The data presented in this article are related to “Odd-even phonon transport effects in strained carbon atomic chains bridging graphene nanoribbon electrodes” . To provide the information that can be utilized in reproducing our results, we provide the fully optimized graphene nanoribbon (GNR)–carbon chain (CC)–GNR junction atomic configurations at different strain values, the computational setting for quantum phonon transport calculations, and several computational data that were not included in the main manuscript. Data on graphene–CC–graphene junctions are additionally presented. Specifications tableValue of the data•The data can be references to the atomistic modeling of carbon atomic chains bridging graphene and graphene nanoribbon electrodes with strain effects.•The data can be references to the quantum phonon transport calculations of carbon atomic chains, graphene nanoribbons, and their heterojunctions.•The data may be generally relevant for the researchers interested in the quantum transport properties of all-carbon devices.1displacement}.xyz”. The simulation cell information are provided in the comment lines within the xyz files. In In this data article, we present the data on the atomic configurations and corresponding lattice thermal conductances of carbon atomic chains (CCs) bridging graphene nanoribbon (GNR) and graphene (GRP) electrodes 22.1−6 eV, and atomic relaxations were performed until the Hellmann–Feynman forces on each atom fall below 10−3 eV/Å.For the junction models described in For the computation of ballistic phonon transport properties, we used an in-house code that implements the atomistic matrix Green’s function (MGF) formalism 2.2The geometries and quantum phonon transport properties of the GNR–CC–GNR and GRP–CC–GRP junction models were obtained following the procedure established earlier for the study of quantum electron transport properties in molecular ΔL = 0.4 Å and 0.8 Å and repeated energy minimization procedures.Expecting the CC parts will adopt similar configurations within the zGNR and aGNR electrodes, junction models based on 7aGNR electrodes were initially prepared by extracting the inner CC parts  in the 4zGNR–CC–4zGNR junction models and replacing the 4zGNRs by 7aGNRs. The scattering-region atoms were energy minimized once again. For the aGRP cases, we repeated a similar procedure to expedite model preparations. After obtaining the optimal geometries of the GNR–CC–GNR and GRP–CC–GRP junction models, we stretched junction model by moving GNR or GRP electrodes outward by


Different clones, protocol conditions, instruments, and scoring/readout methods may pose challenges in introducing different PD-L1 assays for immunotherapy. The diagnostic accuracy of using different PD-L1 assays interchangeably for various purposes is unknown. The primary objective of this meta-analysis was to address PD-L1 assay interchangeability based on assay diagnostic accuracy for established clinical uses/purposes. A systematic search of the MEDLINE database using PubMed platform was conducted using “PD-L1” as a search term for 01/01/2015 to 31/08/2018, with limitations “English” and “human”. 2,515 abstracts were reviewed to select for original contributions only. 57 studies on comparison of two or more PD-L1 assays were fully reviewed. 22 publications were selected for meta-analysis. Additional data were requested from authors of 20/22 studies in order to enable the meta-analysis. Modified GRADE and QUADAS-2 criteria were used for grading published evidence and designing data abstraction templates for extraction by reviewers. PRISMA was used to guide reporting of systematic review and meta-analysis and STARD 2015 for reporting diagnostic accuracy study. CLSI EP12-A2 was used to guide test comparisons. Data were pooled using random-effects model. The main outcome measure was diagnostic accuracy of various PD-L1 assays. The 22 included studies provided 376 2×2 contingency tables for analyses. Results of our study suggest that, when the testing laboratory is not able to use an Food and Drug Administration-approved companion diagnostic(s) for PD-L1 assessment for its specific clinical purpose(s), it is better to develop a properly validated laboratory developed test for the same purpose(s) as the original PD-L1 Food and Drug Administration-approved immunohistochemistry companion diagnostic, than to replace the original PD-L1 Food and Drug Administration-approved immunohistochemistry companion diagnostic with a another PD-L1 Food and Drug Administration-approved companion diagnostic that was developed for a different purpose. Clinical trials have shown that it is possible to successfully restore host immunity against various malignant neoplasms even in advanced stage disease by deploying drugs that target the PD-1/PD-L1 axis . In mostSeveral such fit-for-purpose immunohistochemistry kits are commercially available, but in clinical practice, and especially in publicly funded health care, it is challenging to make all such testing available to patients –9. BecauThe goal of this study is to assess the performance of PD-L1 immunohistochemistry assays based on their diagnostic accuracy at specific cut-points, as defined for specific immunotherapies according to the clinical efficacy demonstrated in their respective pivotal clinical trials.In other words, given an Food add Drug Administration-cleared assay, which other assays can be considered substantially equivalent for that specific purpose? Although comparison of immunohistochemistry assays for their analytical similarities is warranted and useful for clinical immunohistochemistry laboratories, it is an insufficient foundation on which to make an informed decision whether an Food and Drug Administration-approved companion diagnostic with a specific clinical purpose can be replaced by another assay, whether the substitute assay is an Food and Drug Administration-approved companion diagnostic for a different purpose or a laboratory developed test. The more appropriate approach for these qualitative assays would be comparing the results of the candidate assay for its diagnostic accuracy against a comparative method/assay or designated reference standard . We repoMethodology including data sources, study selection, data abstraction, and grading evidence, are detailed in Supplementary Files A systematic review of literature was conducted as a part of a national project for developing Canadian guidelines for PD-L1 testing. The Canadian Association of Pathologists – Association canadienne des pathologistes (CAP-ACP) National Standards Committee for High Complexity Testing initiated development of CAP-ACP Guidelines for PD-L1 testing to facilitate introduction of PD-L1 testing for various purposes to Canadian clinical immunohistochemistry laboratories. This review was also used to guide the selection of publications to be used in this meta-analysis.The purposes identified in the systematic review of published literature were based on either the clinical purpose that was specifically identified in the published study or the intended purpose for which the included specific companion diagnostic assay was clinically validated. Although a large number of potential purposes were identified, only few could be included in this meta-analysis. The selection was based on the type of data available, including which immunohistochemistry protocols and which readout was performed by the authors. The greatest limitation in the accrual of data from these published studies was based on the selection of the readout employed to assess the results; in most studies the readout was limited to tumor proportion score with 1% and 50% cut-offs, which is essentially based on the clinically meaningful cut-offs for pembrolizumab and nivolumab therapy. Hence, these two readouts were selected for our analysis and form the basis for outlining different purposes that are derived from the combination of the readouts and immunohistochemistry kits/protocols that use these readouts and are approved by regulatory agencies  for different clinical uses.Most published studies on PD-L1 test comparison did not include 2 × 2 tables that would allow calculations of either diagnostic sensitivity and specificity or positive percent agreement and negative percent agreement. The CAP-ACP National Standards Committee for High Complexity Testing requested this information from the authors of studies where it was evident that the authors generated such results, but did not include them in their published manuscript. Most studies required generation of multiple 2 × 2 tables, as each one was designed for a specific purpose and set of candidate’ and ‘comparator’ assays. For primary studies that provided sufficient detail, information on study setting, comparative method/reference standard and 2 x 2 tables for different tumor proportion score cut-offs were extracted, from which accuracy results were reported. Studies of PD-L1 immunohistochemistry assay comparisons that did not compare the performance of the assay to any designated or potential reference standard  –22 were Most studies evaluated PD-L1 immunohistochemistry in non-small cell lung cancer, resulting in 337 test comparisons. Comparisons of test performance in other tumors were much less common. These included analysis of urothelial carcinoma (20 test comparisons), mesothelioma (9 test comparisons) and thymic carcinoma (9 test comparisons).2 were used to examine heterogeneity among studies. Funnel plots and Egger’s test were applied to detect possible publication bias  from the individual studies were summarized. Random-effects models were fitted , 45. ForFor the purpose of this study, the immunohistochemistry candidate assays were considered to be acceptable for clinical applications if both sensitivity and specificity for the stated clinical purpose/application were ≥90% .Assuming that laboratories follow the instructions for use provided with Food and Drug Administration-approved or European CE-marked immunohistochemistry kits, the overall results of this meta-analysis could be considered highly representative and generalizable of Food and Drug Administration-approved assay performance and the diagnostic accuracy of that assay against a designated reference standard for the stated specific purpose in any laboratory. However, this assumption cannot be applied to the results of laboratory developed tests, because laboratory developed test immunohistochemistry protocol conditions were often different in different laboratories even when the same primary antibody was used, and the protocol was performed on the same automated instrument with the same detection system . When the results of the meta-analysis for laboratory developed tests were suboptimal, but one or more laboratories achieved ≥90% sensitivity and specificity; we cannot exclude the possibility that with appropriate immunohistochemistry protocol modification and assay validation, other laboratories could also achieve optimal results. This contrasts with the use of Food and Drug Administration-approved assays, where no protocol modifications are allowed. Therefore, where the results of laboratory developed tests are excellent, they are representative of what could be achieved by laboratory developed tests rather than that they are generalizable and that they will automatically be achieved in all laboratories.The number of studies comparing different assays in this meta-analysis was larger than the number of published manuscripts, due to the frequent inclusion of multiple test comparisons in single publication as well as use of different cut-off points for “positive” vs. “negative” test result. Table 2 for sensitivity and specificity across all studies are shown in Supplementary Files Table Cochran’s heterogeneity statistic Q and IWhere the number of studies was less than four or when the data were sparse due to the presence of a zero result in contingency tables , the models did not converge and did not allow for meta-analysis calculations. As summarized in Tables The highest diagnostic accuracy was shown for well-designed 22C3 laboratory developed tests compared to PD-L1 IHC pharmDx 22C3. The sensitivity and specificity were both 100% in 8/9 assays for the 50% tumor proportion score cut-off point Table , Table 2No other candidate assays reached 90% sensitivity and specificity in the meta-analysis for either the 50% or the 1% tumor proportion score cut-off for PD-L1 IHC 22C3 pharmDx Table  and 1B , 27.The highest results were achieved by the Ventana PD-L1 (SP263) assay; it had acceptable accuracy in the meta-analysis compared to PD-L1 IHC pharm Dx 28-8 at the 1% cut-off  Fig. , Table 1No candidate assays achieved required diagnostic accuracy for either the 1% or the 50% cut-off. Most candidate assays achieved acceptable specificity, but the sensitivity was too low for both cut-off points Tables .The most dominant result of this meta-analysis is that properly designed laboratory developed tests that are performed in an individual immunohistochemistry laboratory  and are developed for the same purpose as the relevant comparative reference method standard may perform essentially equally to the original Food and Drug Administration-approved assay, but also generally better than the Food and Drug Administration-approved companion diagnostics that were originally developed for different purposes. For example, to identify patients with non-small cell lung cancer for second line therapy with pembrolizumab where PD-L1 IHC pharmDx 22C3 is not available, the results of our study indicate that it is more likely that 22C3 or E1L3N well-developed, fit-for-purpose laboratory developed tests would identify the same patients as positive and/or negative as PD-L1 IHC pharmDx 22C3, rather than Ventana PD-L1 (SP263), Ventana PD-L1 (SP142), or PD-L1 IHC pharmDx 28-8, which were developed for different purposes –53.The accuracy of laboratory developed tests varied in our meta-analysis. 22C3 laboratory developed tests achieved the best results, with both sensitivity and specificity of 100% in 8/9 studies. E1L3N also showed excellent results, but in only 3/12 comparisons. Its success in 3 separate comparisons illustrates that it is possible to develop an acceptable laboratory developed test with this clone and that this antibody can be optimized for clinical applications for which the PD-L1 IHC 22C3 pharmDx was developed. The successful applications of some of the laboratory developed tests reinforce the importance of considering the original purpose of the immunohistochemistry assay, a point emphasized in the ISIMM and IQN Path series of papers entitled “Evolution of Quality Assurance for Immunohistochemistry in the Era of Personalized Medicine” –57. It sAs so far there are no tools to measure analytical sensitivity and specificity of immunohistochemistry assays; this presents a significant problem in assay development, methodology transfer, and daily monitoring of assay performance, as well as direct comparison of assay calibration. The lack of tools that could assess analytical sensitivity and specificity also hinders attempts of immunohistochemistry protocol standardization/harmonization for the PD-L1 assays; without such tools it is not possible to determine the desirable range of analytical sensitivity and specificity of relevance for diagnostic accuracy for any of the PD-L1 assays. This is one cause that we can identify as a potential source of the discrepancy between previously published works that suggested analytical interchangeability of the several Food and Drug Administration-approved PD-L1 assays, but did not necessarily lead to interchangeability based on calculated diagnostic accuracy as shown in our study.The Ventana PD-L1 (SP263) assay had very high diagnostic sensitivity against all other Food and Drug Administration-approved PD-L1 assays, but its diagnostic specificity was consequently lower. Although several of the studies included in this meta-analysis demonstrated substantial analytical similarity between PD-L1 IHC 22C3 pharmDx, PD-L1 IHC 28-8 pharmDx, and Ventana PD-L1 (SP263), our cumulative results suggest that the diagnostic sensitivity of these various assays  is ordered as follows: PD-L1 IHC 22C3 pharmDx < PD-L1 IHC 28-8 pharmDx < Ventana PD-L1 (SP263).The results of this meta-analysis confirm previous observations that the Ventana PD-L1 (SP142) assay’s analytical sensitivity is significantly lower than that of the three other Food and Drug Administration-approved PD-L1 assays and that the diagnostic sensitivity of Ventana PD-L1 (SP142) against PD-L1 IHC 22C3 pharmDx, PD-L1 IHC 28-8 pharmDx, and Ventana PD-L1 (SP263) assays is prohibitively low for both the 1% and the 50% tumor proportion score in non-small cell lung cancer and other tumor models.Several investigators have evaluated the so-called “interchangeability” of PD-L1 immunohistochemistry assays. The term “interchangeability” has also been used widely by the pharmacological industry to designate drugs that have demonstrated the following characteristics: same amount of the same active ingredients, comparable pharmacokinetics, same clinically significant formulation characteristics, and to be administered in the same way as the drug prescribed . BasicalAlthough they cannot be designated as “interchangeable”, the diagnostic accuracy of assays for a specific clinical purpose may be compared. In this manner, the comparison indirectly generates results that can be used to justify clinical usage of assays other than those included in the clinical trials. We employed ≥90% diagnostic sensitivity and ≥90% diagnostic specificity because these values are often used in other settings, including performance of immunohistochemistry assays –68. WhilThe strengths of this meta-analysis are the focus on diagnostic accuracy, fit-for-purpose approach, and the access to previously unpublished data from a large number of studies, which all resulted in pooled PD-L1 assay comparison in a way that has not been done before.The most significant limitation is that this is a meta-analysis of test comparisons where designated reference standards are other tests rather than clinical outcomes. However, to complete a meta-analysis with clinical outcomes may not be possible for many years, if ever. Other limitations of this meta-analysis are that only two cut-off points were assessed (1% and 50%), no assessment for readout that includes inflammatory cells was included, the impact of pathologists’ readout as potential source of variation between the studies was not assessed, and it is somewhat uncertain how the results apply to tumors other than non-small cell lung cancer due to the smaller number of such studies.The complexity of the PD-L1 immunohistochemistry testing cannot be safely simplified without consideration of the original test purpose. Determination of the diagnostic accuracy and indirect clinical validation of a candidate assay can be achieved by comparing the results of that assay to a previously designated reference standard assay, when direct access to clinical trial data or clinical outcomes is not possible.1) Well-designed, fit-for-purpose PD-L1 laboratory developed test candidate assays may achieve higher accuracy than PD-L1 Food and Drug Administration-approved kits that were designed and approved for a different purpose, when both are compared to an appropriate designated reference standard;2) More candidate assays achieved ≥ 90% sensitivity and specificity for 50% tumor proportion score cut-off than for 1% tumor proportion score cut-off;3) The overall diagnostic sensitivity and specificity analyses indicates that the relative analytical sensitivities of the Food and Drug Administration-approved kits for tumor cell scoring, most specifically in non-small cell lung cancer, are as follows: Ventana PD-L1 (SP142) << PD-L1 IHC 22C3 pharmDx < PD-L1 IHC 28-8 pharmDx < Ventana PD-L1 (SP263).Conflict of Interest (Appendix A)Meta-Analysis MethodologySupplementary Files Table 1Supplemetary Figures 2 - 13Funnel Plots


The final products as filler into carboxylated acrylonitrile‒butadiene rubber (XNBR) improve the dynamic mechanical properties. The improvement in the dynamic mechanical properties of the nanocomposite mainly depends on high interfacial interaction and graphene’s performance characteristics, as well as a good dispersion between functionalized graphene and the XNBR matrix.Graphene has been considered an ideal nanoscale reinforced phase for preparing high-performance composites, but the poor compatibility and weak interfacial interaction with the matrix have limited its application. Here a highly effective and environmentally friendly method for the functionalization of graphene is proposed through an interaction between as-exfoliated graphene and (3-aminopropyl) triethoxysilane (KH550), in which 1-butylsulfonate-3-methylimidazolium bisulfate (BSO Nowadays, damping materials have been widely applied in the mechanical vibration field. Among the numerous damping materials, polymer composites have been extensively researched for their excellent physical and chemical properties ,2. HowevIonic liquids (ILs) have attracted extensive attention due to their unique properties like low toxicity, nonflammability, and recyclability . Liu et 3HMIm)(HSO4) ILs-modified multilayers graphene (MLG) is an efficient platform for further grafting with KH550 to improve compatibility and interfacial interactions in the XNBR matrix. (3-aminopropyl) triethoxysilane (KH550) has been used to increase compatibility in polymer matrixes [3HMIm)(HSO4)/KH550-functionalized graphene was dispersed into a rubber matrix to estimate the dynamic mechanical properties.In this work, a new method for the preparation of functionalized multilayer graphene was proposed. (HSO4) ILs and high-purity graphite rods were obtained from Lanzhou Institute of Chemical Physics . Direct-current power supply was commercially obtained from HYELEC Co., Ltd. . XNBR (1027) with an acrylonitrile mass fraction of 27% was obtained from NANCAR Co., Ltd. . 1-butylsulfonate-3-methylimidazolium bisulfate (BSO3HMIm)(HSO4)/KH550-functionalized graphene was prepared according to four steps. First, two high-purity graphite rods were inserted as electrodes into the solution of water and 1-butylsulfonate-3-methylimidazolium bisulfate with a volume ratio of 10:1. Two high-purity graphite rods were placed parallel to each other and 2 cm apart in a 50-mL beaker. Direct-current power supply was explored to provide a static potential of 5 V. After 4 h, the electrolyte became black due to the exfoliation from the anode graphite surface. Secondly, the black solution with exfoliated graphene was centrifuged at a speed of 4000 rpm. The black precipitate was washed with absolute ethanol and deionized water several times. The as-obtained ILs-modified multilayer graphene was denoted as MLG1. Thirdly, KH550 was used as a grafting agent at 60 °C for 5 h and the product was isolated by centrifugation at 4000 rpm. Finally, the functionalized multilayer graphene was thoroughly washed with deionized water to remove KH550 monomers and dried in vacuum drying oven, and the as-prepared sample was denoted as MLG1-KH550. In order to estimate the reaction efficiency between (BSO3HMIm)(HSO4) and KH550, (BSO3HMIm)(HSO4) with different concentrations was added to regulate its density on MLG’s surface. MLG2-KH550 and MLG3-KH550 were obtained by the same process with mixtures of water and (BSO3HMIm)(HSO4) at 10:2 and 10:3, respectively.. X-ray photoelectron spectroscopy  was employed to explore the chemical state of typical elements on the functionalized multilayer graphene. The morphology of MLG-KH550 and XNBR/MLG-KH550 composites were determined by transmission electron microscopy . The degree of defects in carbon materials was evaluated by Raman microscopy (Horiba LabRam HR800). The thermogravimetric curves of samples were investigated with a Thermal Analyzer  at a heating rate of 20 °C/min in flowing N2. Dynamic thermomechanical analysis was explored by DMA . A twin-roller laboratory mill was employed to mix the rubber and filler. First, the MLG1-KH550 with additional content of 1.0 wt % was added to XNBR and they were mixed at room temperature until the filler had uniform distribution in the matrix. Secondly, the rubber mixture was placed in a metal mold for vulcanization. The vulcanization temperature was set at 145 °C for 10 min and the pressure dependence was under 10 MPa. Finally, the composites with different addition content  were prepared and to explore the performance characteristics. The composites with different content were denoted as XNBR/MLG1-KH550-0.2wt %, XNBR/MLG1-KH550-0.5wt %, XNBR/MLG1-KH550-1.0wt %, and XNBR/MLG1-KH550-2.0wt %, respectively.3H-functionalized ILs can react with amines to stabilize amino silane (KH550) in A possible reaction pathway has been proposed ,33. As s– anions were effectively intercalated into the interlayer spacing of graphite [3HMIm)+ cations in ILs play an important role in the formation of free radicals, like 1-octyl-3-methylimidazolium free radicals that can connect on graphene via physicochemical interaction [The preparation procedure of functionalized-MLG-enhanced XNBR nanocomposites is shown in graphite . (BSO3HMeraction  and coul3HMIm)(HSO4), the surface morphology of graphene can be definitively identified by transmission electron microscopy (TEM). 3HMIm)(HSO4) layer. The diffraction pattern of single-layer graphene shows a typical six-fold symmetry. Observing the TEM images of exfoliated functionalized graphene (as shown in 3HMIm)(HSO4) on the graphene surface (in 3HMIm)(HSO4), the amorphous structure are definitively identified and the families of spots disappear (HSO4) ILs, new absorption peaks at 1230 and 1566 are assigned to O–SO2–O stretch vibration and H–C=C–H scissoring vibration, respectively [−1 are assigned to imidazolium framework vibration, which belong to (BSO3HMIm)(HSO4) ILs. Compared with the spectra of MLG1 and MLG1-KH550, the peak at 2875 cm−1 is assigned to methylene stretch vibration [−1 due to Si–O–C and Si–O–Si bonds [−1 is assigned to the NH3+ stretching mode, indicating that ammonium groups can react with (BSO3HMIm)(HSO4) ILs [FTIR spectra were employed to explore the chemical structure of functionalized MLG. ectively ,35. The ibration ; the strSi bonds , suggestSO4) ILs .3HMIm)(HSO4) were successfully introduced on the interlayer/basal plane of multilayer graphene [3HMIm)(HSO4) and KH550. XPS was used to further analyze the chemical state of typical elements for functionalized graphene. The full-range XPS spectrum of original graphite displays only C 1s peak in graphene ,38,39. C3HMIm)(HSO4), indicating that the KH550 layer is partly covered on the graphene surface. From MLG1-KH550 to MLG3-KH550, the N/O ratio increases from 0.267 to 0.373, close to the theoretical value of 0.33 for KH550. Meanwhile, the Si/C ratio increases from 0.065 to 0.106, close to the theoretical value of 0.111 for KH550. Therefore, it can be assumed that a high concentration of ILs can effectively increase the thickness of the KH550 grafted layer.The elemental ratio of MLG1-KH550, MLG2-KH550, and MLG3-KH550 was explored by XPS spectra (as shown in 2 hybridized carbon [−1 further verifies that (BSO3HMIm)(HSO4) ILs are successful adhered to graphene via physicochemical interaction. Moreover, the 2D band can be used to confirm the layers of graphene, which is sensitive to graphene thickness due to the dispersion of π electrons [Raman spectroscopy plays an important role in the characterization of the features of spd carbon . Raman sd carbon . In Figulectrons . As the lectrons ,43. In Flectrons . It is wlectrons . 3HMIm)(HSO4) are shown in 3HMIm)(HSO4), while pure (BSO3HMIm)(HSO4) shows a serious decomposition (weight loss of approximately 98 wt %), suggesting that the higher the density of (BSO3HMIm)(HSO4) there is on graphene, the more weight loss occurs. After grafting with KH550, the weight loss of MLG1-KH550 is 19 wt %, indicating higher thermal stability of MLG1-KH550 under the same calcination procedure.Thermogravimetric (TG) curves of graphite, MLG1, MLG1-KH550, and (BSO3HMIm)(HSO4) ILs and further grafting with KH550, which could offer excellent dispersion into the XNBR matrix and could enhance the interface interaction between graphene and the XNBR matrix, thereby improving the performance characteristics for practical applications.The combination of the above experimental results confirms that graphene has been functionalized by (BSOg). From g). As shown in DMA is a useful tool to evaluate the interfacial interaction of polymer composites . Thus weg of XNBR/MLG1 and XNBR/MLG1-KH550 are obviously shifted to a lower temperature from g of XNBR/MLG2-KH550 and XNBR/MLG3-KH550 are obviously higher than that of pure XNBR. In previous works [From us works ,48, simiThe dispersity of the functionalized graphene sheets in the XNBR matrix was characterized by TEM images. The darker lines are viewed as a layer structure of functionalized graphene sheets, while the gray regions are identified as the XNBR matrix. As shown in 3HMIm)(HSO4) via an electrochemical exfoliation of the graphite procedure and can be further grafted with KH550 via (BSO3HMIm)(HSO4) ILs as a high-performance platform for interactions. The efficient modification of graphene improves its compatibility in the XNBR matrix. The functionalized multilayer graphene greatly improves the dynamic mechanical properties of XNBR, which we can attribute to the enhancement in the interaction between functionalized multilayer graphene and the XNBR matrix.To allow for practical application of high-performance rubbers, a high-efficiency and environmentally friendly method has been proposed to prepare functionalized multilayer graphene as a filler. This work demonstrates that multilayer graphene can be modified by  Functionalized graphene prepared by electrochemical exfoliation of graphite in an ionic liquids solution and further grafting of KH550 is an environmentally friendly and high-efficiency method for the improvement and development of graphene-like materials;(b) KH550-grafted graphene with a higher density can effectively enhance the dynamic mechanical properties, mainly due to the beneficial interactions of functionalized graphene and the XNBR matrix;(c) KH550-grafted graphene as a nanoscale reinforcement can overcome stacking problems and enhance the dynamic mechanical properties. So, it is of great significance for preparing a high-performance nanocomposite for real-world applications.


Little is known regarding the psychometric properties of computerized long-menu formats in comparison to classic formats. We compared single-best-answer (Type A) and long-menu formats using identical question stems during the computer-based, summative, intermediate clinical-clerkship exams for nine disciplines.In this randomised sequential trial, we assigned the examinees for every summative exam to either the Type A or long-menu format . The primary outcome was the power of discrimination. The study was carried out at the Faculty of Medicine, University of Geneva, Switzerland, and included all the students enrolled for the exams that were part of the study. Examinees were surveyed about the long-menu format at the end of the trial.p = 0.7948) after 22 exams including 88 experimental items. The long-menu format had a similar discriminatory power but was more difficult than the Type A format . Over half of the options (54.4%) chosen by the examinees in long-menu formats were not proposed as distractors in the Type A formats. Most examinees agreed that their reasoning strategy was different.The trial was stopped for futility (In a non-selected population of examinees taking summative exams, long-menu questions have the same discriminatory power as classic Type A questions, but they are slightly more difficult. They are perceived to be closer to real practice, which could have a positive educational impact. We would recommend their use in the final years of the curriculum, within realistic key-feature problems, to assess clinical reasoning and patient management skills. Computer-based assessment facilitates prompt and timely feedback to both students and teachers , it is aPsychometric properties do not differ significantly between the two formats , and morIn addition to the long-menu format, computer-based exams allow the use of other formats, such as key-feature problems where sequential questions need to be answered in a specific order. The sequence mimics real patient management more closely, moving from history-taking to diagnosis and then to treatment. These series of questions can be viewed as simulations  falling p = .005) and more discriminating . However, the retrospective observational design was a limitation to this study: since different questions were used in different formats, the contents and underlying learning objectives were likely to have had an impact on both difficulty and discrimination. Furthermore, all the items were related to a single medical speciality.In a retrospective study assessing the psychometric performance of 553 items used in 13 computer-based paediatrics exams , we founTo the best of our knowledge, little is known about the specific psychometric added value of a long-menu format, compared to a single best answer format, when a teacher chooses how the examinee should answer an exam question: The present study’s main objectives were to compare the level of difficulty and power of discrimination of long-menu questions against single-best-answer multiple choice questions in real-life conditions, i.e. summative undergraduate examinations, by applying both formats simultaneously to the same question stem. The experimental items would thus only differ in the answering modalities: long-menu questions on one hand, and single best answer (best choice), typically from a list of five alternatives , on the other.This prospective study was carried out at the Faculty of Medicine of the University of Geneva, Switzerland. Apart from the selection exam at the beginning of the curriculum, all the written exams for the medical students have been computer-based since 2011, first using desktop computers and then tablets. We included all the written exams taken during the first and second clinical years of the curriculum . In each exam, we identified four Type A questions which could be modified and transformed into long-menu questions (or long-menu questions that could be transformed into Type A questions for the paediatrics exams). For each exam, all enrolled examinees were eligible and randomly divided into two approximately equal sized groups with two different versions of the exam. One group had two questions in the Type A format and the other two questions in the long-menu format, and vice versa for the second group. The question stems for the four questions were kept rigorously identical. The other questions were identical in both versions of the exam, and all questions were included in the computation of the exam score.The administrative staff randomly assigned (computerized procedure) an examination room seat number to every enrolled examinees. Independently and in parallel, the IT staff randomly uploaded  one of the two versions of the exam to every desktop or tablet in the examination room.We used the Item Management System provided by UCAN  combined with CAMPUS (Desktop-based exams) or tEXAM (Tablet-based written exams), which are provided by the same consortium.The primary outcome was the question’s power of discrimination, expressed by the point biserial correlation: this evaluates the item’s ability to differentiate among students on the basis of how well they perform during the exam. It can be viewed as an estimator of the degree to which a single item measures the same underlying construct as all the other items in the exam.The secondary outcome was the difficulty of the question, defined as the average capacity of students to find the correct answer, i.e. for a single best answer the relative frequency of choice of the unique correct answer. By extension, it was for the long-menu format the relative frequency of choice of the correct answer in the long-menu list .For each long-menu question, we determined which distractors the examinees chose. In order to evaluate their perception of the long-menu format, all the examinees received a four-item, online, self-administered questionnaire at the end of the study. The items, measured on a four-point Likert scale  were: “The long-menu format is more difficult than the other formats”; “My reasoning is different whenever I have to answer a long-menu format”; “The situation I am put in when I have to answer a long-menu format is closer to real life than when I have to answer a classic MCQ item”; and “It is more difficult to answer a long-menu question than an open free-text item. We added the latter item although it is not directly related to the main objective of the study because the long-menu format is sometimes criticized as it does not allow, unlike the open-free text, to validate any chain of characters entered through the keyboard or keypad.”Study design followed a sequential procedure . Indeed,Repeated sequential paired Student’s t-test were performed using for every item the difference between the estimates  computed from the two versions of the exam Fig. . With a Unless specified, data were summarised as mean ± standard deviation (S.D.). The difficulty and the power of discrimination were provided either by the item management system, or by some in-house validated programs. No manual data handling or computation was required. Student’s t-tests were used to compare two groups of continuous variables. All analyses were run on TIBCO Spotfire S +® 8.1 for Windows , with the additional S + SeqTrial module.n = 305; 60% of women; mean age ± S.D.: 24 ± 2.4). The mean total number of items per exam was 58 ± 26, with a mean of 107 ± 42 candidates taking an exam. Most Type A questions  included five response options, 14 questions included six options and three included four options. The likelihood estimates had already hit the defined stopping boundaries by the second interim analysis, but the Rao–Blackwell adjusted estimate had not, so it was decided to continue the experiment. The study was stopped after the third interim analysis (88 items from 22 different exams), and we concluded that there was no significant difference regarding the discriminatory powers of the type A and long-menu formats , the long-menu versions were more difficult: − 6.34% .The powers of discrimination and difficulties of both formats are shown in Fig. p-value Student’s t-test = 0.1688 for the comparison with the long-menu format; = 0.3648 with the Type A format). The average percentage of correct answers was 78.1% ± 20.3%, similar to the Type A experimental items , but higher than the long-menu experimental items (p = 0.010).The mean discriminatory power of all the other exam items, i.e. all the items in every exam apart from the four experimental items included either as Type A or long-menu questions, was 0.182 ± 0.202 and showed no evidence of difference with the experimental items  were distractors listed in the Type A questions, and 736 (54.4%) were not proposed as distractors in the Type A questions.The results of the survey conducted at the end of the study  can be found in Table To the best of our knowledge, this study is the first randomised controlled trial in real-life conditions of summative assessment to compare long-menu and Type A formats using the same question items. The long-menu format used to record the examinees’ answers had an equivalent discriminatory power to the classic Type A format and it was more difficult. This higher level of difficulty could be explained, in part, by the straightforward mathematical construction of the long-menu format, which reduces the probability of choosing the right answer at random. This was also observed by Schuwirth  et al. iOur findings did not support the conclusions of our previous retrospective study that had shown a higher discriminatory power for long-menu questions than for other classic formats . The context of our retrospective study was clearly different: psychometric properties were computed from different groups of examinees, and more importantly, the question stems were different, meaning that not only the answer formats were compared but also the topics themselves and the underlying learning objectives. These are likely to be more important elements than the answer format itself , both inIt is interesting to note that more than three quarters of the responding examinees thought that the long-menu format was closer to real life. This assertion is supported by the physicians who often face a long list of alternative diagnoses in their daily practice, but it is also asserted by our results: more than half of the incorrect options chosen by students who had the long-menu format were not among the distractors listed in the type A format. This corroborates the finding of Huwendiek et al.  that thiA vast majority of responding students acknowledged that they had used a different reasoning approach when trying to answer long-menu questions. We may hypothesise that the long-menu format encourages retrieval practice. Therefore, the cognitive effort required by the long-menu format may support mid- and long-term learning and retention of knowledge. However, future research would be needed to address this issue. The students’ perceptions and their observed increased difficulty with long-menu questions also might bring some evidence of a positive educational effect .Almost two thirds of the responding examinees did not find the format more difficult than the free-text open question format, which not only requires a long process of manual marking but is also more liable to a subjective evaluation, and it has never been clearly proven superior to classic multiple choice  or long-Our study has limitations. It was carried out in a single faculty and the results might not be generalizable to other faculties. However, students’ results at the Swiss Federal Licensing Exam have been similar across all faculties in the past few years, showing that there were no significant differences in teaching at the various faculties of medicine and in the students’ performances. Moreover, we included all the students, hence avoiding the selection bias of studies conducted on volunteer students. The two formats were compared in many different medical specialities, decreasing the likelihood that the results were influenced by topics and content. Another limitation was the choice of exam items for our study. The items were not designed especially for the study: in each exam, with the exception of paediatrics, we identified Type A questions which could be modified into long-menu questions. This could have led to a selection bias based, for example, on our knowledge of existing long-menu lists that could be easily re-used and adapted, or on the fact that we could not make any significant changes to the existing question items. Finally, the short survey was conducted to measure the examinees’ general perception regarding some item formats: this does not constitute evidence about differences in psychometric properties or reasoning processes between the item formats, but may represent interesting topics for future studies.They is no evidence that the long-menu questions and Type A question differ regarding the discriminatory power, but the long-menu questions they are slightly more difficult. Long-menu questions are perceived to be closer to real practice, which could have a positive educational impact. Taking into account the increased time required to develop long-menu items when compared to Type A items, we would recommend their use in the final years of the curriculum, within more realistic key-feature problems, which focus on assessing clinical reasoning and patient management skills.


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.