The competence of ChatGPT (Chat Generative Pre-Trained Transformer) in non-English languages is not well studied.This study compared the performances of GPT-3.5 (Generative Pre-trained Transformer) and GPT-4 on the Japanese Medical Licensing Examination (JMLE) to evaluate the reliability of these models for clinical reasoning and medical knowledge in non-English languages.This study used the default mode of ChatGPT, which is based on GPT-3.5; the GPT-4 model of ChatGPT Plus; and the 117th JMLE in 2023. A total of 254 questions were included in the final analysis, which were categorized into 3 types, namely general, clinical, and clinical sentence questions.The results indicated that GPT-4 outperformed GPT-3.5 in terms of accuracy, particularly for general, clinical, and clinical sentence questions. GPT-4 also performed better on difficult questions and specific disease questions. Furthermore, GPT-4 achieved the passing criteria for the JMLE, indicating its reliability for clinical reasoning and medical knowledge in non-English languages.GPT-4 could become a valuable tool for medical education and clinical support in non–English-speaking regions, such as Japan. ChatGPT  is a state-of-the-art large language model (LLM) that can simulate human-like conversations based on user input . As a coHowever, in a previous study, ChatGPT, based on GPT-3.5 (Generative Pre-trained Transformer), performed poorly for 77 out of 79 medical students on a South Korean parasitology examination, which resulted in questions about its ability to provide medically accurate responses in non-English languages . On MarcTherefore, this study compared the accuracy of GPT-3.5 and GPT-4 on the Japanese Medical Licensing Examination (JMLE) . FurtherWe used the default mode of ChatGPT, which is based on GPT-3.5, and the GPT-4 model of ChatGPT Plus. The latest JMLE, number 117, conducted on February 4 and 5, 2023, was also used for this study. The JMLE comprises 400 questions, which were classified into 3 categories: essential knowledge questions, which test the knowledge and ethics required of a doctor; general clinical questions, which cover numerous diseases; and specific disease questions, which test the knowledge of each disease . FurtherQuestions and their multiple-choice answers from the JMLE were used in their original Japanese form, as was the official national examination rubric. Instructions for using ChatGPT were also provided in Japanese. A typical rubric is as follows:We will present questions for the Japanese National Medical Examination. There will be five options from a to e, and you must choose the appropriate option for the question. If there is no specific limit on the number of options to choose, please select one option only.15The definition of “correct” answers to the questions asked to GPT-3.5 and GPT-4 was based on the answers to the JMLE, which were published on the website of the MHLW . Only thWe evaluated the difficulty level of each question and categorized them as hard (n=82), normal (n=112), and easy (n=60) based on the correct response rate published by medu4, a preparatory school for the JMLE ,17. QuesFinally, we simultaneously collected responses from both GPT-3.5 and GPT-4 between March 16 and 18, 2023, and scored them using the definition of correct answers. P<.05.Standard descriptive statistics were used to calculate the numbers, proportions, and means for each data set. The McNemar test was used to compare correct response rates. All analyses were performed using the Stata statistical software (StataCorp LLC) . All tesThis study only used information that was already published on the internet and did not involve human subjects; rather, an analysis of the JMLE was performed. Therefore, approval by the Institutional Review Board of Shimane University was not required.P<.001). In terms of the correct response rate for individual questions, the examinees’ rate for essential knowledge questions was 89.2% compared to 87.2% for GPT-4. Notably, this represents a considerable 32.1% improvement over GPT-3.5, which had a 55.1% correct response rate. Similarly, a 29.5% increase was observed for general clinical questions, and a 25.4% increase was observed for specific disease questions. In all cases, GPT-4 achieved the passing rates for the JMLE. However, none of these rates exceeded the total percentage of correct answers by examinees.A total of 254 questions from the 117th JMLE were used in the experiment. P<.001) and a 29.6% increase for clinical questions (P<.001) compared to GPT-3.5. Notably, a 36.3% increase was observed in the number of correct responses to clinical sentence questions, with a significant improvement in all question types .P<.001), a 31.5% increase for normal-level questions (P<.001), and an 18.3% increase for easy-level questions (P<.001).P<.05). In particular, for hard-level questions, the correct response rate of GPT-4 was 17% higher than the examinees’ average correct response rate.Finally, GPT-4 significantly outperformed GPT-3.5 in all formats in terms of correct response rates (all We compared the correct response rates of GPT-3.5 and GPT-4 on the 2023 JMLE. GPT-3.5 did not satisfy the passing criteria, whereas GPT-4 achieved the required scores. Furthermore, GPT-4 demonstrated a significantly improved correct response rates compared with GPT-3.5 across various question types and difficulty levels. The correct response rate of GPT-4 was particularly enhanced for the challenging hard-level questions and surpassed the average correct response rate of actual examinees. Based on these results, we discuss 2 factors that explain the significant improvement in the correct response rates of GPT-4 on the JMLE.First, we ascribe this enhancement to the augmented NLP capabilities in non-English languages. A performance disparity between English and other languages in LLMs is ubiquitous in NLP . AdditioSecond, since improving the information processing capabilities in professional and academic domains is imperative, OpenAI’s development of GPT-4 aimed to handle more intricate and nuanced tasks beyond those encountered in many real-world situations . The JMLAs the results of this study and several previous studies indicate, LLMs such as ChatGPT have made remarkable progress ,7,13. HoThis study had several limitations. First, the results reflect the capabilities of ChatGPT as of March 17 and 18, 2023, and different results could be obtained even if the same methods were used. The knowledge and interpretation capabilities of ChatGPT will rapidly improve in the future because of user feedback and deep learning. Second, although GPT-4 is a multimodal artificial intelligence that is inherently capable of inputting images and tables, among other things, this study excluded them for an accurate comparison with GPT-3.5, and only text questions were used. Third, the JMLE has a supplementary assessment that states that if an absolute contraindication answer is selected 2 or more times, the applicant will fail the examination, even if they have achieved the passing scores . BecauseGPT-4 passed the 117th JMLE, whereas GPT-3.5 failed the examination. This phenomenon revealed GPT-4’s rapid evolution in Japanese language processing. Investigations are necessary to evaluate its safety, efficiency, and cost-effectiveness for potential application as an LLM artificial intelligence tool for medical practice support, learning in clinical settings, and medical education.


I-V measurements. An increased electrical conductance is observed for the bulk-NCG-based FET, despite its higher defect density, the channel displaying a transconductance of up to ≊The progress of advanced materials has invoked great interest in promising novel biosensing applications. Field-effect transistors (FETs) are excellent options for biosensing devices due to the variability of the utilized materials and the self-amplifying role of electrical signals. The focus on nanoelectronics and high-performance biosensors has also generated an increasing demand for easy fabrication methods, as well as for economical and revolutionary materials. One of the innovative materials used in biosensing applications is graphene, on account of its remarkable properties, such as high thermal and electrical conductivity, potent mechanical properties, and high surface area to immobilize the receptors in biosensors. Besides graphene, other competing graphene-derived materials (GDMs) have emerged in this field, with comparable properties and improved cost-efficiency and ease of fabrication. In this paper, a comparative experimental study is presented for the first time, for FETs having a channel fabricated from three different graphenic materials: single-layer graphene (SLG), graphene/graphite nanowalls (GNW), and bulk nanocrystalline graphite (bulk-NCG). The devices are investigated by scanning electron microscopy (SEM), Raman spectroscopy, and  Field-effect transistors (FETs) have attracted extensive attention in biosensing applications. Unlike traditional sensing methods, FET-based biosensors do not involve extensive photodetectors or fluorescent labeling parameters. A biosensor has two key components: a bioreceptor and a transducer. The bioreceptor is a biomolecule that generates a physical or chemical reaction upon interaction with a target analyte in a biological media. The transducer primarily converts the identification process into a measurable signal, which can be either electrical, electrochemical, optical, or mechanical. One electric-type biosensor that has gained a significant amount of traction over the last few years is the FET. The FET biosensor is composed of a substrate with source, drain, and gate electrodes, as well as a conductive channel that is functionalized with a bioreceptor  and acts as the transducer. The bioreceptor–analyte reactions will cause a change in the electrical conductivity of the transducer, and the FET’s biosensitivity occurs when the respective change in conductivity is large enough to be measured ,2,3.As the size and performance of silicon transistors have approached their physical limits, it is necessary to look for alternative materials to support emerging technologies. One of these materials is graphene, which is an attractive option for the source-drain channel of field effect transistors due to its excellent electrical, optical, mechanical, and thermal properties . In receAs with traditional silicon-based FETs, the GFET’s gate modulates the flow of electrons or holes in the channel. Due to the nanoscale thickness of the GFET’s channel, the current flows mainly on its surface; therefore, the GFETs possess a high sensitivity. The higher electrical and thermal conductivity of graphene results in reduced losses and enhanced heat dissipation compared to silicon. As a result, graphene-based transistors have the potential to deliver enhanced performance and efficiency ,8,9.graphene is used as a general definition for graphene materials, such as graphene (single or multilayer), graphene oxide, or reduced graphene oxide [In the literature, the term ne oxide ,11. Howene oxide . Single-ne oxide  and was ne oxide . Alternane oxide . Their inditions ,16. Overnditions .I-V curve with respect to the gate voltage [One of the first devices to employ graphene-derived materials for biosensing applications was reported by Mohanty and Berry in 2008. They produced cost-efficient chemically modified graphene-based transistor-like devices for the purpose of bacterial and DNA detection with enhanced sensitivity tunability . Exosome voltage .Gold’s standard potential of 1.52 V makes Au nanoparticles (AuNPs) more stable from an electrical standpoint compared to other noble metal nanoparticles such as AgNPs  . Their hThe feasibility of terahertz operation was also determined in 2012 by Mehr et al., with a transistor based on vertically orientated graphene sheets . Later, The GFETs reported in this paper are four-terminal devices , similarThe greater part of their most noteworthy features resides in their electrical properties. Generally, graphene has a very low resistivity at room temperature, as well as high intrinsic mobility, over 100 times that of Si. Theoretically, graphene can transfer electric current with 100% efficiency, and even the earliest manufactured graphene layers, although prepared using relatively modest methods in limited controlled conditions, presented remarkable qualities with reasonably high quasiparticle mobility for FET-like devices (up to 5000 cmV−1 s−1) . Over thV−1 s−1) , and thu2, h-BN)  and over samples ,28. For  samples . Their dubstrate  and up tnded SLG . As it igraphene . Despitef layers ,32. Grapf layers ,33.The SLG thin films were grown on Cu substrates by Chemical Vapor Deposition (CVD), while the GNW and bulk-NCG films were grown directly on SiOThe SLG thin films are grown via thermal CVD on a catalyst substrate, which consists of 35 rocesses ,35,36.The GNW and bulk-NCG films used for the GFETs’ fabrication are grown via PECVD in a capacitively coupled RF reactor from CH process ,38,39, isorption . The PEC4) ratio . The GNW4) ratio ,16, and 4) ratio ,35.The GFETs’ fabrication process differs from the silicon-based FETs’ process as it entails a more complex series of photolithographic steps revolving around both wet and dry etching. The microfabrication of the graphenic-based FETs involves the deposition of the carbonic materials on a thermally oxidized Si wafer, either directly  or by transfer , followed by the metallic contacts’ deposition  and functionalization of the source-drain channel.All presented GFETs were manufactured on In preparation for the back gate deposition, the back-side SiOone bath . The wafIn the case of the SLG-based GFETs, an additional step is required to properly identify the areas onto which the SLG thin films are to be transferred. This is accomplished by wet chemical etching, using a specifically designed mask a within Next, the source and drain electrodes are deposited through e-beam evaporation and lift-off techniques similar to the back gate deposition, except this time a 365 nm thick Au layer and a 30 nm thick Cr adhesion layer are used. Following the subsequent cleaning in acetone and isopropyl alcohol, the graphenic source-drain channels on all wafers are masked with an AZ 4562 photoresist and the rest of the graphenic material is dry etched in an oxygen plasma. The channel patterning is carried out in a capacitively-coupled RF reactor with the Plasma Etcher—Etchlab 200  equipment, at 250 W RF discharge power, a pressure of 20 Pa, and an OThe functionalization of the graphenic drain-source channel is accomplished by AuNPs decoration. The AuNPs’ purpose is to enhance the electron transfer rate and to provide a larger contact surface, thusly improving the sensor’s response.The AuNPs were chemically synthesized by Au (III) to Au (0) reduction: 10 mL of chlorauric acid  investigations confirm the binding of  channel .The graphenic-based channel is deposited on a dielectric layer  at an acceleration voltage of 15 kV. Top-view SEM micrographs showcasing the graphenic source-drain channel and its respective electrodes, along with the top gate electrode, are presented in Additionally, Raman spectroscopy was used to evaluate the source-drain channel and analyze the structure of the different graphenic materials. The spectra  were acqX, respectively, could be explained by defects induced during the transfer process. The appearance of transfer-induced defects is also corroborated by the presence of a small intensity D peak, which can be observed at ≊1340 cmAll Raman spectra are initially vector normalized, and a Savitzky–Golay filter is applied over an 11-point window with a third-order polynomial to improve the signal-to-noise ratio. The spectra are then background corrected by linear subtraction and deconvoluted into individual Lorentzian peaks, with the exception of the G peak in the case of the GNW and bulk-NCG films, which is described by a Breit–Wigner–Fano shape . The coe phonons . Typical phonons ,42. The an bands ,42,43. MI-V readout. The output characteristics of the GFETs at different gate voltages, presented in The electrical performance of the GFETs was investigated at room temperature using the Keithley 4200-SCS Semiconductor Characterization System  coupled with the Manual Wafer Probing Station EP6 . Initially, several attempts were made with varying back gate voltages but, as depicted in I-V transfer curves can be observed in the case of all three materials (The GFETs are unique in that they can act as both n- or p-type channel transistors, depending on the top gate applied voltage. The critical transition between the two regions of the GFETs’ aterials d–f. At tAnother important distinction between the three graphenic channels that can be ascertained from nfluence , as mentThe measured transconductance values of the GFETs and the charge carrier mobility, calculated using Equation , are shoI-V transfer characteristics of the source-drain channel before and after AuNPs decoration, we notice an improved sensitivity. The Dirac point of the bulk-NCG FET showed a As the bulk-NCG channels showed the best electrical characteristics, further investigations were employed in an attempt to improve their sensitivity by AuNP decoration. When comparing the  ≊746.43 .Graphenic materials have proven to be valuable assets in the fabrication of FETs for sensing applications. In addition to their close relation to atomically thin graphene, in terms of their composition and molecular structure, which relate to similar physico-chemical properties, they provide a more feasible fabrication process for the respective GFETs. The PECVD synthesis of the graphenic materials is accomplished directly on an insulating layer, therefore eliminating the requirement of a transfer step from the technological workflow, a step that is still necessary for SLG. Their shared sp


We find that the magnitude of the Seebeck coefficientis up to nine times larger than that of Au–C60–Ptmolecular junctions. Moreover, the sign of the thermopower can beeither positive or negative depending on the details of the bindinggeometry and on the local value of the Fermi energy. Our results demonstratethe potential of using graphene electrodes for controlling and enhancingthe thermoelectric properties of molecular junctions and confirm theoutstanding performance of [60]fullerene derivatives.The thermoelectric properties of molecular junctionsconsistingof a metal Pt electrode contacting [60]fullerene derivatives covalentlybound to a graphene electrode have been studied by using a conducting-probeatomic force microscope (c-AFM). The [60]fullerene derivatives arecovalently linked to the graphene via two  However, functionalization of only one side has also been accomplishedby using a solid substrate where the graphene layer is deposited.13 In this sense, in most of the practical applications of graphene,it is actually supported on a solid substrate which could be easilyincorporated into an electronic device. Therefore, the chemical functionalizationof graphene on a substrate with [60]fullerene may drastically expandthe material capabilities by fine-tuning its chemical and physicalproperties.Graphene and [60]fullerene aretwo Nobel laureated disruptive materials representing new allotropesof carbon whose properties could be fine-tuned upon chemical covalent14 They also open the possibility of designingnew thermoelectric devices with enhanced thermoelectric propertiesthat could find applications in energy management, on-chip cooling,temperature sensing, or energy harvesting.20 All-carbon hybrid devices show great promise in their ability toexploit desirable thermoelectric properties.25 One such property is the delicate control of thesign and magnitude of the Seebeck coefficient. This control has beendemonstrated, for example, by applying tip pressure to modulate thecoupling of the molecule to the electrode26 and by tuning intermolecular interactions between C60 molecules.18Studies of the thermoelectric properties of molecularjunctionsconsisting of organic molecules connected to electrodes provide insightinto the charge transport mechanisms at the molecular level.27 They found that for C60, PCBM, and C70, the Seebeck coefficient was negative,being largest in magnitude for C70 and for the Ag tip (−33μV/K). These are among the highest measured values for molecularjunctions.14 Evangeli et al.18 found that the thermopower of C60 dimers between gold electrodes reaches values of −33 μV/K,almost double that of C60 (−18 μV/K). Thefact that the thermopower is negative for all these fullerene molecularjunctions shows that electron transport takes place through theirLUMOs . In contrast, it hasbeen shown that, in molecular junctions of endohedral fullerenes,the Seebeck coefficient can be either positive or negative dependingon the orientation of the encapsulated moiety and also in responseto pressure; this behavior was explained by the presence of transmissionresonances close to the Fermi level.17The thermopower ofvarious single fullerene molecules between agold substrate and a metal tip of Au, Ag, and Pt was studied by Yeeet al.1–3) bound covalently to grapheneon SiO2/Si  as shown in 60–Ptjunctions. We use density functional theory (DFT) calculations toobtain a detailed understanding of the origin of these values andthe molecular configurations responsible for these amazing behaviors.Here, we investigate the thermoelectric properties of single-moleculejunctions formed by contacting diphenylmethanofullerene molecules led to derivatives GOS-1, GOS-2, and GOS-3 , affording the final amino or diamino-containing diphenylmethanofullerenes 1–3.28 Thecovalent functionalization of GOS was conducted via aryl diazoniumchemistry in the presence of isoamyl nitrite, following a slightlymodified protocol of that previously reported by Tour and co-workers.29 Thus, the corresponding aryl diazonium saltsare generated in advance by adding isoamyl nitrite to a solution of 1, 2, or 3 and subsequently dropcasted on the GOS. As proof of concept and to validate the covalentfunctionalization over the graphene grafted on top of SiO2/Si, we have also synthesized the analogous all-carbon hybrid materialsover few layers graphene (FLG) to achieve a complete characterizationof the final derivatives. Pristine FLG was obtained through graphiteexfoliation following Colleman’s method with a high degreeof purity.30 A suspension of FLG in o-DCB was immediately reacted with [60]fullerene derivatives 1 and 2 in the presence of isoamyl nitrite providing FLG-1 and FLG-2, respectively  oncopper foils, cut into smaller pieces and transferred onto SiOnd GOS-3 1. The syectively 1.2 carbon atoms to sp3 as a result of the covalent attachment of 1, 2, or 3, respectively. Therefore, the ratio withthe G band ID/IG is a way to quantify the covalent functionalization degree of thosematerials. According to this criterion, in our measurement we found ID/IG ratios of 0.26for GOS-1, 0.54 for GOS-2, and 0.23 for GOS-3 . Moreover,a new peak around 1457 cm–1 is clearly observedfor all fullerene aggregates which can be assigned to the pentagonalpinch mode [Ag(2)] of C60.31 These results indicate a higher functionalization in the case of GOS-2, which is also confirmed by the observed ID/IG ratios for FLG-1 and FLG-2 . Thus,this evidence confirms the covalent attachment of 1, 2, or 3 on the FLG or GOS. A characterizationof FLG-1 and FLG-2 employing ATG, FTIR,XPS, and TEM has been carried out and is available in the SI.Raman spectroscopy under 532 nm laser wavelengthsgives insightsof the surface functionalization of FLG and GOS. All aggregates presentan increase in the D-band with respect to the G-band, which is generallyassociated with a higher defect degree on the nanomaterials, alsoattributed to functionalization. This increase is related with a substantialrehybridization from spGOS-1, GOS-2, or GOS-3 that partially cover the graphene sheet with the substrate and held stationarythrough a feedback loop controller. This allows us to measure current–voltage(I–V) characteristics in which the thermoelectricvoltage shows as a voltage-offset ΔV, fromwhich one can obtain the thermopower or Seebeck coefficient S = −ΔV/ΔT. A zero-calibration of the voltage reading is performed in shorttime intervals by disconnecting the c-AFM from the V source and connecting a resistor R instead, facilitatinga precise voltage reading. Then we can determine S and the conductance G (from the slope of the I–V curves) for a number of givenpoints on the substrate.To characterize the thermoelectricresponse of the [60]fullerenederivatives on graphene, we used a conductive atomic force microscope(c-AFM) with Pt-coated tips (Multi75-G from BudgetSensors). S measured with the tip in contactwith the graphene. We obtain a very small average value of 2 μV/K,corresponding to the thermopower of the Pt-tip/graphene/SiO2 junction. This measurement configuration is totally different tothat of previously reported experiments that determine the in-planethermopower of graphene, which is found to be between −50 and+50 μV/K, depending on doping.32GOS-1 yield twodistinct sets of values centered on S = −56μV/K and S = 74 μV/K , as described in the SI.To further understand this behavior,we used density functionaltheory (DFT) combined with quantum transport theory to compute theelectrical conductance SI, the calculatedSeebeck coefficients were found to be sensitive to the location ofthe metallic tip on the surface of the C60 and to the valueof the Fermi energy, which can vary across the film due to the presenceof adsorbates, such as water. Therefore, values of S for a range of tip-C60 contact locations and a rangeof Fermi energies were calculated. These were then used to createthe histograms of Seebeck coefficients shown in GOS-3 values of S being positive,whereas GOS-1 and GOS-2 histograms possesspeaks at both positive and negative values. This suggests that theexistence of two peaks in the histograms cannot be considered as anindication of the presence of molecules bound by one or two legs andthat the precise shape of Seebeck histograms depends on the rangeof Fermi energies and tip-C60 configurations sampled inthe experiments. The sign of the Seebeck coefficient depends on theenergetic location of the frontier orbitals relative to the Fermienergy. If the LUMO is closer to the Fermi energy, then the Seebeckcoefficient is negative, whereas if the HOMO is closer, the Seebeckcoefficient is positive. Bonding of the tip to the unattached phenylring was not considered, since the unattached ring points toward thesubstrate, making it unavailable to the tip.As discussed in the 60 has been considered in a previous work.33 This study showed that intermolecular interactions causesmall changes in the Seebeck coefficient, but these are negligiblecompared to the sample-to-sample fluctuations shown in In the above simulations, each junction containeda single molecule,whereas in the experiments, many molecules are present. The effectof intermolecular interactions in junctions with more than one C1–3) have been covalently linkedto 2Dgraphene either through two meta-connected phenylrings, two para-connected phenyl rings, or just asingle phenyl ring, resulting in the new systems GOS1–3. All the [60]fullerene/graphene hybrids havebeen characterized by spectroscopic and microscopic techniques, revealingthe efficient covalent linkage between the two carbon allotropes.In summary, suitably functionalized [60]fullerene derivatives(GOS-1, GOS-2, and GOS-3 generateexceptionally high quantum thermopowers when covalently anchored tographene, thus suggesting great promise for molecular/graphene compositematerials in thermoelectric applications. The possibility to changethe sign of the most probable Seebeck coefficient when utilizing twoanchoring groups implies potential for tailoring thermoelectric performancein more complex device architectures in which positive and negativecontributions may be combined to build thermoelectric modules.Our work demonstrates that the [60]fullerene derivatives GOS-1 and GOS-2 is very sensitive tolocal variations of the Fermi level, leading to both positive andnegative values of the Seebeck coefficient. In contrast, the calculationspredict that, for the same local variations in the Fermi energy, thethermopower of GOS-3 is more robust when anchored tographene, since its transmission function exhibits a consistentlynegative slope around the energy range of interest. Fluctuations inenergies of the frontier orbitals relative to the Fermi energy aremodeled by sampling a range of Fermi energies. The precise range sampledexperimentally is unknown, because small changes in the bonding configurationand defects in the graphene will change the Fermi energy relativeto frontier orbital energies. Therefore, we sampled a range, whichwas consistent with experiment. In this sense, although the theoryis not predictive, we are able to show that the experiment can beconsistent with theory. In conclusion, the geometries and Fermi energiesvary so much that a broad distribution of Seebeck coefficients areobserved. The sign of the Seebeck coefficient depends on the energeticlocation of the frontier orbitals relative to the Fermi energy. Theseare shifted relative to their gas-phase values by the real part ofthe self-energy, due to contact with the substrate and tip. This shiftdepends on the strength of the contact, which in turn depends on theorientation of the molecule and details of the linker. Random energylevel shifts also occur due to imperfections in the graphene substrate.DFT calculations show that the sign of the Seebeck coefficientof


In the present study, we generalize our recently proposed nomenclature scheme for porous graphene structures to include graphene flakes and (periodic) edges, i.e., nanographenes and graphene nanoribbons. The proposed nomenclature scheme is a complete scheme that similarly treats all these structures. Beyond this generalization, we study the geometric features of graphene flakes and edges based on ideas from the graph theory, as well as the pore–flake duality. Based on this study, we propose an algorithm for the systematic generation, identification, and numbering of graphene pores, flakes, and edges. The algorithm and the nomenclature scheme can also be used for flakes and edges of similar honeycomb systems. Although graphene is a semi-metal, graphene flakes  and graphene nanoribbons, which are the zero- and one-dimensional counterparts of graphene, are semiconductors, due to quantum confinement. This makes them particularly useful for optoelectronic applications  and explDepending on their sizes, shapes, and edge structures, these structures exhibit a variety of different electronic, optical, and chemical properties ,10,22. FThe infinite number of nanographenes and graphene nanoribbons, due to their different edge structures, and the increasing interest in them, give rise to the necessity for a nomenclature scheme, which would have the ability to accurately distinguish them from each other and provide a particular and accurate name for each such different structure.Carbon, regarding the nomenclature of graphene-based two-dimensional materials [So far, attempts for naming nanographenes are restricted to (a) the smaller members of polycrystalline aromatic hydrocarbons and (b) high symmetry flakes. For the former, empirical names have been adopted, for instance, coronene, ovalene, pentacene, pyrene, anthracene, etc. . For theaterials , mainly aterials  suggesteRecently, we proposed a nomenclature scheme for porous graphene structures based on the arrangement of two-fold coordinated atoms (2-FCAs) in the pore boundary . In the It is worth noting that the proposed nomenclature scheme and the generation and numbering algorithm apply to graphene flakes and edges, which are composed of hexagonal rings, including either three-fold coordinated atoms (3-FCAs) or two-fold coordinated atoms (2-FCAs) only. Flakes or edges, including (i) atoms bonded with only one atom, (ii) parts of the flake (or the edge) bonded with the rest of the flake (or the edge) system with only one bond, (iii) helical-like flakes , or (iv)edges (bonds) and vertices (atoms), we showed that each such closed path  can be uniquely defined by the sequence of path lengths In the current section, we show the connection between flakes and edges with pores, which has been studied previously . Considee 2-FCAs .non crossed” means that if we move along that path, each vertex and edge is visited only once. The term “closed path” or “circuit” means that the path begins and ends at the same vertex. In terms of graph theory, these properties describe both an Eulerian and Hamiltonian circuit. The term “path length” is also used from the point of view of graph theory, i.e., it is the number of edges along the path. For convenience, we use the term “2-2 path” for the path along the pore boundary connecting two adjacent 2-FCAs, and the term “2-2 hexagon” for the hexagon of the vacuum space associated with the 2-2 path, which contains the 2-2 path, i.e., the edges of the 2-2 path are edges of the associated 2-2 hexagon. As we showed in our recent work [i is an integer number between 1 and 4  of the pore boundary. The direction of i denotes the 2-2 path and j the edge vector belonging to the i 2-2 path, and considering that I is the In Ref. , for porIn Ref. , we showAs we have shown in Ref. , ei,j ca in Ref. . Therefo5)ϕi,j=k−i+1π/3,a5)ϕi,j=k−i+1π/3,aIn conclusion, cases for The case for i, as shown in i and j are the same as those used in the notation of the edge vectors k is given in Equation 13) are ti and j indices of For Equation  yields Lture see h. In conAs in pores and flakes, a boundary path representing an edge should not be a crossed path. The conditions for a non-crossed path in the edges are the same as those in pores and flakes.It is worth noting that in the case of a path Obviously, a circuit representing a pore boundary can be either considered the boundary of a pore, or the boundary of a flake, depending on the side where the vacancies are. A flake boundary can be considered  the dual of a pore boundary, and vice versa, although, as we have already mentioned in the introduction and will explain below, not all pores have a flake as a dual, and not all flakes have a pore as a dual.The dual of the dual of a pore (flake) is the pore (flake) itself. As a consequence, the dual is unique, i.e., there is a one-to-one correspondence between a pore and its dual flake and between a flake and its dual pore. If this is not true, we consider that the dual of a pore or flake does not exist.The pore (or flake) boundary is the same as that of its dual. The difference is that the edge vectors of the pore (flake) boundary have opposite directions compared to those of its dual flake (pore). This convention for the direction of edge vectors is used so that we have the same determination of the vacuum area and graphene bulk area, regarding their relative position, with respect to the direction of the edge vectors (see above).Excluding the atoms of the boundary, all other atoms are transformed into vacancies and vacancies are transformed into atoms.If the dual exists, then for the transformation of a pore to its dual flake, and the transformation of a flake to its dual pore, we consider the following:L. Moreover, 2-FCAs of a pore (flake) are transformed into 3-FCAs in its dual flake (pore) and vice versa. Thus, The above can be seen in Considering that Equation , for k0=In Ref. , we showsee Ref. ). MoreovLet us now examine cases of flakes that do not have a pore as a dual and pores that do not have a flake as a dual. Pores or flakes that include three adjacent 2-2 paths in their boundaries with length The above cases are not the only cases for which a pore or a flake does not have a dual. In fact, those cases can be considered as special cases of a more general one, where two 3-FCAs of the pore (or flake) boundary, which are not adjacent along the pore (or flake) path, are bonded with each other. The reason is that the transformation from the pore to the flake (or from the flake to the pore) is expected to be accompanied by the conversion of 3-FCAs to 2-FCAs. However, two such 3-FCAs of the pore or flake boundary will remain 3-FCAs in the transformation from the pore to the flake or from the flake to the pore since their bond will not be lost in the transformation. Thus, those atoms will not be converted to 2-FCAs, and the converted structure will have a different boundary compared to the initial one, not allowing the transformed one to be considered as the dual of the initial one. This can be clearly seen in the examples shown schematically in (i)i with The central edge vector (ii)i with The central edge vectors (iii)i and The (iv)i and Two adjacent 2-2 paths (v)i with length A 2-2 path If the dual of a pore (or a flake) exists, the conversion of the pore (or the flake) to its dual implies the replacement of 2-FCA vertices and 2-2 paths of its boundary to other 2-2 paths, according to the following scheme:For example, the dual of the 444 pore is the 112112112 flake. The transformations are shown schematically in L, there are L of the circuit is Considering that in a flake circuit with a total length N of atoms constituting a flake. Using the same strategy as the one used to find the number of vacancies in a pore (see Ref. [An interesting question that needs to be answered has to do with the number see Ref. ), we can in Ref. . This meh of the above notation is used to distinguish the coordinates expressed in the base of N.Let us assume that the Equation  and FiguEquation , the hexEquation , we can A graphene edge can be considered a non-crossed, non-closed, and theoretically infinite path, dividing the honeycomb lattice into two discrete areas. One of them only has vacancies and the other one is the remaining graphene structure. Practically, however, a graphene edge is part of the boundary of a typically large graphene flake and not the boundary of a semi-infinite plane. Therefore, in terms of graph theory, a graphene edge is both an Eulerian and Hamiltonian path, which, contrary to the boundaries of flakes and pores, is a path and not a circuit. Such a path should not form a spiral because a spiral might correspond to a non-crossed and non-closed infinite path, but it does not divide the honeycomb lattice plane into the two discrete areas described above.L is the period of edge lengths at the boundary. This means that after the sequence From the point of view of an infinite path, the graphene edge can be considered periodic. Even if it is not periodic, it can be considered periodic with infinite periodicity. Let us assume that the smallest possible period of a specific edge is represented by the sequence Equation  not to bEquation . As alre(i)(ii)(iii)This means that any combinations of the sets (i) A = {2}, (ii) B = {1, 3} and/or (iii) C = {1, 1, 4} of 2-2 path lengths provide an infinitely periodic path, unless that path crosses itself, and any infinitely periodic path is composed of the combinations of the above sets of 2-2 paths. This does not mean that those sets of 2-2 paths should necessarily follow one another in the periodic A question that rises is whether or not there are specific combinations of path lengths for which It is worth noting that the Equation  . Let us…, Recalling that for flakes Equation  for edgeEquation , one canThe second step of the algorithm checks whether or not the sequence In the third step, the algorithm checks whether or not In the fourth step, the algorithm calculates the Equation , and in Equation , i.e., sEquation , the um If any of the above cases is true, the 2-2 hexagons determined by In the sixth step, which applies to flakes, but not to edges, the algorithm checks if Equation  is satisrelation (19)0≤moThe above procedure is repeated recursively for all possible If our aim is to find and number the N of atoms in a flake, we find the number N. Thus, the eighth step of the algorithm, which will follow each successful i, i,--------The details of these results can be found in Ref. [To find the number  in Ref. . The valConsidering that the hexagonal graphene structure can be constructed by vectors https://github.com/fthenak/Graphene-Pores-Flakes-Edges (accessed on 3 July 2023). The first one with the name “pore_flake_edge_generation.f90” takes as input (i) the system kind  and (ii) the number L of the pore or flake circuit, or periodic edge path. Moreover, for flakes, it provides (iv) the number of hexagons of the flake and (v) the number of atoms constituting the flake, and for pores, (iv) the number of hexagons in the vacuum area and (v) the number of vacancies. The second code with the name “pore_flake_edge_structure.f90” takes as input (i) the system kind , (ii) the number d of atoms around the pore is also an input, considering that pores are periodically arranged in the infinite graphene sheet. If that sequence corresponds to the selected system , it provides as output (i) the structure in the structure.xyz”, (ii) the corresponding minimum image of the sequence, (iii) and the length L of the circuit or path. Moreover, (a) for a flake, it provides (iv) the number of hexagons and (v) the number of atoms in the flake, (b) for a pore, (iv) the number of hexagons in the vacuum area and (v) the number of vacancies, and (c) for an edge, (iv) the number of ribbon atoms and the vector period Using the algorithm described above, we developed two Fortran codes, which can be found at  of Ref. .Using the first code, we find the number of graphene flakes , which appears in some lines of In pore_flake_edge_generation.f90 code, for the generation and numbering of the pore, flake, and edge For completeness, we present in Generalizing our nomenclature scheme for graphene pores, we propose a general, unique, systematic, accurate, and simple nomenclature scheme for naming graphene flakes and edges, which is based on the arrangement of 2-fold coordinated atoms in their boundaries. In the proposed nomenclature scheme, flakes and edges are named after the minimum images of sequences The proposed nomenclature scheme can be easily expanded to name pores, flakes, and edges of other two-dimensional systems , as well as functionalized pores, flakes, and edges, according to the scheme described in our previous work, thus opening up a new window in the nomenclature of such systems.


Due to low vaccination uptake and measles outbreaks across Europe, public health authorities have paid increasing attention to anthroposophic communities. Public media outlets have further described these communities as vaccine refusers or “anti-vaxxers”. The aim of this review was to understand the scope of the problem and explore assumptions about vaccination beliefs in anthroposophic communities. For the purpose of this review, we define anthroposophic communities as people following some/certain views more or less loosely connected to the philosophies of anthroposophy. The systematic review addresses three research questions and (1) collates evidence documenting outbreaks linked to anthroposophic communities, (2) literature on vaccination coverage in anthroposophic communities, and (3) lastly describes literature that summarizes theories and factors influencing vaccine decision-making in anthroposophic communities.This is a systematic review using the following databases: Medline, Web of Science, Psycinfo, and CINAHL. Double-blinded article screening was conducted by two researchers. Data was summarized to address the research questions. For the qualitative research question the data was analysed using thematic analysis with the assistance of Nvivo12.0.There were 12 articles documenting 18 measles outbreaks linked to anthroposophic communities between the years 2000 and 2012. Seven articles describe lower vaccination uptake in anthroposophic communities than in other communities, although one article describes that vaccination coverage in low-income communities with a migrant background was lower than in the anthroposophic community they studied. We found eight articles examining factors and theories influencing vaccine decision making in anthroposophic communities. The qualitative analysis revealed four common themes. Firstly, there was a very broad spectrum of vaccine beliefs among the anthroposophic communities. Secondly, there was a consistent narrative about problems or concerns with vaccines, including toxicity and lack of trust in the system. Thirdly, there was a strong notion of the importance of making individual and well-informed choices as opposed to simply following the masses. Lastly, making vaccine choices different from public health guidelines was highly stigmatized by those outside of the anthroposophic community but also those within the community.Continuing to further knowledge of vaccine beliefs in anthroposophic communities is particularly important in view of increasing measles rates and potential sudden reliance on vaccines for emerging diseases. However, popular assumptions about vaccine beliefs in anthroposophic communities are challenged by the data presented in this systematic review.The online version contains supplementary material available at 10.1186/s12889-023-17081-w. Vaccines save lives . VaccineAnthroposophy is a spiritualist movement that was established by scientist and philosopher Rudolf Steiner born in 1861 in Austria . SteinerAnthroposophic medicine was founded in the early 1920s by Rudolf Steiner and Ita Wegman . DrawingAnthroposophic medicine is practiced in 78 countries worldwide, predominantly in Central Europe. There are circa 24 anthroposophic medical institutions – these include hospitals, departments in hospitals, rehabilitation centres, and other inpatient healthcare centres in six countries  [The 2019 official statement of the international centre of anthroposophic medicine, the Medical Section of the Goetheanum, and the International Federation of Anthroposophic Medical Associations (IVAA) clearly states that they do not support the anti-vaccine movement. Rudolf Steiner did not oppose vaccines, however, vaccination and anthroposophic medicine constitutes a somewhat contentious point . This isTherefore, this review aims to understand the scope of the problem and explore popular assumptions about vaccine beliefs in anthroposophic communities. To achieve this, this systematic review summarizes the existing literature that investigates the relationship between anthroposophy and vaccination beliefs.This is a systematic review, including both quantitative and qualitative studies. The review is based on current best practices utilising the Joanna Briggs Institute systematic review framework , 19.What are the documented outbreaks associated with low vaccination coverage in anthroposophic communities?What is the evidence for vaccination rates in anthroposophic communities?What is the evidence that describes factors and theories for low vaccination uptake in anthroposophic communities?We used the population/concept/context (PCC) framework to guide the development of our research questions . The popNo review protocol exists, and the systematic review has not been registered.A systematic literature search was performed by two researchers in the following databases: Medline, Web of Science, Psycinfo, and CINAHL. The last search was conducted 2022–09-05. The search strategy was developed in Medline (Ovid) in collaboration with librarians at the Karolinska Institutet University Library. For each search concept Medical Subject Headings (MeSH-terms) and free text terms were identified (see appendix). No language restriction was applied. Databases were searched from inception. The strategies were peer reviewed by another librarian prior to execution. De-duplication was done using the method described by Bramer et al. [Independent study selection was completed by two reviewers (SHvW and KA). Inclusion criteria for the first round of screening (title and abstract) were all articles that discussed anthroposophy and vaccination (this was conducted by KA and SHvW). Articles in Swedish and German were only reviewed by SHvW due to language restrictions. Inclusion criteria for the second and more in-depth round of screening – conducted double-blinded by KA and SHvW were all papers relevant to the three research questions, including both quantitative and qualitative studies. Exclusion criteria were, not peer-reviewed papers, opinion pieces, systematic reviews, nor papers that were not relevant to the research question .A quality assessment of the selected papers was conducted double-blinded by two researchers (KA and SHvW). For the qualitative studies, we used the JBI Critical Appraisal Tool for Qualitative Research . This isTo address research questions one and two, the data was summarized in Tables The search revealed 27 papers see Fig. . Twelve Table Table Table The systematic search revealed eight articles examining factors and theories influencing vaccine decision-making in anthroposophic communities  vaccines and meningitis vaccines. Recent studies have highlighted the important effect the HPV vaccine on the reduction of cervical cancer – therefore understanding the views of parents from the anthroposophic community on the HPV vaccine would have important public health implications.In terms of the qualitative findings, the review revealed a broad range of vaccination beliefs and highlighted the importance of individual choice in the vaccine decision process. Although parents consider themselves well-informed, it is unclear from the studies where they obtain their information, although some refer to the use of scientific information. Understanding knowledge and information sources in greater detail would be helpful to understand how certain rumours are maintained. The challenge of reliance on poor information sources to make a vaccine decision was particularly noted during the COVID-19 pandemic and arguably hindered COVID-19 vaccine uptake . Some in“It is not a matter of being in opposition to the school of medicine that is working with the accepted scientific methods of the present time. We fully acknowledge its principles. …we therefore feel compelled to work for an extension of clinical medicine, based on these wider insights into the nature of the world and the human being” [.Although there are currently 27 articles that have investigated the relationship between vaccines and anthroposophy, it remains somewhat nebulous why anthroposophy as a religion or belief system is often considered as an anti-vax movement by popular media. Anthroposophical medicine does not reject vaccines, nor does it reject modern medicine. This was clearly stated at the inception of anthroposophical medicine and it has been a clear statement by Gotheanum. In 1925, Dr Rudolf Steiner and Dr Ita Wegman clearly stated n being” .Sobo’s article described in this review was the only paper that engaged with the anthroposophic movement, particularly the Waldorf school context that arguably cultivated vaccine hesitancy . The notThe anthroposophic community prides itself on being different, communal, and supportive as opposed to following principles of consumerism and individualism. However, none of the studies, except briefly by Sobo, mentioned vaccines as a means for social action and to protect the vulnerable . DistrusLastly, the stigma this group experiences highlights a problem that requires careful attention. This could also be an important finding for other so-called vaccine hesitant groups . One couThere are some limitations to this systematic review. The review only includes peer-reviewed articles; this means that there have probably been other disease outbreaks linked to anthroposophic communities, which were only described in the grey literature. Moreover, some of the studies purely described the outbreaks rather than conducted an analysis; therefore, it is difficult to analyse in depth what actually happened. Regarding the thematic analysis of the qualitative studies, there are limits to conducting such an analysis of results of existing studies, since we could not base our analysis on the full data set.This systematic review showed that there have been several measles outbreaks linked to anthroposophic communities in Europe. Although studies on vaccination coverage in anthroposophic communities are limited, it appears that coverage is lower than in the general population. Monitoring outbreak numbers and vaccination coverage could be important. Popular beliefs about the anthroposophic communities’ vaccination beliefs are challenged in this review. As the evidence shows the communities are not categorically against vaccines. Moreover, there are a myriad of factors that influence vaccine decision-making of parents belonging to an anthroposophic community. The importance of experiencing childhood illnesses and concerns over long-term side effects were mentioned. Moreover, parents want to be able to individually select vaccines for their children. They consider themselves actively engaged in vaccine decision-making and well-informed. Stigma regarding vaccine choices was mentioned repeatedly mostly by people outside of the anthroposophic community but also by people within the community. This review calls for a better understanding of vaccine choices and beliefs for vaccines beyond MMR, in particular HPV vaccines. The review also highlights a potentially important research gap, which constitutes understanding not only a belief system but the role that stigma may play in making decisions about vaccines.Additional file 1.


OpenAI, CA, US), Google Bard , and Bing Chat  [Recent years have witnessed an increasing interest in the application of Artificial Intelligence (AI) and deep learning in Ophthalmology . Large l WA, US) –5. LLMs  WA, US) , 6, 7.We evaluated the performance of LLM-driven AI chatbots on the Fellowship of Royal College of Ophthalmologists (FRCOphth) exams required for autonomous Ophthalmology practice in the UK. We focused on testing the capability of these models in the Part 1 and Part 2 FRCOphth Written exams. These advanced postgraduate exams consist of multiple-choice questions and cover the learning outcomes of the Ophthalmology Specialty Training curriculum in the first two years of training and towards the end of training, respectively.We obtained sample multiple-choice questions from the Royal College of Ophthalmologists website, covering both the Part 1 and Part 2 examinations , 9. We eWe tested each LLM-chatbot three times on the sample questions at different timepoints. Additionally, for Part 2 questions, we evaluated ChatGPT-4.0 using various prompting strategies, such as asking the chatbot to answer the question from the perspective of a pharmacist or statistician. When the LLM-chatbot could not answer the question, it was recorded as incorrect. We did not provide additional instruction or training data.We analysed the association between accuracy and LLM-chatbot using Chi-squared testing and multilevel (mixed effect) logistic regressions. Difficulty and topic were included as fixed effects, and question ID as a random effect. We selected the models with the lowest Akaike information criterion. Part 1 and Part 2 data were analysed separately. All statistical analyses were conducted in R.P < 0.001). Despite a 4% mean difference in accuracy with each iteration, no statistically significant differences in performance were observed for any individual LLM-chatbot.The LLM-chatbots achieved overall accuracies of 65.5% and 67.6% for Part 1 and Part 2 questions, respectively  and Google Bard  in Part 1 questions. No significant associations were found between accuracy and question difficulty or topic. In Part 2 questions, ChatGPT-3.5’s performance was surpassed by both ChatGPT-4.0 and Bing Chat, regardless of whether prompting was used or not  are being developed to aid clinical decision-making through free-text explanations, spoken recommendations, or image annotations.Previous studies have shown that LLMs can achieve accuracies of up to 67.6% in generalist medical examinations using different training data and instruction prompt tuning.This study is the first to demonstrate that LLMs can consistently provide accurate responses to postgraduate Ophthalmology specialty examinations, achieving an impressive accuracy rate of up to 82.9% without prompting or instruction tuning.LLMs outperformed the standards of these specialist examinations, indicating that traditional assessments may not adequately measure clinical competence.Issues related to validation, computational expenses, data procurement, and accessibility must be addressed to ensure the safe and effective integration of AI systems into online learning and clinical practice.Supplementary Materials


We would like to thank the reader for their interest in our paper and for their thoughts on endothelial cell (EC) proliferation and endothelial-to-mesenchymal transition (EndMT) in the Sugen5416/hypoxia (SuHx) model of pulmonary arterial hypertension (PAH).1 which the reader disagreed with. They reasoned this was because the differentially expressed genes between control and PAH were associated with the gene ontology (GO) terms regulation of angiogenesis (GO:0045765), regulation of vasculature development (GO:1901342), and EC proliferation (GO:0001935). We believe that GO term analysis alone is insufficient for making conclusions about cell proliferation. The GO terms highlighted by the reader also contain genes that regulate those processes in either a positive or negative direction, or both. Importantly, angiogenesis involves an intricate coordination and balance of EC proliferation, sprouting, and migration. It is overly simplistic to assume that because angiogenesis is induced, ECs must therefore be proliferating. Various studies have also provided evidence showing that opposing EC proliferative and migratory responses could present together in different pathologies and mechanistic contexts.2–4 The reader also suggested upregulation of Bmpr2, caveolin (Cav) 1/2, and secreted protein acidic and cysteine rich (Sparc) in PAH as markers of EC proliferation. This not only conflicts with our observation of Bmpr2 downregulation in PAH,1 but also with the expression plot in their letter and findings in the wider PAH field of reduced BMPR2 signalling,5 EC hyperproliferation in Cav1 and Cav2 knockout mice,6 and unclear contributions of SPARC in proliferation and angiogenesis.7 Furthermore, we do not believe they are as direct or established a marker of proliferation as cell cycle–related genes . We are thus confident in our finding, which is based on robustly assessing established proliferation/cell cycle markers and the discrete cell cycle phases of each EC.Our study reported no increase in proliferating ECs in SuHx PAH mice,1 though it is unclear from their plot label if it is 24% or 0.24%. Their analysis also showed control and intermittent hypoxia having similar proportions of Acta2+ ECs. This conflicts with their claim and matches our finding, which they had disputed. Although the reader provided immunofluorescent staining of PECAM1 and ACTA2 in lung cryosections from mice in normoxia or SuHx, there was lack of quantification of double-positive cells and statistical evaluation of the difference between groups. Furthermore, they have only evaluated one EndMT marker, ACTA2, where we know that a suite of markers should be used.8In their letter, the reader raised questions about our observation of limited EndMT in control and SuHx-induced PAH, reporting very high levels of Acta2 + control ECs in their analysis of murine whole lungs. We do not expect ECs to express any mesenchymal markers in control. As such, the 24% of Acta2 + control ECs they reported seem unexpectedly high, unlike the 1% Acta2 + control ECs we observed,et al.8 have extensively reviewed the challenges posed by a lack of standard molecular definition. We used a panel of genes that could mark the following EndMT characteristics: reduced endothelial phenotype, increased mesenchymal phenotype, and increased expression of EndMT mediators.8 Our comprehensive assessment did not reveal any EC populations distinctly expressing these markers, suggesting EndMT was not present in our data. We also have no reason to believe that the tamoxifen induction efficiency and the fluorescence-activated cell sorting strategy we used are limitations in this context. EndMT has previously been studied in tamoxifen-induced condition.9 We found 95.5% of highTdTomato cells in our Cdh5-CreERT2-TdTomato mouse line, which were also highCdh5 and highPecam1 and were independently annotated as ECs by SingleR.1 Considering how EndMT could present as a transient phenomenon9 and how scRNA-seq only reveals a static snapshot of the cell state, we acknowledge that it is possible for EndMT to have occurred in earlier stages of our model, but this was not captured in our study. Importantly, we would like to emphasize that EndMT in PAH, and the wider cardiovascular disease field, has thus far been largely observational (i.e. being associated with a disease) and not causal.8EndMT is in itself a highly complex phenomenon and field of study, and Kovacic 10 However, the exact contribution of CD74 to the non-immune components of EC dysfunction in PAH remains unclear. We thus chose to extend our investigation of CD74 beyond inflammation, through targeted assays of EC proliferation and barrier function, with HUVECs as an initial in vitro model system, before extending these findings further into other EC types and ultimately into in vivo models.The final point raised by the reader was that our functional validation of CD74 should have focused on its immune function, rather than its impact on EC proliferation and barrier integrity. They also proposed using pulmonary artery ECs, instead of human umbilical vein ECs (HUVECs). PAH is a complex disease that involves not only an immune component, and the immune contribution of CD74 in ECs has been previously studied.


Graphene has been under intense scientific interest because of its remarkable optical, mechanical and electronic properties. Its honeycomb structure makes it an archetypical two-dimensional material exhibiting a photonic and phononic band gap with topologically protected states. Here, we assemble colloidal graphene, the analogue of atomic graphene using pseudo-trivalent patchy particles, allowing particle-scale insight into crystal growth and defect dynamics. We directly observe the formation and healing of common defects, like grain boundaries and vacancies using confocal microscopy. We identify a pentagonal defect motif that is kinetically favoured in the early stages of growth, and acts as seed for more extended defects in the later stages. We determine the conformational energy of the crystal from the bond saturation and bond angle distortions, and follow its evolution through the energy landscape upon defect rearrangement and healing. These direct observations reveal that the origins of the most common defects lie in the early stages of graphene assembly, where pentagons are kinetically favoured over the equilibrium hexagons of the honeycomb lattice, subsequently stabilized during further growth. Our results open the door to the assembly of complex 2D colloidal materials and investigation of their dynamical, mechanical and optical properties. Colloidal particles bonding via attractive patches mimic the bonding of atoms in atomic compounds and materials. By assembling patchy particles into the graphene lattice, the authors obtain insight into lattice defects in this important 2D material. The most prominent representative, graphene, consists of a monolayer of carbon atoms bonded in a honeycomb lattice. The strong covalent bonds within the honeycomb lattice make the material particularly strong and light, while the honeycomb structure gives rise to a photonic and phononic band gap5. On a larger length scale, micrometre-size particles assembled into colloidal graphene, the analogue of atomic graphene assembled from colloidal particles, would open the door to two-dimensional multifunctional materials with photonic and phononic band gap for applications as 2D photonic and phononic crystals. However, producing large defect-free single-crystalline graphene of both atoms and colloids remains a great challenge, crucially limiting its applications.Two-dimensional materials have attracted intense scientific interest, both from an application and a fundamental point of view, offering applications from light-weight materials to optoelectronic devices. These materials combine extraordinary mechanical, optical and electronic properties compared to bulk materials6. However, while defects are introduced unavoidably during growth or added on purpose to tune mechanical and electronic properties, a comprehensive understanding of their formation is missing: because the trivalent carbon atoms or particles can arrange into a variety of polygons and structures, a coherent lattice exists even with defects, and rearrangements can take many paths. Despite advances in direct visualization of graphene defects using electron microscopy8, defect kinetics and healing remain poorly understood, and defect-free graphene challenging to produce.Structural defects are known to be central to all of graphene’s properties, enabling among others band-gap tuning in graphene-based electronic devices11. One advantage of colloidal systems is that defect formation14 and dynamics16 can be conveniently studied directly in real time with single particle resolution using optical microscopy18, which remains challenging in atomic systems, especially under the harsh high-temperature atomic deposition used for graphene growth19. The recently-gained ability to synthesize anisotropic particles23, in particular colloidal particles with attractive patches providing specific valency and bond angles, has opened a design space for assembling more complex structures such as molecule analogues26 and covalently bonded crystals28.Although colloidal particles are several orders of magnitude larger than atoms, their phase behaviour and dynamics are governed by the same thermodynamics principles. Phase behaviour of both atoms and colloids is largely governed by thermal forces, which means we can use colloidal aggregation and crystallization as a simple model for atomic crystallization29. Experimentally realizing these structures remains challenging, as they require fine control over specifically coordinated interactions, or purposeful geometric design to block kinetically favoured nonequilibrium routes, as recently shown for the realization of colloidal diamond31. In contrast to tetrahedrally coordinated diamond, graphene relies on the trivalent coordination of particles. Patchy particles with patches at 120∘ angles can mimic these covalent bonds; yet, achieving such valency and controlling these directed bonds on the scale of kBT, the thermal energy, remains challenging, but would open up the assembly of structurally complex 2D materials, and investigation of their structural and mechanical properties.Simulations and experiment have shown that these colloidal molecules can grow into larger assemblies, yielding rich structures ranging from the kagome lattice to buckyball-like clustersHere, we assemble colloidal graphene and elucidate the kinetic pathways of crystallization and defect formation of this 2D material. We form the graphene lattice using pseudo-trivalent patchy particles adsorbed at a substrate, and directly follow the crystallization, defect formation and healing with great temporal and spatial resolution using confocal microscopy. Fine control of the patch-patch bond strength allows observation of near-equilibrium assembly in analogy to high-temperature deposition of atomic graphene. From the number of saturated bonds and the bond strain, we determine the configurational energy of the lattice and follow its evolution during lattice rearrangement and healing. Our results reveal that the most prominent defect motif of colloidal and atomic graphene, a pentagon, is kinetically favoured in the early stages of graphene growth, and acts as seed for extended defects during subsequent growth. These results hint at the importance of the early stages of assembly in generating defect-free graphene.23. The synthesis yields tetrahedrally coordinated particles with a PS bulk and fluorescently labelled TPM patches  and 3-(trimethoxysilyl)propyl methacrylate (TPM) spheres by colloidal fusionhes Fig. . The parcL = 0.25 close to the critical volume fraction cL,c = 0.2736, and solvent demixing temperature Tcx = 33.95 °C, and add 1 millimolar of MgSO4 to screen the particles’ electrostatic repulsion and enhance the lutidine adsorption of the hydrophobic patches25. The suspension is injected into a glass capillary with hydrophobically treated walls  in Fig. 43. However, CVD-grown atomic graphene does not normally show vacancy defects, even though experiments show that vacancies are generated during the early stages of the CVD process44. Unlike the substrate-adsorbed colloidal particles, carbon atoms can approach vacancies from outside the plane during CVD growth, filling the vacancies with feedstock carbon44. Hence, unlike vacancies in colloidal graphene, vacancies in atomic graphene anneal in the CVD process, and irradiation or chemical treatment is used to induce them. These generated vacancies typically reconfigure to a (slightly) lower-energy structure that contains fewer dangling bonds. For instance, a divacancy can reconfigure into two 5-membered and one 8-membered ring45. In contrast, the divacancy in Fig. 46, making reconfigurations in colloidal graphene unlikely due to high energy barrier  51, or aligning graphene islands before merging57. While crystallization and defect dynamics in atomic graphene are ultimately governed by quantum mechanics, we expect that in the high-temperature limit studied here, where the quantum-mechanical states become quasi-continuous, our colloidal system provides a good model. Yet, differences arise from the different form of the potential and the different nature of the in-plane  versus interlayer bonding . Despite these differences, the energy scales are similar: at typical high-temperature deposition (~800 K) the covalent bond energy of graphene on a metal substrate is ~1 eV58, corresponding to 14.5kBT - similar to our critical Casimir bond strength of 12–15kBT. During higher temperature annealing procedures of graphene (~2000 K) the corresponding energy (6kBT) is lower, allowing together with the higher atomic attempt frequency for faster dynamics, with rearrangements occurring on the order of seconds59 rather than hours as in our colloidal case. The assembly of colloidal graphene demonstrates the increasing control over bottom-up assembly of complex materials. The honeycomb lattice is of specific interest because it is the simplest metamaterial exhibiting a photonic and phononic bandgap61, and topologically protected states. While achieving the structural complexity of macroscopic mechanical metamaterials remains still a challenge, our results demonstrate that the necessary structural motifs can be assembled using patchy particles, opening the door to microscale mechanical metamaterials4.Trivalent colloidal particles adsorbed at a substrate form the colloidal analogue of graphene, allowing direct observation of its crystallization and defect dynamics. The fine interaction control opens near-equilibrium crystallization and annealing pathways. We find that colloidal graphene defects originate in the early stages of crystallization from pentagonal motifs that are kinetically favoured over the equilibrium hexagonal motifs, further stabilized by adjacent heptagonal motifs, together forming stable grain boundaries. These results are reminiscent of high-resolution electron microscopy observations of atomic graphene, which however are limited to fully grown graphene, and cannot access the initial stages of crystallization. Grain boundaries and extended polyvacancies reconfigure towards lower-energy states by an interplay of dangling-bond saturation and lattice distortions, ultimately increasing the number of hexagons. Strategies for synthesizing large atomic graphene crystals tend to circumvent this issue by either generating as few graphene seeds as possible4 . The particles are washed several times in the water-lutidine mixture. The resulting particle dispersion is injected into a silanized hydrophobic glass capillary  and sealed with teflon grease (Krytox GPL-205). The full silanization procedure is given in Supplementary Method Tetrapatch particles  are dispersed in a binary solvent of 25% 2,6-lutidine  and 75% milliQ water with 1 millimolar MgSOT ≊ 0.6 °C), which causes one of the particle patches to attach to the sample wall. For heating, we use a well-controlled temperature stage in combination with an objective heating element to obtain a relative temperature accuracy of about 0.01 °C with minimal temperature gradients.Particles are left to sediment to the bottom of the sample at room temperature before measurements. We tilt the sample slightly during sedimentation, so a small density gradient is present in the sample. We then heat the sample to approximately 33.35 °C  to determine the centre of each patchy particle. Because one of the patches is attached to the glass, each particle centre is approximately given by this lower patch. The three remaining patches are suitable for bonding, and form a single blob with another bonded patch if bonded. This tells us if two nearby particles are actually bonded or merely randomly meeting. For more tracking details, see Supplementary Note We image the assembled structures using confocal microscope image stacks, sometimes alternating with bright field images. The images are processed using particle tracking software , and the composition of the binary solvent, as detailed in ref. 46. By formulating the critical Casimir potential model presented in ref. 46 for patchy particles and benchmarking it onto our patchy particles as shown in ref. 63, we determine the attractive minimum of the pair potential to be 12–15kBT in the studied temperature range of ΔT = 0.25–0.1K. The full potential is given in Supplementary Note The critical Casimir potential is short ranged, its magnitude and range are set by the temperature offset ΔThe bond-bending potential is harder to estimate theoretically, but can be determined from experimental measurements by following clusters of three particles, and tracking the fluctuations of the internal angle of these particles. The resulting angle probability distribution can then be converted to a potential, as shown in Fig. Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2