomb lattice, has become one of the most promising materials for designing THz absorbers due to its tunability, broadband response and high carrier mobility17. Various types of graphene-based absorbers have been proposed to achieve near-unity absorption. Many of these absorbers are based on periodically structured graphene like disks18, microrings19, fishnets20, ribbons with gradient width21 and multilayered ribbons with asymmetric voids22. Nevertheless, edge effects of structured graphene are evident disadvantages of these structures23. To avoid this problem, absorbers using nonstructured graphene are put forward, such as metal-dielectric-graphene sandwich structure24, graphene loaded with periodical arrays of dielectric bricks25, multilayer graphene sheets on quartz substrates26, graphene with metasurface comprising of plasmonic structures27, and multilayer graphene with uneven dielectric slab structure28.Terahertz (THz) devices have attracted increasing attention due to the unique properties of the THz region (0.1–10 THz), which share similarities with both the microwave and the far infrared rangesIn this letter, we propose an alternative broadband mechanism of multi-band continuous plasmon resonances, and demonstrate that the relative bandwidth of over 90% absorption can reach about 65% in the THz range by using nonstructured graphene loaded with geometrically gradient dielectric structures. Here, we expound the physical mechanism firstly. Then, we present numerical simulations and results. Finally, we investigate the effects of some relative parameters.W along x-axis, the periodic interval L along y-axis, the semi-minor axis R1, the semi-major axis R2 and the thickness H2 of the elliptic dielectric cylinder, the Fermi level EF of graphene, the thickness H1 of the dielectric substrate and the thickness Hm of the metallic film. The material of the dielectric substrate and the elliptic dielectric cylinder are dielectric 1 and dielectric 2, respectively. In this structure, the metallic film can reflect waves and the nonstructured graphene loaded with periodic arrays of geometrically gradient dielectric can also provide array local resonant reflection (ALRR)29, which form two mirrors of an asymmetric Fabry-Perot (FP) cavity30. The transmission of waves can be blocked as long as the metallic mirror is thick enough compared with the typical skin depth at THz frequencies. The reflection of waves can be eliminated if the critical coupling condition is satisfied27 or the impedance of the absorber is matched to that of the free space31. When both the transmission and reflection channels are suppressed, the near-unity absorption can be realized. Since the near-unity absorption is decided by the critical coupling condition or impedance matching condition that is closely related with the graphene plasmon resonances (GPRs), we can broaden the absorption band through broadening GPRs. However, generally, the GPRs are narrowband. So, in order to achieve broadband absorption, the key problem is to find a method to broaden the GPRs. In this configuration, the GPRs are related to the formation of standing-wave patterns of graphene plasmons with the wave vector33.e is the charge of electron, ħ is the reduced Planck’s constant, ω is the angular frequency of graphene plasmons, EF is the Fermi level of graphene, τ is the carrier relaxation time in graphene, ε0 is permittivity of vacuum, ε01 is the relative permittivity of the dielectric substrate, and ε02 is the relative permittivity of dielectric cylinders above the graphene film. When the structures are illuminated by a plane wave with the electric field linearly polarized along the x direction, the excited graphene plasmons propagate along the x direction and experience multiple reflections from the interfaces between the dielectric cylinders and air gap. In this structure, based on the thin element approximation in scalar Fourier optics, the elliptic dielectric cylinders can be divided into infinite infinitesimals with the profile of isosceles trapezoids along the y-axis, as shown in Figure Lp(y), namely the FP cavity lengths, between two mirrors at a specific position y is approximated askGP) is the real part of wave vector kGP, N is a positive integer determining the order of a resonance mode, ϕ is the phase of the reflection coefficient for graphene plasmon reflection at two mirrors. In general, ϕ is non-zero that depends on the structural, material parameters and position y. The physical explanation for this non-zero f is related to extension of the plasmon field beyond the interfaces of the dielectric cylinders and air gap.The proposed structure of the broadband THz absorber is presented in Fig. N can be set as different positive integers. We can also see that, for each discrete resonance, there exists a set of corresponding continuous plasmon resonances in the structure because y varies continuously in the elliptic dielectric cylinders. Under certain conditions, the frequency region of the continuous resonances can be larger than the corresponding frequency interval between two discrete neighbor resonances. So, the overlapping resonant frequencies can spread over a wide range, which is referred to as multi-band continuous GPRs.From eqs –3), by , by 3), β between two mirrors of a FP cavity is a key factor determining the performance of resonance, and an angle of zero or close to zero is a condition for obtaining strong resonances. In our proposed structure, the angle β between two mirrors at a specific position y can be approximately given byAs we know, the angle R1 and R2, the angle β decreases with a decrease of position y, which shows the FP-type resonances are most likely to occur at smaller position y and can always occur at position y = 0 particularly. For a fixed R1 and position y, the angle β becomes smaller and tends to zero as R2 increases. This implies that it is reasonable to use the elliptic dielectric cylinders rather than usual dielectric cylinders to act as the geometrically gradient dielectric to obtain multi-band continuous GPRs. Since the near-unity absorption is closely related with the GPRs, the achieved multi-band continuous GPRs imply that the broadband near-perfect absorption may be obtained under certain conditions.From equation , we can εr1 = 4 (dielectric 1) and εr2 = 12 (dielectric 2) are selected to show the physical mechanisms of broadband absorption. In the THz range, εr1 = 4 and εr2 = 12 can be approximatively provided by silicon dioxide and silicon, respectively34. The metallic material in this structure is gold, which is described by Drude model. The relative permittivity of gold is expressed by ε∞, ωP and γ are 1.0, 1.38 × 1016 rad ⋅ s−1, and 1.23 × 1013 s−1, respectively29. Graphene is modeled as an anisotropic layer with thickness hg = 1 nm. The relative permittivity of graphene comprises the out-of-plane component εout = 2.5 and the in-plane component εin = 2.5 + iσ(ω)/(ωε0hg), where the graphene optical conductivity σ(ω) is derived using the random-phase approximation (RPA)36. σ(ω) is calculated by σ(ω) = σinter(ω) + σintra(ω), where σinter(ω) and σintra(ω) are the interband and intraband contributions, respectively. In the THz range, the interband part σinter(ω) is negligible compared with the intraband part σintra(ω)37, hence we can use σintra(ω) to approximate σ(ω) here, which is described asω is the frequency of the incident wave, kB is the Boltzmann constant, e is the charge of an electron, T = 300 K is the temperature, & planck; is the reduced Planck’s constant, τ is the carrier relaxation lifetime, and EF is the Fermi level. Here, relaxation lifetime is expressed by 27, where the mobility μ is 10000 cm2/(V ⋅ s) and the Fermi velocity vF is 106 m/s. In the simulation, the open boundary condition is adopted in z direction and the unit cell boundary condition is adopted in x and y directions. The absorption is calculated from the obtained S parameters using A = 1 − R − T, where R is the reflection and T is the transmission.To verify the theoretical prediction, we next conduct full-wave numerical simulations employing frequency domain solver in CST Microwave Studio. In the simulation, EF = 0.5 eV, H1 = 15 μm, H2 = 11 μm, Hm = 2 μm, R1 = 10 μm, R2 = 36 μm, L = 80 μm, W = 24 μm. Figure First, we consider the case where R2, incident angle θ and Fermi level EF. It is necessary to stress that in each group of simulations, all the other parameters and conditions are kept the same as the above simulations.In order to investigate the characteristics of the broadband THz absorber, we sweep a few parameters, including semi-major axis R2 from 1 to 40 μm while fixing R1 = 10 μm. Figure R2/R1 under normal incident wave with the electric field parallel to x-axis. It can be observed that the absorption is very sensitive to ellipticity R2/R1. When the values of R2/R1 are very large (e.g. R2/R1 > 3), the characteristics of broadband absorption maintain well. On the other hand, when R2/R1 is relatively small (e.g. R2/R1 < 1.5), the broadband absorption tends to be torn. This is in agreement with that, decreasing the ellipticity R2/R1 results in an increase of the angle β between two mirrors of FP-type GPRs at specific positions in the elliptic dielectric cylinders, which eventually makes the corresponding resonance strength weaker and even disappearing. As a result, the corresponding absorption becomes small and the broadband absorption is torn. In order to further confirm the broadband mechanism, the typical distributions of the z component of the electric field at the distance 50 nm above the interface between the graphene and the elliptic dielectric cylinders for R2/R1 = 1 and R2/R1 = 0.4 are shown in Fig. R2/R1 = 3.6. Comparing the typical distributions of electric field in three different R2/R1 ratios, we can visually find that the larger R2/R1 can really provide more GPRs. For example, at the case of R2/R1 = 0.4, the FP-type GPRs only occur at the position y = 0. However, for R2/R1 = 3.6, the FP-type GPRs can occur at a wide region of position y. These field distributions are consistent with the prediction.We first investigate the effect of ellipticity of elliptic dielectric cylinders by varying θ from 0° to 89.9° while maintaining the incident plane wave in the x-z plane and the magnetic field parallel to y-axis to examine the incident angular sensitivity. The incident angle θ is defined as the angle between the incident plane wave and the positive z-direction. Figure θ. It can be seen that the dependence of the absorption on the incident angle is relatively weak when the incident angle varies between 0° and 60°. The reason for this is that the near-unity absorption of this type of absorber is closely related to the GPRs. However, the excitation of the GPRs is insensitive to the incident angle38. As the incident angle increases beyond 60°, the absorption decreases rapidly. This can be understood through the following qualitative analysis. The near-unity absorption is also closely related to destructive interference. But, in the case of large angle incidence, the reflection amplitudes from two mirrors of the asymmetric FP cavity formed by the metallic film and the nonstructured graphene loaded with periodic arrays of geometrically gradient dielectric have a large mismatch, and the destructive interference amplitude-matching condition is destroyed. It is noticeable that there is an abnormal absorption phenomenon near the incident angle 85°. The reason of this phenomenon is: for the structure consisting of a single layer of lossy two-dimensional (2D) material separated from a mirror by a dielectric spacer layer, there always exists an angle of incidence, at which the external radiation decay equals to internal absorption decay and thus complete absorption of the 2D material can be achieved39. For our absorber, at 3.6 THz, the above condition of complete absorption is satisfied when the incident angle is near 85°.Next, we vary the incident angle EF on the absorption in Fig. EF increases, which is because the plasmon resonance frequencies of the doped graphene increase with increased Fermi level40. It can also be seen that the absorption decreases rapidly when EF varies from 0.2 to 0 eV, which results from the fact that the metallic character of graphene decreases when EF is relatively small. However, there is an interesting and abnormal phenomenon that a narrowband absorption still exists near the upper absorption band even as EF approaches 0, where GPRs do not exist. To explain this phenomenon, we extract the absorption for EF = 0.0 eV from Fig. Then, we show the effect of Fermi level In summary, we propose a broadband THz absorber based on multi-band continuous plasmon resonances sustained by geometrically gradient dielectric loaded with graphene. The simulation results show that the relative bandwidth for the absorption above 90% can reach about 65%. The broad bandwidth mainly originates from two key aspects. One is that the nonstructured graphene loaded with geometrically gradient dielectric can provide multiple discrete graphene plasmon resonances with large relative frequency interval. The other is that, for each discrete resonance, there exists a set of continuous plasmon resonances because the width of dielectric structure varies continuously and gradiently. Besides, FP resonances of the dielectrics also contribute to the absorption. Based on nonstructured graphene, the absorber avoids processing graphene and destroying the unique properties of graphene. The approach of combining geometrically gradient dielectric structures and nonstructured doped graphene facilitates the application of the broadband THz absorbers and related devices.