The study of processing characteristic and property optimization of ultra-high-molecular-weight polyethylene (UHMWPE) pipe is increasingly performed, mainly focusing on difficulties in the melting process and poor thermal-mechanical properties after forming, which have limited the wider engineering application of UHMWPE pipe. In this study, organo-montmorillonite (OMMT)-modified UHMWPE pipe with good thermal-mechanical properties was prepared by screw extrusion molding. First, high-density polyethylene was subjected to fluidity modification so that the screw extrusion molding of UHMWPE pipe was feasible. Then, OMMT-modified UHMWPE pipes under different addition amounts of OMMT were innovatively prepared by extrusion. Furthermore, the effects of the addition amounts of the compatibilizer HDPE-g-MAH and the silane coupling agent γ- propyl trimethoxy silane (KH560) on the thermal properties of OMMT-modified UHMWPE pipe were investigated for the first time. Compared with those of pure UHMWPE pipe, the Vicat softening temperature (from 128 to 135.2 °C), thermal deformation temperature (from 84.4 to 133.1 °C), bending strength (from 27.3 to 39.8 MPa), and tensile strength (from 20.8 to 25.1 MPa) of OMMT-modified UHMWPE pipe were greatly increased. OMMT-modified UHMWPE pipe with good thermal-mechanical properties was able to be prepared by extrusion for the first time. The compatibilizer method of HDPE-g-MAH was slightly more effective than the coupling agent method of KH560. Ultra-high-molecular-weight polyethylene (UHMWPE) is a polymer that has been used in pipelines because of its good physicochemical and tribological properties ,2. NowadCompression molding without flow, which features simple operation and low cost, can overcome the disadvantage of the high viscosity of UHMWPE ,6,7,8,9.Screw extrusion molding, which features continuous production, can overcome the disadvantage of the low production efficiency of compression molding . By the However, the fluidity modification of UHMWPE will inevitably induce a degree of decrease in thermal and mechanical properties. Thus, the strengthening modification of UHMWPE is necessary for good thermal-mechanical properties. Strengthening modification methods of UHMWPE mainly include physical modification ,21,22, cIn this study, the fluidity modification of UHMWPE was performed so that the screw extrusion molding of UHMWPE pipe was feasible. Then, with different addition amounts of organo-montmorillonite (OMMT), OMMT-modified UHMWPE pipes were innovatively prepared in a single screw extruder. In addition, the effects of the addition amounts of HDPE-g-MAH and KH560 on the thermal properties of OMMT-modified UHMWPE pipe were investigated for the first time. With the management of thermal and mechanical properties, the Vicat softening temperature, thermal deformation temperature, bending strength, and tensile strength of OMMT-modified UHMWPE pipe were greatly increased. OMMT-modified UHMWPE pipe with good thermal-mechanical properties could be prepared by extrusion for the first time.UHMWPE (granule/GUR4152) was supplied by Ticona Co., Ltd., Frankfurt, Germany. OMMT (powder/I.44P), used as filler, was provided by Nanocor Co., Ltd., Arlington Heights, IL, USA. HDPE (powder/5070), used for the fluidity modification of UHMWPE, was provided by Beijing Yanshan Petrochemical High-Tech Co., Ltd., Beijing, China. HDPE 5070 was chosen because the melt flow index (MFI) of this HDPE is high (6.1~8.0). HDPE-g-MAH (powder/ME5506), used as a compatibilizer, was obtained from Nantong Rizhisheng New Polymer Material Technology Co., Ltd., Nantong, China. A silane coupling agent (liquid/KH560), used to improve the interaction between UHMWPE/HDPE and OMMT, was supplied by Hangzhou Jiexika Chemical Co., Ltd., Hangzhou, China. The chosen silane should have both an inorganic reactive group and an organic reactive group so that it can enhance the interaction between the filler OMMT (inorganic phase) and the matrix UHMWPE/HDPE (organic phase). Ethanol (liquid) was supplied by Dongguan Jintai Chemical Technology Co., Ltd., Dongguan, China.A single screw extruder (with a screw diameter of 45 mm), used for ultra-high-molecular-weight material, was developed by Beijing University of Chemical Technology, China. A high-speed mixer (SHR-25A) was provided by Zhangjiagang Yongli Mechanical Co., Ltd., Zhangjiagang, China. An electric constant temperature drying oven (SFG-02.400) was provided by Hengfeng Medical Device Co., Ltd., Huangshi, China.The preparation of OMMT-modified UHMWPE pipe samples is summarized as follows:Step 1. The mixtures of materials were prepared with a compatibilizer method and a coupling agent method.(a)For the compatibilizer method, the OMMT was kept in a drying oven at a constant temperature of 110 °C for four hours. Then, UHMWPE, HDPE, OMMT, HDPE-g-MAH, and other additives were added into a mixer for homogeneous mixing.(b)For the coupling agent method, a solution of KH560 and ethyl alcohol was prepared with a volume ratio of 1:10. Then, the solution was premixed uniformly with OMMT, and the mixture was kept in a drying oven at a constant temperature of 110 °C for four hours. Lastly, UHMWPE, HDPE, OMMT, KH560, and other additives were added into a mixer for homogeneous mixing.The mixer has two levels of revolving speed: 725 and 1440 rpm. At room temperature, the mixer works at 725 rpm for five minutes and then at 1440 rpm for another five minutes.Step 2. The mixtures were processed into pipe samples see  in a sin3 for the Vicat softening temperature and 80 × 10 × 4 mm3 for the thermal deformation temperature. The pressure 0.45 MPa was used to test the thermal deformation temperature. The sample bars were obtained from pipe samples by cutting and flattening according to China national standards (the same below).A tester of Vicat softening temperature and thermal deformation temperature  was used to test the Vicat softening temperature and thermal deformation temperature of OMMT-modified UHMWPE pipe according to China national standard GB/T1633-2000. The lengths, widths, and thicknesses of the sample bars were 10 × 10 × 4 mm3.A Shore D durometer  was used to test the Shore hardness of OMMT-modified UHMWPE pipe according to China national standard GB/T2411-2008. The length, width, and thickness of the sample bar were 50 × 50 × 4 mmSEM  was used to analyze the microstructures of OMMT-modified UHMWPE pipe. Cooled with liquid nitrogen, the sample was sprayed with gold after a brittle fracture.DSC  was used to measure the melting enthalpy, melting point, and crystallinity of OMMT-modified UHMWPE pipe. Under the protection of nitrogen gas, each sample of 5 milligrams was heated from room temperature to 200 °C at a rate of 10 °C/min and then cooled to room temperature.3 for the bending strength and 120 × 10 × 4 mm3 for the tensile strength.A universal material testing machine  was used to test the bending strength and tensile strength of OMMT-modified UHMWPE pipe according to China national standards GB/T9341-2000 and GB/T1040-2006. The lengths, widths, and thicknesses of the sample bars were 80 × 10 × 4 mmAn X-ray diffractometer  was used to measure the diffraction angle at the diffraction peak of OMMT-modified UHMWPE pipe. The diffraction test was performed under CuKα-ray radiation with a tube voltage of 40 kV, a tube current of 50 mA, and a scanning speed of 5°/min.Firstly, the thermal and mechanical properties of pure UHMWPE pipe were tested and are shown in In order to study the effect of the addition amounts of the filler OMMT on the thermal and mechanical properties of UHMWPE pipe, OMMT-modified UHMWPE pipes were prepared using different addition amounts of OMMT. The other additives in wOMMT ≤ 8%. When the nano-filler OMMT is introduced, it disperses in the matrix of UHMWPE/HDPE. With an increasing amount of OMMT, more polymer macromolecules should be adsorbed on the surface of the nano-filler OMMT. As a result, UHMWPE/HDPE entanglements will be strengthened. The strengthened entanglements will enhance the intermolecular forces and the rigidity of the OMMT-modified UHMWPE. According to its testing principle, the Vicat softening temperature can be regarded as the hardness of materials under heating conditions, i.e., the rigidity of materials. Therefore, the Vicat softening temperature increases with an increasing amount of OMMT for wOMMT ≤ 8%. When the OMMT-modified UHMWPE is heated, OMMT around the molecular chains of UHMWPE/HDPE plays the role of a physical cross-link, which limits the thermal motion of UHMWPE/HDPE molecular chains. Therefore, the thermal deformation temperature increases with increasing amounts of OMMT for wOMMT ≤ 8%. Moreover, with the filling of OMMT, the OMMT-modified UHMWPE is denser in molecular chain distribution, more complex in space structure, and more difficult to invade than the matrix UHMWPE/HDPE. Therefore, the Shore hardness increases with increasing amounts of OMMT for wOMMT ≤ 8%.As shown in wOMMT = 8%, and then decrease. Therefore, a reasonable addition amount of OMMT is 8%. At wOMMT = 8%, the Vicat softening temperature, thermal deformation temperature, and Shore hardness are 135.2 °C, 133.1 °C, and 90 HD, respectively.In wOMMT = 5%, 8%, and 10% were compared, as shown in wOMMT = 5% and 8%, while aggregation of OMMT is found at wOMMT = 10%. At wOMMT = 8%, OMMT disperses uniformly in the matrix UHMWPE/HDPE without aggregation; however, excessive amounts of OMMT give rise to aggregation, which can be a material defect and weaken the thermal and mechanical properties of OMMT-modified UHMWPE. In wOMMT > 8%.To study the deterioration of the Vicat softening temperature, thermal deformation temperature, and Shore hardness, SEM micrographs of the fracture surfaces at wOMMT = 5%, 8%, and 10%. The melting enthalpy (ΔH) and melting point (Tm) of the OMMT-modified UHMWPE, which can be obtained from the DSC curves, are also shown on the left and right sides in H and the melting enthalpy of fully crystallized UHMWPE (ΔH0) known, the crystallinity (Xc) of the OMMT-modified UHMWPE can be obtained by the following equation and as shown in In addition, wOMMT = 8%, and then decreases with increasing amounts of OMMT. For wOMMT ≤ 8%, the addition of OMMT gives rise to the heterogeneous nucleation crystallization of UHMWPE/HDPE, which increases the crystallinity and the melting point of OMMT-modified UHMWPE; however, for wOMMT > 8%, an excessive amount of OMMT results in aggregation, which will weaken the effect of the heterogeneous nucleation crystallization of UHMWPE/HDPE. It is also found that the bending and tensile strengths of the OMMT-modified UHMWPE have the same trend as the crystallinity  were compared, as shown in wOMMT = 8%) occur at 2θ = 3.314 and 2.538 deg, respectively. Then, the layer distances can be obtained by the Bragg equation:wOMMT = 8%) are 2.663 and 3.477 nm, respectively.To better understand the changes in the bending and tensile strengths, small-angle X-ray diffraction (XRD) curves of OMMT and OMMT-modified UHMWPE (wOMMT = 8%) moves to a smaller angle, which indicates that the molecular chains of UHMWPE/HDPE enter the lamellar structure of OMMT. With more molecular chains of UHMWPE/HDPE entering the lamellar structure of OMMT, the OMMT-lamellar-coated molecule chains of UHMWPE/HDPE are able to sustain larger bending loads. In addition, under tension loads, the molecular chains of UHMWPE/HDPE in the lamellar structure of OMMT extend along the lamellar structure, which gives rise to a uniform distribution of tensile stress. As a result, both the bending and tensile strengths of the OMMT-modified UHMWPE increase with an increasing amount of OMMT for wOMMT ≤ 8%. However, for wOMMT > 8%, an excessive amount of OMMT induces aggregation, which will be a material defect and weaken the effect of OMMT-lamellar-coated molecular chains.Compared with that of OMMT, the diffraction peak for the crystal (001) of the OMMT-modified UHMWPE  than by using KH560 (wKH560/wOMMT = 1%).A further comparison between wHDPE-g-MAH = 3%) and using KH560 (wKH560/wOMMT = 1%), small-angle XRD curves of the corresponding OMMT-modified UHMWPE were compared, as shown in θ = 2.538 and 2.584 deg, respectively. The layer distances of OMMT for the corresponding OMMT-modified UHMWPE can be obtained by Equation (2), and the calculated layer distances are 3.477 and 3.415 nm, respectively. A longer layer distance by using HDPE-g-MAH than by using KH560 indicates that more molecular chains of UHMWPE/HDPE enter the lamellar structure of OMMT. The distance between molecular chains is shorter in the lamellar structure than outside the lamellar structure. With more molecular chains in the lamellar structure, the intermolecular force becomes stronger. Thus, the strong intermolecular force limits the thermal motion of molecular chains of UHMWPE/HDPE and increases the thermal deformation temperature and Vicat softening temperature.To better understand the differences in thermal properties between using HDPE-g-MAH , thermal deformation temperature (from 84.4 to 133.1 °C), bending strength (from 27.3 to 39.8 MPa), and tensile strength (from 20.8 to 25.1 MPa) of OMMT-modified UHMWPE pipe were greatly increased. Our results showed that(1)screw extrusion molding of UHMWPE was feasible by the modification of UHMWPE;(2)wKH560/wOMMT = 1% for the coupling agent method;for the process parameters in this study, the reasonable addition amounts of OMMT and HDPE-g-MAH were 8% and 3%, respectively, for the compatibilizer method, and the reasonable addition amount of KH560 was (3)with the management of thermal-mechanical properties, OMMT-modified UHMWPE pipe with good thermal-mechanical properties was able to be prepared by extrusion for the first time; and(4)the compatibilizer method of HDPE-g-MAH was slightly more effective than the coupling agent method of KH560.


We have previously observed that tropomyosin receptor kinase B (TrkB) induces breast cancer metastasis by activating both the Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) and phosphatidylinositol-3-Kinase (PI3K)/AKT signaling pathways and inhibiting runt-related transcription factor 3 (RUNX3) and kelch-like ECH-associated protein 1 (KEAP1). These studies indicated that TrkB expression is crucial to the pathogenesis of breast cancer. However, how TrkB regulates bone morphogenetic protein (BMP) signaling and tumor suppression is largely unknown. Herein, we report that TrkB is a key regulator of BMP-mediated tumor suppression. TrkB enhances the metastatic potential of cancer cells by promoting cell anchorage-independent growth, migration, and suppressing BMP-2-mediated growth inhibition. TrkB inhibits the BMP-mediated activation of SMAD family member 1 (SMAD1) by promoting the formation of the TrkB/BMP type II receptor complex and suppresses RUNX3 by depleting BMP receptor I (BMPRI) expression. In addition, the knockdown of TrkB restored the tumor-inhibitory effect of BMP-2 via the activation of SMAD1. Moreover, the TrkB kinase activity was required for its effect on BMP signaling. Our study identified a unique role of TrkB in the regulation of BMP-mediated growth inhibition and BMP-2-induced RUNX3 expression. The bone morphogenetic protein (BMP) family, a member of the transforming growth factor-β (TGF-β) superfamily, consists of over 20 multifunctional growth factors with a broad range of regulatory functions, including embryonic development, apoptosis, chemotaxis, proliferation, and differentiation ,2,3,4. ASimilar to the functions of TGF-β in the cancer microenvironments, BMPs, and their receptors play a dual tumor-suppressive and -promoting role in cancer. For example, in an analysis of large patient cohorts, Gremlin 1, a BMP-antagonist, was found to be significantly increased in the tumor tissues of breast cancer patients . In anotBrain-derived neurotrophic factor (BDNF), a neurotrophin, interacts with and activates tropomyosin receptor type B (TrkB) and plays a key role in the survival, differentiation, and maintenance of neurons . Recent To understand the correlation between TrkB and BMP signaling, we examined whether TrkB expression can modulate the transcription activity of a luciferase gene driven by the BMP response element (BRE). RIE-1, HeLa, and NMuMG cells were transiently-transfected with TrkB, which reduced BRE-luciferase activity relative to that of the control . Thus, TAs expected, the BMP-2-mediated transcriptional activity was reduced by TrkB overexpression in both TrkB-overexpressing cell lines . In normTo further substantiate the role that BMP signaling plays in regulating tumor invasion and the function of TrkB, we knocked down TrkB expression in MDA-MB-231 and Hs578T cells, which have high TrkB expression . We founOur results suggest that TrkB suppresses BMP-mediated tumor inhibition by regulating the processes upstream of SMAD1. We speculate that the mechanism underlying this process might be associated with TrkB-BMP receptor interactions. We found that TrkB directly interacted with BMPRII, but not with BMPRI A,B. In aMoreover, when the cytoplasmic and N-terminal regions of BMPRII, which included the extracellular and transmembrane domains, were deleted, the binding affinity for TrkB remained unchanged. However, when an area between amino acids 203 and 350, which corresponded to the tyrosine kinase domain of BMPRII, was removed, this abolished its binding affinity E. Thus, We assessed the effects of a TrkB kinase inhibitor on BMP-mediated tumor inhibition in RIE-1 and RIE-1-TrkB cells. The inhibition of TrkB kinase activity with K252a significantly increased BRE transcriptional activity in RIE-1-TrkB cells upon treatment with BMP-2, whereas no change to BRE transcription was observed in RIE-1 cells, with or without K252a treatment A. SimilaSimilarly, in MDA-MB-231 and Hs578T breast cancer cells, which expressed endogenous TrkB, the BRE transcriptional activity increased in response to K252a and BMP-2 treatments C,D. ConsTo assess the effects of TrkB activation on BMP-associated transcriptional activity, we generated RIE-1 TrkB kinase-dead (KD) cell lines using vectors that expressed a kinase-dead mutant of TrkB (K588M). As expected, both RIE-1-TrkB and HeLa-TrkB cells exhibited reduced reporter activity in response to BMP-2 treatment, whereas in cells that expressed the TrkB KD, BMP-2 treatment increased reporter activity A,B. In aWe were also interested in the possibility that TrkB KD and BMPRII form a complex. Wild-type TrkB was strongly associated with BMPRII; however, the interaction between BMPRII and the TrkB KD mutant was significantly reduced with or without BMP-2 treatment. Furthermore, BMPRII phosphorylation following BMP-2 treatment was observed only in TrkB wild-type cells D. BecausBMP-2 induces the expression of the tumor suppressor RUNX3 and consequently decreases c-Myc expression when RUNX3 binds to the c-Myc promoter. Moreover, in comparison to the constitutively-active BMPRIA (CA-ALK3), the transfection of a dominant-negative version of BMPRI (DN-ALK3), in the presence of BMPRII, does not suppress c-Myc transcription activity , indicatOur results show that BMPRI-BMPRII complex formation was significantly suppressed in the presence of TrkB E,F. UndeOur results suggest that TrkB expression might enhance the tumorigenic potential of the tumor by countering BMP-2-mediated growth inhibition. Based on the above findings, we evaluated whether TrkB overexpression inhibited RIE-1 cell metastasis. To metastasize to distant organs, cancer cells must overcome anoikis as part of cancer progression . We firsWe have previously shown that TrkB induces tumorigenesis and metastasis via the induction of the JAK2/STAT3 and PI3K/AKT pathways. In this study, we showed that TrkB modulates BMP-mediated the growth inhibition of cancer cells. BMP-2 treatment induced SMAD1 phosphorylation and downstream transcription in RIE-1 and HeLa cells, which were significantly reduced by TrkB. In contrast, the loss of TrkB in highly metastatic breast cancer cell lines (MDA-MB-231 and Hs578T) restored BMP signaling. Moreover, growth inhibition induced by BMP-2 treatment was rescued when TrkB was also knocked down in these cells. Our results indicate that the TrkB-mediated suppression of BMP activities is necessary for the induction of tumorigenicity and cancer metastasis, which also supports results from our previous study. We have previously demonstrated that TrkB expression is dramatically increased in triple-negative breast cancers (TNBCs) relative to other subtypes and induces the growth and metastasis of breast cancer cells in vivo ,25. In aFurthermore, when the TrkB+ CSCs were eradicated, TNBC recurrence was prevented, which increased patient survival. In our study, TrkB overexpression in RIE-1 cells markedly increased anchorage-independent growth, migration, and mammosphere formation. These results suggest that TrkB may regulate cancer progression and recurrence by acquiring CSC traits.We also investigated whether TrkB can regulate the expression of BMP antagonists to inhibit BMP signaling. We found that TrkB significantly upregulated the expression of Gremlin 1 (a BMP antagonist). A previous study demonstrated that BMP signaling prevents TGF-β-mediated CSC transition in breast cancer and TGF-β-induced epithelial-to-mesenchymal transition (EMT) in renal fibrosis. In a mesenchymal subpopulation (MSP) of the human breast mammary epithelia, BMP antagonists (Chordin-like 2 and Gremlin 1) were significantly increased, whereas BMP ligands (BMP-2 and BMP-4) were markedly decreased. Additionally, BMP-4 treatment inhibited the capacity of HMLE, an MSP that had arisen from immortalized human mammary epithelial cells (MECs), to migrate and form mammospheres. The twist is overexpressed in HMLE cells ,29. MoreOur studies (both past and present) showed that TrkB represses RUNX3 expression by activating the PI3K/AKT signaling pathway and inhibiting the canonical BMP signaling. Introducing BMPRI in the presence of TrkB upregulated RUNX3 expression, which is further enhanced by the addition of BMP-2. Furthermore, upon transfection with BMPRI, RUNX3 expression is increased in MDA-MB-231 TrkB-shRNA but not in MDA-MB-231 control cells, and this increase is further enhanced by BMP-2 treatment. The activity of RUNX3, a tumor suppressor, is inhibited in various cancers by mislocalization, hypermethylation, or loss of heterozygosity ,33,34. RAdditionally, BMP signaling plays a role in the Hippo signaling pathway by inducing the phosphorylation of YAP (inactivated form) and stimulating the Ras association domain family  to inhibit tumor development and progression. BMP-2 treatment activates the Hippo kinase cascade, which includes mammalian Ste20-like kinase 1 (MST1), MOB kinase activator 1 (MOB1), and YAP (inactivation), and this action is tumor suppressive. BMP-2 also induces BMPRII expression, apoptosis by inhibiting the nuclear translocation of YAP, and RASSF1-MST1 complex formation by disrupting AKT-RASSF1 interaction . AnotherTo date, the link between TrkB and BMP signaling is unknown, and our study is the first to report this association. We have identified a unique role for TrkB in the regulation of BMP and RUNX3-mediated growth inhibition of cancer cells.RIE-1, HeLa, NMuMG, 293T, and human highly metastatic cancer cells (Hs578T and MDA-MB-231) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). BMP-2 from PeproTech (500-P195BT) used at the final concentration of 50 ng/mL for the indicated time. The protein kinase inhibitor K252a was purchased from Abcam.RIE-1, HeLa, and NMuMG cells were infected with a V5-TrkB using a pLenti6.3/V5-TOPO TA Cloning Kit  to generate TrkB overexpression cells  and the Proteins extracted from human breast normal and tumor samples obtained as previously described ,41.We performed Western blotting, immunoprecipitation, and immunofluorescence analysis, as previously described . phospho3 cells/well seeded into 6-well cell culture plates. For the wound healing assay, 1 × 106 cells/well seeded into 6-well cell culture plates. For the invasion assay, 1 × 104 cells seeded into 24-well BD Matrigel invasion chambers with 8-µm pores . For anoikis assay, RIE-1 and RIE-1-TrkB cells were seeded into an Ultra-Low Cluster plate  at 1 × 105 cells per well in a six-well plate and photographed at 7 days. For mammosphere assay, 1 × 103 cells/well were seeded into 96-well ultra-low adhesion plates in DMEM medium.All the assays were performed as previously described ,43,44. F4 cells were transfected with a BRE-Luciferase reporter plasmid using Lipofectamine 2000 . The cell lysates collected 48 hr after transfection, and the luciferase activities were measured using the Enhanced Luciferase Assay Kit .3 × 10Total RNA was isolated using RNeasy Mini Kits , and RT-PCR analysis was performed using a One-Step RT-PCR kit  according to the manufacturer’s instructions.For quantitative RT-PCR, reverse transcription performed with the Superscript Ⅳ First-strand synthesis system  and amplification performed with SYBR Green Mix I , and All PCR analyses were conducted in triplicate using the 7900HT Fast Real-Time PCR System . The primer sequences used to amplify the investigated genes listed in 5 cells/well in 0.5 mL culture medium. After waiting 4 hr for surface attachment, we incubated the cells in the presence or absence of porcine BMP-2 (5 ng/mL) for 24 hr. The cells were then pulse-labeled with 0.5 µCi [3H]-thymidine for 2 hr, fixed with 1 mL methanol/acetic acid, 3:1 (vol/vol), for 1 hr at 25 °C, washed twice with 2 mL 80% methanol, incubated at 37 °C with 0.2 mg/mL trypsin for 30 min and then solubilized with 0.5 mL 1% sodium dodecyl sulfate (SDS). Incorporated activity levels were measured with a scintillation counter.Actively growing asynchronous cells were plated in 24-well plates at a density of 0.5 × 10t-test (two-tailed). Differences were considered statistically significant at p < 0.001.Data expressed as the means ± SEM. Statistical analyses of the data conducted via the Student’s TrkB promotes the formation of TrkB/BMPRII complex by interacting between the TrkB kinase domain and then TrkB suppresses activation of SMAD1. Eventually, TrkB suppresses BMP and RUNX3-mediated growth inhibition of cancer cells.


In this study, graphene was synthesized on the Si(100) substrates via the use of direct microwave plasma-enhanced chemical vapor deposition (PECVD). Protective enclosures were applied to prevent excessive plasma etching of the growing graphene. The properties of synthesized graphene were investigated using Raman scattering spectroscopy and atomic force microscopy. Synthesis time, methane and hydrogen gas flow ratio, temperature, and plasma power effects were considered. The synthesized graphene exhibited n-type self-doping due to the charge transfer from Si(100). The presence of compressive stress was revealed in the synthesized graphene. It was presumed that induction of thermal stress took place during the synthesis process due to the large lattice mismatch between the growing graphene and the substrate. Importantly, it was demonstrated that continuous horizontal graphene layers can be directly grown on the Si(100) substrates if appropriate configuration of the protective enclosure is used in the microwave PECVD process. Graphene is a monolayer or several layers of hexagonally shaped carbon atoms . This 2DA complicated graphene transfer process is one of the main limitations preventing the broader use of graphene in semiconductor device technology. In this instance, graphene is grown on the copper of nickel catalytic foils ; followe2, Al2O3, MnO2, HfO2, and TiO2 films 2D/IG rat highest  at the sature in . A decreorted in ,51, simiThus, the different effects of the gas flow ratio, plasma power, and temperature on the graphene structure were found in various studies. The discrepancy of the results reported by other authors was more considerable than the results reported in the present study. The graphene structure’s dependence on the technological synthesis process conditions found in the present research can be explained by considering the main graphene growth-related physical and chemical processes. Notably, the decrease in the number of graphene layers with increasing process time can be explained by the hydrogen etching prevailing over the growth of additional graphene layers as it was reported in . 2D/IG ratio with plasma power ubstrate , and SiOtrate [2 . Thus, otrate [2 ,69.In conclusion, the transfer-less and catalyst-less synthesis of graphene on Si(100) substrates via a combination of direct microwave plasma-enhanced chemical vapor deposition and protective enclosures was performed.4/H2 gas flow ratio, temperature, and plasma power on graphene structure revealed that the most significant technological parameter used in the present study was methane and hydrogen gas flow ratio. Plasma power effects were the least pronounced. It seems that if a temperature is sufficiently high for graphene synthesis, the crucial process is a competition between plasma etching by hydrogen and carbon-containing active species influx towards the surface. If hydrogen flow is too high and/or methane flow is too low, etching prevails against growth and no graphene is formed. Afterwards, the number of graphene layers increases with carbon species flow and/or with decreased hydrogen flow. Hydrogen species density is a much more critical etching factor than increased plasma power. The thermally stimulated desorption of carbon atoms is important, while the formation of the plasma-induced radiation defects has less influence on graphene growth and defect density. A study of the effect of the CHA study of the enclosure effects revealed no top hole size effects for investigated enclosures. The absence of top holes in the middle of the enclosure reduced the plasma effect on the growing graphene and decreased defect density. The graphene was successfully synthesized using just a rectangular steel sheet folded in two places as a simplified protective enclosure, considering these results. Graphene was grown at a temperature of 700 °C using a protective sheath with such a simple structure. Analysis of the positions of 2D and G peaks revealed unintentional n-type doping of the graphene. It was explained by charge transfer from the Si(100) substrate to the graphene. The presence of compressive stress was found in graphene. It was supposed that the large lattice mismatch between the growing graphene and the silicon induced thermal stress.An atomic force microscopy study confirmed the growth of continuous horizontal graphene layers.


Nanoscale photothermal effects enable important applications in cancer therapy, imaging and catalysis. These effects also induce substantial changes in the optical response experienced by the probing light, thus suggesting their application in all-optical modulation. Here, we demonstrate the ability of graphene, thin metal films, and graphene/metal hybrid systems to undergo photothermal optical modulation with depths as large as >70% over a wide spectral range extending from the visible to the terahertz frequency domains. We envision the use of ultrafast pump laser pulses to raise the electron temperature of graphene during a picosecond timescale in which its mid-infrared plasmon resonances undergo dramatic shifts and broadenings, while visible and near-infrared plasmons in the neighboring metal films are severely attenuated by the presence of hot graphene electrons. Our study opens a promising avenue toward the active photothermal manipulation of the optical response in atomically thin materials with potential applications in ultrafast light modulation. A new breed of ultrafast light modulators that operate across the visible to terahertz regions may be possible by harnessing photothermal control of plasmons in graphene and thin-metal films. Theoretical analysis performed by Eduardo Dias and coworkers from the ICFO research institute in Barcelona, Spain suggests that ultrafast optical excitation of thin gold/silver-graphene structures can significantly raise the electron temperature in the carbon layer and as a result dramatically modify the plasmonic response. Calculations indicate that strong changes (>70%) in the optical reflection of such structures are possible, with a strength that increases with the fluence of the excitation pulses. Importantly, the fluence is below the damage threshold of the materials. The photothermal modulation approach could have useful applications ranging from all-optical light modulation to optical sensing. These important applications have been extensively investigated in conducting materials due to the substantial enhancement produced in the strength of nanoscale photothermal processes as a result of the excitation of their collective electron oscillations, known as plasmons12. Among plasmonic materials, graphene offers additional appealing properties, such as large field confinement and enhancement15, remarkably low optical losses16 and the ability to tune its plasmons electrically27, which have generated expectations for applications in optical modulation31, light detection33, and sensing36. Unfortunately, plasmons in graphene have been observed only in the mid-infrared (mid-IR) spectral range, far from the technologically appealing visible and near-infrared (vis-NIR) spectral regime. Photothermal excitation has been proposed as a way to reach the vis-NIR plasmonic regime37, but experimental attempts in this direction have only been limited to the mid-IR39. As an alternative approach to extrapolate the appealing plasmonic properties of graphene to vis-NIR, both monolayer noble metals40 and hybrid systems formed by graphene in close proximity to few-nanometer metal films41 have been predicted to display relatively low losses and a large electrical tunability in the vis-NIR domain. Progress in this direction has been recently made with the experimental demonstration of plasmons in laterally patterned sub-2-nm crystalline silver42 and thicker amorphous gold43 films. In particular, epitaxially grown silver films serve as a novel platform for ultracompact nanophotonic devices that can further benefit from the comparatively high quality factor of plasmons in defect-free crystalline silver samples42.Heat generation driven by light absorption in nanostructures has proven useful for photothermal therapy44. Importantly, due to the small electronic heat capacities of graphene45 and ultrathin noble metals46 compared with their total heat capacities, the lattice remains close to ambient temperature for optical pump-pulse fluences as high as ~10 J/m2, which is the upper limit of the range typically used to investigate thin-film dynamics through pump-probe spectroscopy48. In combination with the strong temperature dependence of the graphene optical conductivity, the transmission and reflection of a light probe can undergo radical variations produced by heating49, while intense photothermal effects should be expected due to the exceptionally small electronic heat capacity and generally weak electron-phonon coupling in this material45. Metals present a weaker thermal dependence, although the effects of intense pumping have also been observed to affect the probe as heat propagates away from the pumped region48. Consequently, graphene, thin metal films and hybrid systems comprising these materials hold strong potential to achieve large photothermal modulation over a broad spectral range extending from the visible to the THz region. Despite the vast range of applications that such modulation could open, the ultrafast photothermal response of these systems has been poorly explored.Under ultrafast light irradiation, the absorbed energy is first deposited on conduction electrons, which remain at an elevated temperature for ~1 ps before transferring a substantial fraction of heat to the atomic latticeIn this paper, we theoretically investigate the thermal control of plasmons assisted by light absorption in graphene, thin metals and graphene/metal hybrid films. Based on a realistic description of the temperature-dependent optical properties of these materials, combined with the light-electron-lattice heat flow under ultrafast optical pumping, we predict a remarkable thermally driven attenuation of the plasmon strength in mid-IR graphene plasmons and vis-NIR metal plasmons of hybrid systems. This attenuation is directly revealed by plotting the reflection coefficient of planar structures. We further propose a practical scheme to exploit this dependence, consisting of laterally patterning the metal so that propagating external light can directly couple to the noted plasmons. Our results predict a large modulation of the light reflection produced by optical pumping using attainable intensities below the damage threshold level. Graphene plays an important role in these structures because in its absence the photothermal response is much weaker; we relate this result to the small heat capacity of the carbon layer. Our findings open a promising route towards ultrafast photothermal light modulation over a broad spectral range extending from vis-NIR to far-IR.We illustrate the main concept explored in this paper in Fig. 37. This effect is thus an ideal ingredient for the pump-probe scenario envisioned in Fig. EF  for realistic levels of doping, and the same is true for noble metals. A femtosecond optical pump can then raise the electron temperature Te to 1000′s K, while heat transfer to the lattice, which takes place over a picosecond timescale44, does not substantially move the lattice temperature T0. In fact, we estimate that for fluences of the order of 1–5 mJ/m2, which roughly correspond to ultrafast pulses of ~100 fs with peak intensities of 0.1–0.5 GW/cm2, the electron temperature in graphene increases up to ~5000 K52, whereas electrons in the metal stay within a few 100′s K, as shown in Fig. 53. Therefore, in what follows, unless otherwise stated, we assume Graphene displays stronger photothermal effects than noble metals see Fig.  due to iTe through the chemical potential μ, the Drude weight μD and the inelastic scattering rate 1/τ, which enter its optical conductivity σ, as shown in Eq. . In contrast, the plasmon frequencies in noble metals exhibit a very mild dependence on the electron temperature (see Methods).The optical response of graphene is highly dependent on see Fig. . The nonTe, as shown in Fig. EF because the phase space for electron-phonon interactions is enlarged. Similar trends of the temperature dependence of the scattering rates are found in noble metals, as shown in Fig. The inelastic scattering rate of graphene also depends strongly on Te, as calculated using methods discussed in previous studies54. In all cases considered in Fig. Te as a result of the increase in Re{σ}. For the mid-IR graphene plasmons considered in Fig. Te rises , while thermal smearing of Dirac fermions shifts down the chemical potential μ for the Te values under consideration and diffuses the quenching within an energy interval of roughly ±kBTe  around 2μ .In Fig. see Fig. . We note57) or additional elements . Recently, an alternative route to excite graphene plasmons has been proposed49, where two interfering optical pump beams create a periodic spatial temperature profile in graphene to assist the coupling of a probe beam to plasmons. The characteristic diffusion time of the temperature profile created by the optical pump is of the order of a few hundred femtoseconds58, which should allow a probed optical pulse to excite plasmons, as considered in this study. Here, we further demonstrate that not only graphene plasmons but also metal plasmons or even subwavelength acoustic plasmons can be excited using this approach. We assume a periodic pattern of electron temperature Te created by the optical grating pump only inside the graphene sheet with a modulated temperature range extending from 300 to 8000 K. Due to the strong temperature dependence of the graphene conductivity σ  in 1-nm-thick (four atomic layers) gold gratings, which is a smaller value than in hybrid structures, despite the large fluences under consideration compared with those needed to heat graphene electrons to the values of Te used in Fig. We finally explore in Fig. see Fig.  in SI, w37, but their applicability has been limited to the mid-IR regime. We have shown that the addition of a noble metal film in close proximity to graphene enables similar levels of modulation >70% to be reached over a wider spectral range extending down to the visible domain. More precisely, optical pumping can bring graphene electrons to a high temperature, while the large heat capacity of the metal prevents similar heating inside it; then, high-energy metal-like plasmons are strongly affected by the opening of additional loss channels ; additionally, lower-frequency acoustic plasmons supported in these hybrid structures also become more lossy. A practical way to exploit these effects consists in laterally patterning the metal , thus breaking the energy-momentum mismatch that otherwise prevents light coupling into the plasmons of planar films. We envision applications in ultrafast optical modulation, which could be potentially pushed to the single-photon level through plasmon blockade in sufficiently small graphene nanostructures60. Spectrally resolved optical sensing could also be carried out by exploiting the temperature-dependent plasmon shifts reported here, which would enable a continuous sweep of the probe frequency as the system cools down over a picosecond timescale. Although the achieved thermally-induced spectral shifts can be limited by the presence of a finite diffusion time in the temperature profile, the amplitudes of the associated changes in the reflectivity provide an alternative sizeable signature for optical sensing in the ~300–3000 K temperature range.In summary, the remarkably small electronic heat capacity of atomically thin systems, such as graphene and few-atomic-layer noble metal films, allows us to elevate their electron temperature via ultrafast optical pumping in such a way that their optical responses are dramatically modified with moderate pump intensities below the damage threshold. This effect, which constitutes the basis for ultrafast thermo-optical modulation, is particularly strong in graphene due to the large photothermal response of this material, in which the conical electronic band structure causes large changes in the optical behavior for moderate amounts of deposited electronic heat. The photothermal modulation of the frequency and strength of graphene plasmons has been extensively studied in the past62We use the local random-phase approximation to calculate the optical conductivity of graphene asτ is a phenomenological inelastic scattering lifetime, Te, energy E and chemical potential μ, kB is the Boltzmann constant and 63where EF is the Fermi energy defined at zero temperature. The graphene conductivity has two contributions corresponding to the electronic intraband (first term in Eq. (μD) and interband . However, at higher electron temperatures, the chemical potential becomes increasingly smaller than EF  transit EF Fig. , allowinτ depends on both electron and lattice temperatures. For clean graphene samples , the inelastic scattering rate is dominated by electron-phonon scattering. Following previous studies65, the energy-dependent scattering rate associated with the b-type phonon is given to first order byIn general, the inelastic scattering rate 1/kk′ is the scattering angle between incoming and outgoing wave vectors k and k′, l = 1) and hole (l = −1) energies in the conical band dispersion regime, vF ≈ 106 ms–1 is the Fermi velocity andwhere θb-phonon scattering probability. Here, q = k′−k, b-type phonon that can be readily evaluated using Eqs. (16–21) and Table IV of ref. 64. Then, the total DC inelastic scattering rate is given byis the electron-where Te-dependent electronic heat as63The specific heat capacity of graphene Fig.  is calcuwhereis a thermal coefficient.59. In addition, the Drude model is in agreement with recent observations of plasmons supported by few-atomic-layer crystalline silver films42. We therefore use the Drude model and write the metal permittivity as48For ultrathin metal films, a recent study shows that the Drude response works extremely well compared with the random-phase approximation incorporating film electron wave functions and atomic-plane corrugationε∞ is a background accounting for the polarization of interband transitions, the plasmon frequency B  and68where mentclass2pt{minimmentclass2pt{minimγ0 is found by matching the total inelastic scattering rate γm to the experimental results at ambient temperature T0 = 300 K . The rightmost approximate expression in Eq. (γm in Fig. Te dependence of the plasmon resonance frequency of metallic nanostructures is mostly controlled by that of the classical metal plasma frequency ωp, which is robust against the electron temperature range considered in this work (see Fig. where n in Eq.  is validsee Fig.  in SI.Supplementary Information


Microorganisms can survive in extreme environments and oligotrophic habitats thanks to their specific adaptive capacity. Due to its severe and contrasting climate conditions, the cold mountain desert in Eastern Pamir provides a unique environment for analyzing microbial adaptation mechanisms occurring within colonization of endolithic habitats. This study aims to investigate the composition and structure of endolithic microbial communities and analyze the interactions between microorganisms and colonized lithic substrates. Endolithic biofilms were examined using scanning electron microscopy in backscattered electron mode (SEM-BSE) and next-generation sequencing (NGS) applying amplicon sequence variants (ASVs) approach. The investigation of the V3–V4 region of 16S rRNA gene revealed that endolithic communities are dominated by Actinobacteria (26%), Proteobacteria (23%), and Cyanobacteria (11.4%). Cyanobacteria were represented by Oxyphotobacteria with a predominance of subclasses of Oscillatoriophycidae, Synechococcophycideae, and Nostocophycidae as well as the rarely occurring Sericytochromatia. The positive correlation between the contribution of the orders Synechococcales and Rhizobiales to community structure suggests that some functionally closed taxa of Cyanobacteria and Proteobacteria can complement each other, for example, in nitrogen fixation in endolithic communities. The endolithic communities occurring in Eastern Pamir were identified as complex systems whose composition and structure seem to be influenced by the architecture of microhabitats and related microenvironmental conditions. Microorganisms play a crucial role in biogeochemical processes occurring in desert ecosystems . DespiteMicroorganisms can supply barren soil with nutrients such as carbon (autotrophs) and nitrogen (diazotrophs), which can be used by other organisms ,14. At hSome microbial species are more adaptable and can colonize different habitats in a poor desert ecosystem, while others can subsist only in particular microniches; these can be seen as habitat specialists. Understanding the role of particular taxonomic and functional groups of microorganisms in limited niches could help find their application in biotechnology and medicine ,15.There are numerous studies on endolithic microbial communities from the cold desert of Antarctica ,10,16,17Our preliminary study reported the diversity of endolithic microbial communities in granites, limestones, and gneiss , while tThis study was designed to assess the diversity of endolithic microbial communities inhabiting granites and quartzites from Eastern Pamir. The study is part of a wider project targeting taxonomic and functional diversity at multiple niches in the cold mountain desert of Pamir (Tajikistan) based on the V3–V4 hypervariable region of 16S rRNA gene which allows for a comparative examination of the microbial profile along multiple ecological niches in a high mountain cold desert ecosystem. The research aims of this study were to (1) investigate the composition and structure of endolithic microbial communities; (2) study the complexity of the investigated communities ; and (3) explore cyanobacterial diversity and estimate their potential roles in the functioning of the investigated communities.The Pamir Mountains lie between the world’s highest mountain ranges, namely, the Himalayas, the Tian Shan, Kunlun, Hindu Kush, Karakoram, Suleiman, and Hindu Raj . In termSamplings were performed in July 2017. The collected samples were stored in sterile plastic bags and were divided into 4 pieces for the following analysis: the first was powdered and used for DNA extraction; the second was prepared and analyzed under an optical and scanning electron microscope (SEM); the third was kept for isolation and cultivation of cyanobacteria; the fourth was frozen and stored in the freezer. Samples selected for research were collected from the sites which are characterized by rather similar physical and chemical parameters of the soil, humidity, altitude, and annual temperature.A).Granites collected from the slopes situated close to lakes Khargush (southern part of Eastern Pamir), and Rangkul (eastern part of Eastern Pamir): TAKHG and TARG.B).Granites collected from sampling sites located nearby Lake Karakul (northern part of Eastern Pamir): TAKAG1, TAKAG2, and TAKAG3.C).Quartzites collected from the same sampling area as the granites from group B which is nearby Lake Karakul: TAKAW1, TAKAW2, and TAKAW3 .Sample groups:Most of the investigated samples were collected from mountainsides with a similar altitude (between 3901 and 4244 m a.s.l.) except for sample TAKAG1, which was collected from a sampling site located at 5019 m a.s.l. The rock pieces with green, blue-green, and brown visible colonization were implanted on Petri dishes containing WC or BG11 medium ,27 solidThe powdered rock samples were treated with liquid nitrogen and the DNA was extracted using a Soil DNA Purification Kit . The DNA extraction was conducted following manufacture protocols and was repeated at least three times for each sample. The concentration and quality of the DNA were checked with Hybrid Multi-Mode Reader . The V3_V4_341f and V3_V4_785r primers  targetinThe V3–V4 sequences identified as cyanobacterial according to the SILVA  databasev/v) and later with osmium tetroxide solutions (1% w/v), dehydrated in a graded ethanol series (from 30% to 100% v/v), and embedded in LR-White resin. Later, the resulting blocks of resin-embedded rock colonized samples were finely polished, carbon-coated, and observed using a FEI INSPECT 105 SEM microscope.Small fragments of rocks with endolithic colonization were prepared for observation under the scanning electron microscope (SEM) in backscattered electron mode (SEM-BSE) according to a procedure developed by Wierzchos and Ascaso  in the lHierarchical clustering analysis and non-metric multidimensional scaling (NMDS) have been conducted using the R environment . The alpThe endolithic communities from strongly weathered granites and less porous quartzites were rich in photoautotrophic microorganisms and were characterized by blue-green and green to brown colonization. Biofilms occurring in quartzites were smooth and softly attached to the substratum, while those from granites were mostly covered by tiny mineral particles . In the Overall, the biofilms occurring in granites were characterized by brighter blue-green color, while those biofilms which developed in the cracks of the quartzites were of a darker yellowish or dark green to brown color. The presence of yellowish pigment in cyanobacterial sheaths was also observed under a light microscope during the examination of the biofilms.The results of amplicon-based analysis of Pamirian endoliths revealed that the communities were dominated by Actinobacteria (26% on average), Proteobacteria (23%), and Cyanobacteria (11.4%) including Sericytochromatia which were represented by 0.1% (47 reads) ASVs (amplicon sequence variants). Other bacterial phyla were less abundant, sharing about 39% altogether. The percentage of bacteria identified to phylum level only was 0.5% and unassigned 0.03% .Actinobacteria were the principal contributors to the endolithic community in the TARG (group A) TAKAW2 and TAKAW3 samples (group C). Proteobacteria, Actinobacteria, and Bacteroidetes were abundant in the samples from group B and quartzites from group C (TAKAW1). The distribution of Oxyphotobacteria was irregular, with the highest percentage in samples TAKHG (27.3%) and TAKAG2 (26.6%). No statistically significant correlation was found between Cyanobacteria and other bacterial phyla.The results of non-metric multidimensional scaling, based on the structure of microbial communities, revealed that endolithic communities from studied groups were sharing only some ASVs, while most of the sequence variants seem to be group-specific. The NMDS plot showed a triangle-like arrangement of the communities’ structures. Thus beta-diversity analysis did not reveal a high resemblance between particular communities. .Desmochloris halophila (70 representative sequences), and Picocystis salinarum (11 representative sequences). Some sequences which could not be further classified were identified as “chloroplast” only.The total number of representative sequences originating from chloroplast DNA was 587. Some of the sequences belonged to Chlorophyta, Alpha diversity metrics calculated for studied communities demonstrate significant differences between the communities . The ShaThe clustering based on the alpha diversity revealed the presence of two main clusters of communities . ClusterCyanobacterial communities were represented by 26 groups of amplicon sequence variants (ASVs). The distribution between the communities was mosaic . The phyr = 0.76 , however the correlation index was only calculated for eight studied communities here. The community TARG that was characterized by the absence of Synechococcales, but a greater contribution of Oscillatoriales and Nostocales according to the SILVA database contained also only 1% of Rhizobiales.According to the SILVA database, the contribution of the order Synechococcales was high compared to those mainly consisting of species that can fix atmospheric nitrogen, such as Nostocales and Chroococcidiopsidales . The samThermosynechococcaceae uncultured were located on the branch of Chroococcopsis gigantea with a like weight ratio of 0.9. The sequences of Aliterella CENA 595 according to the SILVA database were mapped on the branch of Synechocystis sp. with like weight ratio 1 (maximum). The results of the phylogenetic placement of the sequences with a low like weight ratio (about 18% with like weight ratio under 0.2) suggested the possibility of a novel taxa of endolithic cyanobacteria. Sequences belonging to one of the ASVs identified as “chloroplast” were placed on a branch of Chlorogloea microcystoides with a 0.83 weight like ratio.Analysis of cyanobacterial sequences placed on the Cydracil phylogenetic tree revealed that the sequences identified as Oscillatoria, Phormidium, and Leptolyngbya) and different phyla with a high abundance of Proteobacteria (mainly represented by Gammaproteobacteria) and Bacteroidetes. In two smaller clades (red and blue clades) representatives of Oxyphotobacteria  were associated with different genera of Actinobacteria and Alphaproteobacteria. Oxyphotobacteria (Cyanobacteria) from different orders were dispersed between the three biggest clades. The smallest clade (marked yellow in Aliterella genus occurred in the clade with the genus Rubrobacter (including Rubrobacter radiotolerans) and Frankiales bacterium (Actinobacteria). Generally, communities were diverse and represented by different types of ASVs belonging to the same classes or phyla; therefore, a clear pattern of microbial associations has not been detected.The analysis of microbial association at the endolithic niche revealed the presence of four main clusters of endolithic communities . The bigThe results of 16S metabarcoding were compared with morphological analysis of isolated and cultured cyanobacteria in sampled communities.Chroococcidiopsis-like taxa prevailed in cyanobacterial microbial communities in both types of mineral substratum, the granites, and quartzites. The granites were inhabited by more diverse cyanobacterial morphotypes than quartzites . Other less abundant morphotypes were Microcoleus-like (TAKHG and TAKAW1), Synechococcus-like (TAKHG and TAKAG2), Gloeocapsa- and Phormidium-like cyanobacteria (TARG), and Cyanosarcina-like cyanobacterium (TAKAHG)  ix.The study using the SEM-BSE technique revealed the presence of two types of microbial communities based on dominants: (1) “cyanobacteria-dominated” communities , TARG.Aliterella which belongs to Chroococcidiopsidaceae and was present in the samples TAKAW2 and TAKAW3.The SEM-BSE analysis allowed the identification of different lithobionthic niches; three types of endolithic colonization are differentiated: chasmoendolithic , TARG. ILichen-dominated communities colonized granite and quartzite substrates in two samples  , althougThe communities from group B granites were characterized by a similar contribution of Actinobacteria, Proteobacteria, and Bacteroidetes. However, these phyla were represented by the different families in individual samples. The Eastern mountains represents an environment rich in diverse rocks with different architecture and unique chemical properties that are colonized by cyanobacteria. We detected some similarities in the composition of Eastern Pamir communities to the endolithic communities from the cold desert of Antarctica ,14,42, oThe granites, characterized by heterogeneous porosity, were colonized by more diverse cyanobacterial communities than quartzites, with similar physicochemical properties. Bioreceptivity, weathering processes, and the microhabitat architecture of rocks are significant factors in shaping the structure of endolithic communities ,46. The However, some similarities between the colonization of granites and quartzites were also found. Lichens and coccoid cyanobacteria were detected in both lithic substrates and the dominance of certain taxonomic groups were not associated with a specific substrate. The 16S-based structure of endolithic cyanobacterial communities demonstrated that the quartzites were dominated by specific ASVs, while the contribution of representative cyanobacterial taxa occurring in granite’s inhabiting cyanobacteria was more balanced. The dominance of aggregates of coccoid cyanobacteria in endolithic communities has been previously reported for hot deserts such as Atacama ,44,45 orContrasting differences and similarities of the composition and structure of the communities could be explained by the fluctuating chemical composition within the same type of rocks ,9. HowevSlight differences detected in the color of studied biofilms developing in both types of mineral substratum could be considered as an adaptation to the colonization of rocks with different light regimes reaching the different microhabitats. The dark yellowish color of the biofilm TAKAW3 was detected by light microscopy, suggesting the presence of some accessory pigments in cyanobacterial sheaths. One of the UV-protecting metabolites in cyanobacterial sheaths is scytonemin. The production of this pigment was considered a strategy gained by lithobionths to cope with UV-radiation, especially for those occurring in more transparent types of rocks such as quartzites . SimilarDifferent biotic interactions are visualized by SEM-BSE concerning the microbial aggregates observed in endolithic habitats from Eastern Pamir. Microorganisms develop naturally in consortia in the environment, sharing some functions which are essential to survive in deserts or needed to cope with harsh conditions . The invInsight into the diversity and distribution of microbial taxa in extreme and in nutrient-limited environments, as well as an exploration of the relationships between abiotic and biotic components at different niches, could be key elements of understanding the functional diversity and the role of organisms in ecosystems ,48. Morp


Next, the promising applications of graphene-based electrodes in promoting this two-electron oxygen reduction reaction are considered and this is followed by an account of the various graphene-based materials that have been used successfully to give highly efficient graphene-based cathodes in electro-Fenton. In particular, graphene-based composites that have been combined with other carbonaceous materials, doped with nitrogen, formed as highly porous aerogels, three-dimensional materials and porous gas diffusion electrodes, used as supports for iron oxides and functionalised with ferrocene and employed in the more effective heterogeneous electro-Fenton, are all reviewed. It is perfectly clear that graphene-based materials have the potential to degrade and mineralise dyes, pharmaceutical compounds, antibiotics, phenolic compounds and show tremendous potential in electro-Fenton and other advanced oxidation processes.In recent years, graphene-based materials have been identified as an emerging and promising new material in electro-Fenton, with the potential to form highly efficient metal-free catalysts that can be employed in the removal of contaminants from water, conserving precious water resources. In this review, the recent applications of graphene-based materials in electro-Fenton are described and discussed. Initially, homogenous and heterogenous electro-Fenton methods are briefly introduced, highlighting the importance of the generation of H These oproducts . Conventproducts ,4,5. Theing AOPs ,6,7. 2+ to Fe3+ facilitates the conversion of H2O2 to the highly oxidising OH•, with a standard reduction potential of 2.56 V vs. SCE . Consequently, these OH• radicals can be employed to mineralise a large number of organic contaminants. The sustained production of OH• requires both Fe2+ and H2O2. The Fe2+ ions can be regenerated through the reduction of Fe3+ at the cathode (Equation (2)), provided the Fe3+ ions do not form hydroxide precipitates in the solution phase, to give a near-continuous supply of Fe2+. The H2O2 is produced by the two-electron reduction of dissolved oxygen; see Equation (3).The main principle of the E-Fenton process is summarised in Equation (1), where the oxidation of Fe2 and Fe(OH)3, the system must be operated under stringent pH control as these hydroxide species are only soluble at pH values lower than about 4.0. Therefore, the pH of water samples or effluents must be acidified and brought to an acidic pH value of approximately 2.0 to 3.0. Moreover, the OH• radicals are not continuously generated and require a supply of pure oxygen. However, the classical homogeneous E-Fenton process suffers from a number of limitations. The generation of secondary sludge, as ferric and ferrous ions in the treated wastewater, gives hydroxide precipitates and these must be removed, adding cost and reducing the overall efficiency. To limit the formation of solid Fe(OH)3O4, into a suitable electrode material. In 2+ is generated from a sacrificial anode and reacts with the H2O2 in the bulk solution. An iron salt can also be added to the cell to facilitate this reaction. In contrast, the aim in heterogeneous E-Fenton ) [2O2 is achieved at pH values between 2.0 and 3.0 [+, while the lack of protons with increasing pH reduces the rate of the reaction.As high yields of H process . Consequd oxygen ,30. Despd oxygen . In addid oxygen . The rednterface  (Equatioreaction  (Equatioion (7)) . It is g and 3.0 , with mo2O2 production rate of 7.7 mg h−1 cm−2.In an attempt to overcome these complex issues, various carbon-based materials have been considered, as these are generally cost effective and facilitate the two-electron reduction of oxygen. In particular, carbon in different forms, such as amorphous carbon, glassy carbon, graphite, fullerenes and carbon nanotubes have all been evaluated . Howeversp2-hybridised carbon atoms arranged in a two-dimensional honeycomb monolayer to produce a one-atom-thick sheet. Graphene can be produced using a variety of both top-down and bottom-up approaches, including exfoliation, sonication, ball milling, chemical vapour deposition and epitaxial growth [3, KMnO4 and H2SO4, which is widely known as the modified Hummers method [Graphene is a two-dimensional material with l growth . Howeverl growth . One of l growth ,43,44. Gs method ,46. Once2O2 in E-Fenton applications. The reduction methods employed are generally thermal or chemical reduction processes [2, giving a reduction of GO and the formation of rGO. Various reducing agents, such as borohydride, hydrazine or hydrogen iodide can be employed at room temperature or with mild heating to give rGO. However, some of these chemicals have environmental concerns and they can introduce impurities to the carbon matrix. These health and environmental issues can be overcome by using ascorbic acid as the reducing agent. This has been successfully employed to reduce GO and the resulting rGO was shown to have good conductivity [This oxidation process introduces a number of negatively charged oxygen-containing groups, such as hydroxy and carboxy groups, to the GO sheets and as a result, the GO sheets have good hydrophilic properties. GO exhibits good stability as a colloidal solution and this is very useful for solution processing approaches. The GO sheets can be subsequently reduced to give rGO, which has a much higher conductivity, and it is this member of the graphene family that is most suitable for electrochemical applications, such as the reduction of dissolved oxygen to produce Hrocesses . The theuctivity . The eleuctivity ,50,51. F2O2. These approaches include various electrophoretic and electrodeposition routines to generate graphene-based cathodes, graphene-based materials deposited from slurries or suspensions at graphite or carbon felt electrodes which act as supports, graphene-based inks, porous graphene-based aerogels, three-dimensional graphene-modified electrodes, heteroatom doped graphene composites, graphene composites combined with iron oxides and graphene-based diffusion electrodes. These are now described and discussed in the following sections.Different approaches have been used in forming graphene-based cathodes in an attempt to give enhanced and selective production of HOne of the most common approaches is to use carbon or graphite felt as a support for GO or rGO as these three-dimensional felt cathodes have low cost, high surface area, excellent conductivity and high porosity. The exfoliated GO/rGO can be deposited at the carbon or graphite electrodes using electrophoretic deposition , coated 2O2 with low energy consumptions of 9.7 kWh kg−1 [−1 [Indeed, these electrochemical approaches, employing combinations of electrochemical exfoliation and electrophoretic deposition, followed by the reduction of GO to rGO, have been used to coat carbon felt electrodes , while ikWh kg−1  and 3.08kg−1 [−1 , sulfadikg−1 [−1 . 2O2 (Equations (8) and (9)). For example, Gao et al. [2O2; see Equation (12).On the other hand, graphene-containing slurries can be readily formed using exfoliated or electrochemically exfoliated GO and these have been combined with quinones (AQ), which have the ability to generate Ho et al.  used GO,2O2 when GO/rGO is employed as the cathode material. Indeed, Nambi and co-workers [2O2 and the degradation efficiency of E-Fenton to quinone functional groups on electrochemically exfoliated rGO.It is well established that a number of oxygen-containing functional groups, such as epoxides (C–O–C), hydroxy (OH), carboxylic (COOH) and carbonyl groups (C=O) are present on GO nanosheets . However-workers  attribut−1 for the ferrocene-modified rGO electrode, significantly higher than the value of 0.004 min−1 obtained for the unmodified graphite felt and also higher than 0.010 min−1, which was the rate constant observed with the reduced rGO-modified felt electrode. The authors concluded that the rGO and ferrocene participated in sequential steps, with rGO facilitating the production of H2O2, while the Fe2+ centre in ferrocene catalysed the decomposition of H2O2 to form OH• and Fe3+-centred ferricenium. The same group studied the ferrocene-functionalised rGO felt electrode as an E-Fenton catalyst using rotating disc voltammetry for the removal of ciprofloxacin and carbamazepine [• concentrations of 644 µM, 264 µM and 163 µM at pH values of 3.0, 7.0 and 9.0, respectively, facilitating the removal of contaminants over a wide pH range. Graphene oxide can be easily functionalised with ferrocene ,67, and mazepine . Using r3 and Ce and deposited the hybrid composite, rGO/Ce/WO3, on carbon felt. This composite was employed in E-Fenton to remove ciprofloxacin with complete degradation within 1 h, and a mineralisation degree of 98.55% within 8 h. It was suggested that the Ce3+ catalysed the decomposition of H2O2 to give the OH• radical species (Equation (13)), while superoxide was generated from the reaction between Ce3+ and dissolved O2 (Equation (14)) and between W5+ and O2 (Equation (15)). The Ce4+ was recycled to Ce3+ through a simple reduction step (Equation (16)), while Fe2+ ions added to the E-Fenton cell also facilitated the reduction of Ce4+ (Equation (17)).Using another approach, Mi et al.  combined2O2 production and greater efficiency in the degradation of methylene blue in heterogeneous E-Fenton compared with the GO-free PEDOT:NaPSS. This was attributed to a synergistic effect between PEDOT and GO, promoting higher electron transfer rates.Graphene/GO has also been combined with conducting polymers and used in E-Fenton. In a recent study, a simple electropolymerisation method was employed to deposit poly  (PEDOT), sodium polystyrene sulfonate (NaPSS) and GO on graphite felt electrodes . This GO2O2 generated and a three-fold increase in the rate of phenol degradation. A graphene-based paste cathode has also been employed to degrade a pharmaceutical product [In addition to electrophoretic deposition and coating from graphene-containing slurries, there has been considerable interest in graphene-based inks that can be painted or printed onto substrates ,70. This product . The pas product  used ano product , and an  product , while a product .2O2 generation to give a more efficient removal of the contaminants. Interestingly, it was shown by Wang et al. [2O2. This suggests that the graphene-modified electrodes are not only more superior than the conducing graphite or carbon substrates used in their fabrication, but they may also be more efficient in producing H2O2 than carbon nanotubes. The impressive performance of the graphene-modified carbon/graphite felt electrodes is clearly illustrated in 2O2 generated is expressed differently, sometimes as rates and in other cases as mass per volume, it is evident that a graphene-based system can be employed to give the more efficient generation of H2O2 with relatively high rate constants for the removal of a variety of contaminants.In most of these approaches, the graphene-modified electrode is compared to the conducting carbon or graphite felt or carbon cloth substrate electrodes and the addition of rGO clearly enhances the rate of Hg et al. , who empPorous graphene-based composites that can be employed as aerogels, foams and as gas diffusion electrodes are interesting and these new materials are beginning to emerge in E-Fenton. 3O4 nanoparticles can be embedded and protected within the aerogel [Aerogels are three-dimensional highly porous materials and graphene-based aerogels exhibit interesting properties of high thermal stability, surface area and electrical conductivity . They ar aerogel . A numbe aerogel ,87,88,892+, EDTA-Ni. Using surface analytical techniques and adsorption measurements, the authors confirmed that the three-dimensional structure possessed homogenous macropores with a high surface area of 280.15 m2 g−1. The cathode showed enhanced electrocatalytic activity, leading to the efficient generation of H2O2 and regeneration of Fe2+. Likewise, Nazhif Mohd Nohan et al. [For example, Wen et al.  preparedn et al.  used a o2O3 nanoparticles have also been successfully fabricated and employed in the heterogenous E-Fenton system for the degradation of Rhodamine B [2O3 nanoparticles enhanced the degradation rate and this was attributed to the generation of H2O2 within the aerogel and the good diffusion and electrosorption of Rhodamine B within the aerogel to give high concentrations of the accumulated contaminant. The H2O2 was decomposed in the presence of Fe2O3 to give oxidising OH• with little quenching, as high concentrations of the pollutant were available. Chemical oxygen demand elimination rates up to 82% were observed, while a degradation rate of 99% was reached after 30 min. Low iron leaching rates were observed even in acidic media and there was no significant loss in the catalytic activity over six cycles. In a separate study, a carbon aerogel with rGO, CNT and iron oxide nanoparticles, Fe3O4, was formed successfully using a sol–gel process and then employed in an E-Fenton cell as a cathode in the degradation of methyl blue [Graphene-based aerogels containing trapped Fedamine B . The addhyl blue . The goo2O2 electrogeneration yield, degradation and mineralisation rates [In terms of three-dimensional graphene-based materials, there is some evidence that three-dimensional graphene-modified foams may be suitable in E-Fenton. In a comparison of graphene-based monolayer, multilayer and three-dimensional graphene-based foams as cathode materials in E-Fenton for wastewater treatment and the mineralisation of phenol, it was found that the three-dimensional foam exhibited the highest Hon rates . The supon rates  have sho2O2 [+ from the aqueous phase, while the conducting catalyst provides the electron, facilitating the formation of H2O2. Recently, this approach is finding applications in E-Fenton [Gas diffusion electrodes (GDE) are being used increasingly in fuel cells , batteri2O2 ,95. TheyE-Fenton ,97,98. H2O2 through the oxygen reduction reaction, for the removal of Rhodamine B [2O2. The porous structure of the graphene-based diffusion electrode provided a reaction chamber for the efficient conversion of oxygen to H2O2. Liu et al. [2O2 production was significantly improved. The apparent rate constant of dimethyl phthalate degradation was computed as 0.0322 min−1. The graphene-based diffusion electrode showed excellent recovery and could be reused. However, the degradation rate decreased slightly with increasing use and this was attributed to the blocking of the microspores and channels by iron sludge as the E-Fenton reaction proceeded. In another study, a gas diffusion electrode, consisting of a cylindrical body with a built in air diffuser, was fabricated using carbon cloth treated with PTFE and coated with electrochemically exfoliated rGO for the removal of industrial electronic wastewater [2O2 concentrations of 495 mg L−1 were achieved to give a mineralisation rate of 80% over 80 min. Gas diffusion electrodes have also been fabricated by combining rGO with Fe3O4 [3O4 particles adjacent to the electrogenerated H2O2 facilitated the efficient generation of OH• and the removal of Bisphenol A [2O2, and they have been employed in the removal of several pollutants.For example, a graphene–graphite diffusion electrode with high conductivity and electrocatalytic activity was formed for the continuous in situ generation of Hdamine B . A removu et al.  used a gstewater . High H2th Fe3O4  and by uth Fe3O4  for the phenol A . A compa2O2. In a comparative study, tert-butyl-anthraquinone (TBAQ) was used to modify four different carbon materials, carbon aerogel, CNT, carbon black and graphene-doped carbon black to fabricate gas diffusion electrodes for the production of H2O2 [2O2 production and current efficiency were achieved with the CNT-gas diffusion electrode modified with 2% TBAQ, giving 2.15 mg h−1 cm−2 compared to 1.97 mg cm−2 h−1 of H2O2 for the corresponding graphene-based material. However, rates higher than 2.15 mg h−1 cm−2 can be seen in While many studies indicate the superiority of graphene/rGO in fabricating gas diffusion electrodes, there are examples where other carbon-based systems exhibit a somewhat higher catalytic activity for the generation of H of H2O2 . In this2O2 [2O2 [The doping of graphene/rGO, using heteroatoms such as N, B, P and Fe, has been widely studied for the development of catalysts for the oxygen reduction reaction ,107. Dep2O2 , while F2O2 . Likewis2O2 [2O2 . As N ha2O2 [2O2 ,111. How2O2 [2O2 ,113. The2O2 [2O2 ,114, whi2O2 [2O2 . It has 2O2, but on reduction with NaBH4, the same materials exhibit more selectivity for the four-electron pathway [2O2 is connected with epoxy or ether groups in the N-doped rGO, while N-doped graphene composites, with abundant quaternary nitrogen species, show the selectivity of the two-electron reduction pathway; however, with pyridinic species, the four-electron pathway is preferred [The four-electron transfer pathway is normally observed in alkaline solutions , while,  pathway . Kim et  pathway  suggestereferred . These s3C nanoparticles was used for the removal of phenol, methanol, acetone, dichloromethane and diethyl phthalate from real samples [3C nanoparticles were encapsulated and protected, preventing the aggregation or leakage of the Fe, while the high-surface-area porous framework, with abundant channels and pores, provided access for the pollutants, while facilitating the formation of H2O2. A gas diffusion cathode was formed with N-doped graphene composites and CNTs for the removal of dimethyl phthalate [2O2 at a relatively low potential of 0.2 V vs. SCE. Yang et al. [•, with a high yield of 6.2 mg h−1 cm−2 of H2O2. Su et al. [2O2, reaching a value of 8.6 mg h−1 cm−2, with a selectivity of 78% at neutral pH and a low energy consumption of 9.8 kW h kg−1. In another recent study [2O2 and the generation of OH•. This N-doped graphene-modified graphite felt electrode was used in the mineralisation of 2,4-dichlorophenoxiacetic acid with an impressive rate of 88% at a pH of 7.0 after 480 min. A number of N-doped graphene-based electrodes have been employed successfully in E-Fenton. Li and Zhang  employed samples . The auththalate . The autg et al.  employedu et al.  obtainednt study , N-doped3O4 catalyst, giving a removal rate of 71%, a mineralisation rate of 51% with good stability and low iron leaching of 0.33 mg L−1. Interestingly, it was observed that the N/S-doped graphene facilitated the four-electron oxygen reduction reaction, while it was mainly the two-electron pathway that was observed with the N-doped graphene-based aerogel. While N-doped graphene appears to be superior in the promotion of the two-electron oxygen reduction reaction, Wu et al. [Graphene-based aerogels, where the graphene was doped with N and also co-doped with N and S were compared as cathodes in a heterogeneous E-Fenton cell . The higu et al.  have shoGraphene/GO can be easily combined with a variety of other components such as metal oxides , metal n2+/Fe3+ couple to catalytically decompose H2O2 into OH•, a potent oxidant . While the Fe2+ and Fe3+ are soluble in acidic solutions, with any increase in pH, for example through the four-electron reduction at the cathode (Equation (4)), insoluble iron hydroxides begin to form, reducing the concentration of Fe2+ ions that are needed for the E-Fenton reaction. This is readily seen from the Pourbaix diagram presented in 2+ and Fe3+ are shown as a function of pH. The dashed lines show the pH dependence of the oxygen reduction and hydrogen ion reduction reactions.As detailed earlier, the homogeneous E-Fenton reaction uses the Fe3+ to Fe2+ is approximately 6000 times slower than the oxidation of Fe2+ to Fe3+, which inhibits efficient recycling between the Fe2+ and Fe3+ ions [3+ to Fe2+ in the solid-state catalyst. As shown in 2O2 or OH• concentrations formed, where it is seen that relatively low leaching rates can be achieved.Moreover, the reaction rate of Fee3+ ions ,132. In e3+ ions ,133. In 2O3, Fe3O4 and FeOOH, and ferrocene, which all contain the Fe2+/Fe3+ redox pair, have been employed [3O4 [2+. This system was employed at a neutral pH to give a total organic carbon removal efficiency of 89%. Zero-valent iron has also been encapsulated within a three-dimensional graphene-based network to give a catalyst for the adsorption and degradation of sulfadiazine [2O3 as the iron source have also been employed in heterogenous E-Fenton [−1) in acidic solutions.The oxides Feemployed ,139,140,yed [3O4 ,142, FeOyed [3O4 , zero vayed [3O4  and ferryed [3O4 . In a reyed [3O4  fabricatadiazine . Iron oxE-Fenton . In this3O4, has been used more widely as the iron-containing catalyst [3O4 nanoparticles was employed for the continuous electrogeneration of H2O2 and OH•. A degradation efficiency of 98% was observed for the removal of Bisphenol A within 90 min at neutral pH with less than 1% of iron leaching [3O4 nanoparticles on GO and rGO to enhance the removal of two dyes, methylene blue and acid red. Shen et al. [3O4 hollow hybrid microspheres, while graphene oxide–Fe3O4 was employed as a heterogeneous catalyst for the E-Fenton degradation of two antibiotics, chloramphenicol and metronidazole [3O4 particles have also been utilised with N-doped graphene-based aerogels for the degradation of acetaminophen with a low iron leaching of 0.33 mg L−1 in heterogeneous E-Fenton [3O4 particles at N-doped rGO as catalytic particle electrodes. The Fe3O4/N-rGO also served as the heterogeneous catalyst, resulting in the rapid regeneration of Fe2+ and high concentrations of OH• oxidants [The iron oxide, Fecatalyst ,144. Forleaching . Akerdi leaching  have alsn et al.  have empnidazole . Fe3O4 pE-Fenton . These iE-Fenton . In a mooxidants .2/Ce0.75Zr0.25O2 composite was fabricated and used for the effective removal of ciprofloxacin. The decomposition of H2O2 to generate OH• was facilitated by the redox pair Ce3+/Ce4+ to give complete removal of ciprofloxacin within 5 h, with a mineralisation rate of 77% in 3 h [+, which then catalysed the decomposition of the electrogenerated H2O2 to give OH• and as a result the Cu+ was converted to Cu2+. The conducting catalyst layer, serving as the cathode, facilitated the reduction of Cu2+ to generate Cu+, leading to the efficient recycling of the Cu+/Cu2+ redox couple. Metal-free E-Fenton has also been recently proposed, as graphitic and pyridinic N sites on graphene-based materials appear to function as active sites for both the electrogeneration of H2O2 and the activation of OH• radicals [There is also increasing interest in finding iron-free or, indeed, metal-free catalysts for heterogeneous E-Fenton. An iron-free rGO/MoS% in 3 h . HKUST (% in 3 h . It was radicals ,126,148.2O2 is considered as an undesirable product in many research fields, such as batteries and fuel cells, tailoring the properties of the graphene-based composite with doping or by combining it with other materials to give a two-electron oxygen reduction reaction has significant potential not only in E-Fenton, but there are a number of other technologies, such as antimicrobial, medical, bleaching, gas scrubbing and refinery applications, that would benefit from the in situ generation of H2O2, eliminating the need for its storage.Graphene-based materials have attracted considerable interest, both from a fundamental viewpoint and in terms of their potential applications, and this family is one of the most studied, surpassing all other two-dimensional materials. It is no surprise that these materials are now finding applications in E-Fenton and, when combined with other carbon-based materials, they have the potential to give true iron- and metal-free E-Fenton catalysts. This would have a significant impact in terms of cost and environmental concerns. As shown earlier, rGO can be employed effectively in several ways, supported onto carbon or graphite electrodes or cloth, combined with CNTs, iron and other metal oxide catalysts, formed as aerogels and used in gas diffusion electrodes and doped with N and other elements, with N-doping appearing to be the best option in E-Fenton. It certainly appears that GO, with its very good conductivity when reduced to give rGO, high surface area, and good stability, will be used increasingly in future research in E-Fenton, leading to new and exciting developments. While the electrocatalytic generation of H2O2, while catalysing the efficient conversion of H2O2 into OH•, reducing or eliminating the release of metal ions, such as Fe3+ or Fe2+, into the water, are required. These catalysts will also need to exhibit high stability, enabling their use over multiple E-Fenton cycles, with low energy demand. This will require the development of new Fenton catalysts and graphene composites have a clear role to play in these developments, most likely in terms of composites formed using other new and emerging materials, including other two-dimensional materials, and by doping.In order for E-Fenton to emerge and be well integrated into real wastewater treatment facilities, new and more efficient Fenton catalysts are required. While heterogenous E-Fenton addresses many of the limitations of homogeneous E-Fenton, more efficient catalysts that are capable of generating high yields of HIn terms of possible new materials that could, in the future, be combined with graphene-based materials, MXenes deserve a special mention. These are exciting two-dimensional materials ,150 thatHowever, the application of graphene-based materials, as composites, or combined with other materials, and/or with doping, in E-Fenton has a number of challenges that must be overcome before significant advances are made. While remarkable progress has been made in the synthesis of graphene flakes, GO and rGO, a cost-effective large-scale synthesis is needed before these applications become a reality. The cost effective production of graphene and its oxides is required, but control over reproducibility and quality is equally important. Wet graphite exfoliation methods, including chemical and electrochemical processes, are promising in terms of scalability and cost, but concerns still remain over the reproducibility of these approaches and the quality of the final graphene product. Nevertheless, there is evidence to show that the oxygen reduction reaction is promoted at graphene edges, rather than basal planes, making these wet graphite exfoliation methods possibly suitable for the scaling up and production of graphene-based cathodes that can be employed in E-Fenton.It is also difficult to precisely control the doping levels of graphene-based materials. At another level, there are some concerns in terms of the environmental impact of GO and its potential adverse effects on aquatic ecosystems . In partNevertheless, with further advancements in the synthesis, scale-up and processing of graphene-based composites and electrodes, it is very likely that new Fenton catalysts and, indeed, metal-free Fenton catalysts can be fabricated, leading to innovations in advanced oxidation processes for the protection of water resources.