High-quality graphene-based van der Waals superlattices are crucial for investigating physical properties and developing functional devices. However, achieving homogeneous wafer-scale graphene-based superlattices with controlled twist angles is challenging. Here, we present a flat-to-flat transfer method for fabricating wafer-scale graphene and graphene-based superlattices. The aqueous solution between graphene and substrate is removed by a two-step spinning-assisted dehydration procedure with the optimal wetting angle. Proton-assisted treatment is further used to clean graphene surfaces and interfaces, which also decouples graphene and neutralizes the doping levels. Twist angles between different layers are accurately controlled by adjusting the macroscopic stacking angle through their wafer flats. Transferred films exhibit minimal defects, homogeneous morphology, and uniform electrical properties over wafer scale. Even at room temperature, robust quantum Hall effects are observed in graphene films with centimetre-scale linewidth. Our stacking transfer method can facilitate the fabrication of graphene-based van der Waals superlattices and accelerate functional device applications. The large-scale fabrication of twisted van der Waals heterostructures remains challenging due to the formation of defects and contaminations during the transfer process. Here, the authors report a transfer method to fabricate graphene-based van der Waals superlattices at the wafer scale, showing controllable twist angles and robust quantum Hall effect. Based on the vdWS assembled from 2D conductors, semiconductors, ferromagnets and superconductors, a series of functional devices have been constructed, such as vertical field effect tunnelling transistors3, superconducting Josephson junctions4, spintronic memories5 and photodetectors6. Nowadays, there are mainly two strategies to build 2D vdWS with wafer scale, including the bottom-up stacking growth8 and the top-down transfer10. Therein, chemical vapour deposition (CVD) growth of various large-sized transition metal dichalcogenides (TMDCs) heterostructures has been demonstrated8. Moreover, benefiting from the weak coupling between the wafer-scale TMDCs with the growth substrate, one layer-by-layer transfer method of namely programmed vacuum stack has also been developed for building the TMDCs-based vdWS10.Two-dimensional (2D) van der Waals superlattices (vdWS), which are vertically stacked by individual 2D layered materials, offer a platform for exploring physical properties and functionality11. Furthermore, graphene-based vdWS have also revealed numerous fresh physical properties. Such superlattice minibands of Hofstadter’s butterfly13, unconventional superconductivity14, correlated insulator behaviour15, ferromagnetism16, quantized anomalous Hall effect17 etc., are stimulated in the stacked graphene/hBN or double-layer graphene by precisely controlling their twist angles. Graphene-based vdWS offer possibilities for creating a generation of ultrathin transparent functional devices18. However, there are a few effective methods to stack growth double-layer graphene with controllable twist angles20, but the stacking growth strategy is usually hindered by the harsh growth conditions. Wafer-scale graphene films are typically grown via CVD method at a relatively high temperature 21, which is much higher than most of other 2D materials. Therefore, graphene is commonly used as the underlying layer while growing in the stack manner23, which makes it difficult to integrate graphene into the programmed order of vdWS. In contrast, the layer-by-layer transfer shows more flexibility to build diverse graphene-based vdWS. Actually, most graphene-based superlattices with specific properties are fabricated through the modified dry transfer technique24, where the graphene or other 2D materials are limited to dozens of microns. Until now, it is relatively easy to realize the transfer of wafer-scale monolayer graphene34, and there are also a few approaches for manipulating the twist angles between the adjacent graphene layers37. However, stacking homogeneous graphene-based vdWS at wafer size remains a challenge, and the homogeneous vdWS with controlled twisted angles is more difficult.Graphene, as the first discovered and highly conductive 2D material with remarkable mechanical and electrical properties, is likely to be the most compatible component of future vdWS and their functional devices39. Wrinkles or some folds, which are formed during CVD growth at high temperature, can be eliminated by the proton-assisted or low-temperature methods40. However, most folds, cracks and tears are induced by the inappropriate transfer operation of pasting graphene on the target substrate or other 2D materials38, where the trapped water or residual nanoparticles (NPs) should be the main obstacles41. Once graphene is firmly attached to substrate, it is difficult to reduce the trapped water, NPs and consequent folds through the process like nitrogen gas blow42. The adsorbed water also causes the heavily doping state of graphene and degenerates their electrical performance43. Thus, the transfer defects and the residues on the bottom 2D materials should be completely eliminated if stacking transfer a homogeneous vdWS. Furthermore, the hydrophilic substrates show more helpful for transferring defect-free graphene films than the hydrophobic ones30, the substrates with flatter surfaces are also better than the rough ones44, and the residual NPs trapped in the interlayers of stacked vdWS are considered to be avoided through the layer-by-layer hydrogen annealing45. However, most 2D materials are approximately hydrophobic, and the wetting angle of DI water on intrinsic graphene (θ1) is more than 80 degree46, hence the high-quality stacking transfer of wafer-scale graphene-based vdWS is still plagued by the trapped air, water or NPs in the interlayers.During traditional operations, the macroscopical transfer defects are the main trouble for the homogenous feature and the subsequent stacking transferIn this work, we develop a flat-to-flat transfer method, i.e. transferring flat graphene films onto the flat substrate, which consists of the spinning-assisted process and the proton-assisted treatment (PAT) process which is based on the permeable protons and recombined hydrogen, to exhaust the trapped water and decouple the transferred graphene. The wafer-scale monolayer graphene and graphene-based vdWS with ultra-flat surface and scarcely transfer defects are obtained through adjusting the appropriate wetting angles in different aqueous solutions. The twist angles of double-layer and triple-layer graphene can be precisely adjusted by the macroscopically stacking angles according to the wafer flats of graphene. The transferred graphene and graphene-based vdWS exhibit excellent morphological, structural, optical, and electrical homogeneity over the wafer size. Robust quantum Hall effect (QHE) arises in the transferred graphene films, and even appears at room temperature  with linewidth of centimetres.2/Si under wet condition. Because the wetting angle of DI water on SiO2/Si (θ2) is usually less than 60 degree30, the approximative hydrophilia makes DI water can be used as the transfer solution here . Atomic force microscope (AFM) measurements show that the thickness of the DI water layer trapped between graphene and SiO2/Si is ~40 nm and the roughness of PMMA/graphene/DI water/SiO2/Si is ±25 nm  trapped in the interface between graphene and substrate, which are difficult to be eliminated through the thermal annealing  over 100 × 100 μm2 of transferred graphene after different treatments are shown in Fig. 2D/IG of the as-transferred graphene is concentrated at the values of 1.6 ± 0.3, demonstrating its monolayer nature. The I2D/IG decreased to 1 ± 0.2 after vacuum annealing, mostly caused by the annealing introduced substrate doping47. In contrast, the distribution of I2D/IG after PAT is more uniform and exhibits minimal variation. Typical Raman mapping for I2D/IG of the as-transferred graphene and graphene after annealing are shown in supplementary Fig. D/IG of the graphene after PAT are plotted in supplementary Fig. 21. Figure ω2D), and it shows that the as-transferred graphene presents the least value of Δω2D, indicating the weakest coupling between graphene and the substrate. After vacuum annealing, the Δω2D become the largest, meaning that the interaction is significantly enhanced. In contrast, Δω2D after PAT process present much smaller, indicating the PAT graphene remains the similar coupling state with the as-transferred film, and PAT is able to decouple graphene from the substrate and weaken their coupling. The detailed spectra are plotted in supplementary Fig. Subsequently, the statistical distribution of Raman intensity ratio of 2D to G peaks (IVbg), and the inset shows the typical optical image of the Hall bars with 200 μm linewidth. The as-transferred graphene films show the heaviest p-doping level with the carrier density of 4.4 × 1012 cm–2, where the Dirac point is located at Vbg of 58 V. This should reasonably be attributed to the large amounts of residual NPs and the adsorbed H2O molecules. After the vacuum annealing, the Dirac point of graphene film shifts to 16 V, i.e. the p-doping level is significantly reduced. In contrast, the graphene films after PAT process exhibits the weakest doping level with the carrier density of 2.9 × 1011 cm–2 and Dirac point at 3.8 V, indicating that PAT process can truly decouple graphene and neutralize the doping level. The statistical distribution of Dirac points and the corresponding carrier density of the transferred graphene films are compared in supplementary Fig. 2 V–1 s–1, and the hole mobility increases to ~6950 cm2 V–1 s–1 after vacuum annealing. In contrast, the PAT graphene presents the best state, where the hole and electron mobilities reach ~13,000 cm2 V–1 s–1 and ~10,600 cm2 V–1 s–1 at 1.5 K, and they also reach ~7450 cm2 V–1 s–1 and ~7250 cm2 V–1 s–1 at RT, respectively.We continue to measure the doping levels of transferred graphene films after different treatments via the back-gate field effect transistor (FET) devices, which are batch fabricated by the simple etching through a shadow mask and present the same Hall bar configurations. Figure Rxx) and Hall conductivity (σxy) for the same Hall devices in Fig. B┴) of 6.5 T and the temperature of 1.5 K are plotted in supplementary Fig. Rxx and σxy at different temperatures, where the first σxy plateaus at ±2e2/h easily appears even at RT and the fifth integer plateaus is still obvious under B┴ of 7 T and at 1.5 K. The robust QHE plateaus emerged under relatively mild measurement conditions demonstrate the high homogeneity and high quality of the flat-to-flat transferred films on a wafer scale. Moreover, the hole mobilities of this centimetre-sized graphene film are still up to ~12,000 cm2 V–1 s–1 at 1.5 K and ~9300 cm2 V–1 s–1 at RT, as shown in supplementary Fig. The realizations of QHE usually represent the high electrical quality of transferred graphene films. The longitudinal resistances  and the substrate (θ2) are both hydrophobic, the folds are frequently formed in the stacked graphene films, and the typical optical image is shown in the top inset of Fig. The flat-to-flat method is easily generalized to the stacking transfer of wafer-scale few-layer graphene-based vdWS. Figure 2O clusters could be reduced and excluded from the graphene edges during the PAT process. Furthermore, the residues trapped in the interlayer of double-layer graphene can also be reduced and the double-layer graphene is further flattened through the PAT process. Double-layer graphene films during the multi-cycled PAT processes are non-destructive, and the Ar plasma treatment seems ineffective in eliminating the intralayer residues. All the evolutional results are compared in supplementary Fig. After spinning-assisted transfer, the stacked graphene film is also treated by PAT process. Typical AFM images of the stacking transferred double-layer graphene are shown in Fig. ωG and ω2D for double-layer graphene films, and their distributions are both within the range of ±2.5 cm–1 in large area, indicating high homogeneity. The typical Raman mapping of I2D/IG, FWHM of G and 2D bands, ωG and ω2D across the monolayer, double-layer and triple-layer transition regions are shown in Fig. 48. We further count hundreds of double-layer graphene films which are stacked randomly and find their twist angles approximately follow the normal distribution, as shown in Fig. Figure α) between the upper and bottom graphene films, we can preliminarily obtain a double-layer graphene film with a controlled twist angle, illustrated in Fig. Furthermore, this stacking transfer can be also applied to control the twist angles between the double-layer graphene films at wafer scale. Benefiting from the approximately single crystalline graphene films grown on Cu111)/sapphire  patterns at random locations. Two pieces of 2-inch double-layer graphene films with designed stacking angles of ~6° and ~38°, as well as corresponding SAED patterns, are shown in supplementary Fig. Rxx of the double-layer graphene with ~10° twist angle changes with varying Vbg at 1.5 K, 77 K and RT, and the inset is the typical optical image of the Hall bar with 50 μm linewidth. This double-layer graphene film present slight p-type doping and the hole and electron mobilities reach ~7750 and ~7500 cm2V−1s−1 at 77 K, ~5950 and ~6250 cm2 V–1 s–1 at RT, respectively  and 10° (2nd and 3rd) also shows QHE plateaus at 1.5 K under B┴ up 2 T  films on c-plane sapphire, growth temperature of 650 °C, pressure of 6 Pa, CH4/H2 ratio of 1:20, plasma power of 15 W and growth time of 5 min. The wrinkled graphene films are grown in a tube furnace by the traditional CVD method, typical parameter is as follows: sputtered 800 nm Cu-Ni(111) films on c-plane sapphire and then annealed at 1050 °C under the hydrogen atmosphere for 60 min to flatten the surface. During growth, growth temperature of 1050 °C, CH4/H2/Ar ratio of 0.1:10:500 and growth time of 10 min. To simplify writing, we use Cu(111) instead of Cu-Ni(111) alloy in our descriptions below.Ultra-flat graphene films are grown by the proton-assisted CVD methods as reported before2 and MoSe2 films are grown by the two-step vapour deposition method, which includes the typical physical vapour deposition (PVD) and CVD processes, typical parameters are as follows: for PVD process, sputtered 0.8 nm Mo films on SiO2/Si substrate; then for CVD process, Mo films and S or Se powders are located in a two-zone tube furnace, Mo films are heated to 750 °C and S powder is heated to 150 °C for forming MoS2 films, Mo films are heated to 750 °C and Se powder is heated to 280 °C for MoSe2. The H2/Ar mixture gas is used as carrier gas and their flow rate is both 100 sccm. The growth times are all 30 min. Wafer-sized hBN films are grown in a three-inch tube furnace and assisted with the proton process, typical growth parameter is as follows: sputtered 800 nm Cu(111) films on c-plane sapphire and then annealed at 1050 °C under the hydrogen atmosphere for 60 min to flatten the surface. During growth, flattened Cu(111) substrate is heated to 650 °C, the set pressure is 1 Torr, H2/Ar ratio is 100:10, ammonia borane is used as the precursor and loaded into a sealed chamber at the upstream of the growth substrate with the constant temperature at 70 °C. The plasma power is 40 W and the growth time is 10 min. The grown MoS2, MoSe2 and hBN films are all polycrystalline, and they all have ultra-flat surfaces.Wafer-sized MoS2, MoSe2 films on SiO2/Si are spin-coated with double PMMA films  as the protection layer. Then, 1 M (NH4)2S2O8 aqueous solution is used to etch the Cu(111) substrate and 1 M KOH aqueous solution is used to etch the SiO2. The PMMA/2D material, like PMMA/graphene, is then pulled out from the etchant by a clean polyethylene terephthalate (PET) sheet and moved to DI water for cleaning. The cleaning is normally needed to repeat three times. After that, a two-step spinning-assisted process is developed during pasting the graphene or other 2D materials films onto the flat target substrates (like SiO2/Si). The slow spinning rate of 90–150 rpm is used for the first stage with a spinning time of 6 min, and the fast spinning rate of 300–600 rpm is used for the second stage with a time of 10 min. Then, all the pasted PMMA/graphene on the target substrate is backed at 80 °C for 30 min and at 150 °C for 15 min on a hot plate in sequence. Finally, PMMA is removed by acetone, then the film is dried by a gentle nitrogen gas blow. To further clean the transferred graphene films and decouple them from the substrate, vacuum annealing or PAT is performed. The annealing temperature is 450 °C, the annealing time is 40 min and the vacuum degree is less than 5 × 10–5 Pa. The PAT to decouple graphene is performed at 450 °C under 6 Pa, the H2 plasma power is constant 15 W and treatment time is usually 5 min, and the PAT to clean hBN, MoS2 and MoSe2 needs the temperature of 450 °C, 350 °C, and 350 °C, respectively.The as-grown graphene, hBN films on Cu(111)/sapphire and the as-grown MoS2/graphene, the range of IPA is from 10 vol% to 22.5 vol%, and 15 % of IPA is preferred. For MoS2/MoSe2, the range of IPA is from 7.5 vol% to 17.5 vol%, and 12.5% of IPA is preferred. After the removal of PMMA, the PAT is also performed to clean the surface and the interface between the stacked graphene films. In order to avoid the appearance of C-H bonds during the PAT process, the treatment parameter of double-layer and triple-layer graphene is as follows: H2 plasma of 15 W, treatment time of 5 min, constant pressure of 6 Pa and temperature of 550 °C.During the stacking transfer process, the as-transferred 2D materials like graphene are used as the flat target substrate. A two-step spinning-assisted process which consists of the same transfer process for monolayer graphene is used for stacking the double layers of graphene films. The difference is that the DI water is replaced by mixing IPA or acetic acid in DI water as the transfer solution, which ensures the wetting angle of solution on graphene falls within the range of 40°–60°. For double-layer graphene, the optimal wetting angles for IPA/DI water volume ratio range from 10 vol% to 25 vol%. For hBN/graphene, the range of IPA is from 15 vol% to 25 vol%, and 20 % of IPA is preferred. For MoS–8 Pa), and the probe electron beam size is about 1 mm.The optical images are captured by the optical microscope (Nikon LV100ND). Large-area photographs and wetting angles are captured by a high-resolution digital camera. AFM measurements are carried out by a Bruker Dimension Fastscan system at tapping mode under ambient conditions. Raman spectroscopy is carried out by a Witec/alpha 300R confocal microscope with 532 nm laser under ambient conditions, and the in situ variable temperature Raman is performed in a temperature-controlled stage (Linkam THMS600) with introducing pure Ar. All the laser power is set below 2 mW to avoid heating. SAED is performed by a transmission electron microscope (TECNAI F20) with an accelerating voltage of 200 kV, and the sizes of the selected area are all chosen to be ~1 μm. LEED  is measured using electron beam energy of 170 eV at RT under an ultrahigh vacuum of (1 × 104He cryostat with a superconducting magnet (Oxford Teslatron 8T and lowest temperature 1.5 K) with source metre (Keithley 6430) and lock-in amplifier (Stanford SR830). All the transport data are transformed by tensor inversion. The field effect mobility is extracted by μFE = C-1 (dσ/dVg), where C is the gate capacitance per unit area and calculated as 1.21× 10-4 F/m2 for 285 nm SiO2 layer.The electrical transport measurements are performed in a Supplementary InformationPeer Review File


Covalently interconnected graphene (CIG) nanoscaffoldswith a preadjusted finely tuned graphene layer–layer distancefacilitated the formation of sandwiched US–AuNPs . The elemental composition analysis byX-ray photoelectron spectroscopy displayed an aniline group additionper ∼55 graphene carbon atoms. It provided information on covalentinterconnection via amidic linkages, while Ramanspectroscopy offered evidence of covalent surface functionalizationand the number of graphene layers (≤2–3 layers). High-resolutiontransmission electron microscopy images indicated a layer–layerdistance of 2.04 nm, and low-angle X-ray diffraction peaks (2θat 24.8 and 12.5°) supported a layer–layer distance increasecompared to the characteristic (002) reflection (2θ at 26.5°).Combining covalent bridging with NP synthesis may provide precisecontrol over the metal/metal oxide NP size and arrangement between2D layered materials, unlocking new possibilities for advanced applicationsin energy storage, electrochemical shielding, and membranes.The covalent functionalizationof graphene for enhancing theirstability, improving their electrical or optical properties, or creatinghybrid structures has continued to attract extensive attention; however,a fine control of nanoparticle (NP) size between graphene layers  The gold-filledsandwiches showed the lowest sheet resistance, a red-shifted broadoptical absorption spectrum, and a shifted work function, suggestiveof distinct properties from simple graphene laminations or graphenewith metallic overlayers. In addition, a concept study that followsgraphene growth, nanoparticle decoration, and graphene layer transferto prepare sandwich structures of graphene (or other 2D sheets) wasreported by using various nanoparticles, including Au (∼10–24nm), Pd (∼15 nm), and PbSe (∼20 nm).17 Although this method offered a route for preparing sandwichnanoparticles (≥10 nm) between graphene layers, the size controlof nanoparticles was highly dependent on the optimization of reactionparameters, such as precursor concentration and reducing agent. Asacrificed template-based technique was also developed to sandwichlarge cobalt (Co) nanoparticles and their oxides between carbon nanosheets via high-temperature calcination of glucose-based polymer/(NH4)2Co8(CO3)(OH)6:4H2O under an inert atmosphere.18 Although ultrasmall nanoparticles (USNPs), with sizes of 1 to 3nm, are known to have unique properties highly distinct from largercounterparts,19 the precision-tuning ofNPs’ size between layered nanomaterials has been studied less.For example, the US–AuNP (2–4 nm)-decoratedgraphenenanocomposites, prepared by laser irradiation, were reported as promisingmaterials for photothermal therapy and the efficient conversion ofsolar energy into useable heat for a variety of thermal, thermochemical,and thermomechanical applications.Herein, we report for the first time the synthesis of ultrasmallgold nanoparticles (mean particle size of 1.94 ± 0.20 nm) (US–AuNPs)sandwiched between graphene layers with a preadjusted finely tunedlayer–layer distance of ∼2.04 nm.20 Natural graphite flakes  were used for bulk-functionalization experiments. N,N-Dimethylformamide (DMF) , 1,4-diaminobenzene , pyromellitic dianhydride (PMDA) , and sodiumnitrite (NaNO2)  were purchasedfrom Sigma-Aldrich and used as received. High-purity distilled waterwas obtained from an Elga PURELAB Prima deionized water machine (15Ω).Graphite was provided by GraphexelLimited (graphite grade 2369) and used as received to prepare highlyexfoliated graphene flakes following the literature.21 In a typical procedure, a cooled solution (0 °C) ofNaNO2 was added dropwise to 1,4-diaminobenzene . The stoichiometric ratio wasadjusted to 1 equiv/1 equiv to ensure that only one amine group is diazotized.22 The resulting yellow solution was stirred for 30 min andmaintained at 0 °C. A cooled solution (0 °C) of highly exfoliatedgraphene flakes in DMF20  was then added dropwise. The reaction mixture was heldat 0 °C, stirred for 4 h, and then mixed for 15 h at room temperature.The surface-modified graphene flakes were filtered through a PTFEmembrane (0.5 μm), redispersed in 2 M HCl (20 mL), and filteredand washed with a copious amount of water until pH was neutral. Theresultant solid material was dispersed in 2 M sodium hydroxide (NaOH)(10 mL) and stirred overnight to convert the produced aniline hydrochloridesalt to aniline groups. The aniline-modified graphene flakes werethen isolated by filtration through a PTFE membrane (0.5 μm),washed with water until pH neutral, redispersed, and filtered usingTHF (2 × 10 mL), acetone (2 × 10 mL), and ethanol (2 ×10 mL), respectively, and dried at 80 °C in an oven. A bifunctionalmolecule, PMDA, was used to covalently interconnect aniline-modifiedgraphene flakes. A two-step reaction was followed to ensure the formationof a molecular bridge between the aniline-modified graphene flakes.Initially, the aniline-modified graphene flakes (10 mL) were reactedwith excess PMDA in dry DMF at 60 °C to enable only the one-sideaddition of PMDA to the aniline-modified graphene flakes. UnreactedPMDA was removed by membrane filtration, and the wet cake was washedwith DMF (100 mL) and THF (100 mL). The aniline-PMDA-modified grapheneflakes were redispersed and filtered using dry DMF (4 × 10 mL)and dry THF (4 × 10 mL), respectively, and then dried under N2 flow. The resultant aniline-PMDA-modified graphene flakeswere redispersed in dry DMF to prepare a highly stable dispersion.Then, 10 mL of well-dispersed aniline-modified graphene flakes indry DMF was added to the aniline-PMDA-modified graphene flakes’solution. The mixture was heated overnight to 60 °C under nitrogen.The above-mentioned purification steps  were repeated to isolate theprepared covalently interconnected graphene nanoscaffolds. For bulkfunctionalization, a graphite dispersion (instead of a stable graphenedispersion) in DMF  wasused to prepare aniline-functionalized graphene nanosheets followingthe modified literature procedure described above,21 and the resulting aniline-functionalized graphene was usedfor nanoscaffold preparation. AuNPs were prepared in the presenceof the covalently linked graphene nanoscaffolds following the Turkevichmethod.24 In addition,two control experiments were performed with and without aniline-modifiedgraphene. In a typical procedure, ∼10 μg/mL of covalentlylinked graphene nanoscaffolds was prepared in high-purity water (20mL) using bath sonication (30 min). Then, 2 mL of freshly preparedaqueous HAuCl4 (0.005 M) was added to the covalently linkedgraphene nanoscaffold solution. This mixture was initially stirredat room temperature for 30 min, and the solution temperature was raisedto 90 °C. After that, 1.2 mL of hot (90 °C) Na3Cit solution (0.05 M), required to prepare the citrate-to-gold molarratio of 6-to-1, was added to the HAuCl4/covalently linkedgraphene nanoscaffold solution at 90 °C. The mixture was stirredfor an additional 2 h at the same temperature. After cooling to roomtemperature, it was filtered through a PTFE membrane (0.5 μm)and washed with copious amounts of water to remove the unreacted goldsalt precursor. The resultant material was dried at 80 °C ina vacuum oven to give a gold nanoparticle/covalently linked graphenenanoscaffold hybrid structure.A modified procedure was usedto functionalize the surface of the graphene flakes.–1. Purchased graphite flakes and the covalently linked graphene nanoplateletswere tested in powder form. A stable DMF dispersion of the covalentlylinked graphene nanoplatelets was deposited on the substrate surfaceand tested in powder form. Raman analysis was performed on a cleanSi-wafer substrate to prevent the substrate’s interferencewith the spectra and to observe any peak shift relative to the Sireference material at 520 cm–1. Ultraviolet visible(UV/vis) spectra were obtained from stable dispersions of the producedgraphene in DMF using a Shimadzu UV-2550 UV/vis spectrophotometer.High-resolution X-ray photoelectron spectroscopy (XPS) was performedby a Thermo Scientific K-alpha photoelectron spectrometer with monochromaticAl Kα radiation; peak positions were referenced to the C 1sline at 284.5 eV, and the CasaXPS software with Shirley backgroundwas used while deconvoluting N 1s spectra. Transmission electron microscopy(TEM) and high-resolution transmission electron microscopy (HRTEM)were performed using Jeol JEM-1010 and JEOL-2010F coupled with EDSdetector (Oxford Instruments) instruments, respectively. All TEM sampleswere prepared in DMF and dropped on carbon-coated copper grids, whichwere dried in air before imaging. The particle size distribution ofAuNPs was estimated from TEM data by using ImageJ software. X-raydiffraction (XRD) analysis was performed using a Bruker D8 ADVANCE5–90°, 0.02 step size, Cu Kα (1.5406 A) at 40 kV10 mA, and carbon nanomaterials dispersed in ethanol were used toprepare XRD samples. All graphs, statistical analyses, and Raman spectralfittings were performed using OriginLab software (Origin 9.1).Raman spectra were obtainedfrom a Renishaw InVia Raman Microscope using a 514.5 nm excitationlaser and wavenumbers ranging between 100 and 3000 cmvia chemical reaction-mediated self-assemblyof functionalizedgraphene nanoplatelets. Stable graphene dispersion (∼15 μg/mL)in DMF  was usedfor functionalization experiments, and it was prepared following aliterature method,20 which produces defect-freesingle-layer graphene via microwave solid exfoliation.In a typical CIG nanoscaffold preparation, exfoliated graphene nanosheetswere first functionalized with aniline groups following a modifieddiazonium salt addition procedure (21 The stoichiometricratio between sodium nitrite (NaNO2) and 1,4-diaminobenzenewas 1 equiv to ensure that only a single amine was diazotized.22 The aniline-functionalized graphene nanoplateletswere covalently interconnected using a bifunctional linker [pyromelliticdianhydride (PMDA)] to prepare CIG nanoscaffolds.The molecularly designed CIG nanoscaffoldswere prepared rocedure 1.21 The 27 The D-band observedat ca. 1350 cm–1 is widely regardedas a reliable measure of surface defects and chemical functionalization.Therefore, the area ratio of the D-band to the G-band (AD/AG) is used to determinethe degree of surface defects on a graphitic material.25 Raman spectra of pristine graphite and CIG nanoscaffoldsare shown in AD/AG ratio of 0.36, compared to0.07 and 0.11 for graphite and exfoliated graphene, respectively,indicating the successful covalent addition of aniline groups to thegraphene surface  compared to the pristine graphite with an I2D/IG ratio of ∼0.14at ca. 2728 cm–1  suggests the formation of rotationally reordered single-layergraphenes.29 On the other hand, the 2D-bandof the CIG nanoscaffolds displays an intense peak at ca. 2723 cm–1 (I2D/IG at 1580 cm–1 = 0.41)  1a, and a = 0.41) 1d, chara30 In such reactions, primary aminegroups convert into amide linkages; thus, one can expect a chemicalenvironmental change in the nitrogen atom of aniline. Furthermore,adding a bulk group such as the oxygen-rich PMDA (C10H2O6) results in a considerable difference in theelemental composition of the functionalized graphene compared withthat of the pristine one.Thecovalent molecular interconnection between the graphene layerswas further characterized by X-ray photoelectron spectroscopy (XPS).The aniline-functionalized graphene flakes shown in Supporting Information Figure S2 for high-resolution XPS spectraof C 1s and O 1s). The elemental composition of nitrogen (1.48 at%) corresponds to approximately 1 aniline group per 55 graphitic carbonatoms. In addition, the XPS elemental analysis of the CIG nanoscaffoldsshows 81.96, 17.32, and 0.72 atom % C (284.68 eV), O (532.28 eV),and N (400.38 eV), respectively . There was a significant decrease in the elemental nitrogen(0.72 at %) and carbon (81.96 at %) compositions in the CIG nanoscaffolds,while there was an apparent increase in the oxygen content (17.32at %) compared to that in the aniline-functionalized graphene. Theobserved changes were attributed to the covalent addition of oxygen-richPMDA (C10H2O6) groups per approximately55 graphitic carbon atoms. The N 1s spectra of both aniline-functionalizedgraphene and CIG nanoscaffolds were deconvoluted to give an idea aboutthe covalent interconnection between graphene layers  and 399.20 (cont. 19.14%) eV,attributable to O=C–N (amide)32 and unreacted NH2 groups,31 respectively , O(532.78 eV), and N (399.78 eV), respectively  precursors inan aqueous solution following the Turkevich method24 TEM is about 7–8 nm . However,a few large aggregates (≥200 nm) are also present on the graphenesurface (0 (111).19 The experimentallyderived mean particle size of US–AuNPs (1.99 ± 0.20) isin very close agreement with the estimated layer–layer distanceof the CIG nanoscaffolds (∼2.02 nm). In addition, there areAuNPs (∼10–25 nm) and some large aggregates (≥200nm) on the CIG surface, which are comparable to the results obtainedfor the aniline-functionalized graphene. However, no US–AuNPis observed in HRTEM images of the aniline-functionalized graphene,suggesting that they can be produced only when the layer–layerdistance is finely tuned. Overall, HRTEM results underscore that thefine-tuning of AuNPs between graphene nanoplatelets can be achieved via precise covalent chemistry.Analysis of the graphene dispersion by transmission electron microscopy(TEM) shows randomly stacked few-layer graphene nanoplatelets 3a, whichngements 3b, attringements 3c,g.On ngements 3d,h,whi39 In addition, the spacing between graphene layers can be altered via intercalation,20 or covalentcross-linking chemistries,41 and the covalentlyinterconnected graphene architectures can be characterized by HRTEM42 using lateral distances.Further evidence of the finely tuned graphene layer–layerdistance and the accompanying US–AuNP formation comes fromthe lattice spacing distance of the CIG nanoscaffolds analyzed byHRTEM. Theoretically, the distance between two graphene layers is∼0.335 nm, and the thickness of a single layer of graphenemeasured by atomic force microscopy (AFM) varies from 0.4 to 1.0 nm.42 Assuming that three-layergraphene has a thickness of ∼1.0 nm (by AFM),38 the measured distance of ∼4.8 nm well agrees withthree-layer graphene, which is covalently interconnected by two PMDAbridges. Another complementary HRTEM image of the CIG nanoscaffolddisplays US–AuNP-decorated surfaces, and the graphene layersare visibly separated  and (004) reflections was observed after exfoliationand aniline functionalization of graphite flakes; however, the intensityof the (002) peak significantly decreased, attributable to less-layerednanosheets, compared to that of the as-received graphite flakes. ThePMDA-bridged CIG nanoscaffold displayed no (002) and (004) reflections;in contrast, two new peaks appeared at ca. 24.8 and12.5°, attributed to covalently bridged graphene layers withPMDA spacers, which correlates well with an increase in the layer–layerdistance between graphene nanosheets. Such a low-angle shifting phenomenoncompared to the (002) reflection at ∼26.5° was also observedin previous studies, which used rigid linkers  to preparecovalently connected graphenes.41 AfterAuNP incorporation into the CIG nanoscaffold, there was no changein the peaks at ca. 24.8° and 12.5°; however,AuNP-related five peaks at ca. 38.2, 44.4, 64.6,77.6, and 81.7° were attributed to standard Bragg reflectionsof (111), (200), (220), (311), and (222) for the face-centered cubiccrystal structure, respectively.44Complementary X-ray diffraction data of natural graphite,exfoliatedgraphite, aniline-functionalized graphene, CIG nanoscaffold, and AuNP-CIGnanoscaffold are presented in 17 and sacrificedtemplate18 techniques reported in the literature.The linker molecule, PMDA, principally predetermines the layer–layerdistance of graphene nanoplatelets and the size of the sandwichednanoparticles. When ease of access to commercially available dianhydrides andother bifunctional cross-linking agents was combined with well-practicedcarbon nanomaterials covalent surface modification approaches,46 an acceptable size control on sandwiched nanoparticles in betweengraphene nanosheets can be realized for several applications fromenergy storage materials49 to electromagnetic shielding50 and membranes.51 Inaddition to 2D graphene nanoplatelets, given the available covalentsurface modification techniques, a library of 2D materials such asMoS2,53 h-BN,54 TiS2,55 MoSe2,55 and Bi2Te355 can be processed similarly to prepare hierarchicalnanoscaffolds of other 2D-materials. This elegant approach may provideprecise size control while producing sandwiched metal/metal oxidenanoparticles between 2D-layered nanostructures, whose surfaces areprone to covalent chemistries.In summary, a hybrid US–AuNP (mean particle size of 1.94± 0.20 nm)/graphene nanoplatelet sandwich was fabricated usingthe CIG nanoscaffolds with a pretuned graphitic layer–layerdistance of ∼2.04 nm. The precision covalent chemistry strategyto prepare sandwich structures significantly differs from the sequentiallayer-by-layer stacking


We investigate the dynamics of high energy dual regime unidirectional Erbium-doped fiber laser in ring cavity, which is passively Q-switched and mode-locked through the use of an environmentally friendly graphene filament–chitin film-based saturable absorber. The graphene–chitin passive saturable absorber allows the option for different operating regimes of the laser by simple adjustment of the input pump power, yielding, simultaneously, highly stable and high energy Q-switched pulses at 82.08 nJ and 1.08 ps mode-locked pulses. The finding can have applications in a multitude of fields due to its versatility and the regime of operation that is on demand. The 1.5 µm band is best known as the standard telecommunication band, of which the losses in the silica fiber would be minimal compared to shorter or longer wavelength bands. Mutually, mode-locking operations in this region play an irreplaceable role in commercial and non-commercial fields, including in metrology and holography ,3. The aTwo-dimensional (2D) layered materials are among the most well exploited SAs, owing to their high nonlinearity, high saturable absorption, and high damage threshold, with graphene and carbon nanotubes (CNTs) still spearheading the generation of ultrashort pulses due to their performance reproducibility and their facile fabrication. Their sub-picosecond relaxation time is beneficial because it makes them a reliable alternative to the conventional use of a semiconductor saturable absorber mirror (SESAM), which requires a complicated post-fabrication process. Compared to CNT, graphene works as a fast SA owing to its relaxation time of approximately 100–200 fs, and, thus, is more capable of having high a mode-locking pump power threshold. Additionally, there is a limitation on the working wavelength of the diameter-dependent CNTs. Having an independent wavelength absorption also means that graphene resonantly absorbs light [Although graphene has been extensively exploited in terms of its performance parameters in the years that follow its first demonstration as SA in 2009 , such asWe have reported the successful implementation of a biodegradable and biocompatible chitin in a graphene-based SA device in the eye-safe region , and thiPleurotus ostreatus) were extracted to produce the chitin and 42 g of nanofibers were successfully extracted from 3 kg of whole mushrooms. Initially, the mushrooms were blended for 5 min using a regular domestic blender before undergoing hot water extraction to remove any water-soluble components. Following that, the Pleurotus ostreatus tissues underwent alkaline deproteinization treatment (1M NaOH) at 65 °C for 3 h. This is effective at removing proteins, lipids, and alkali-soluble glucan. Water was then added to the slurry in order to increase its volume to 1.5 l. The suspension was stirred for 30 min at 85 °C. Through 15 min of centrifugation , at 7000 rpm, the soluble components and excess water were removed to produce precipitate (cake), which was then soaked in alkaline solution. The suspension was heated to 65 °C for 3 h while stirring continuously before it was neutralized by re-centrifugation in excess water. The neutralized cake was then re-suspended in water (1:40 w/v ratio) and dispersed by final blending for another 1 min. The suspension was stored at 4 °C until further use. The produced graphene-THF suspension and chitin was mixed together with a one-to-one (1:1) ratio, totalling up to 5 mL. A 1-h ultrasonication process was then conducted to break down any agglomeration and to stack graphene , producing a well-dispersed graphene in chitin. A thin yet sturdy film was obtained after a 36-h ambient temperature drying in a petri dish, revealing an SA with 50 µm thickness, as measured using a 3D laser microscope. Sturdy film was obtained after a 36 h ambient temperature drying process in a petri dish.The graphene–chitin film used in this study was fabricated following the same procedure as reported in ,23. The The graphene–chitin film was first characterized using field emission scanning electron microscope (FESEM) and Raman spectroscopy for their physical morphologies and signature peak profiles, respectively, as shown in 2 with a saturable loss of around 85%.A twin-balanced detector, as illustrated in The transmission and absorption of the graphene–chitin film was recorded at 39% and 42%, respectively, in the 1500 nm region, as shown in Additionally, the thickness of the graphene–chitin film was investigated and found to be approximately 50 μm, as shown in The experimental setup of the Erbium-doped fiber laser (EDFL) is shown in A wavelength shift of around 7.05 nm and a widening of the spectral band of approximately 1.03 nm was observed upon the insertion of the graphene-chitin SA, as shown in The input pump power was then increased steadily, recording a Q-switched operation as the pump power reached 80.63 mW, and it remained steady up until 163.16 mW, with no damage inflicted on the film, indicating that the film was able to withstand high input power and can self-start a Q-switched lasing at low power.Typical Q-switched laser output train and its single envelope under the maximum input pump power of 163.16 mW is shown in The pulse width obtained was lower than many of the previously reported single-regime Q-switched operation using graphene-PVA based Sas, and it was comparable to the one employing a graphene-silica hybrid waveguide and optically deposited graphene oxide ,33,34. IAs the pump power increased further than 163.16 mW, the pulse train observed in the oscilloscope began to become unstable, and by the point that the pump power reached 167.75 mW, a mode-locking operation can be seen to start taking over, though with a very unstable operation. At a high pumping level, the thermal accumulation and supersaturation of the SA produced a sharp decrease in the transmission of the laser pulse passing through the SA, leading to the generation of short pulses ,40. EvenThe central wavelength slightly shifted from the continuous wave (CW) of the laser cavity which was initially at 1564.48 nm, mainly due to the loss introduced by the insertion of the SA. Several pairs of Kelly sidebands can be observed from the spectrum, indicating a clear characteristic of a traditional negative dispersion soliton wherein the group velocity of the signal in the fiber is higher at a shorter wavelength. This resulted in a stable pulse that maintained its shape and its amplitude as it propagated through the fiber due to the balance between non-linear effects and dispersion. With high peak power as one of its characteristics, this explains why the peak power of several magnitudes is higher than that of Q-switching operation ,45,46, a2 fitting, the pulse duration at its FWHM was estimated to be about 0.70 ps, resulting in a pulse duration of 1.08 ps (τFWHM/1.54). The autocorrelation trace revealed that the result of the experiment follows the sech2 fitting closely. The calculated output power and pulse energy of the mode-locked EDFL was 39.75 W and 0.043 nJ, respectively.The mode-locked pulse was observed to be stable from the threshold pump power of 172.33 mW to the maximum pump power of 181.50 mW. The usual limitation of film-based SA in withstanding high input power ,48 was nThe RF spectrum of the generated mode-locked pulse was recorded with a span of 500 MHz and 50 MHz, respectively, as shown in Graphene–chitin film-based SA was used to generate simultaneous Q-switching and mode-locking operations in the telecommunication region. The lasing produced a Q-switched 1.54 μs pulse at a high input pump power of 163.16 mW. This operation was able to produce a high peak power and a pulse energy of 53.3 mW and 82.08 nJ, respectively. Beyond the input pump power of 163.16 mW, the pulse started to become unstable, and upon reaching 172.33 mW, the mode-locked pulse can be observed. The 26.11 MHz pulse correlated to a 1.08 ps pulse width with a peak power of 39.75 W. Although progressive works are still needed to improve the performance of the Q-switched and the mode-locked EDFL––such as by optimizing the cavity configuration through output coupling efficiency, by gaining medium doping concentration, and by improving the SA quality––this finding will be of interest due to their application in material processing, optical spectroscopy, and telecommunication.


UCL Open: Environment is committed to sharing peer-reviewed knowledge about environmental issues, including climate change. In this special series we are encouraging contributions from authors engaged in producing knowledge about climate change in and with local communities. The published articles and case studies will meet our usual standards of quality and originality, whilst fulfilling the need for wider recognition of transdisciplinary, community-based knowledge.This special series will include research articles, case studies and commentaries about community-level responses to climate change. We hope that all submissions will be based on work done in collaboration with or led by non-traditional research communities. The special series will provide a platform to record and disseminate the insights from many community-scale and community-led researchClimate change is arguably the best-known environmental and societal challenge of our time and while calls to respond to the ‘climate emergency’Local knowledge of climate change impacts, adaptation and mitigation are often overlooked by large research and policy institutions, despite vast potential to contribute to sustainable responses . Where lAt the same time as awareness of the issues linked to working with communities on environmental topics has grown, scientific knowledge about future climate impacts has also become progressively granular, with more information now available and accessible that is meaningful for local communities . The chaCommunity-scale and community-based responses to climate change have burgeoned in recent years. In some cases, regions, cities and communities have acted ahead of and in the absence of national and international leadership on key issues . CommuniGlobal governance mechanisms, such as the Paris Agreement , provideThe need to respond to the immediate and future impacts of climate change is now widely accepted, and is becoming more urgent. The world is currently about 1°C above the pre-industrial average temperature . This avThanks to a combination of observations and modelling at local scales  we know that some communities are facing much higher temperatures than they are accustomed to: the UK is subject to a growing chance of experiencing daily maximum temperatures of over 40°C. . The impFor communities to respond they will probably have to move through several stages of development if they are to help mitigate climate change and also adapt to it. One way that communities may become better able to adapt, and support other communities in the process, is by curating and sharing knowledge and experiences. Little of that has been done so far and doing so might make it easier to:overcome barriers to progress that arise from, say, issues linked to laws, governance finance and the psychological frames that people see climate change through and, then, to develop the necessary engineering and behavioural responses, and;assist and enable communities to be resilient and thrive, particularly perhaps, those communities facing compound risks (such as those facing flooding from both sea and rivers or extreme heat and drought).We suspect that some of the most grounded, creative, innovative and transformative work on preparing for and responding to climate change impacts is and will be coming from communities around the world who are themselves foreseeing or currently grappling with the impacts of climate change. Communities of all kinds hold and constantly evolve important knowledge, skills and insights highly relevant to addressing climate change. This special series aims to share knowledge about if and how these are being brought to bear in climate responses in a range of international settings, to understand how communities and researchers might better respond to these coming challenges.We welcome and look forward to submissions on any aspect of how communities might be addressing climate change as it is a core issue for the world and hope to attract the attention of many actors outside the academic research community as we run up to the COP26 meeting in November 2021.


PurposeThe purpose of this study was to evaluate the changes in capabilities between the Generative Pre-trained Transformer (GPT)-3.5 and GPT-4 versions of the large-scale language model ChatGPT within a Japanese medical context.MethodsThe study involved ChatGPT versions 3.5 and 4 responding to questions from the 112th Japanese National Nursing Examination (JNNE). The study comprised three analyses: correct answer rate and score rate calculations, comparisons between GPT-3.5 and GPT-4, and comparisons of correct answer rates for conversation questions.ResultsChatGPT versions 3.5 and 4 responded to 237 out of 238 Japanese questions from the 112th JNNE. While GPT-3.5 achieved an overall accuracy rate of 59.9%, failing to meet the passing standards in compulsory and general/scenario-based questions, scoring 58.0% and 58.3%, respectively, GPT-4 had an accuracy rate of 79.7%, satisfying the passing standards by scoring 90.0% and 77.7%, respectively. For each problem type, GPT-4 showed a higher accuracy rate than GPT-3.5. Specifically, the accuracy rates for compulsory questions improved from 58.0% with GPT-3.5 to 90.0% with GPT-4. For general questions, the rates went from 64.6% with GPT-3.5 to 75.6% with GPT-4. In scenario-based questions, the accuracy rates improved substantially from 51.7% with GPT-3.5 to 80.0% with GPT-4. For conversation questions, GPT-3.5 had an accuracy rate of 73.3% and GPT-4 had an accuracy rate of 93.3%.ConclusionsThe GPT-4 version of ChatGPT displayed performance sufficient to pass the JNNE, significantly improving from GPT-3.5. This suggests specialized medical training could make such models beneficial in Japanese clinical settings, aiding decision-making. However, user awareness and training are crucial, given potential inaccuracies in ChatGPT's responses. Hence, responsible usage with an understanding of its capabilities and limitations is vital to best support healthcare professionals and patients. Artificial intelligence (AI) represents rapid growth, transforming many aspects of our lives, including the medical field. Utilizing advanced algorithms and machine learning, AI potentially aids physicians in making more accurate diagnoses, identifying potential health risks, and providing patients with personalized treatment plans -5. NotabMoreover, OpenAI released GPT-4, the latest version of the large-scale language model that powers ChatGPT, on March 14, 2023. Internal evaluations reported that GPT-4 saw an 82% decrease in the likelihood of responding to unauthorized content requests and a 40% increase in the probability of producing fact-based answers compared to GPT-3.5 . One sigIn order to examine the changes in capabilities between GPT-3.5 and GPT-4 in the context of a Japanese-speaking medical setting, we used the 112th Japanese National Nursing Examination (JNNE) . AccordiChatGPTChatGPT  is an artificial intelligence language model that became widely available in November 2022. It is an AI that generates instant, natural conversation-style responses to queries . The numJapanese National Nursing Examination (JNNE)The JNNE has a total examination time of five hours and 20 minutes, divided into morning and afternoon sessions with 120 questions each, totaling 240. Eleven subjects are covered in the examination, including the structure and function of the human body, understanding disease and promoting recovery, health support and social security systems, basic nursing, adult nursing, geriatric nursing, pediatric nursing, maternal nursing, psychiatric nursing, home care nursing theory, and integrated and practical nursing.The examination comprises 50 compulsory questions that probe fundamental medical knowledge, 130 general questions in a question-and-answer format covering all 11 subjects established in the test criteria, and 60 scenario-based questions. The scenario-based questions set up scenarios that one could face in a nursing setting for seven subjects  and probe the examinee's understanding and judgment in response to these situations.The basic format of JNNE is multiple-choice with four options, but a few questions with five options (select one or select two) are also presented. A few calculation problems are included, and some conversation questions, which simulate dialogues with patients in the actual clinical setting, are also part of the exam. Questions are deemed 'inappropriate' if they have multiple correct answers, are excessively difficult, or the situation is not adequately set up to obtain a correct answer despite being a multiple-choice question. Depending on the reason for being labeled as inappropriate, measures include excluding all examinees from scoring, only including correct responses for scoring (incorrect responses are excluded), or treating multiple options as the correct answer. According to the official announcement, one question was excluded from scoring in the 112th JNNE, and another question was considered to have multiple answers . TherefoCompulsory and general questions are scored one point per question, while scenario-based questions are scored two points. The passing criteria are set as answering correctly to over 80% of compulsory questions and meeting the annual standard for the total score of general or scenario-based questions. Specifically, about 65% of the points are required for the latter. In the 112th National Nursing Examination, which we used for our analysis and was conducted in February 2023, the passing criteria were 40 points or more for compulsory questions and a total score of 152 points or more for general and scenario-based questions. The exam had 64,051 candidates, 58,152 of whom passed, making the pass rate 90.8% .AnalysisThe test questions from the 112th JNNE . All tesEthical approvalThis study solely utilized data previously published online and did not involve any human subjects. Instead, an analysis of the JNNE was conducted. Therefore, ethical considerations were not applied to this study.Out of 238 questions posed in Japanese, both versions of ChatGPT, GPT-3.5, and GPT-4 were able to generate some form of answers for the same 237 questions. The question for which an answer could not be generated came from a general question that required image information to be answered and was excluded from the analysis. Thus, the assessment was carried out with a total of 237 questions. Among them, one question was a mathematical problem, and 15 were conversation questions.The overall accuracy rate was 59.9% (142/237) for GPT-3.5 and 79.7% (189/237) for GPT-4, showing a statistically significant difference (p<0.01). In terms of scoring, for both compulsory and general/scenario-based questions, GPT-3.5 achieved 58.0% (29/50) and 58.3% (144/247), respectively, failing to meet the passing standard in both categories. In contrast, GPT-4 scored 90.0% (45/50) and 77.7% (192/247), respectively, satisfying the passing standard in both categories.Table For compulsory questions, the accuracy rates were 58.0% (29/50) for GPT-3.5 and 90.0% (45/50) for GPT-4 (p<0.01).For general questions, they were 64.6% (82/127) for GPT-3.5 and 75.6% (96/127) for GPT-4 (p=0.014). For scenario-based questions, they were 51.7% (31/60) for GPT-3.5 and 80.0% (48/60) for GPT-4 (p<0.01). In all categories, GPT-4 demonstrated a higher accuracy rate than GPT-3.5.Table Both GPT-3.5 and GPT-4 successfully generated correct answers for a total of 131 questions (46.4%). Additionally, there were 58 questions (24.5%) where GPT-3.5 gave incorrect answers that GPT-4 provided correct answers for, and there were 37 questions (15.6%) where both versions provided incorrect answers. On the other hand, there were 11 (4.6%) questions where GPT-3.5 provided correct answers while GPT-4 returned incorrect ones. In the category of compulsory questions, there were 17 instances (34.0%); in general questions, 21 instances (16.5%); and in scenario-based questions, 20 instances (33.3%), where questions that were incorrectly answered by GPT-3.5 were correctly answered by GPT-4.In terms of conversation questions, the accuracy rate was 73.3% (11/15) for GPT-3.5 and 93.3% (14/15) for GPT-4 (p=0.248). Neither achieved a correct answer to the mathematical problem. The mathematical problem was not exported with the correct answer in both versions.In this study, we assessed the accuracy of ChatGPT versions GPT-3.5 and GPT-4 using the multiple-choice format of the JNNE administered in February 2023. Although the accuracy of randomly chosen answers for this exam stands at approximately 20%-25%, the overall accuracies for GPT-3.5 and GPT-4 were recorded at 59.9% (142/237) and 80.2% (190/237), respectively, both significantly exceeding the likelihood of random selection. While GPT-3.5 did not meet the passing requirements of the JNNE, GPT-4 exceeded the passing criteria. This suggests the potential utility of the latter in assisting with clinical diagnoses and treatment decisions in real-world Japanese medical settings, provided its use is approached with caution and an understanding of its inherent characteristics.The documented 80% accuracy rate in scenario-based questions, in conjunction with a 93.3% accuracy rate in conversation questions, bolsters the proposition that ChatGPT could potentially augment medical professionals in delivering personalized treatment plans and facilitating remote patient care , 16. MorThe accuracy of ChatGPT has been reported to improve with a certain degree of training . Indeed,Additionally, while direct comparisons are challenging, it is worth noting that GPT-4 recorded an 80.2% accuracy rate on the JNNE in this study, while its accuracy rate on the United States Medical Licensing Examination Educational (USMLE) entrance examination was reported to exceed 90% . This imFurther, the discrepancy in accuracy rates might be attributable to cultural nuances inherent in the Japanese examination system. The Japanese medical examination may require significant degrees of implicit understanding from the examiners to attain high scores, given that the exam may not always explicitly indicate correct or incorrect responses. Japan, recognized as a high-context communication society, often involves interpreting the atmosphere and anticipating unexpressed intentions due to shared cultural norms . This cuThis study has several limitations. First, we did not scrutinize the basis of the answers in this analysis. Since most of the exam questions are multiple-choice, there is a possibility that the correct answer could be reached by chance. In the future, it will be important to verify the validity of the basis of ChatGPT's correct answers and reveal its performance in more detail. Second, in this study, the trial was conducted only once, based on other studies conducted in a similar manner, and the results were analyzed. However, ChatGPT may output different answers each time a question is asked due to the language model behind the main architecture. Therefore, in order to reduce errors caused by this, it may be possible to measure ChatGPT performance more rigorously by attempting the JNNE several times and comparing the percentage of correct answer rates in each of these attempts. Third, a detailed evaluation of questions based on correct or incorrect responses, or a detailed analysis by field, was not performed. Investigating the characteristics of problems where GPT-3.5 was incorrect and GPT-4 was correct, as well as differences in accuracy rates by domain, through trials based on larger datasets, could potentially reveal areas in need of further enhancement. Therefore, it is important to focus on these aspects as areas of consideration for future studies. Fourth, it is important to acknowledge that ChatGPT versions are constantly evolving. With subsequent versions beyond GPT-3.5 and GPT-4, modifications to the LLMs could potentially lead to significant changes in the time required for generating responses and their quality . DespiteOur study demonstrated that while the GPT-3.5 version of ChatGPT did not meet the criteria, the GPT-4 version already exhibits the performance required to pass the JNNE. The fact that the accuracy rate dramatically improved in less than half a year suggests that by executing training specialized in the medical field in Japanese, such large language models could potentially become beneficial sources of information in clinical decision-making in Japanese clinical settings, assisting medical professionals in diagnosing and determining treatment plans, and helping patients decide whether to visit a hospital or not. However, given that accuracy is not assured and the hallucinations generated by ChatGPT can be convincingly naturalistic, there is a potential for erroneous judgments to be made unless the messages are meticulously examined by an experienced healthcare professional. Therefore, when implementing in a clinical environment, not only the training of the language model but also the training and awareness-raising of the users will be important. It is crucial to ensure that these tools are used responsibly and with a clear understanding of their capabilities and limitations to provide the best support to healthcare professionals and patients alike.


Toxocariasis has been listed among the most neglected parasitic diseases worldwide, with approximately one fifth of the global population exposed, particularly those living under poverty. In Brazil, communities of descendants of enslaved blacks (quilombola) have historically had some of the highest rates of vulnerability and poverty, characterized by lack of health assistance, poor quality of life, and nutritional insecurity.Toxocara spp. IgG antibody detection was carried out on dog feces and hair, and soil samples were surveyed for presence of Toxocara spp. eggs.A cross-sectional sampling of quilombola individuals living in four communities of southern Brazil, as well as their dogs and the soil, was carried out from December 2021 to March 2022. Sociodemographic and other information such as water source, alimentary habits, and dog and cat ownership were gathered using a semi-structured questionnaire for assessing toxocariasis risk factors. Human serum samples were tested by ELISA for anti-(P = 0.029), educational level (P = 0.026), and drinking water source (P = 0.043) were associated with seropositivity by univariate analysis. Final logistic regression revealed increased odds  to have seropositivity in individuals > 50 years old (< 10 years old). As expected, individuals with soil contact were more likely seropositive . Although retrieved in only 5/96 (5.2%) dog feces, Toxocara spp. eggs were found in 18/60 (30.0%) soil samples.Overall, 172/208 individuals  were seropositive, the highest seroprevalence rate to date in Brazil. Male gender Toxocara spp. in both human and dog populations.The high vulnerability and seroprevalence observed in quilombola communities clearly demand a One Health approach for detection, monitoring, and prevention of infection by The online version contains supplementary material available at 10.1186/s13071-023-06010-w. Toxocariasis has been a prevalent zoonosis with a significant socioeconomic impact, particularly on impoverished communities worldwide , and hasToxocara canis or Toxocara cati infective eggs has been considered the primary transmission route of human toxocariasis  to predict seropositivity for anti-Toxocara spp. antibodies in individuals of quilombola communities of Paraná State, southern BrazilAdditional file 2:  Raw data of associated risk factors of toxocariasis in quilombola communities of southern Brazil..


The presence of soluble human programmed cell death-ligand 1 (shPD-L1) in the blood of patients with cancer has been reported to be negatively correlated with disease prognosis. However, little information exists about the mechanisms underlying high levels of shPD-L1 for promoting disease progression.In this study, we first analyzed the correlations between shPD-L1 and apoptosis of T cells in patients with cancer, then tested the effect of shPD-L1 on T-cell functions and the production of regulatory T cells.We found that the apoptosis of human peripheral PD-1+CD4+ T cells was significantly elevated in patients with cancer compared with healthy donors and was positively correlated with circulating PD-L1 levels in patients with cancer. In vitro, monomeric shPD-L1 significantly inhibited the proliferation, cytokine secretion, and cancer cell-killing activity of peripheral blood mononuclear cells (PBMCs) activated by either agonist antibodies or HATac (high-affinity T cell activation core)-NYE (NY-ESO-1 antigen). It also promoted CD4+ T cells to express forkhead family transcription factor 3 (FoxP3) for the conversion of induced T regulatory cells, which was more significant than that mediated by soluble human PD-L1 fusion protein (shPD-L1-Fc).These results confirm that soluble PD-L1 could be a candidate for inhibiting the functions of activated T cells, promoting peripheral tolerance to tumor cells, and implicating in system tumor immune escape in addition to the tumor microenvironment. This is an important mechanism explaining the negative correlation between peripheral blood PD-L1 levels and cancer prognosis. Therefore, understanding the roles of hPD-L1 in peripheral blood will be helpful for the development of precision immunotherapy programs in treating various tumors. The opemic tail , 3. PD-Lmic tail . PD-L1 imic tail . Programmic tail , 6. It imic tail , 8. The + T cells into forkhead family transcription factor 3 (Foxp3)+ Tregs. This binding suppresses the killing of tumor cells by cytotoxic lymphocytes within the tumor microenvironment , which results in T cell exhaustion and dysfunction. This prevents excessive immune response and maintains immune tolerance to self-antigens –11. In aironment , 12, 13.Recently, soluble PD-L1 (sPD-L1) has been detected in the supernatants of membrane PD-L1-positive cells, including tumor cell lines and immune cells , 15. Thein vivo and in vitro by evaluating their molecular weights , in which the extracellular domain of hPD-L1 is fused to the crystallizable fragment (Fc) of immunoglobulin to form the hPD-L1 dimer, has been shown to suppress T cell activity , 26. Nev weights , 17, 27.22.1A total of 16 patients with hepatocellular carcinoma, 7 patients with cholangiocarcinoma, and 3 patients with pancreatic carcinoma who were newly diagnosed and treated at the Department of Hepatobiliary Surgery, Nanfang Hospital, Southern Medical University, Guangdong, China from September 2017 to November 2017, and 13 healthy donors who served as controls were included in this analysis. Blood samples were drawn prior to any treatment. All patients had histologically confirmed carcinoma. Staging was based on clinical assessment and histopathological analysis according to the International Union Against Cancer (UICC) TNM staging system as recommended by the American Joint Committee on Cancer  and the Japanese Gastric Cancer Association (JGCA) guidelines. The Barcelona Clinic Liver Cancer (BCLC) staging system was used to determine the appropriate treatment for patients with hepatocellular carcinoma. The clinicopathological characteristics of the patients were collected from the hospital’s electronic patient records. The study was approved by the Ethics Committee of Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences . Informed consent was obtained from each patient and healthy donor.2.2- NY-ESO-1+) were purchased from the Chinese Academy of Sciences Shanghai Cell Resource Center (TCHu160) in 2010, and Mel624 tumor cells (HLA-A*0201+ NY-ESO-1+) were kindly provided by Prof. Cassian Yee . NCI-H1299 tumor cells (HLA-A*0201+ NY-ESO-1+) were generated via transduction with HLA-A*0201 gene and utilized in this study. The cell lines were treated with M-Plasmocin (InvivoGen) for one week after revival, cultured continuously for less than two months, and authenticated for characteristic markers and growth properties. Mycoplasma testing was performed monthly using PCR. The tumor cells were grown in Dulbecco’s modified Eagle’s medium  supplemented with 10% heat-inactivated fetal bovine serum  at 37°C in a humidified 5% CO2 incubator. Peripheral blood samples from anonymous healthy donors were obtained from the Guangzhou blood center. Peripheral blood mononuclear cells (PBMCs) were isolated via Ficoll-Hypaque gradient centrifugation and maintained in RPMI-1640 medium (Gibco) supplemented with 10% FBS at 37°C in a humidified 5% CO2 incubator.NCI-H1299 tumor cells  were obtained from GenScript to produce soluble monomeric human PD-L1 (smhPD-L1). smhPD-L1 was prepared as previously described  was fused with human IgG4  to construct hPD-L1-Fc. That fusion gene was synthesized and inserted into the EcoRI and NotI cloning sites of the vector pcDNA3.1(+) (GenScript). The recombinant plasmid pcDNA3.1(+)/hPD-L1-Fc was transfected into HEK293T cells using Lipofectamine 2000 (Invitrogen). The supernatant was collected after culture for three days and purified using an anion QHP column  and Superdex™ 200 10/300GL gel filtration column . Proteins were treated with Triton X-114 to remove endotoxins.The gene sequence of human 2.5157-165 peptide presented by HLA-A*0201 (Escherichia coli as inclusion bodies. These protein chains were refolded and purified to obtain HATac (high-affinity T cell activation core)-NYE (NY-ESO-1 antigen), as previously described (The high-affinity T cell receptor (TCR) named 1G4 was generated from a wild-type TCR that can recognize a NY-ESO-1A-A*0201 . The betescribed , 31.2.6The serum was obtained from blood samples of 26 patients with cancer and 13 healthy donors, centrifuged , aliquoted, and stored at -80°C until analysis. The serum level of sPD-L1 was measured using a commercially available sandwich enzyme-linked immunosorbent assay (ELISA) kit for PD-L1 (USCN Life Science), according to the manufacturer’s protocol. The minimum detectable level of sPD-L1 was 0.066 ng/mL and the detection range was 0.156-10 ng/mL. Samples were measured in duplicates.2.7® non-radioactive cytotoxicity kit according to the manufacturer’s instructions (Promega). In addition, the cells were collected and stained with Annexin V PE Apoptosis Detection Kit I (BD Pharmingen), according to the manufacturer’s instructions. For the IncuCyte® assay, PBMCs were co-cultured into flat-bottom plates pre-plated with Mel624 cells (incubated overnight) in the presence of HATac-NYE reagent (1 nM), YOYO®-3 Iodide , and smhPD-L1 (1.2 μM or 2.4 μM respectively) or shPD-L1-Fc (0.6 μM). The plates were incubated in an IncuCyte® ZOOM live cell analysis system for 72 hours. Images were taken every two hour and the number of apoptotic cells per mm2 was quantified using YOYO®-3 iodide and the IncuCyte® ZOOM software (Essen Bioscience). All conditions were assayed in triplicate.For proliferation assays, PBMCs were pre-stained with carboxyfluorescein diacetate succinimidyl ester  at a final concentration of 1 μM, as described previously . Pre-sta2.8® 647-conjugated mouse anti-human Foxp3 (BD Pharmingen), APC-conjugated mouse anti-human CD4 (BioLegend), APC-conjugated mouse anti-human CD8 (BioLegend), PE-conjugated mouse anti-human PD-1 (BD Pharmingen), FITC-conjugated Annexin V (BD Pharmingen), and an Annexin V PE Apoptosis Detection Kit I (BD Pharmingen) with the matched isotype controls. Fluorescence was evaluated via FACS analysis using a BD Accuri™ C6 system within one hour of cell staining. The data were analyzed using FlowJo 7.6 software (Tree Star).Cell surface and intracellular staining were conducted as previously described . The exp2.9t-test was used to determine the statistical significance between groups, with p<0.05 considered significant. Correlations between the serum sPD-L1 levels and phenotypes of apoptotic T cells were analyzed using linear correlation analysis (Pearson’s coefficient). The size of the samples and the number of replicates were given in the corresponding figure legends.Statistical analysis and graphical presentations were performed and generated, respectively, using GraphPad Prism V5.0 software (GraphPad Software Inc.). The data were expressed as the mean ± SEM. Unpaired student’s 33.1+ T cells , which were consistent with previous reports . In addition, Pearson’s correlation analysis demonstrated that sPD-L1 expression levels were significantly and positively correlated with the apoptosis of PD-1+ cells . Therefore, non-immobilized smhPD-L1 demonstrated similar inhibitory effects as those of immobilized PD-L1-Fc on plastic plates or beads  in combination with anti-CD28 antibody in the presence or absence of smhPD-L1 or shPD-L1-Fc. As shown in or beads , 9, and 3.3p<0.05, To evaluate the inhibitive effects of smhPD-L1 and shPD-L1-Fc on cytokines production, smhPD-L1 and shPD-L1-Fc were supplemented to the PBMCs stimulated with anti-CD3/CD28 antibodies. The intracellular expression levels of IFN-γ, IL-2, TNF-α, and IL-10 in PBMCs were analyzed. IFN-γ levels were reduced by 55.3% upon supplementation of smhPD-L1, while only a 15.1% reduction was observed for shPD-L1-Fc supplementation p<0.05, , 9. IL-23.4in vitro system using a reagent named HATac-NYE to monitor the tumor cell-killing ability of PBMCs. HATac-NYE was previously demonstrated to have potent efficacy for redirecting T cells to lyse tumor cells in vitro  on Tregs was significant (p<0.05) (p<0.05). Its inhibitory effect was maintained at 1.2 μM of shPD-L1-Fc  are some hallmarks of exhausted T cells . In somen tumors . Recentln tumors , 20–25. in vitro to obtain soluble monomeric hPD-L1 (smhPD-L1), and detected its effect on T-cell functions. Our results showed that smhPD-L1 inhibited the anti-CD3 antibody-activated PBMCs from secreting IL-2  development by suppressing Akt-mTOR signals and enhancing and maintaining the Foxp3 expression of piTreg, which is critical for maintaining peripheral tolerance  for the publication of any potentially identifiable images or data included in this article.YL and ZL designed the study. ZL collected and analyzed the blood samples. ZL, WFC, YG, YR, YT, WXC, YB, QL, and PD performed the immunological experiments. YT conducted the statistical analysis. YL and ZL analyzed and interpreted the data. ZL and YL wrote the manuscript. All authors contributed to the article and approved the submitted version.


IntroductionWhile Google is frequently used to access internet-based health resources, the quality of online health information remains variable. Our purpose was to assess suggested resources identified through Google search features for common symptoms related to carpal tunnel syndrome (CTS).MethodsTwo searches were performed. The first, labeled "symptom-related," included the terms "hand numbness," "hand tingling," and "hand falling asleep." The second, labeled "CTS-specific," included "carpal tunnel syndrome," "carpal tunnel surgery," and "carpal tunnel release." A novel feature of Google’s search engine is to display similar searches made by other users ("People Also Ask" snippet). For each search, the first 100 results snippets and the associated website links were recorded. A list of unique questions was compiled and classified into 1 of 3 categories using the Rothwell classification: fact, policy, or value. Questions were also classified based on the diagnoses suggested by the query. Website authorship was determined, and the corresponding links were categorized by two independent reviewers.ResultsThe "symptom-related" searches yielded 175 unique questions and 130 unique website links, and the "CTS-specific" searches yielded a total of 243 questions and 179 unique links. For "symptom-related" searches, 65% of questions suggested a diagnosis, with CTS being suggested as a diagnosis for only 3% of questions. In contrast, CTS was suggested by 92% of questions in "CTS-specific" searches. In both searches, nearly 75% of questions were classified as "facts." Commercial websites were the most common in both searches.ConclusionGoogle searches for common symptoms of median nerve compression rarely yield information related to CTS. The landscape of how patients obtain information regarding medical conditions and treatments has changed rapidly in the 21st century . ApproxiCarpal tunnel syndrome (CTS) is the most common compressive neuropathy of the upper extremity, affecting one to three patients per 1,000 per year . Costs aThe purpose of this investigation was to quantify and assess suggested resources identified through Google search features for common symptoms related to CTS as well as for direct searches regarding CTS and related surgery. In addition, we aimed to characterize the suggested resources provided by Google’s "People Also Ask" snippet. We hypothesized that suggested information found using symptom-related search terms would be related to CTS and would be predominantly produced by academic centers.www.google.com) that was clean-installed with the browser history removed to eliminate bias from Google’s personalized search algorithm. The first search was performed in September 2021, and the second search was performed in December 2022. For each search, the first 100 results of the "People Also Ask" snippet and the associated website links were recorded. A list of unique questions was compiled. Each question was categorized, according to Rothwell’s classification, into fact (is the given question true?) or policy (will a course of action solve a specific problem?) or value  [Two groups of searches were performed. The first, labeled "symptom-related," included the terms "hand numbness," "hand tingling," and "hand falling asleep." The second, labeled "CTS-specific," included "carpal tunnel syndrome," "carpal tunnel surgery," and "carpal tunnel release." Each term was individually queried into a Google Web Search (or bad?) .Questions populated by the "People Also Ask" snippet were classified based on any diagnosis suggested by the question, post hoc. These categories included cardiovascular, psychological, diabetes, nutrition, hydration, multiple sclerosis, nerve injury/damage, carpal tunnel syndrome, COVID-19, and none for the searches related to symptoms of carpal tunnel. Similar to previous studies ,11, eachThe questions and the corresponding links were categorized by two independent reviewers. Interobserver reliability was calculated using Cohen’s kappa coefficient. Discrepancies were resolved via consensus discussion with the senior author, where the category was discussed amongst all parties and the final decision was made by consensus regarding the definitive category. When discussing agreement levels for Cohen’s kappa coefficient, the following guidelines were utilized: slight (0.01-0.20), fair (0.21-0.40), moderate (0.41-0.60), substantial (0.61-0.81), and near perfect (>0.81) ,14.The "symptom-related" searches yielded a total of 230 unique questions and 130 unique website links for review. Fifty-five (24%) questions were deemed irrelevant and excluded, leaving 175 questions for evaluation. Irrelevant questions often related to common suggested diagnoses but did not allude to any of the queried symptoms. Cohen’s kappa coefficient was 0.954 for question classification and 1.000 for website classification.The "CTS-specific" searches yielded a total of 243 unique questions and 179 unique links for review. No questions were deemed irrelevant. Cohen’s kappa coefficient is 0.815 for question classification and 0.844 for website classification.For "symptom-related" searches, a diagnosis was suggested in 65% 120/175) of questions  of questions  of questions were classified as fact questions  of questions were classified as fact questions. Value questions represented 14.4% (n=35) of search questions, and policy questions represented 13.17% (n=32). The most common website categories were commercial , followed by medical practice  and academic .Contrary to our hypothesis, search terms representing the classic symptoms of CTS do not commonly yield resources pertaining to CTS. While nearly two-thirds of FAQs alluded to a diagnosis, less than 5% were related to CTS. Other common causes of these symptoms within orthopedics, such as radiculopathy or other compressive peripheral neuropathies, were not represented at all. The more commonly recommended diagnoses from this study, such as nutritional deficiencies, dehydration, and diabetes, rarely present with hand numbness -17. MostFor "symptom-related" searches, over half of all recommended questions were associated with a commercial link. Similarly, commercial websites were the most common category for "CTS-specific" searches. These findings are in contrast to previous studies on Google’s "People Also Ask" snippet ,11. SudaThese data highlight the limitations of patient-driven searches for online health information. The issue remains that resources from academic centers and professional societies are not prioritized by Google’s search engine, as evidenced by two different search queries. Patients searching online highly value convenience when selecting websites to review . TherefoSeveral limitations must be considered when interpreting our results. First, while we selected our "symptom-related" search terms based on common symptoms of CTS, our terms may not be representative of all common search terms used by patients. For example, we used the term "hand numbness" but not "why are my hands numb." Also, while Google is the most common search engine , our resGoogle searches for common symptoms of median nerve compression rarely yield information related to CTS. Information produced by academic institutions and professional societies is infrequently accessed via these searches, and the suggested information has a strong commercial influence. Suggested FAQs by Google may not be a reliable source for health information.


The previous epidemiological and experimental evidence has implied the linkage between chronic inflammation to idiopathic pulmonary fibrosis (IPF). However, it was still unclear whether there were casual associations between circulating inflammatory cytokines and IPF development. The objective of present study was to examine whether altered genetically predicted concentration of circulating cytokines were associated with IPF development using a two-sample Mendelian randomization (MR) analysis.The causal effects of 23 circulating inflammatory cytokines were evaluated on IPF using MR analysis. The primary approach of MR analysis was the inverse variance-weighted (IVW) method. The sensitivity analyses were conducted by simple median, weighted median, penalized weighted median and MR-Egger regression methods.The present MR study found suggestive evidence that a higher circulating IL-14 level was associated with an increased risk of IPF . The sensitivity analysis yielded directionally similar results for IL-14. There was no significant association found between other circulating inflammatory cytokines and IPF.The high level of IL14 predicted by genes had a casual relationship with the increased risk of IPF. This finding provided epidemiological evidence for drug therapy targeting inflammatory factors in the prevention and treatment of IPF. It’s warranted further exploration to validate the clinical significance of IL14 associated with developmental risk of IPF.The online version contains supplementary material available at 10.1186/s12890-023-02658-3. Idiopathic pulmonary fibrosis (IPF) is a prototype of chronic, progressive, and fibrotic lung disease with replacement of altered extracellular matrix and destroy of alveolar architecture, leading to decreased lung compliance, disrupted gas exchange, and ultimately respiratory failure and death . AlthougMendelian randomization (MR) is an observational study design that seeks to understand causal relationships between an exposure  and an outcome  by using genetic variants as instrumental variables, which has been widely applied to mitigate certain significant drawbacks encountered by conventional observational studies . Specifihttps://gwas.mrcieu.ac.uk/datasets/ebi-a-GCST90018120/.We employed a two-sample MR analysis model in order to assess the causal impact of circulating inflammatory cytokines on IPF, as depicted in Fig. https://gwas.mrcieu.ac.uk/datasets/ebi-a-GCST90018120/ [We employed a rigorous approach to identify SNPs that exhibit a significant association with circulating inflammatory cytokines. These identified SNPs were subsequently utilized as instrumental variables (IVs) in our analysis. The selection criteria for these IVs included a stringent significance threshold (P < 5 × 10 − 6), as well as specific considerations pertaining to linkage disequilibrium (LD), whereby the LD measure (r2) was required to be below 0.001 and the LD distance to exceed 10,000 kb. Subsequently, we endeavored to locate information regarding the aforementioned IV SNPs within the IPF dataset sourced from large-scale GWASs. Specifically, this IPF dataset was acquired from the prominent GWAS conducted by Duckworth, as identified by the unique identifier (id: ebi-a-GCST90018120). Readers looking to access the detailed data for IPF can refer to the following resource: 0018120/ . The stu0018120/ .In this study, a comprehensive array of seven MR analysis methods were employed. These methods encompassed the inverse-variance weighted (IVW) technique, both in the fixed-effect and random-effect frameworks, alongside the simple median, weighted median, penalized weighted median, MR Egger, and MR Egger (bootstrap) approaches. The IVW method, serving as the primary analytical tool, was utilized due to its ability to yield reliable estimates of causal effects, even in the presence of heterogeneity. To further ensure robustness and explore potential sources of bias, two additional methods, namely the weighted median estimator and MR-Egger, were implemented for sensitivity analyses. The weighted median estimator was particularly proficient in furnishing dependable causal assessments when a majority of the instrumental variables adhered to MR assumptions. On the other hand, the MR-Egger estimate remained unbiased under the proviso that the genetic instrument exhibited no dependency on pleiotropic effects. Moreover, in order to comprehensively evaluate the presence of pleiotropic effects and heterogeneity among individual SNPs, we employed the IVW method in conjunction with MR Egger intercept and Cochran’s Q statistics. The absence of pleiotropic effects was determined if the intercept did not significantly deviate from 0 (p > 0.05). Heterogeneity, on the other hand, was assessed based on the value of Cochrane’s Q statistic. When the p-value of this statistic was less than 0.05, the IVW method with a multiplicative random-effects model was selected as the primary outcome. Conversely, if the p-value was greater than or equal to 0.05, the IVW method with a fixed-effects model was considered the primary outcome. To further account for potential pleiotropy and derive a causal effect assessment while addressing the issue of directional horizontal pleiotropy, MR-Egger regression was also performed in this study. Furthermore, a leave-one-out analysis was conducted to assess the robustness of the MR analysis results by examining the influence of individual outlier SNPs. In line with a previous study, a causal relationship was deemed significant if three conditions were met: (1) the p-value of the IVW method was less than 0.05, (2) the estimates from the IVW, MR-Egger, and weighted median methods were consistent in direction, and (3) the p-value of the MR-Egger intercept test was greater than 0.05. All statistical analyses were undertaken using the “TwoSampleMR” package in R version 3.4.1 , and a two-tailed p-value of less than 0.05 was considered statistically significant.We included 555 SNPs significantly related to the circulating inflammatory cytokines variation as IVs for circulating inflammatory cytokines-IPF causal estimations. We used a 2-sample MR model to evaluate the causal effect of circulating inflammatory cytokines on IPF Fig. . The genGenetically predicted higher plasma CRP levels showed a suggestive inverse association with IPF using IVW method   were performed as the complement to IVW, confirming the robustness of the IVW analysis results. The MR analysis by MR Egger and IVW showed that IL2 was not significantly associated with risk of IPF  Table ; Fig. 3.The MR analysis by IVW models and other analytic methods  showed that MCP1 was not significantly associated with development risk of IPF (P > 0.05) Table ; Fig. 4.The present study carried out a series of sensitivity analyses to evaluate the potential horizontal pleiotropy. The results of MR-Egger Intercept-test indicated that there was not significantly impact caused by horizontal pleiotropy (P > 0.05). The results of the leave-one-out sensitivity analysis suggested that the association between inflammatory cytokines and IPF was not substantially driven by any individual SNP .The present study performed a two-sample MR analysis to explore genetic evidence to support causal relationships of circulating inflammatory cytokines with the risk of IPF. The current MR study demonstrated that genetically proxied higher circulating IL14 level was causally associated with an increased risk of IPF, whereas no significant associations were found between genetically higher exposures of other circulating inflammatory cytokines levels  and IPF. These findings provided epidemiological evidence for drug therapy targeting inflammatory factors in the prevention and treatment of IPF.IPF is characterized as a profoundly debilitating interstitial lung disease, with a rapid deterioration in lung function and a mortality rate of 50% within 3–5 years following diagnosis , 24. WhiInflammation, recognized as a critical component in numerous autoimmune disorders, is widely implicated in various significant pathogenic processes of IPF. These include epithelial-mesenchymal transition, epithelial cell apoptosis, impaired fibrinolytic system, and myofibroblast accumulation, which are considered pivotal in the pathophysiology of IPF –30. The In our study, we successfully established a causal link between serum IL14 levels and an elevated risk of developing IPF. However, it is important to note that the incremental risk associated with IL14 levels was not substantial, and its clinical significance may be limited. As for the relationship between other inflammatory cytokines and IPF, no causal link has been established thus far. Therefore, based on the current body of research, it is our belief that the pathogenesis of IPF is not triggered by fluctuations in serum inflammatory cytokine levels. Instead, these fluctuations may be a consequence of the progression of IPF. Moreover, our study yielded robust null results for circulating inflammatory cytokines , indicating that prolonged exposure to elevated levels of these circulating inflammatory cytokines does not increase the risk of IPF. Presently, the blocking of inflammatory cytokines appears to hold promising potential as a therapeutic approach in IPF drug therapy. A phase 2, randomized, double-blind, placebo-controlled trial conducted by Maher et al. in 2021 has recruited about 500 patients with IPF to evaluate the efficacy and safety of lebrikizumab , alone or with background pirfenidone therapy . Interes− 6), which may introduce bias when weak IVs are included. Fourthly, the scope of our study was limited to individuals of European descent, thereby limiting the generalizability of our findings to individuals of other ancestral backgrounds. Consequently, the present study does not provide support for a causal relationship between levels of 27 inflammatory cytokines and IPF. Considering the restricted dataset of inflammatory factors included in our analysis, alternative datasets examining inflammatory factors are needed to validate our findings. Therefore, we anticipate that future studies incorporating updated GWAS data on inflammatory factors will serve to further validate the results of our investigation.Several limitations warrant consideration in our study. Firstly, although no causal association was observed, it is possible that the effect size is too small to be detected comprehensively, although this cannot be definitively ruled out. Secondly, the lack of available data pertaining to the pathological classification of IPF hindered our ability to conduct further analyses specifically examining IPF subtypes. As such, further investigation into IPF subtype analysis is necessary to elucidate the potential correlation between levels of circulating inflammatory cytokines and IPF across different subtypes. Thirdly, the identification of IVs was conducted using a relatively stringent threshold (p < 5 × 10The high level of IL14 predicted by genes had a casual relationship with the increased risk of IPF. No significant associations were found between genetically higher exposures of other circulating inflammatory cytokines levels and IPF. These findings provided epidemiological evidence for drug therapy targeting inflammatory factors in the prevention and treatment of IPF. It’s still warranted further exploration to validate the clinical significance of IL14 associated with developmental risk of IPF.Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2Supplementary Material 3Supplementary Material 4


Venetoclax is a small molecule inhibitor of BCL-2 used in the treatment of acute myelogenous leukemia and chronic lymphocytic leukemia. Recent post-marketing studies on venetoclax suggested that it may predispose to opportunistic infections (OIs). We sought to systematically review the randomized controlled trial (RCT) evidence on venetoclax to assess whether it predisposes patients to infectious adverse events (IAEs) and neutropenia.i.e., a regimen with additional anti-cancer agents) or placebo controls in patients with hematologic malignancies, irrespective of treatment line. Data on IAEs and neutropenia were pooled by Bayesian meta-analysis and we computed the probability of any increased risk in infectious or neutropenic complications (Risk Ratio [RR] >1). Subgroup analyses were conducted based on treatment line, use of an inactive comparator, malignancy type, and prophylaxis strategy. Additionally, we pooled the incidence rates of fatal OIs between venetoclax and comparator arms and compared them by univariate meta-regression.We systematically reviewed the RCTs comparing venetoclax therapy to active . The probabilities of venetoclax increasing the risk of grade 3-5 and fatal IAEs were 86.6%  and 81.1% , respectively. There was a 97.2% likelihood that venetoclax increased the risk of grade 3-5 neutropenia . Venetoclax treatment did not increase the incidence of fatal OIs (P=0.55).Our results suggest that venetoclax likely increases the risk of high-grade neutropenia and IAEs overall, but not fatal OIs. However, our analyses did not identify any specific IAEs that would benefit from routine anti-infective prophylaxis or pre-emptive testing.Michael Sebag, MD, PhD, Amgen: Advisor/Consultant|BMS: Advisor/Consultant|Focus Therapeutics: Advisor/Consultant|Gilead: Advisor/Consultant|Janssen: Advisor/Consultant|Novartis: Advisor/Consultant|Pfizer: Advisor/Consultant|Sanofi: Advisor/Consultant Matthew Cheng, MD, Amplyx Pharmaceuticals: Grant/Research Support|AstraZeneca: Advisor/Consultant|AstraZeneca: Honoraria|Cidara Therapeutics: Grant/Research Support|GEn1E lifesciences: Advisor/Consultant|GEn1E lifesciences: Stocks/Bonds|Kanvas Biosciences, Inc.: Board Member|Kanvas Biosciences, Inc.: Pending patents|Kanvas Biosciences, Inc.: Ownership Interest|Merck: Honoraria|nomic bio: Advisor/Consultant|nomic bio: Stocks/Bonds|Pfizer: Honoraria|Scynexis Inc.: Grant/Research Support|Takeda: Advisor/Consultant|Takeda: Honoraria