We report the near atomic resolution (3.3 Å) of the human polycystic kidney disease 2-like 1 (polycystin 2-l1) ion channel. Encoded by PKD2L1, polycystin 2-l1 is a calcium and monovalent cation-permeant ion channel in primary cilia and plasma membranes. The related primary cilium-specific polycystin-2 protein, encoded by PKD2, shares a high degree of sequence similarity, yet has distinct permeability characteristics. Here we show that these differences are reflected in the architecture of polycystin 2-l1. Primary cilia house key downstream elements of the sonic hedgehog pathway, which regulates embryonic development and some cancers. Recently, new tools have enabled measurements of other signaling elements within cilia. Primary cilia have elevated resting internal Ca0–700 nM ), and positus inversus phenotype (Xenopus oocytes (2+ permeability (PCa/PNa ~6) (PKD2 and PKD2L1 share high degrees of sequence identity (52%) and similarity (71%). Mutations in PKD2 account for ~15% of cases of individuals afflicted with Autosomal Dominant Polycystic Kidney Disease (ADPKD). In contrast, there are no diseases currently linked to PKD2L1. Deletion of PKD2 is lethal in mice , but delhenotype . Finallyhenotype , 2016, p oocytes  and dire oocytes . These s/PNa ~6) , 2016. DIn a similar fashion, structural information about membrane proteins has undergone a profound advance. This is largely due to the explosion of structures from single particle electron cryo-microscopy (cryo-EM) in recent years. Accordingly, multiple structures have been solved for the TRP ion channel family, including the TRPP family to which polycystin-2 and polycystin 2-l1 belong.Here we present a structure of the full-length human polycystin 2-l1 protein at 3.3 Å resolution in cryo-EM. The overall architecture conforms to that of other TRP channels, and in particular to the TRPP polycystin-2 structures. We establish the core structure and point out differences in regions and residues that may account for polycystin-2 and polycystin 2-l1’s distinct permeation and gating.- cells and the BacMam system   using HEm system . To detem system    and sharThe intracellular face of polycystin 2-l1 is too poorly resolved to model the N terminus and the S4-S5 linker densities. Also, despite expression of full-length protein  the C te10-helix configuration (I571 – F579). The density of the S4 helix of polycystin 2-l1 is incomplete at this region so a 310 configuration is not observed. Similarly, the equivalent residues of polycystin-2 that are thought to form a salt bridge between the S3 and S4 helix   , do not The polycystin mucolipin domain (PMD) rests on top of the VSLD of the same monomer and the adjacent pore domain , with a Grieben et al.,  describeDespite moderately low identity and an increase in charged residues in polycystin 2-l1’s TLC3 (SPDKEE (residues 228–232) versus SVSSED in polycystin-2), the loop appears essentially the same as in polycystin-2. This element appears to reach under the PMD’s β-sheet. Proximity of the returning loop of TLC1 towards TLC2, as well as the S5-PH1 loop of the monomer and the PH2-S6 loop adjacent monomer creates four lateral openings at the base of the PMD in polycystin 2-l1, as in polycystin-2. These could present an alternative route for ion permeation . This arPolycystin 2-l1’s PMD also interacts with the adjacent subunit’s upper pore domain. In the polycystin-2 multiple- (pdb 5MKF) and single-ion (pdb 5MKE) structures, Wilkes et al.,  observedAs in polycystin-2, polycystin 2-l1’s selectivity filter is flanked by two pore-helices, which are themselves each flanked by a transmembrane spanning helix (S5 and S6). The S6 helix of polycystin 2-l1 remains α-helical throughout its length, whereas the S6 helix of polycystin-2 is broken by a middle π-helix element, before continuing to the C terminus  Figure . The S6 Relative to polycystin-2’s pore helix 1 (PDB 5T4D), the angle between the beginning of pore helix 1 and the top of the selectivity filter is similar (77° for polycystin 2-l1 and 75° for polycystin-2) compared to the 10° pitch difference between polycystin-2 and TRPV1 . However2+ ion with an average ~2.4 Å metal-oxygen distance   was 18-fold, and polycystin 2-l1(LGDFD525: D525N) 9-fold less calcium-permeant (PCa2+/PCs+) than wt polycystin 2-l1. Also, and significantly, the D525N mutation reduced outward current block by Ca2+, while D523N did not  are conserved, leading us to examine other explanations as to why polycystin 2-l1 conducts Ca/PNa ~6) . If the and Ca2+ . Point mand Ca2+ . However did not . These eof CaVAb ), and mo2+-selective TRPV5 and TRPV6 (PCa2+/PNa+> 100)   structura+> 100)    in whichAn additional mechanism to explain different selectivities of TRPPs is a local bonding environment difference at and near the selectivity filter between polycystin-2 and polycystin 2-l1. A conserved salt bridge between D523 and R518 in polycystin 2-l1 exists in polycystin-2 (D643 and R638). Yet a hydrogen bond between residues D525 and A528 in polycystin 2-l1 is not present in polycystin-2; the residues are not conserved . Local hIn both observations, we rely on one structure, which may change with gating. The use of molecular dynamics (MD) to observe the simulated trajectory of ions and their relationship to the energy landscapes of the selectivity filter is an excellent starting point for future experiments. Coupling future structures with functional mutagenic studies  should answer these questions.Although polycystin 2-l1 and polycystin-2 structures are very similar, there are notable differences that may explain reported functional differences in their selectivity and permeation. For discussion, we divide polycystin 2-l1 into three regions to highlight these differences; the voltage sensing-like domain (VSLD), the pore mucolipin domain (PMD), and the pore (PD). In the VSLD, polycystin 2-l1’s S3 helix extends into the PMD as a complete α-helix. In addition, the top of the S3-S4 helical region abuts the PMD. Comparing the PMDs, polycystin 2-l1 lacks oligosaccharides that act as bridges between adjacent subunits. Finally, although similar, polycystin 2-l1’s pore diameter is slightly larger.2+, while polycystin-2 is monovalent-selective P termini . The curThe explosion of structures available for TRP channels lays the groundwork for interpreting future detailed characterization. The structure of polycystin 2-l1 presented here, will help in understanding what drove evolution to create the diversity that exists in the TRPP subfamily, but only when we more completely understand their biophysical and physiological functions. The recent progress made in cryo-EM underscores the importance of discovering these features.- cells at 10% v/v at 37°C and 8% CO2 with shaking. After 24 hr, sodium butyrate was added to a final concentration of 10 mM and cells were harvested after 48 hr.Cloning, expression, and purification of full-length human polycystin 2-l1 were completed using the BacMam strategy . Briefly2 5; pH 7.5) with an EDTA-free protease inhibitor cocktail (Roche) tablet for 2 hr at 4°C using gentle rotation. Ultracentrifugation of the sample was completed at 40,000 RPM for 1 hr at 4°C. The resulting supernatant was retained and used for a batch incubation with amylose resin overnight at 4°C. Resin was collected, washed in HKN buffer with higher K+ (500 mM) and 2x CMC of n-Dodecyl β-D-maltoside: cholesteryl hemisuccinate 10:1 (DDM:CHS) for 10 bed volumes followed by a normal HKN buffer for 10 bed volumes with 2xCMC of DDM:CHS. The protein was eluted using 40 mM maltose in HKN buffer at 2xCMC DDM: CHS. All fractions were collected, concentrated and subjected to 1 round of size exclusion chromatography using an Increase Superose6 (GE Lifesciences) column pre-equilibrated with HKN and 2xCMC DDH: CHS. Protein-containing fractions, as determined by A280 and western-blotting against maltose binding protein (MBP), were collected, concentrated and used for further preparation. The sample’s concentration was again determined using A280 and incubated with a 1:3 ratio (mass) of poly  propylamine; PMAL C8) overnight with gentle rotation at 4°C. A final round of size exclusion chromatography using an Increase Superose6 column equilibrated with HKN buffer was performed, fractions collected, and concentrated using a Vivaspin Turbo4 100,000 molecular weight cutoff (MWCO) centrifugation device before being applied to grids and freezing.Protein was extracted directly from pellets using 40x critical micellar concentration (CMC) of the detergent C12E9 in HKN buffer . Grids were blotted for 3 s at 95% humidity and flash frozen in liquid nitrogen-cooled liquid ethane bath using a Vitrobot . Samples were stored in liquid nitrogen until acquisition.- pixel−1 s−1 at 0.2 s/frame for a total dose of approximately 60 e- Å2 s−1 using SerialEM  at 300 kV with a Gatan Quantum Image Filter (20 eV slit) and K2 Summit detector at the Janelia Research Campus  using the following parameters: A total of 3814 image stacks were acquired at a sampling rate of 1.04 Å/pixel with an 8 s exposure of 8 eSerialEM .ab initio from a data set acquired for polycystin 2-l1 and processed using cisTEM . Eight class averages representing different orientations were selected for further iterative 3D classification and refinement in C4 symmetry     . Local ralk.edu)   . ExamplePolyalanine α-helices and β-sheets were built for each transmembrane section and the PMD using pdb 5T4D as a final guide in Coot . Densiti2 1), pH 7.4; the pipette contained the same buffer with 1 mM MgCl2. Solution osmolarity was measured at 400–405 mOsm. Data was acquired at 10 kHz and a low-pass Bessel-filtered at 1–2 kHz. For each recording, pipette offset and capacitance were corrected. Approximately 2 min after a GΩ seal was achieved, the patch was excised, and capacitance corrected before recording.Liposomes containing full-length polycystin 2-l1 protein were voltage clamped to record single channel currents. Protein was reconstituted following the dilution method  into lip In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by Richard Aldrich as the Senior Editor, a Reviewing Editor, and three reviewers. The reviewers have opted to remain anonymous.Thank you for submitting your article "Cryo-EM structure of the polycystin 2-l1 ion channel" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:In this study, Hulse et al., presented a structure of the full-length human polycystin 2-l1 protein at 3.1Å resolution using single particle electron cryo-microscopy (cryo-EM). Although it shares high sequence similarity to polycystin 2, whose structure has been determined by multiple labs recently, polycystin 2-l1 exhibits distinct ion permeability. The study aims to provide a detailed view of the structural differences between polycystin 2-l1 and polycystin 2, and these structural differences may underlie the functional difference between the two channels.Essential revisions:1) The manuscript is structured from the viewpoint of understanding the function of the channel but, at present, it is not clear at this point whether the structural differences contribute to function. The main impact of the present study would be to compare the structures of polycystin 2-l1 and PKD2 in as much detail as possible. Oddly, the introduction to the manuscript barely mentions the fact that a structure for PKD2 has already been reported by this and other groups, but the trajectory of the manuscript would be better served by addressing this up front and moving on from there. In this context, figures showing direct comparisons or overlays of these two TRPP structures would be very useful and help convey important points that are difficult to ascertain from the current format. We are not expecting new functional data but the new structures at the least should help develop a cogent hypothesis for further testing.2) There seems to be a disconnect between the stated resolution of the structure and the quality of the maps. This is suggested by the inability to resolve many regions and, in particular, side chains. From the figures provided , it is not clear how well the model fits the density map in various regions. The maps look more to be in the 4 – 4.5Å range, and it isn't clear where the possible discrepancy lies. Overfitting is a known issue with cisTEM program for EM structure determination. The authors should re-evaluate their data processing and analysis.3) Related to the above point, please provide the map for this structure which would help reviewers assess the quality of the data and thus the validity of the conclusions.4) Another question concerns the unusual protocol for channel protein purification, which is very different from that described for PKD2 and which involves initial extraction into C12E9 detergent, batch overnight incubation with amylose resin, and multiple gel filtration steps. Could this be enhancing protein instability that degrades resolution?5) Overall, the manuscript is difficult to follow. Most figures are not properly labeled and many of the descriptions in the paper are not adequately supported by figures, making it difficult to comprehend, particularly for readers who are not familiar with the TRPP family of channels. Specifically:Results section: "We conclude that these elements are either unstructured or connected with flexible regions, which, along with the lack of cytoplasmic elements in 2D classification , prevents model building of these regions." This sentence is unclear and I am not sure what the phrase "cytoplasmic elements" refer to.The Voltage Sensing-Like Domain”: "This S3 helix exhibits greater secondary structure than the presumed closed polycystin-2 structure of Shen et al." What does greater secondary structure mean here – longer S3?Subsection “Subsection "The Polycystin Mucolipin Domain". The authors spend so much effort pointing out very detailed structural differences between polycystin 2-l1 and polycystin 2. However, these differences do not seem to have any functional relevance.Subsection “Three leaf clovers of the polycystin mucolipin domain”: "However, the nature of the interaction differs in that F216 of TLC1 is near W308 of the adjacent subunit's PMD, representing a possible pi-pi stacking interaction or a hydrophobic pocket". A hydrophobic pocket is not a proper way to describe this interaction.Subsection “Fenestrations and TLC3”: If I recall correctly, the fenestration appears to be sealed in polycystin-2 by an extended loop based on Shen's structure. If there is a fenestration in the present structure, a proper figure should be provided here.Subsection “Fenestrations and TLC3”: What does "upper pore domain" refer to: the extracellular side of the pore domain? What do "the loop pore helices" refer to here: the loop and/or the pore helices? What does "mutually exclusive glycosylation states" refer to? In the last sentence of this section: "This difference may help rationalize the intermediate diameter observed in the polycystin-2 multiple-ion structure (1.4Å)  and the single-ion structure (1.0Å)  where such interactions do not exist, and our polycystin 2-l1 structure (3.1Å)." What diameters are the authors describing here and what does "this difference" refer to? Again, there is no figure to support all the descriptions in this paragraph.Subsection “The Pore Domain”:I am not sure how the figures are ordered. Figure 2 is described after Figure 4. Also, there are no proper labels on Figure 2.First paragraph of subsection “The Pore Domain”: "Notably, one element, K511 in polycystin 2-l1, is highly variable among TRPPs." Where is this K511? Again, there is no figure to support it.Second paragraph subsection “The Pore Domain”: I simply don't understand what is discussed here and again there is no figure to support the description. Should the filter diameter be radius? Does 5K47 refer to a PDB code?2+ and oxygen. These two distances are not comparable.Third paragraph of subsection “The Pore Domain”: The narrowest distance of 2.1 Å appears to be the narrowest filter radius based on Figure 2A (right panel), which excludes the van deer Waals radius of the G522 carbonyl oxygen. The "2.4 Å metal-oxygen distance" refers to atom-to-atom distance between Ca2+ ion would be 4.4 Å. If so, the argument of 2.4 Å metal-oxygen distance is no longer correct. Basically, the whole description about the pore domain is messy and incomprehensible.Third paragraph of subsection “The Pore Domain”: Here the author repeated the same discussion as the last paragraph, except using atom-to-atom diagonal distances. If the diagonal distance between the two G522 carbonyl oxygen atoms is 8.7Å, then the minimum distance between the G522 carbonyl oxygen and CaSubsection “Ion permeation differences between polycystin-2 and polycystin 2-l1”. The values of relative permeability ratio should be provided.2+ and/or monovalents that are occupying the pore with high certainty ), and realistic molecular dynamic simulations, should shed light on these issues." What is realistic MD simulation? What do "these issues" refer to here?"Higher resolution Cryo-EM structures that identify Ca2+ permeability of the channel? Also, how do they compare the relative Ca2+ permeability among four different channels?I simply don't understand the logic behind the electrostatic argument. Are the authors suggesting that the net positive charge of the polycystin 2-l1 pore helices contributes to the CaDiscussion section "The relatively larger diameter of polycystin 2-l1's pore (0.6Å – 1.1Å) may enable partially […]" Where is this distance value from? It does not match with the pore radius plot! Is it 0.6-1.1 Å larger than the pore of polycystin 2?Figure 1A the pore radius and the solvent accessible pathway does not match, particularly the lower gate (green) part. Essential revisions:1) The manuscript is structured from the viewpoint of understanding the function of the channel but, at present, it is not clear at this point whether the structural differences contribute to function. The main impact of the present study would be to compare the structures of polycystin 2-l1 and PKD2 in as much detail as possible. Oddly, the introduction to the manuscript barely mentions the fact that a structure for PKD2 has already been reported by this and other groups, but the trajectory of the manuscript would be better served by addressing this up front and moving on from there. In this context, figures showing direct comparisons or overlays of these two TRPP structures would be very useful and help convey important points that are difficult to ascertain from the current format. We are not expecting new functional data but the new structures at the least should help develop a cogent hypothesis for further testing.2+ permeation physiologically: polycystin-2l1 is permeant to calcium, but polycystin-2 is not. We have included a comparison of the profile of the selectivity filter and mapped the RMSD difference onto the structure as well as provided an overlay .We have taken your point and extensively discuss the PKD2/PKD-2l1 structures. We believe that the most relevant comparisons are made in Figure 5 with PKD2 and TRPV5 and TRPV6. We emphasize that despite PDK2’s similarity with PKD2L1 , there are differences in Ca2) There seems to be a disconnect between the stated resolution of the structure and the quality of the maps. This is suggested by the inability to resolve many regions and, in particular, side chains. From the figures provided , it is not clear how well the model fits the density map in various regions. The maps look more to be in the 4 – 4.5Å range, and it isn't clear where the possible discrepancy lies. Overfitting is a known issue with cisTEM program for EM structure determination. The authors should re-evaluate their data processing and analysis.We agree. Further evaluation of the data after consultation with Niko Grigorieff’s group revealed that the data was slightly overfit. To address this concern, we reviewed our data processing strategy and now have limited the resolution during refinement to 8Å. This decreased the nominal resolution but improved map density, with associated improved 3D FSCs (see below). We’ve re-evaluated the data with new tools from the Lyumkis group using 3D FSC plots to characterize the preferred orientation observed in our data Tan et al., 2017. The 3D FSC results are consistent with the angular distribution plot  which show that while the Z-resolution is high (labeled side view), the in-plane (x/y) resolution is weaker. This is also reflected in the fits of side chains in the map such as elements seen in Figure 1—figure supplement 5B. We have included the results of this analysis as well as new maps and models. We hope that our analysis and the new tools will help other ion channel groups facing similar problems with their structural efforts using cryoEM. While we cannot eliminate the preferred orientation of our channel, we believe that Lyumkis’ methods enable a better understanding of the effect on the data. Additional metrics, such as the fit of the map , the high degree of similarity to Erhu Cao’s polycystin-2 model , and the similarity to the mouse polycystin 2-l1 model that was released very shortly after we submitted this work  all verify that our model is not significantly affected. The current estimation from cisTEM and the 3D FSC is 3.3Å and 3.2Å respectively. We report our resolution at the more conservative 3.3Å. The model was reevaluated with the new maps, and several minor adjustments were made including the ability to build in a side chain for residue D523, a critical component of the selectivity filter. We also reveal a more complete loop in the PMD, and some improved density fitting at linkers between transmembrane helices. All figures and the manuscript were updated with this information.3) Related to the above point, please provide the map for this structure which would help reviewers assess the quality of the data and thus the validity of the conclusions.Done.4) Another question concerns the unusual protocol for channel protein purification, which is very different from that described for PKD2 and which involves initial extraction into C12E9 detergent, batch overnight incubation with amylose resin, and multiple gel filtration steps. Could this be enhancing protein instability that degrades resolution?Our protocol is based on empirical evaluation of extraction, stabilization and exchange using standard techniques. C12E9 was selected as an efficient extraction detergent on an empirical screen using western blot against MBP. Stability is evident from the exchange into DDM:CHS during amylose chromatography and in the SEC  and the ability of the protein to remain in solution for up to 72 h with stable SEC profiles (not shown).5) Overall, the manuscript is difficult to follow. Most figures are not properly labeled and many of the descriptions in the paper are not adequately supported by figures, making it difficult to comprehend, particularly for readers who are not familiar with the TRPP family of channels. Specifically:Results section: "We conclude that these elements are either unstructured or connected with flexible regions, which, along with the lack of cytoplasmic elements in 2D classification , prevents model building of these regions." This sentence is unclear, and I am not sure what the phrase "cytoplasmic elements" refer to.Polycystin 2-l1’s C terminus is cytoplasmic, likely forming a coiled-coiled motif like other homotetrameric channels. In Shen, 2016, a truncation of the C terminus was required for a stable biochemical preparation. We were able to express full-length protein, but the C terminus was still unresolvableSubsection “The Voltage Sensing-Like Domain”: "This S3 helix exhibits greater secondary structure than the presumed closed polycystin-2 structure of Shen et al." What does greater secondary structure mean here – longer S3?Yes, S3 in polycystin 2-l1 has more α helical content and is thus continuously longer than the interrupted C terminus of Shen et al., 2016. We have clarified the text.Subsection "The Polycystin Mucolipin Domain". The authors spend so much effort pointing out very detailed structural differences between polycystin 2-l1 and polycystin 2. However, these differences do not seem to have any functional relevance.We believe that the PMD will turn out to be a ligand binding domain, but to date, none has been found (for the PMD of any type II TRP). Since disruption of the PMD results in nonfunctional channels, it will take some time to understand its function.Subsection “Three leaf clovers of the polycystin mucolipin domain”: "However, the nature of the interaction differs in that F216 of TLC1 is near W308 of the adjacent subunit's PMD, representing a possible pi-pi stacking interaction or a hydrophobic pocket". A hydrophobic pocket is not a proper way to describe this interaction.We have corrected the manuscript.Subsection “Fenestrations and TLC3”: If I recall correctly, the fenestration appears to be sealed in polycystin-2 by an extended loop based on Shen's structure. If there is a fenestration in the present structure, a proper figure should be provided here.The fenestration seen in polycystin 2-l1 is a small hole at the interface of the top of the VSLD and bottom of the PMD. Polycystin-2 has a similar configuration, with a small hole (5T4D). Whether the PMD is truly sealing this entry is unclear in our model since we can only approximate the position relative to the membrane.Subsection “Fenestrations and TLC3”: What does "upper pore domain" refer to: the extracellular side of the pore domain? What do "the loop pore helices" refer to here: the loop and/or the pore helices? What does "mutually exclusive glycosylation states" refer to? In the last sentence of this section: "This difference may help rationalize the intermediate diameter observed in the polycystin-2 multiple-ion structure (1.4Å)  and the single-ion structure (1.0Å)  where such interactions do not exist, and our polycystin 2-l1 structure (3.1Å)." What diameters are the authors describing here and what does "this difference" refer to? Again, there is no figure to support all the descriptions in this paragraph.The comparison of polycystin-2 and polycystin 2-l1 in the supplementary section helps to clarify this discussion.Subsection “The Pore Domain”:I am not sure how the figures are ordered. Figure 2 is described after Figure 4. Also, there are no proper labels on Figure 2.First paragraph of subsection “The Pore Domain”: "Notably, one element, K511 in polycystin 2-l1, is highly variable among TRPPs." Where is this K511? Again, there is no figure to support it.Second paragraph of subsection “The Pore Domain”: I simply don't understand what is discussed here and again there is no figure to support the description. Should the filter diameter be radius? Does 5K47 refer to a PDB code?2+ and oxygen. These two distances are not comparable.Third paragraph of subsection “The Pore Domain”: The narrowest distance of 2.1 Å appears to be the narrowest filter radius based on Figure 2A (right panel), which excludes the van deer Waals radius of the G522 carbonyl oxygen. The "2.4 Å metal-oxygen distance" refers to atom-to-atom distance between CaWe apologize for the confusion. Our attempt to discuss both the radii of the measurements from the HOLE program  and measurements from Pymol (as either Cα or CO distances) present different information. HOLE is better suited to this discussion. We’ve removed the Pymol distances. We’ve also corrected the specific questions, improved the figure legends and ensured the order is correct.2+ ion would be 4.4Å. If so, the argument of 2.4Å metal-oxygen distance is no longer correct. Basically, the whole description about the pore domain is messy and incomprehensible.Third paragraph of subsection “The Pore Domain”: Here the author repeated the same discussion as the last paragraph, except using atom-to-atom diagonal distances. If the diagonal distance between the two G522 carbonyl oxygen atoms is 8.7Å, then the minimum distance between the G522 carbonyl oxygen and CaSubsection “Ion permeation differences between polycystin-2 and polycystin 2-l1”. The values of relative permeability ratio should be provided.2+ and/or monovalents that are occupying the pore with high certainty ), and realistic molecular dynamic simulations, should shed light on these issues." What is realistic MD simulation? What do "these issues" refer to here?"Higher resolution Cryo-EM structures that identify Ca2+ permeability of the channel? Also, how do they compare the relative Ca2+ permeability among four different channels?I simply don't understand the logic behind the electrostatic argument. Are the authors suggesting that the net positive charge of the polycystin 2-l1 pore helices contributes to the CaWe have added the relative permeabilities from previous lab work  and cited literature .2+ relative to polycystin-2.Yes, we believe that the overall difference in charge at this region contributes to the moderate increase in Ca2+ (with weak selectivity over Na+). TRPV5 and TRPV6 have a much higher selectivity (>10x) for Ca2+ over Na+ than polycystin 2-l1. We believe that both the local energy landscape and geometry of the selectivity filter are at least two variables that distinguish polycystin 2-l1 from other TRPP and TRPV channels.Polycystin-2 is nonselective among monovalents and does not conduct calcium, whereas polycystin 2-l1 is nonselective among monovalens but also conducts CaDiscussion section "The relatively larger diameter of polycystin 2-l1's pore (0.6Å – 1.1Å) may enable partially […]" Where is this distance value from? It does not match with the pore radius plot! Is it 0.6-1.1 Å larger than the pore of polycystin 2?This was an error and has been fixed.Figure 1A the pore radius and the solvent accessible pathway does not match, particularly the lower gate (green) part.This figure was updated with the new model.


Furthermore, the generated THF GPs can be efficiently guided along low-loss GP waveguides that are connected to both sides of grating section. Our results can stimulate making graphene-based light sources for mid- and far-infrared silicon photonics.Benefiting from the large third-order nonlinear susceptibility of graphene and significantly enhanced field intensity of graphene plasmons (GPs), graphene has shown great potentials to enhance plasmonic third-harmonic generation conversion efficiency. However, it still lacks an effective configuration that can excite the fundamental frequency (FF) GPs and guide the generated third-harmonic frequency (THF) GPs simultaneously. Here, we have proposed a diffractive silicon grating underneath a graphene sheet to generate and transmit THF GPs. The FF GPs are efficiently excited by illuminating a normal-incidence plane wave due to guided-mode resonance and then are converted to the THF GPs with a large conversion efficiency, originating from the giant field intensity of the FF GPs. We numerically demonstrate that, a large third-harmonic generation conversion efficiency of 3.68 × 10 As a means to extend coherent light sources to short wavelengths, third-harmonic generation (THG) has attracted tremendous research interest. Conventionally, high-efficiency harmonic generation is realized in exotic crystals, but which compromises high-density photonic integration  and imaginary [Im(neff)] part of the effective indices and absorption on light wavelength with different values of τ, μc, anddg. It apparently explains how the parameters of graphene influence the excited FF GPs under the illumination of a normal-incidence x-polarized plane wave of FF, where the grating period is fixed at 4 μm. Both the real [Re(neff)] and imaginary parts [Im(neff)] of effective refractive indices of the FF GPs decreases with the increase of light wavelength within the considered wavelength range  and the excitation wavelength of FF GPs, but greatly affects the Im(neff) and absorption  and Im(neff) simultaneously, hence reduces the resonance wavelength of FF GPs accordingly , Im(neff), and the resonance wavelength of FF GPs reduce with the increase of the graphene thickness, corresponding to the number of graphene layers . In contrast, the absorption efficiency is kept at a low level (below 2%) over the entire spectral range considered if the grating is not taken into account. The noticeably enhanced absorption for the former case can be attributed to the excitation of the GPs at λ = 28.62 μm. We can find from the |E| distributions at λ = 28.62 μm . One can see from Fig. In the following, we take  μm Fig. c that thνF = 9.5 × 104 m/s.It should be noted that, a greatly enhanced plasmonic field on the graphene surface occurs due to the significant reduction of the group velocity of the FF GPs . The plasmonic field undergoes electric field enhancement 5 times as high as the illuminating plane waves, which can be highly expected to generate THF GPs with a significantly enhanced CE, in combination with the large third-order optical nonlinearity of graphene , 17. The2 and the central wavelength of 28.62 μm. Here, the NEFI is obtained by normalizing the electric field intensity to its value at 28.62 μm (FF) with the grating structure. It can be observed that, an apparent peak occurs at THF in the NEFI spectrum with grating structure (GSSDG), compared to the NEFI spectrum without grating involved. Defining the CE as y component of poynting vector at THF, and PFF is the power density of the incident light, the CE reaches as high as 5.71 × 10−7 for the GSSDG. It can be easily inferred that, the excitation of the FF GPs contributes to the enhancement of the CE of THF GPs. The field distributions of real part of Ey at THF shown in Fig. We then compare the electric field intensity of the THF GPs on the graphene surface when the graphene sheets are sustained with and without grating. The boundary conditions in FDTD simulations are the same as those used in Figs. y and z directions in the modeling. We simulated a normal-incidence FF light wave that impinges the grating section, and showed the electric field distributions for the FF and THF GPs  at the grating boundary and exponentially attenuates along the propagation direction due to the ohmic absorption loss from graphene.We next consider the use of the GSSDG plasmonic wavelength converter to directly provide light source for the silicon integrated photonic circuits. As an example shown in Fig. GPs Fig. b, c. A t−7 even if the power density of incident light waves is as low as 0.19 MW/cm2, which is 6–7 orders of magnitude smaller than those within the same spectral band [Np, affects the CE in the GSPWs as well. When Np is increased, a reducing portion of the THF GPs generated in the center of the grating reaches the GSPWs because of the enhanced propagation loss induced by graphene absorption. Nevertheless, the input power, associated with Np, presents linear enhancement. Therefore, the maximum CE of the THF GPs decreases with the increased Np. We emphasize here that the absolute output power density of THF GPs should be more meaningful to guide the design of a THG wavelength converter for practical applications, once the incident power density is fixed. Although the maximum CE of the THF GPs lies at Np = 2 in our case, the output power density of THG approximates the maximum when Np ≥ 3  is achievable in the experiment [τ= 0.5 ps) shown in (Figs. The physical characteristics of graphene may also affect the device performance of THF GPs once the studied structure shown in Fig. GPs Fig. f, i and GPs Fig. c. Howeve. τ Fig. b, which periment , our simin Figs. , 5 and 6Finally, it is worth discussing the influences of surface roughness of graphene sheets on the device performance. The surface roughness of graphene could potentially scatter plasmon, and hence, the plasmon loss will be enhanced . The proWe have numerically demonstrated the generation of the THF GPs in a graphene sheet on silicon gratings with the normal-incidence plane waves in mid- and far-IR regions. It was shown that THF GPs are generated and transmitted on the graphene surface, and the CE is dramatically enhanced due to the significantly increased field intensity of the excited FF GPs in combination of the large third-order nonlinear susceptibility of graphene. The generated THF GPs can be conveniently coupled to a GSPW, which greatly facilitates the integration of the graphene-based wavelength converter on a silicon platform. Our proposal can stimulate making graphene-based light sources for mid- and far-infrared photonics on a silicon platform and hence broaden the functionalities of silicon photonics, such as signal processing, spectroscopy, and sensing.


Immune checkpoint inhibitors (CPIs) are associated with a number of immune-related adverse events and low response rates. We provide preclinical evidence for use of a retroviral replicating vector (RRV) selective to cancer cells, to deliver CPI agents that may circumvent such issues and increase efficacy. An RRV, RRV-scFv-PDL1, encoding a secreted single chain variable fragment targeting PD-L1 can effectively compete with PD-1 for PD-L1 occupancy. Cell binding assays showed trans-binding activity on 100% of cells in culture when infection was limited to 5% RRV-scFv-PDL1 infected tumor cells. Further, the ability of scFv PD-L1 to rescue PD-1/PD-L1 mediated immune suppression was demonstrated in a co-culture system consisting of human-derived immune cells and further demonstrated in several syngeneic mouse models including an intracranial tumor model. These tumor models showed that tumors infected with RRV-scFv-PD-L1 conferred robust and durable immune-mediated anti-tumor activity comparable or superior to systemically administered anti-PD-1 or anti PD-L1 monoclonal antibodies. Importantly, the nominal level of scFv-PD-L1 detected in serum is ∼50–150 fold less than reported for systemically administered therapeutic antibodies targeting immune checkpoints. These results support the concept that RRV-scFv-PDL1 CPI strategy may provide an improved safety and efficacy profile compared to systemic monoclonal antibodies of currently approved therapies. A retroviral replicating vector (RRV) platform based on an amphotropic gamma retrovirus that preferentially infects and replicates in tumor cells has been developed to address historical challenges with viral-based treatments for cancer. One example of the RRV approach is Toca 511, (vocimagene amiretrorepvec), an RRV encoding optimized cytosine deaminase derived from yeast) used in combination with an oral prodrug, Toca FC (extended-release 5-fluorocytosine) which has demonstrated durable, complete responses and a favorable safety profile in a Phase 1 trial , 3. FurtImmune checkpoint inhibitors (CPI) such as therapeutic antibodies targeting cytotoxic T lymphocyte antigen 4 (CTLA-4), programmed death-1 (PD-1) and its ligand programmed death-1 ligand 1 (PD-L1) have represented a breakthrough for immunotherapies in treating cancer patients and have specifically made a significant impact in the fight against melanoma, lung, bladder head and neck cancer as well as in Hodgkin lymphoma . HoweverPD-1 is one of the major inhibitory receptors highly expressed on tumor infiltrating lymphocytes where upon its ligation with PD-L1 reduces cytokine production and T-cell proliferation resulting in immune escape from an active anti-tumor T-cell response in the TME . In addiin vitro. This activity was further supported by data from multiple syngeneic mouse models showing that tumors infected with RRV-scFv-PDL1 conferred robust and durable immune-mediated anti-tumor activity comparable or superior to systemically administered anti-PD1 and anti-PD-L1 monoclonal antibodies. Most importantly, our data show that only nominal level of scFv-PD-L1 was found in the circulation in mouse models where significant efficacy was observed. These results support the concept of an RRV-based platform for delivery of an immune CPI for an improved therapeutic window compared to systemic monoclonal antibodies currently approved for clinical use in many cancer types.We have previously reported a new RRV configuration utilizing the “self-cleavage” 2A peptide (RRV-2A) as an alternative of the internal ribosomal entry sequence (IRES) described in Toca 511 , 22. TheWe have previously reported a new RRV configuration utilizing the viral-derived “self-cleavage” 2A peptide for transgene expression  and demoWe have also previously shown that transgenes targeted for different cellular compartments encoded in-frame with the viral envelope (Env) protein in the RRV-2A configuration, are efficiently separated from Env-transgene polyprotein . BecauseWe next evaluated the viral function of both epitope tagged (HA and Flag) and untagged RRV-scFv-PDL1 and RRV-scFvFc-PDL1 vectors, as proper viral Env processing is an important determinant for viral replication . We firsAnother key factor which could influence the therapeutic effect of the transgene is vector stability. We have previously showed that the RRV-2A can tolerate a large transgene(s) insertion up to 1.2 kb . In the D) of scFv PD-L1 for recombinant human PD-L1 and mouse PD-L1 were determined to be 0.426 nM and 4.78 nM, respectively,  followed by anti-HA antibody for detection. The data show that scFvHF PD-L1 and scFvFc-HF PD-L1 bind to PD-L1 expressed on cell surfaces in human and mouse cell lines as indicated by a marked shift in mean fluorescent intensity (MFI) with an anti-HA antibody  and preliminary data demonstrated a failure of anti-PD-1 treatment as a monotherapy to prolong survival  , 31. Desin vitro . In thisin vitro . Furtherin vitro . Moreovein vitro . TogetheSince RRV-scFv-PDL1 is delivered as a virus rather than a therapeutic antibody and that scFv PD-L1 is produced from the infected tumor cells, scFv PD-L1 serum level is anticipated to be significantly lower than the levels reported for therapeutic antibodies delivered intravenously. Given the possibility that scFv PD-L1 may distribute into circulation during tumor lysis, we collected serum from tumor-bearing mice in both subcutaneous and intracranial models that demonstrated anti-tumor activity and measured scFv PD-L1 using a surface plasma resonance method. As expected, our data reveal that the scFv PD-L1 serum concentrations from tumor-bearing mice expressing scFv PD-L1 were comparable to that of negative control groups in both models Figure . AlthougCheckpoint inhibitor blockade has proven to be a promising treatment across many cancer types. However, they are not always effective and are associated with systemic immune toxicities which can lead to discontinuation of treatment. In this study, as a proof of principle, we have demonstrated that PD-1/PD-L1 mediated immune suppression can be rescued by RRV delivery of a secreted scFv targeting PD-L1 in tumors and immune infiltrating cells in the TME resulting in increased survival and a protective anti-tumor memory response associated with minimal serum exposure of scFv PD-L1. Most importantly, in contrast to oncolytic virus, the anti-tumor activity observed in tumor-bearing mice treated with RRV-scFv-PDL1 in our studies is not associated with or primed by innate antiviral immunity as shown by 100% RRV-GFP infected tumor’s growth kinetics (included as one of the control groups rather than naïve tumors).http://ir.cytomx.com/news-releases/news-release-details/cytomx-therapeutics-presents-preliminary-clinical-proof-concept). However and notably, grade 3/4 immune-related toxicity was still identified in some patients.The advantages of using tumor-selective RRV for delivering scFv PD-L1 in the TME are at least two fold. One is the localized and sustained delivery of therapeutic payload in the TME with relatively short half-life compared to recombinant therapeutic antibodies. These features allow for engagement of the checkpoint to promote anti-tumor activity in TMEs that may be difficult to reach with conventional systemic delivery of CPI monoclonal antibodies and consequent efficacy in otherwise poorly responsive tumors as well as avoidance of undesirable binding to healthy tissue leading to systemic immune toxicity . Anotherin vitro can fully occupy PD-L1 on bystander cells which translates to a high level of anti-tumor activity in several mouse tumor models. Also, RRV-scFv-PDL1 efficacy was evaluated in several syngeneic mouse tumor models in pre-transduced setting to evaluate dose effect and via intratumoral vector injection to mimic a potential clinical setting. A dose-dependent anti-tumor activity was observed in all three tumor models tested, indicating that 2% transduction level led to similar therapeutic effect as the anti-PD-1 antibody treated group and that a higher transduction level demonstrated anti-tumor activity that is superior than anti-PD-1 antibody group, suggesting the level of PD-L1 occupancy by anti-PD-1 antibody infusion may be suboptimal at the tumor site.We and others have previously demonstrated PD-1/PD-L1 blockade using the RNAi and aptamer approach –41. NeveD = 0.426 nM vs 0.77 µM) [1/2 = ∼77 min for scFv PD-L1/PD-1 vs. 0.9 sec for human PD-L1/PD-1) [Given that RRV has been demonstrated to be cancer-selective both in preclinical and clinical settings , 42, an 0.77 µM)  as well L1/PD-1)  may provet al., OncoImmunology, https://doi.org/10.1080/2162402X.2018.1501137), their tumorigenic property is not induced by carcinogen and thus are most likely to have low tumor mutational burden which closely resembles human GBM.Immunotherapy studies in brain tumor have been limited by the lack of representative murine models. The anti-tumor activity observed in the Tu-2449 syngeneic orthotopic glioma model brings a second model other than GL-261 reported to respond to checkpoint inhibitor as a monotherapy , 45, 46.RRV-scFv-PDL1 could potentially be applicable in a neoadjuvant setting prior to tumor resection; this type of use with nivolumab, was recently reported to induce expansion of antigen-specific clones in lung cancer . In addiIn conclusion, our study demonstrates that RRV-scFv-PDL1 checkpoint inhibition with a tumor-selective delivery and a high bystander index localized within the TME provides an attractive treatment regimen to target immune checkpoints.Two pairs of two different configurations of single-chain variable fragment (scFv) against PD-L1 were designed. One pair consists of scFv with and without the Fc from human IgG1, designated scFv-PDL1 and scFvFc-PDL1, respectively. The other pair consists of scFv-PDL1 and scFvFc-PDL1 with HA and Flag epitope incorporated at the C-terminus, designated scFv-HF-PDL1 and scFvFc-HF-PDL1. The coding sequence of each configuration contains the 3′ coding sequence of the viral envelope gene followed by the gT2A peptide sequence  and was Human 293T cells , human glioma cell line U87-MG , mouse colorectal cancer cell line CT26 , mouse breast cancer cell line EMT6 , mouse glioma cell line Tu-2449 [Jurkat T cells and Raji B cells overexpressing human PD-1 (Jurkat/PD-1) and PD-L1(Raji/PD-L1), respectively, were generated by transducing the cells with lentiviral vectors coding human PD-1 or PD-L1 at MOI of 1 followed by puromycin selection (1.6 μg/mL) 2 days post transduction. PD-1 expressing Jurkat T cells and PD-L1 expressing Raji B cells were enriched under puromycin selection for 14 days and confirmed by cells surface staining with anti-PD-1 and anti-PD-L1, respectively.Twenty micrograms of plasmid DNA encoding the RRVs were used for transient transfection using the calcium phosphate method as previously described . The virFor viral replication kinetics, 20 μL of collected supernatant from each time points during the course of infection were obtained to extract viral RNA and measure particle titer as described . For vecFor the assessment of the transgene protein expression, separation efficiency of the polyproteins, and viral envelop protein processing, cells lysates were prepared as described .EMT6 cells maximally infected with RRV-scFv-HF-PDL1 (HA-tagged scFv-PD-L1) or RRV-GFP at 90% confluency were treated with recombinant human IFNγ at 250 IU/mL for 24 hours to upregulate PD-L1 expression on the cell surface. One million of IFNγ-treated EMT6 cells maximally infected with RRVscFv-HF-PDL1 or RRV-GFP at indicated ratios were split into 2 sets. One set of cells was stained with Alexa Fluor 647-conjugated anti-HA antibody  and the second set of cells was stained with PE-conjugated anti-human PD-L1 antibody . HA-positive, PD-L1-positive, and GFP-positive cell populations were measured by flow cytometric analysis . HA-positive vs GFP positive cell populations and PD-L1-positive vs GFP-positive cell population were calculated by 2-color compensation for proper gating . Cytometry data analysis was performed using the FlowJo v10 software (FlowJo LCC).Wells in a 96-well microtiter plate were coated with 100 μL of 1.5 µg/mL recombinant human PD-L1-Fc  or mouse PD-L1-Fc  overnight at 4°C, followed by 2 hour blocking at room temperature with blocking buffer (1X PBS + 10%FBS + 0.05% Tween 20) the next day. Subsequently, PD-L1-Fc coated wells were co-incubated with 1 µg/mL His-tagged recombinant human PD-1  or His-tagged recombinant mouse PD-1  in competition with supernatant of undefined protein concentration collected from CT26 cells maximally infected with RRV-scFv-PDL1 or RRV-scFvFc-PDL1 , anti-PDL1 antibody  or purified scFv PDL1 protein (Accelagen Inc.) for 2 hours at room temperature. For interaction between scFv PD-L1 and human PD-L1, bound His-tagged recombinant human PD-1 was detected using an HRP-conjugated anti-6X His tag antibody . For binding interaction between scFv PD-L1 and mouse PD-L1, bound His-tagged recombinant mouse PD-1 was detected by performing an additional signal amplification step using an biotin-conjugated anti-6X His tag antibody ,  followed by HRP-conjugated streptavidin , . Color development was performed by adding 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate solution  and terminated by TMB STOP solution  after 6–10 minutes incubation. Optical density was read by a plate reader (Molecular Devices iD5) at 450 nm (with 570 nm correction) using the Soft Max Pro software. The percentage of inhibition was calculated with respect to a positive control which supernatant from CT26 maximally infected with RRV-GFP (non-scFv-PD-L1) was used in the competition.5 cells/0.5 mL. Raji B cells were washed and resuspended in serum-free RPMI-1640 medium at 1 × 106 cells/0.5 mL followed by addition of super antigen SEE , (Toxin Technology #ET404) or SEE plus anti-human PD-L1 or SEE plus purified scFv PD-L1 (10 μg/mL) followed by incubation at 37° C for 1 hour. At the completion of incubation, SEE loaded Raji B cells were washed in serum-free RPMI-1640 medium and resuspended in RPMI-1640 medium containing 20% human AB serum  at 5 × 105 cells/0.5 mL. Both rested Jurkat T cells and SEE-loaded Raji B cells were then co-cultured with equal volumes to reach 1 × 106 cells/mL/well in a non-coated 24-well plate and incubated at 37° C for stimulation.Jurkat T cells were washed and rested in serum-free RPMI-1640 medium at 37° C for 3 hours at 5 × 10For IL-2 ELISA, the supernatants of the co-culture cells were collected 20–22 hours after stimulation and separated from the cells by centrifugation at 300 × g for 5 minutes. IL-2 production was measured by using the Quantikine ELISA Kit  according to the manufacturer’s protocol.6 of Tu-2449SC cells maximally infected with RRVscFv-PDL1 or RRV-GFP at indicated ratios were subcutaneously implanted on the right flank of 8-week-old female B6C3F1 mice (Harlan Sprague Dawley Inc.) and assigned to 8 groups (n = 10 per group). Control Group 1 and 2 which are mice implanted with 100% Tu-2449SC/RRV-GFP cells received PBS or anti-PD-1 antibody. Anti-PD-1 antibody (Clone J43) was intraperitoneally (i. p.) administered on day 0 (300 µg per mouse), day 3, day 6 and day 9 (200 µg per mouse). Group 3, 4, and 5 received ratios of 3/97, 30/70 and 100/0 of Tu-2449SC/RRV-scFv-PDL1 and Tu-2449SC/RRV-GFP cells, respectively. Group 6, 7, and 8 received ratios of 3/97, 30/70 and 100/0 of Tu-2449SC/RRV-scFvFc-PDL1 and Tu-2449SC/RRV-GFP cells, respectively. Tumor sizes were monitored 3 times a week. Animals were removed from the study if tumors became necrotic or reached 2000 mm3 in volume. All mice that cleared their initial tumor implant from RRV-scFv-PDL1 or RRV-scFvFc-PDL1 treated groups (n = 15) and naïve group (n = 5) were challenged with 2 × 106 Tu-2449SC on the left side of the flank. Tumor growth and measurement were monitored and recorded over time.All animal protocols and experiments were approved by the IRB/IACUC of Explora BioLabs . For the Tu-2449SC subcutaneous tumor model, 2 × 10n = 10) were intracranially (i.c.) implanted with 1 × 104 of Tu-2449 cells. Survival analysis was monitored for approximately 90 days. Mice in the experimental groups were injected with purified RRV-scFv-PDL1 of 1 × 105 or 1 × 106 transduction unit (TU) on day 4 post tumor implant. Control groups are mice bearing 100% pre-transduced RRV-scFv-PDL1 tumor cells and Tu-2449SC tumor cells treated anti-PD-1 antibody  or isotype control. Survival data were plotted by the Kaplan–Meier method. Statistical significance of survival between mice treated with isotype and 100% pre-transduced scFv-PD-L1 or injection-treated RRV-scFv-PDL1 groups were determined by the Log-rank (Mantel-Cox) test using GraphPad Prism 7 . Mice which had survived from initial tumor implant from RRV scFv-PDL1 treated groups were challenged with 2 × 106 Tu-2449 cells on the right flank. Tumor growth and measurement were monitored and recorded over time.For the orthotopic glioma model, 8-week-old female B6C3F1 mice underwent surgical implantation of the tumor cells by Hamilton syringe. The stereotaxic coordinates were anteroposterior (AP), 0.5 mm; mediolateral (ML), 1.8 mm; and dorso-ventral (DV), 3.5 mm (from bregma). Mice in each group  and single-cell suspensions were obtained using gentleMACS™ C Tubes with gentleMACS™ Dissociator , and enzymatic solution containing 50 µg/mL final Liberase™ TM  and 200 U/mL final Deoxyribonuclease I . After filtration through a 70 µm cell strainer and washing, erythrocytes were removed following incubation with eBioscience 1X RBC Lysis Buffer . Viable cell counts were determined using a Cellometer K2 (Nexcelom Bioscience). For subsequent flow cytometry analyses, cells were first incubated with TruStain FcX antibody  and Zombie UV  before being subjected to surface staining with fluorochrome-conjugated antibodies against mouse CD45 , CD3ɛ , CD4  and CD8α . Acquisition was conducted on an LSRFortessa X-20 flow cytometer and analysis by using FlowJo software (version 10) with flowAI (1.7) plugin as described .20 (Tween 20), and 0.5 mg/mL BSA at 25° C. Purified scFv PD-L1 (>95% purity) at concentrations ranging between 0.01 200 nM (∼0.0028 µg/mL-5.6 µg/mL) was spiked into 1:20 (v/v) diluted naïve serum to generate a standard curve for every 10 regeneration cycles. Experimental samples were also prepared at 1:20 dilution and injected in at a flow rate of 5 µL/min for a 6-minute contact time. Raw data was analyzed in the Scrubber2 software (BioLogic Software). The lower limit of quantification of this assay is determined to be 0.011 µg/mL. The concentration of scFv PD-L1 level in serum was determined using the SoftMax Pro 5.45 software (Molecular Devices) with the standard curves plotted using a 4-parameter fit algorithm.Recombinant human PD-L1 (R&D Systems # 156-B7) was immobilized on the CM5 sensorchip surface standard amine coupling chemistry using a Biacore 3000 instrument. The running buffer comprised 25 mM HEPES, pH 7.5, 150 mM NaCl, 0.005% P


Semiconductor pn junctions are elementary building blocks of many electronic devices such as transistors, solar cells, photodetectors, and integrated circuits. Due to the absence of an energy bandgap and massless Dirac-like behaviour of charge carriers, graphene pn junction with electrical current rectification characteristics is hardly achieved. Here we show a graphene pn junction diode can be made exclusively from carbon materials by laminating two layers of positively and negatively charged graphene oxides. As the interdiffusion of oppositely charged mobile counterions, a built-in potential is created to rectify the current by changing the tunnelling probability of electrons across the junction. This graphene diode is semi-transparent, can perform simple logic operations, and since it has carbon nanotubes electrodes, we demonstrate an all carbon materials pn diode. We expect this graphene diode will expand material choices and provide functionalities (e.g. grafting recognition units on graphene oxides) beyond that of traditional semiconductor pn junctions. Chemically functionalized graphene oxide-based pn junction diodes have potential for future electronic device applications. Here, the authors report an all carbon pn diode with graphene oxide and carbon nanotubes electrodes showing excellent current rectification and efficient logic gates. In contrast to conventional semiconductors, its conduction and valence bands meet at Brillouin zone corners and, therefore, fabricating electronic components from this zero-gap semiconductor should find an unusual way11. Techniques such as cutting graphene into nanoribbons12, placing bias on bilayer graphenes13, and applying mechanical strains14 were developed to open a bandgap and turn off the current of graphene field-effect transistors. In addition, graphene diodes have received particular attention because diodes are the basic building blocks in a wide variety of electronic/photonic systems. Since graphene is metallic at a sufficiently large Fermi energy, Schottky diodes were fabricated by bringing metallic graphenes and semiconductor materials into contact16. With a gate modulation of localized energy levels, tunable light-emitting diode was demonstrated by using a semi-reduced graphene oxide17. Although pn junctions with a planar geometry could be realized by multiple electrostatic gating18, local chemical doping19, and engineering of substrates20, no current rectifying effect was observed owing to the Klein tunnelling of relativistic Dirac fermions21. In the vertical structure pn junctions, current rectifying was achieved by inserting/forming a thin layer of insulating or semiconducting barrier material between p and n graphene layers, showing behaviours of a typical resonant tunnelling diode23. So far, there is still lack of robust technique in making graphene diodes especially the diode as a similar structure to semiconductor pn junction. On the other hand, graphene oxide (GO) contains various reactive oxygen groups , which provide possibility for chemical modification and produce functional materials for a variety of applications25.Graphene, an atomically-thin hexagonal lattice, has currently attracted enormous research interest because of its great promising use for a wide range including electronic devicesHere, the graphene pn junction diode was fabricated by laminating two layers of such chemically functionalized positively (p) and negatively (n) charged graphene oxides on the transparent substrates. As the contact of two layers, mobile counterions diffused across the interface while the anchored charges on graphene oxides frame were immobile. An equilibrium condition was reached in which an internal electric field was established to tune the electron tunnelling probability through the junction. The coupled movement of ions and electrons as well as the current rectification were modelled by using a combination of Nernst–Planck diffusion and Poisson’s equations. Since carbon nanotubes were used as conductive electrodes, we demonstrated an all carbon materials pn diode. To evaluate the utility of these graphene diodes to build the processing units needed for simple computation, in combination with graphene resistors, all carbon materials AND and OR logic gates were constructed.3)4N+OH−, TMAH, pH = 10), while the positively charged GO was synthesized via a two-step procedure27  and O–H (3415 cm−1) stretching peak were greatly suppressed while amide-I band, amide-II band, and amide-III band appeared at 1631 cm−1, 1460 cm−1, and 1384 cm−1, respectively, which were attributed to C=O stretching vibrations in RNH–CO and the coupling of C–N stretching vibration to N–H bending vibration  were seen while these peaks were not observed prior to positive modification  and charged GOs solutions were successively filtered through a mixed cellulose ester membrane . (Note: 1) the CNT electrodes were also chemically functionalized by the similar procedures to the GOs (see Methods) to eliminate possible dipole formation at the interface between CNT and GO layers; (2) the conductivity of charged 20 nm-thick CNT electrodes was over 10 the CNT I–V) characteristics of the graphene pn junction diode was shown in Fig. A typical current–voltage 4N+, see Fig. In the GO layers, the charges anchored onto the GO frames are jammed and restricted to migration, but the counterions (Cl29). This model combined Nernst–Planck diffusion and Poisson’s equations, in which both counterions and electrons could diffuse in response to concentration gradients and migrate due to local electric fields. The interdiffusion of counterions  characteristics cs Figs. .Fig. 3MoTo demonstrate the utility of the graphene pn junction diode to build the processing units needed for simple computation, AND and OR logic gates were constructed Fig. . For theIt must be noted that the cutting-off capability of the AND gate  the interdiffusion happened at the mixing zone rather than the depletion region in semiconductor pn diode, (2) the formation of internal electric field was entropically driven, unlike in semiconductor pn junctions where the voltage arose from equilibration of the electron chemical potential, (3) the internal electric field of graphene diode increased the probability of electron tunnelling/hopping from n to p layer, in contrast to a semiconductor pn diode where the band alignment allowed electrons to flow from n type region to p type region, but not in the reverse direction, and (4) the interdiffusion-created electric field was pointing from p to n layer while in the depletion region of a semiconductor pn diode, this field was reversed from n to p region. On the other hand, diodes based on pure ionic charge transport32 are often labelled as P or N based on the sign of conducting charge carriers: mobile cations are given as P while conducting anions are denoted by N. If positive biases are applied to the P (GO(−) with positive counterions) side of the device—i.e., forward bias—high currents are ordinarily expected. In fact, this bias corresponds to low current in the present diode. Moreover, either the device structure or rectifying principle of the graphene pn junction diode is distinct from other graphene diodes such as Schottky diodes16, light-emitting diode17, and resonant tunnelling diodes23. These differences strongly suggest the use of charged GOs as the electron-carrying phase leads to a different mechanism for diode behaviour than those that have been reported previously. Based on Zeta potential measurements  diffuse/drift across the interface and at the equilibrium condition, a depletion region was created and an internal electric field is established pointing from n region to p region in the absence of an external applied biashowever,  the inteFinally, we wish to emphasize on the relatively low rectification ratio Fig.  of the g33, systems for recording and manipulating tissue behaviours34, and even smart self-powered devices with multi-functionalities35. Also, we wish to integrate functions of GOs  25 into our graphene circuit which is hard to achieve in semiconductor electronics.In summary, we have demonstrated a semi-transparent all carbon materials pn junction diode by laminating two layers of oppositely charged GO layers. This diode is distinct from previous devices where the current rectification happens because an internal field controls the tunnelling and/or hopping probability of conduction electrons. The observation of current rectification in graphene pn junctions greatly expands the class of materials and physical phenomena that can be used to build electronic components. Looking forward, we envision our graphene pn diode being inkjet-printed from aqueous carbon inks on flexible, transparent, and even stretchable substrates in a wide range of applications. These applications could be skin-like electronics to monitor the physiological conditions−1, Graphenea Inc.) under constant stirring. 5 min later, the mixture solution was heated to 35 °C and kept reacting for 3 h36. 63 mL DI water was then poured into the mixture. 15 min later, additional 200 mL DI water and 3.5 mL hydrogen peroxide (30 wt%) were added to stop the reaction. The yellowish product was thoroughly washed with 200 mL dilute hydrochloric acid (3.5 wt%) for 5 times and then re-dispersed in DI water . This further oxidization procedure produces GOs with more –COOH groups .In a typical synthesis, 35 mL sulphuric acid (98 wt%) and 2.60 g potassium permanganate were carefully added into an ice-bath-cooled flask with 50 mL commercially available GO solution , and 60 mg HATU (O–(7-Aza-1H-benzotriazol-1-yl)–N,N,N’,N’-tetramethyluronium hexafluorophosphate) were firstly mixed and reacted at 65 °C for 6 h in 60 mL N,N-Dimethylformamide (DMF). This step was repeated for three times to ensure the majority of –COOH groups were amidated. Next, 500 μL hydrochloric acid (36.5%) and 5.5 g 3-Chloro-1,2-propandiol (98 wt%) were added into an aqueous solution of above amidated GOs . The mixture was heated to 85 °C and kept reacting for 7 h under constantly stirring and refluxing. The grayish products were washed with DI water for 4 times and finally dispersed into DI water (0.25 mg mL−1).A two-step procedure was employed to synthesize positively charged GOs2SO4 and 3 M HNO3 with 3 to 1 volume ratio) and refluxed for 2 h at 80 °C under stirring. This step produces CNTs with more –COOH groups. The products were collected and washed with DI water for 5 times and then re-dispersed into DI water by the assistance of Sodium deoxycholate and sonication . To produce negatively charged CNTs, Tetramethylammonium hydroxide was only used (pH = 10). For the synthesis of positively charged CNTs, the same procedure with GO(+) was employed, while Hexadecyltrimethylammonium bromide (CTAB) was added to improve their dispersity.100 mg carboxyl functionalized CNTs powder  was added into 100 mL mixed acid solution (3 M H−1. 15 mL CNT(−) was firstly vacuum filtered through a mixed cellulose membrane  and thoroughly washed with 20 mL DI water to remove surfactants. 10 mL GO(−) was subsequently added. The MCE-carbons film was peeled off and cut into desired pieces (15 mm × 10 mm) when all the carbons were dried. The MCE was then removed by floating the film onto DMF solvent for approximately 120 min and the negatively charged GO layer was transferred onto the transparent glass slide (20 mm × 14 mm). The layer was finally dried by using nitrogen gas. This produces a layer of approximately 20 nm CNTs and approximately 40 nm GOs  for 1 h. Gradient centrifugation was then used to collect highly dispersed carbon solutions. The concentration of all carbon materials is approximately 0.03 mg mLThe structure of carbon materials was characterized by using scanning electron microscopy  and transmission electron microscopy . The composition of GOs was evaluated by using X-ray photoelectron spectroscopy  and Fourier-transform infra-red spectroscopy . Ultraviolet-visible spectroscopy  was used to evaluate the light transmission capability. Zeta potential is tested on Zetasizer Nano ZS. The surface roughness and thickness of carbon layers were measured by using Dektak XT Stylus Profiling System. The conductivity of carbon layers was characterized by a four-probe technique (MST 4145-B). The measurement of graphene pn diode was conducted under ambient condition with a computer-interfaced high precision electrometer (Keithley 6517). To drive logic circuits, an additional 6517 electrometer was applied.29. For simplicity, we considered one dimensional domain which contain four regions: CNT(−), GO(−), GO(+), and CNT(+). We assumed that the CNT electrode layer (either CNT(−) or CNT(+)) is equipotential37 and the electrons are free to move out or into the layers. The accumulation of mobile counterions in CNT layers was neglected. The flow of electrons as well as the rearrangement of ionic charges throughout the layers produced the electric current J. The dimensionless electric current density (L represents the thickness of GO(+) and GO(−) layers and ni and ne are the number density of mobile ions and conduction electrons respectively, Di and De are the ion and electron diffusivities, Zi is the valence of the mobile ions, e is the elementary charge, kB is the Boltzmann constant, T is the absolute temperature, u is the electrostatic potential, ε0 is the vacuum dielectric constant, and the ε is relative dielectric constant; Di = De = 1.0 × 10−9 cm2 s−1, ε = 4.5. The coupling Eqs.29 were then solved by using a commercial finite element solver (COMSOL 5.1).The graphene pn junction diode was simulated by using a continuum charge-transport model, which combined Nernst-Planck diffusion and Poisson’s equations. The details of this model was presented in referencesmentclass2pt{minimSupplementary Information


We aimed to determine the function of pyrroline-5-carboxylate reductase 1 (PYCR1) on progression of papillary renal cell carcinoma (PRCC) and related mechanism.The TCGA database provided us expression profiles of PYCR1 and overall survival rates. Small interfering RNA (siRNA) was used to knockdown PYCR1; quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting were conducted to identify the expression levels of mRNA and protein. The cell counting kit-8 (CCK-8) and colony formation assays were used to explore cell viability in Ketr-3 cells. The migration and invasion of Ketr-3 cells were investigated by transwell assays.We found that PYCR1 was over-expressed in PRCC tissues and cells, causing poor outcomes. Moreover, reduction of PYCR1 played a negative role on cell proliferation, migration and invasion in tumor cells. The important Akt/mTOR pathway proteins, phosphorylated Akt (p-Akt) and phosphorylated mTOR (p-mTOR), also showed lower levels compared with control groups.These findings showed that disordered expression of PYCR1 could modulate PRCC progression through the Akt/mTOR pathway, implying a theoretical basis for PYCR1 as a potential therapeutic target in future clinical PRCC treatment. Complic3) genes . Consideet al. confirm a close relationship between PYCR1 expression and non-small-cell lung carcinoma (NSCLC) progression [PYCR1 plays an important role in proline biosynthesis and conversion of pyrroline-5-carboxylate (P5C) into proline also depends on the involvement of PYCR1 . Prolinegression . Additiogression , lung cagression , 16, andgression , 18.In the present investigation, we first aimed to characterize the function of PYCR1 in PRCC. The expression level of PYCR1 in tumor tissues and cells was assessed, and prognosis relying on its expression level also was analyzed using statistical methods. Cell behaviors were measured using vitro experiments and the underlying mechanism was explored by key protein detection. Taken together, these data may provide new mechanistic insights into the function of PYCR1 for PRCC treatment in future.22.12. Additionally, 10% fetal bovine serum (FBS) , 100 U/mL penicillin and 100 μg/mL streptomycin  were added to RPMI-1640 medium for cell growth.The American Type Culture Collection  provided us with cell lines including HRCE, 786-O, OS-RC-2, Ketr-3 and A498. RPMI-1640 medium  was used to culture these cell lines and environmental conditions were set at 37˚C and 5% CO2.2-ΔΔCt analysis and GAPDH was regarded as standard control.To determine the expression of PYCR1 in all cell lines, we isolated total RNA using Trizol . Then SuperScript III RNase H Reverse Transcriptase  was utilized to reverse transcribe total RNA into cDNA. PCR reactions were carried out on a Bio-Rad Connect Real-Time PCR machine with SYBR Green PCR Master Mix . The PCR procedure was run as follows: 95˚C for 5min, 95˚C for 30s with 40 cycles, 60˚C for 45s and 72˚C for 30min. The primer sequences: PYCR1: F, 5’-TGACCAA-CACTCCAGTCGTG-3’, R, 5’-GTCCAGCTTCACCTTGTCCA-3’; GAPDH: F, 5’-GGAGCGAGATCCCTCCAAAAT-3’, R, 5’-GGCT-GTTGTCATACTTCTCATGG-3’. The relative expression of PYCR1 was assessed by 22.3Lipofectamine2000 transfection Kit  was used to inject si-PYCR1 into cells according to the manufacturer’s instructions. In order to control the unique variables and nonspecific interference, si-con without target was used as internal control. The sequences were as follows: PYCR1 siRNA 1 : F: 5’-CTTCATCCTGGATGAAAT-3’, R: 5’-GAAG-TAGGACCTACTTTA-3’; siRNA 2 : F: 5’-TGCTCATCAACGCT-GTGG-3’, R: 5’-ACGAGTAGTTGCGACACC-3’; si-con: F: 5’-AATTCTCCGAACGTGTCACGT -3’, R: 5’-TTAAGAGGCTTG-CACAGTGCA-3’. Primers were synthesized by GenePharma Co., Ltd .2.4CCK-8 kit  was used to investigate cell vitality. Transfected cells were placed into 96-well plates with 1,000 cells per well and incubated in a carbon dioxide incubator. 10 μL CCK-8 regent was added before cell activity was examined at 0 h, 24 h, 48 h and 72 h. Subsequently, cells were cultured at 37˚C for 1.5 h and then the OD values were read under a microscope at 450 nm.2.52. After washing with 1×PBS, cells were fixed and stained with 4% paraformaldehyde and 0.1% crystal violet respectively. Finally, the clones were counted by naked eye.Colony formation assays were used when the cells entered the log phase and these cells were trypsinized to make into suspensions. Cell suspensions were put into 60 mm dishs which contained 5 mL warmed medium at a density of 1,000 cells/dish and maintained for two weeks until clones appeared on the culture dish at 37˚C with 5% CO2.6Transwell assays were performed to explore cell migration and invasion. For cell invasion, Matrigel was diluted with medium six times after overnight thawing and then put into the upper chamber. Cells (1×105/well) were seeded into the upper chamber pre-coated with Matrigel and to the bottom chamber 500 μL complete medium was added . After 24 h incubation, cells that did not invade were removed by cotton swabs while invasive cells were fixed using 4% paraformaldehyde for 30 min, stained by 0.1% crystal violet for 20min. Next, PBS was used to wash off the floating color and the status was imaged under an optical microscope. Cells were calculated in several random fields. For cell migration, the density was 5, 000 cells per well and the chamber without Matrigel was utilized. The experiment was repeated at least three times and all the results were present as mean ± standard deviation (SD).2.7Proteins were extracted from transfected cells with RIPA buffer and quantified using a BCA kit . Each tank was filled with 20 μg proteins and then 12% SDS-PAGE was used to separate proteins. When proteins were transferred onto PVDF membrane, 5% non-fat milk was utilized to block these membranes for 1 h. Subsequently, PVDF membranes were incubated with primary antibodies  against Akt, p-Akt, mTOR, p-mTOR and GAPDH overnight at 4˚C. After washing with 1×TBST three times, PVDF membranes were cultured with secondary antibodies . Protein images were captured through ECL and the relative expression of protein bands were measured by Quantity One software.2.8Data analyses were performed with SPSS22.0 and Graph-Pad Prism version 5.0. Student’s t-test was conducted to compare the difference in two samples and one-way ANOVA with Dunnett’s post hoc test utilizing in diverse groups. Pearson’s analyses were applied to reveal the association between PYCR1 and PYCR2 or PYCR3. Significant statistical differences were defined as P < 0.05. All these results were expressed as the mean ± standard deviation (SD) in at least three replications. Ethical approval: The conducted research is not related to either human or animal use.33.1To determine PYCR1 expression level and confirm association between PYCR1 expression level and papillary renal cell carcinoma, we first analyzed the expression level of PYCR1 in PRCC tissues and corresponding tumor cell lines. Data about PYCR1 expression in PRCC tissue was downloaded from TCGA database and revealed that compared to normal tissues, PYCR1 showed a higher level in the respect of mRNA (3.2In order to explain the role of PYCR1 accurately, we knocked down PYCR1 using siRNA in Ketr-3 cells. The effective reduction was exhibited at both the mRNA level and protein level, especially si-PYCR1-2 (3.3Next, transwell assays were implemented to explore cell migration and invasion in Ketr-3 cells after PYCR1 knockdown. The experimental results demonstrated that deficiency of PYCR1 inhibited Ketr-3 cell activity in the aspect of invasion while there was no significant difference in the control group . Meanwhi3.4Considering the function of PYCR1 knockdown on PRCC cells, we further studied the underlying molecular mechanism involved in PRCC. We found that the introduction of si-PYCR1 dramatically hindered p-Akt and p-mTOR protein expression in Ketr-3 cells when compared to the si-con group, but that Akt and mTOR implied no difference (4We discovered that PYCR1 was over-expressed in human PRCC tissues compared with controls. Highly regulated PYCR1 expression led to poor prognosis in PRCC patients. All the data suggest that PYCR1 could promote the tumorigenesis of PRCC in the view of cell proliferation, migration and invasion.et al. searched for different genes that were consistent in nineteen cancers and found that PYCR1 is proposed as a novel enzyme [Previously, metabolism is required in tumors and mediates cellular processes, such as proliferation, redox homeostasis and reprogramming . Recent l enzyme . In addil enzyme , 25. Redl enzyme . Moreovel enzyme , 28.in vitro and found that PYCR1 could accelerate the progression of PRCC. There is a study about skin cancer that identified that PYCR2 was abundantly present in melanoma cells and regulated the Akt/mTOR pathway [We performed experiments  pathway . Epiderm pathway , 31.In order to determine how PYCR1 regulates the physiological processes of tumor cells, we first examined the effect of silencing PYCR1 on the Akt/mTOR pathway. The results of western blotting showed p-Akt and p-mTOR were impaired owing to knockdown PYCR1. On the contrary, the normal control exhibited no difference. However, here we did a preliminary investigation about the relationship between PYCR1 and PRCC. To thoroughly understand the underlying mechanism, a series of in-depth studies including in vivo experiments need to be followed up.In total, all the data in this present study confirm that PYCR1 was highly regulated in PRCC and its over-expression could induce poor prognosis in patients with PRCC. Based on the results of the silencing tests, PYCR1 stimulated cell behaviors including proliferation, migration and invasion. The vitality of colony formation was also inhibited because of silencing PYCR1. The key proteins, phosphorylated Akt and mTOR, revealed a downtrend at the level of protein expression level. We predict that PYCR1 might be an independent risk element for patients prognosis in PRCC via regulating Akt/mTOR pathway.


Supercapacitors are a highly promising class of energy storage devices due to their high power density and long life cycle. Conducting polymers (CPs) and organic molecules are potential candidates for improving supercapacitor electrodes due to their low cost, large specific pseudocapacitance and facile synthesis methods. Graphene, with its unique two-dimensional structure, shows high electrical conductivity, large specific surface area and outstanding mechanical properties, which makes it an excellent material for lithium ion batteries, fuel cells and supercapacitors. The combination of CPs and graphene as electrode material is expected to boost the properties of supercapacitors. In this review, we summarize recent reports on three different CP/graphene composites as electrode materials for supercapacitors, discussing synthesis and electrochemical performance. Novel flexible and wearable devices based on CP/graphene composites are introduced and discussed, with an eye to recent developments and challenges for future research directions. The continued use of fossil fuels, combined with limited reserves, has resulted in two serious and urgent issues to be addressed in our daily lives; environmental pollution and concern around continued energy supply. To address these problems, scientists focus on looking for clean and renewable energy sources such as solar energy and wind energy. However, these renewable energy sources are difficult to store, meaning that the development of novel, efficient, and low cost energy storage technology is very significant. In recent years, fuel cells, ionic batteries and electrochemical supercapacitors have been investigated ,2,3. ComBased on the different mechanisms of energy storage, supercapacitors can be broadly classified into two types: electric double layer capacitors (EDLCs) and pseudocapacitive enhanced devices, sometimes referred to as pseudocapacitors ,8,9,10. 2, NiOx, etc.) are typical active materials for pseudocapacitors  and iron (III) chloride (FeCl3), or sodium persulfate (Na2S2O8) and iron (III) sulfate [Fe2(SO4)3] are added as oxidants to catalyze the polymerization . . 113]. F2 hybridization of carbon atoms in the graphene, so it may possess a better conductivity [As mentioned above, most organic molecules can be used to functionalize graphene by non-covalent or covalent methods. Each has its own advantages; the covalent approaches may be easy to control and are stable, but in non-covalent methods organic molecules adsorb on the surface of graphene by π–π stacking interactions . When couctivity . FurtherIn recent years many flexible energy storage devices have been investigated and designed by researchers for applications in flexible displays, smart electronics, medical devices and other flexible devices. Given the advantage of being highly integratable and potentially even wearable, flexible energy storage architechture is considered to be one of the most prominent emerging disruptive-technologies that may completely transform our lives ,123,124.Typical flexible CP/graphene supercapacitors not only have excellent electrochemical properties, but also exhibit favorable mechanical performance. There are five commonly explored methods for fabricating flexible composite electrodes; electrodeposition, in situ polymerization, direct coating, chemical vapor deposition (CVD), and the vacuum filtration technique . −2 at 0.5 A g−1 in 1 M H2SO4. Because of the high electrical conductivity and good chemical stability, graphene papers have been used as a flexible substrate for electrodes in recent years, but it is difficult to fabricate large-scale graphene paper materials via vacuum filtration. Inkjet printing technique address this challenge. Chi et al. used well-controlled full inkjet printing method to fabricate a freestanding graphene paper (GP) supported 3D porous graphene hydrogel-polyaniline (GH-PANI) nanocomposite [−1 at 400.33 W kg−1 combined with remarkable mechanical flexibility.Shu et al. synthesized rGO-PPy hybrid paper for flexible supercapacitors by electropolymerization on a paper-like graphene gel . This hyomposite . GO ink In the traditional fabrication of supercapacitor electrodes, active materials are mixed with carbon black and a binder (such as PVDF or PTFE). However, binder materials are generally non-conducting and therefore decrease the electrical conductivity of electrodes, inhibiting electrochemical performance. For flexible supercapacitors the graphene may act as a flexible provider of mechanical strength, and as an active charge-storing part of composites. Furthermore, PANI or PPy, for example, are able to be polymerized directly on the graphene surface, without any binder, which is a good way to reduce the mass of material in an electrode which is not contributing to the electrochemical performance.−1 and 990 F cm−3 at 1 A g−1 in 1 M H2SO4 and 85% capacitance retention after 60,000 cycles in a three-electrode cell configuration. Out of this 3D-G/PANI composite film, flexible all-solid-state supercapacitors (ASS) were prepared, without the need for any current collectors or supporting substrates  composite electrode . The G/Pbstrates d. The ASbstrates e. Moreovbstrates f. In the−1 and an energy density of 23.2 Wh kg−1 at a power density of 399 W kg−1. Furthermore, in the bent or stretched state, the specific capacitance and capacitance retention are close to the normal state, demonstrating that this wavy shape device can tolerate mechanical deformation and maintain good electrochemical performance at the same time.Xie et al. synthesized graphene/PANI composite film with a wavy shape, able to behave as a stretchable upercapacitance electrode . Porous Recently, Ren et al. combined CVD and chemical interfacial polymerization methods, to fabricate stretchable electrodes based on a graphene foam/PPy . Pure grCompared with the liquid electrolytes commonly used in supercapacitor research, flexible devices often use a PVA based gel impregnated with electrolytes. This provides the possibility of omitting the rigid packaging that energy storage devices generally require to prevent electrolyte leakage. Therefore, the development of flexible leak-proof electrolyte systems is also a critical aspect of producing flexible and/or wearable energy storage, however detailed discussion of such systems is beyond the scope of this review which is centred on electrode materials. Nevertheless, when these components are brought together the uniquely novel mechanical properties of flexible and stretchable supercapacitors show strong potential as a disruptive technology to a wide range of established markets.In the above discussion, flexible supercapacitors are presented as an emerging energy storage device architecture with highly desirable mechanical properties. Wearable energy storage generally refers to devices fabricated within flexible and lightweight fibers or yarns, which are then woven into textile fabrics. To satisfy the needs of wearable energy storage, devices must not only show excellent electrochemical performance and possess all the mentioned flexible behaviors  but must also have added durability in order to resist the destructive forces that worn textiles are exposed to in application. 2@rGO hierarchical structure via electrodeposition [−2 in 1 M Na2SO4 (three-electrode cell), and 411 mF cm−2 in an all-solid-state two-electrode cell with PVA/H3PO4 electrolyte. Mechanical deformation tests were performed, consisting of bending at 90°, knotting, and twisting, each repeated 1000 times. Following each of these tests, the devices had retained capacitances of 80%, 91% and 103%, respectively. In a practical demonstration of the devices’ capabilities, 7 yarn supercapacitors were woven into a 15 × 10 cm2 piece of cloth, connected in series, and then used to light 30 LEDs. This provides clear evidence that yarn-based supercapacitors have the potential to possess excellent electrochemical and mechanical properties, giving them  a bright future in various wearable electronics.Huang et al. synthesized stainless steel yarns coated with a PPy@MnOposition . The proAlthough the electrochemical performance of yarn supercapacitors has been established, it still lags behind the energy requirement for conceivable applications, such as heated clothing, or charging personal devices. In fact the amount of charge stored via the EDL capacitance mechanism is proportional to the surface area of an electrode interfacing with an electrolyte. In the case of yarn supercapacitors, it may not be clear how this can be increased without either longer or thicker yarn. If it were possible to create additional inner interface (i.e. within the core of the yarn), the specific capacitance of yarn supercapacitors could be increased. This is what researchers have set out to do.−2 at 0.08 mA cm−2 and a high energy density of 27.1 μWh cm−2 at a power density of 66.5 μW cm−2. Due to the nematic state of rGO sheets which arrange along the length of a fiber, and π–π interactions between rGO and PEDOT, the fiber showed an outstanding tensile strength of up to 631 MPa. After bending 500 times the CV curve was almost same as the initial curve. It is suggested that the hollow structure not only increases the capacitance but also provides favourable flexibility and stability.Qu et al. fabricated a hollow graphene/CP fiber electrode with a high energy density when tested as a yarn supercapacitor . As show2 modified polyester textile electrodes, as shown in 2 modified polyester fiber (M-PEF) and then PPy is deposited on the substrate of graphene/SnCl2 modified polyester yarn (rGO/M-PEFY). The large-area textile electrodes and ultralong yarn electrodes fabricated by this material showed excellent electrochemical and mechanical properties. This material showed an outstanding areal capacitance of 1117 mF cm−2 at a current density of 1 mA cm−2 in 1 M Na2SO4 (three-electrode cell) and a capacitance retention of 100% after 10,000 cycles at 20 mA cm−2. The textile electrode achieved a capacitance retention of 98.3% when bent from 0° to 180° after 1000 cycles. The polypyrrole/graphene/SnCl2 modified polyester textile (PPy/rGO/M-PEFT) electrodes exhibited outstanding electrochemical performance, which is attributed to the synergistic effect of the pseudocapacitive PPy, and highly conductive electric double layer forming rGO. The results reveal this method is suitable for fabricating textile and yarn electrodes, with both the desirable electrochemical and mechanical behaviors demanded by wearable electronics.High capacity performance and robust mechanical properties are both essential characteristics for wearable supercapacitor electrodes. In some cases, to improve the mechanical properties, flexible textile substrates were chosen ,141. How−3 at the power density of 30.77 mW cm−3. There were no changes in the CV curves under normal, knotted, and twisted states  produced via a self-assembly technique, at room temperature . PANI nad states a. In addd states b,c. Figud states e. The reIn the above studies, graphene and CPs are successfully used to fabricate yarn electrodes and woven textile electrodes for wearable supercapacitors. Graphene not only acts as a highly conductive, electric double-layer capacitance material, but also connects the CPs with a flexible, highly conductive scaffold. On the one hand, the CPs contained in the reviewed composite electrodes contribute pseudocapacitance to the electrochemical performance. On the other hand, they play an important role in the porous structure formation of graphene and may also work as a coating to protect the inner material. The synergistic effect of graphene and CPs effectively improves the electrochemical and mechanical properties of textile based devices for wearable electronics. With continued development of textile energy storage technology, it is anticipated that supercapacitors will be integrated into commercialized wearable electronics within the next few years.As low-cost, high pseudocapacitance materials, CPs have attracted lots of attention in next generation supercapacitor electrode research. While pure CPs show many problems, such as relatively poor electrical conductivity, poor cyclic stability, and low energy density, the combination of graphene and CPs demonstrates how these problems may be solved. This paper has reviewed graphene and CPs including PANI, PPy and PEDOT, as composite electrodes for supercapacitors. It has been shown that graphene/CP composites may be constructed to possess desirable 3D porous structures. In summary of the synergistic effects, graphene is able to reduce the volume change during charge/discharge cycles, improving the cyclic stability of CPs via reducing the mechanical stresses on them, while at the same time the graphene improves the electrical conductivity and resultant power handling. Meanwhile, CPs can be grown in situ on the surface of graphene which typically reduces the aggregation of the GO precursor during reduction processes, maintaining higher interfacial area within the electrode for improved double layer capacitance. Furthermore, under certain synthesis conditions the excellent mechanical properties of some graphene/CPs composite films demonstrate that the materials can be drawn into yarns suitable for flexible and wearable energy storage devices. In recent years, the functionalization of graphene with organic molecules has been investigated for energy storage. There are a number of reports where functionalized graphene based electrodes show outstanding specific capacitances due to the high pseudocapacitance contribution of the organic molecules. Although the number of reports discussing organic molecules for energy storage devices is growing, up to now, only a few amino or hydroxyl group-containing molecules have been investigated, leaving many other molecules remaining to be studied. In addition, details of the energy storage mechanisms of these molecules is still to be clearly understood. In most cases discussed, the reported high energy densities and favourable specific capacitances were obtained using small amounts of active materials as electrodes in lab-scale test cells and/or devices. However, for practical applications, supercapacitor devices are typically much larger and require more active materials constructed into larger electrodes. Scaling-up the production of many of the active material combinations discussed in this review is a serious challenge for researchers and engineers. In addition to this broad limitation, the composites in most of the cited literature can only be used in aqueous electrolyte systems, whose potential window is limited to around 1.2 V. This may be compared to organic-solvent or ionic liquid electrolyte systems which may reach around 3 V. Although the relationship has not been discussed in detail, lower maximum voltage severely limits the energy density of devices. While aqueous systems may find some genuine niches, such as for biological implants, the relatively lower energy density generally limits their application. Thus, to improve the energy able to be stored in supercapacitors, electrolyte systems that are able to support a wider potential window are always being sought, especially for graphene/CP composites where the present choices are more restricted by the chemistry of the active materials. In conclusion, graphene/CP composites are promising materials for supercapacitor applications. However, currently the available CPs for supercapacitors are limited to PANI, PPy, and PEDOT. Hence their drawbacks in terms of poor cycling stability and narrow voltage window in aqueous electrolyte are anticipated to be solved by the synthesis of new-type CPs. Building asymmetric supercapacitors by using two dissimilar CPs, one as the negative electrode and the other one as the positive electrode, can be an efficient way towards high performance supercapacitors with high specific capacitance and broad voltage window beyond the crytical voltage of water splitting (~1.23 V). With continued effort made to overcome the identified obstacles, and continued research into new-type CPs with better cycling stability, higher specific capacitance, and broader voltage window in aqueous electrolyte, completely novel energy storage devices based on graphene/CPs composites could become commercially available in the not-too-distant future.