These findings are confirmed through various spectroscopy and surface characterization techniques. Controlling flake size, thickness, and yield are demonstrated via optimization of the sonication process, centrifuge time, and H2SO4 pretreatment.Graphene is a 2D material with promising commercial applications due to its physicochemical properties. Producing high-quality graphene economically and at large scales is currently of great interest and demand. Here, the potential of producing high-quality graphene at a large scale via water-phase exfoliation methods is investigated. By altering exfoliation parameters, the production yield of graphene and flake size are evaluated. Pretreatment of the precursor graphite powder using acidic solutions of H Additionally, it has high electron mobility in the range of 10,000–50,000 cm2 V−1 s−15 and excellent thermal conductivity of above 3000 W/m K8. These superior properties allow graphene to be used in a range of applications, from drug delivery and chemical sensors to energy absorption and flexible electronics. Recent exciting applications include 3D printing of graphene-reinforced metal matrix composites for tailored properties that involve enhancing the mechanical or electrical properties of the metal matrix9. Graphene’s attractive ability to be used as a reinforcing material was also seen in lightweight cement composites where the compressive strength and specific energy absorption were increased significantly10. Further, graphene’s unique nanostructure allows it to be used as a robust material platform for quantum computing13. Another potential application of graphene is in membrane technology for water desalination15.The discovery of graphene and its remarkable properties has fascinated the scientific community. Single-layer graphene (SLG) has exceptional mechanical properties, with a Young’s modulus in the order of 1 TPa16 and the mass-scale production of high-quality graphene in an economical way. In fact, graphene commercialization has been inactive in the past decades due to the issues related to the supply chain in providing high-quality and reliable graphene material. Regardless of all the challenges, a sharp increase in graphene use in commercial products in recent years has been observed and is predicted to grow significantly in the coming years18. Finding an easy-scalable technique to produce graphene is now, therefore, more important than ever.Despite having remarkable properties and promising applications, the commercialization of graphene has been rather slow, mainly due to the zero-band gap nature that hinders graphene from being integrated into the semiconductor industry20. On the other hand, a plasma enhanced CVD (PECVD) process could provide monolayer to few layers terminated 3D graphene sheets22. However, a major issue in using CVD/PECVD was in the production cost and the mass scale production inefficiency in a reasonable amount of time. Recently, few groups have attempted to overcome such problems by inducing roll-to-roll concept for continuous production of graphene23. Despite the lab-scale success, commercially, it remains a challenging issue. The production up-scaling of the roll-to-roll (R2R) method is limited by the efficiency in the change of the carbon phase, complexity of infrastructure, need for high temperature, and high vacuum25. Slow manufacturing and high equipment costs are also considered as the major bottlenecks for the mass production of graphene using the R2R method26. In parallel, several graphene exfoliation routes from bulk graphite material are investigated and explored. A few examples are graphene exfoliation using layer engineered method27, electrochemical exfoliation process29, laser ablation method30, liquid phase exfoliation method32, etc. Out of the aforementioned methods, liquid-phase exfoliation (LPE) has been a popular approach for graphene synthesis due to its straightforward nature in processing, synthesis, and inherent capability for mass-scale synthesis at a relativity low cost. LPE works by extracting graphene from graphite by overcoming the interlayer van der Waals force for mass-scale graphene synthesis33. This can be done by applying ultrasound to produce a few layer-multilayer graphene sheets at a large scale35. Additionally, the process is economical and is a potential route to produce high-quality graphene for commercial applications36.A promising route to synthesize large-scale, high-quality monolayer to few layers graphene is via chemical vapor deposition (CVD) approaches35. The solvent choice38, sonication mode39, time, and power could be considered as the critical parameters in this case. By tuning these parameters, properties, including graphene dimension and thickness, can be altered for the desired application. The use of the exfoliation method needs to be better understood so it can be used on a larger scale.Despite recognizing LPE as a low-cost graphene production method, there are still various critical parameters that could control the quality of the exfoliated graphene39. However, mostly, few-layer nanosheets of approximately 1–10 monolayers are regularly produced40. Furthermore, the sizes range between 10 nm and 10 µm41. Nevertheless, this method allows for an easier scale-up for industrialization, making it highly important to refine. Therefore, by exploring ways to ease the exfoliation process between the layers, such as introducing functional groups44 in a simple and economical way to reduce the van der Waals force between the layers, the process can become extremely efficient.The LPE process still needs to be polished to produce a few layers of graphene characterized by a uniform and intact size. Furthermore, the flake thickness and size distribution of the produced flakes ranges greatly for liquid phase exfoliated graphene. It has been observed that this exfoliation method could give nanosheets of thickness of approximately 1–2 nm46. The use of water as a liquid phase exfoliation medium using probe sonication with acid pretreatment has not been widely studied. Morton et al.47 analysed in-depth the degree of exfoliation, quality, and stability of graphene exfoliation in a mixture of water and ethanol. Tyurnina et al.48 focused on ultra-sound assisted LPE technique using a dual frequency approach in pure water. Further assessment to analyse the optimum solution temperature was done by the same group to highlight the importance of temperature and its effects49. Gao et al.50 designed a novel cyclic ultrasound-assisted exfoliation apparatus that utilized cavitation due to abrupt pressure fluctuations from water bubbles. Kim et al.51 utilized a bath sonicator to exfoliate and disperse 2D materials and found that the dissolution of graphite in water would be around 40 °C. These studies have shown the promising nature of using water as a potential solvent for graphene exfoliation and its vast area of optimization.The use of pure water as a medium for LPE can be very suitable as using deionized (DI) water helps to avoid any foreign chemical , which could be unfavorable and adds an additional task to remove them during the post-process part. Additionally, synthesizing powder material with inherent morphology of the synthesized material using freeze drying adds further advantage with water solution. The exfoliation of graphene sheets in water is highly desirable as the formation of small active cavitation bubbles in water can stimulate the exfoliation as well as support a stable dispersionIn this work, we have systematically investigated the graphene exfoliation in deionized water using the probe sonication method. The impact of pretreatment role of bath sonication in acid solution and the effect of centrifuging process has been explored.2SO4 : 3 M and 5 M were considered for the pretreatment process. The bath sonication method was carried out at a continuous ultrasonic frequency of 40 kHz for 60 min. The sonication was carried out at room temperature (RT) water. However, during the experiment, the bath temperature was observed to increase, which was then flushed with RT water while pausing the sonication process. The temperature variation was kept within 5–7 °C. Probe Sonicator manufactured by Hielscher Ultrasonics GmbH (UP400St) was implemented to homogenize and exfoliate the graphene flakes. The Sonotrode was made of Titanium with an amplitude ratio of approx. 1:2.55, Ø14 mm (173 mm2), and an approximate length of 100 mm. The pulse sonication was performed at 57 W, 40% continuous, and 28% Amplitude parameters.Commercially available high-quality graphite powder (IMERYS: Graphite & Carbon) was used as a source material for the exfoliation. The median particle size reported by the manufacturer was 17 µm. Two different molar concentrations of HA centrifuge model Sigma 3-18K manufactured by Sigma Laborzentrifugen GmbH was used to separate the graphene thin layers from the thicker layers. The centrifuging step was carried out at 5000 rpm in a swinging type rotor at a g force of 4332*g and at a temperature of 19 °C for different duration of time. The tool that we have been using is a refrigerated centrifuge type (Sigma 3-18K). The refrigerated centrifuges counteract the temperature fluctuations that could occur during the centrifugation process. Additionally, we have not observed any noticeable fluctuation in the set value in the temperature monitor. Samples (a few drops from the top portion) were collected at different intervals of the centrifuging time of 0 min, 15 min, 30 min, 60 min, 120 min, and 180 min for characterization and analysis of the exfoliated flakes.Various characterization tools were employed to evaluate the quality of the exfoliated graphene layers. As a first step, a scanning electron microscope (SEM) (Scios 2) manufactured by ThermoFisher Scientific was implemented to have an initial understanding of the particle size and morphology. The tool, also equipped with an energy dispersive spectroscopy (EDS) detector manufactured by Oxford Instrument (Ultim Max 40), was used for compositional analysis. The crystalline nature of the pristine graphite powder was assessed by X-ray diffraction . Additionally, a Raman microscope (LabRAM Soleil) was used with an excitation laser of 532 nm. X-ray photoelectron spectroscopy (XPS) analysis was performed using a PHI 5000 VersaProbe Scanning X-ray Photoelectron Spectrometer. The system was equipped with a monochromated Al X-ray source (1486.6 eV) which was used as a probe for the experiments. Further, transmission electron microscope (TEM) images were collected using a Titan G2 model (ThermoFisher Scientific) to evaluate the thin flakes. Atomic force microscopy (AFM) was also used to analyze the thickness of the exfoliated thin layers. The AFM measurements were conducted in tapping mode using a Jupiter-XR system manufactured by Asylum Research Oxford Instruments. The scan rate was set to 1 Hz, with a high setpoint of ˂ 500 mV. The absorption study of the solutions was carried out in a PerkinElmer LAMBDA 1050 + UV–Vis–NIR spectrometer.40.Figure 40. On the contrary, the pulse mode can reduce sample heating if proper cycle times are used. It is worth mentioning that the probe sonication process also produces strong convection of the solution/medium. Since we have been using a lab scale beaker in our experiments, the observed convection is strong enough to distribute the temperature rise (produced by the probe tip) within seconds throughout the solution/medium. The probe induced temperature rise was then mitigated by keeping the dispersion in ice bath, replenishing the ice every two hours. Additionally, air flow at ~ 19 °C and 0.5 m/s was kept during the process as a further measure to keep the temperature rise in check. Hence, in the current study pulse mode with proper amplitude and cycle was implemented. Gradually, intermediate steps of acid treatment and centrifuging were integrated.Probe sonication was performed in continuous and pulse modes. Pulse mode was found to be a more efficient exfoliation method to produce graphene flakes compared to continuous sonication. The continuous mode quickly raised the temperature of the sample dispersion, and it was difficult to stabilize even with ice bath. Controlling overheating is crucial to avoid chemical degradation and reduced concentration of the nanosheets51. Generating shock waves, liquid jets from bubble implosion and rapid oscillating forces all add to the powerful shear forces that proliferate layer delamination. It is also seen that within the cavitation zone/under the probe the bulk graphite flake layers were subject to layer tearing47. Therefore, ultrasonic cavitation can be used to physically exfoliate 2D materials by breaking the bulk lamellar materials into thinner flakes, which is the main mechanism for exfoliating graphene. After sonicating the sample for a specific time in pulse mode, the dispersion was left unperturbed during periods of time to separate the graphene flakes via sedimentation. Drops from the top portion of the dispersion were collected after 1, 24, and 94 h, drop-casted on precleaned Si substrates, and dried on a hot plate at 100 °C. The samples are named 1hr_sed, 24hr_sed and 94hr_sed, respectively. Once dried, the samples were investigated by SEM  water. During sonication, the probe is in direct contact with the sample where it utilizes ultrasound energy. This ultrasonic agitation creates energetic cavitation bubbles due to the abrupt pressure fluctuations from the creation and bursting of these small bubblesAfterward, the supernatant was further subjected to sonication with the previous parameters. Every two hours, the sonication was stopped, the dispersion was sedimented for an hour, and a sample was collected from the top portion for characterization. These samples are denoted as 4hr_son, 6hr_son, and 8hr_son, as shown in Fig. Further, a TEM sample was prepared from the 8hr_son sample. Figure 52 and so a trade-off between the sonication time and flakes with lesser defects must be made. In our study, a sonication time of 6 h was considered for further experiments  generates high number of defects in the graphene dispersion even though thinner flakes are created. This is in accordance with previous studies2SO4. The ionic size of sulfate ion is 0.46 nm, which is comparable to the graphite interlayer spacing (0.335 nm). This makes the sulfate ions a favorable candidate for the intercalation process for effective exfoliation of the graphene layers57. Sulfate-based ions are also used during anodic exfoliation of graphene from graphite58. Therefore, H2SO4 is chosen as the most appropriate pretreatment medium allowing sulfate ions intercalation between the graphene sheets and assisting in the exfoliation. The obtained acid pre-treated solution was rinsed with DI, filtered, and dried. The dried powder was resuspended in DI for further sonication following the parameters discussed above. After centrifugation, the samples were analyzed by SEM. Figure 2SO4 solutions. Here, it is important to point out that during imaging we noticed that it was easier to find separated flakes in the pre-treated samples, suggesting an increased yield. However, the lateral size of the flakes was consistently reduced when increased H2SO4 concentration was used  from analyzing graphene’s baseline absorbance at 660 nm and applying Beer-Lambert’s law:To further quantify graphene’s yield due to the pretreatment of the graphite powder, absorption study of the graphene solutions was performed. Figure −1 m−1, was within the range reported in the literature61. The pretreatment step has been found to increase the exfoliation yield by 24% as seen in Fig. 62, see supplementary Table 2 and this could impact the sulfate ion intercalation. Besides, the flake size is likely to impact as well. The sulfate ion intercalation starts from the edges which is the c-axis of the graphite structure. Coverage of the sulfate ion in the material/flake can be insignificant in the case of large flakes and this can decrease the graphene exfoliation. However, extensive parametric studies should be carried out in order to establish a flake size-dependent graphene exfoliation process.The absorption coefficient, calculated to be 1415.7 ml mg−163. In the case of pre-treated 0 M, 3 M, and 5 M samples, the D bands are localized at 1347, 1350, and 1349 cm−1, respectively. The G bands shift to 1577 (0 M), 1581 (3 M), and 1581.5 (5 M) cm−1 with respect to graphite  in stage-2  after acidic pretreatment69. This suggests the formation of GICs in our samples, although partial rather than an ideal full intercalation  may produce gaseous molecules such as, CO2, SO2 and H2O that can further push the layers to separate from each other75.To better understand the effect of the acidic pretreatment on the exfoliation process, the samples were analyzed by Raman spectroscopy. As shown in Fig. 76. Besides, a high oxygen content was found on specific regions of the flakes, indicating a partial oxidation of these  during the chemical process. The high resolution C1s peak with different acid concentrations, elemental analysis, and corresponding peak deconvolution are shown in Fig. 77, while the peaks at around 286, 288 and 290 eV corresponds to hydroxyl or epoxide groups (C–OH or C–O–C), carboxyl group (C=O), and the pi–pi* peaks, respectively78. XPS analysis confirmed that the atomic concentration of oxidized carbon increases from 3% for the 3 M to 29.7% for the 5 M.The survey scans revealed the presence of three main components in all samples, which are denoted as: C1s at 286 eV, O1s at 532 eV and Si at 99 eV and 120 eV Fig. a. The ap79. According to several applied research80 and DFT simulations, the XPS peak at 285.2 eV indicates the existence of defects, while the peak broadening is directly related to the size of the defect83. Barinov et al.84 demonstrated that the line broadening and extra peaks are caused by the point defects present in the hexagonal carbon lattice. Additionally, as the defect density increases significantly, the form of the C1s line broadens significantly. The evolution of defect bands at the lower BE regime of C1s band requires an extra peak with BE of 285 eV to accommodate the substantial line broadening, and this peak was attributable to the existence of C adatoms or sp3-C bond over the surface. Additionally, the peak at 285 eV was attributed to defects such as nonconjugated carbon (nc-C) bonding in multi-walled CNTs and C–H bonding in hydrogenated graphite85.XPS measurements have been also used to evaluate the defect density in graphene89.As observed in Fig. 90. The quantity of C=C sp2 bonding in the hexagonal lattice determines the conjugation strength, which relates to the relative amplitude of this ShU signal. The relative area under the curve (AShU) was greater in 5 M sample, as shown in Fig. 2SO4, has the lowest total defect concentration whereas the sample 5M, pre-treated with higher acid concentration, has the largest vacancy and nc-C defects.As shown in Fig. C/O was calculated from the XPS data and found that it was about 1 for the 0 M sample, which increased to about 1.54 for 3 M and 2.98 for the 5 M, in agreement with the ID/IG value estimated from the Raman analysis  without the addition of any surfactant is a promising path for graphene exfoliation. We have also shown that the addition of an acid pretreatment is a simple and easy approach to increase the exfoliation yield of graphene. It is found to be very effective in overcoming the van der Waals forces and could generate thinner graphene flakes. As demonstrated, acid treatment with optimum molar concentration, such as 3 M or 5 M could introduce a sufficient number of functional groups to the graphene flakes, which can be further considered as a source material for the production of useful other graphene derivatives, such as holey graphene2.There are many techniques for the production and processing of graphene, but LPE is the simplest and most versatile method to produce stable dispersions. However, as Backes et al.The use of probe sonication allows the exfoliation of the graphene layers effectively. Increasing sonication time proves to be useful in thinning down the graphite flakes; however, it comes at the cost of reduction of the flake lateral sizes. This highlights the deteriorating side of probe sonication and limits its indefinite use in exfoliating graphene layers with high surface area. Despite the limitation, a trade-off could be made for an efficient use of the sonicating method. This could be beneficial for certain applications, such as controlling thermal reduction and pore creation or changing the inherent properties of the flakes.In this work, we have studied the exfoliation of graphene layers from graphite powder. Probe sonication was largely employed where parameters such as time, power, and centrifuge time were optimized to produce few-layer graphene flakes with size ˂ 1 µm. An intermediate sulfuric acid treatment was incorporated to increase the exfoliation yield. Increasing acidic concentrations produces thinner flakes but also increases the number of defects. Acidic pretreatment also introduces functional groups, which can be evident in the XPS studies. Thus, although sulfuric acid could help achieve thinner flakes, it inadvertently introduces certain functional groups and defects. Further, since the lateral size of the flakes decreased with increased acid concentration, we can conclude that the pretreatment with probe sonication not only promotes exfoliation between layers but also reduces their lateral size. This proves to be the major bottleneck for the full use of ultrasonic exfoliation, and a trade-off must be made between the flake size and thickness. Lastly, pretreatment with 3 M offers a 24% increase in graphene exfoliation yield with respect to the 0 M samples. The results also indicate that 3 M sample could be an ideal trade-off for obtaining thinner flakes with the highest yield and lower defect density. Further, the study and the understanding of the exfoliation process could be extended to other van der Waals materials for gaining thinner layers of 2D materials.Supplementary Information.


The production of highly stable, defect-free, and electricallyconducting 3D graphene structures from graphene oxide precursors ischallenging. This is because graphene oxide is a metastable materialwhose structure and chemistry evolve due to aging. Aging changes therelative composition of oxygen functional groups attached to the grapheneoxide and negatively impacts the fabrication and properties of reducedgraphene oxide. Here, we report a universal strategy to reverse theaging of graphene oxide precursors using oxygen plasma treatment.This treatment decreases the size of graphene oxide flakes and restoresnegative zeta potential and suspension stability in water, enablingthe fabrication of compact and mechanically stable graphene aerogelsusing hydrothermal synthesis. Moreover, we employ high-temperatureannealing to remove oxygen-containing functionalities and repair thelattice defects in reduced graphene oxide. This method allows obtaininghighly electrically conducting graphene aerogels with electrical conductivityof 390 S/m and low defect density. The role of carboxyl, hydroxyl,epoxide, and ketonic oxygen species is thoroughly investigated usingX-ray photoelectron and Raman spectroscopies. Our study provides uniqueinsight into the chemical transformations occurring during the agingand thermal reduction of graphene oxide from room temperature up to2700 °C. These steps can be performed simultaneously orone after the other. Although the GO-based methods are simple andversatile, there are challenges associated with them.Several synthesis methodshave been developed using GO as a startingmaterial for the preparation of 3D graphene materials  into graphene oxide aging and chemical transformationsto graphene occurring during the annealing from room temperature upto 2700 °C. We demonstrate that the high-temperature annealingat 2700 °C completely removes all the residual oxygen speciesand repairs intrinsic structural defects in the materials withoutaffecting their structure. As a result, we obtain highly mechanicallystable and conductive graphene aerogels with extremely low defectdensity. We demonstrate a practical and universal strategy to healdefects and improve the electrical properties of complex 3D graphenestructures without affecting their morphology.2 plasma environment for120 s, at 50 W, process pressure of 50 Pa with 50 sccm of O2 gas, and frequency f = 8.0 MHz (TESLA Rožnov)to produce pGO.The graphene aerogelswere prepared using different kinds of graphene oxide precursors:new GO, aged GO (aGO), and plasma-treated aged GO (pGO). The new GOpowder was purchased commercially from XFNano. The GO aging was performedby keeping the GO powder for 12 months in a sealed container to makeaGO. Then aGO was exposed to an O–1 mbar) at −70 °Cfor 16 h to remove the residual water content. The rGO aerogels beforethe annealing are termed nonannealed rGA.A GO and deionized (DI) water solution was preparedby mixing 2 mg/mL of the GO powders in DI water. A homogeneous dispersionsolution was obtained after ultrasonication for 30 min. Then the solution(30 mL) was sealed in a Teflon-lined autoclave of 50 mL size. Theautoclave was heated at 180 °C for 6 h to yield a self-assembledinterconnected 3D microporous network of reduced graphene oxide (rGO)hydrogels. The 3D rGO hydrogels were subsequently freeze-dried ina vacuum  was utilized with a graphite paperheating element that surrounded the annealed samples. All the annealingwas performed in the vacuum furnace at 2.3 × 10–4 mbar for 30 min. The annealing temperature was monitored by a pyrometer.The obtained freeze-driedrGA samples were annealed at differenttemperatures, i.e., 400, 750, 1000, 1300, and 2700 °C, to obtaingraphene aerogels (GA). For annealing, a homemade vacuum furnace (see Scanning electron microscopy(TESCAN MAIA3) was used to characterize the rGA and GA morphology.The materials were measured by X-ray photoelectron spectroscopy  and Raman spectroscopy (Renishaw inVia setupusing a 442 nm laser). Zeta potential and flake size measurementswere performed with a ZetaNano ZS  device equippedwith a He/Ne laser operating at 633 nm as a light source and an avalanchephotodiode as a detector. The DLS analysis used in this work is basedon the model which considers spherical particles. DC conductivitymeasurements were performed by linear four-point probe method by applyinga constant current of 0.05 mA using a constant current source  and measuring the voltage via a multimeter . The measurement was done by slightly pressing thesample (≤5% strain) on the top of predeposited electrodes toachieve good electrical contact.17 GO used in this study was purchased from commercial sources. Thatis why its properties can vary from batch to batch due to aging ordifferent storage conditions. In most cases, the purchased GO enabledformation of a stable hydrogel using hydrothermal synthesis. However,older GO affected by 1 year aging (aGO) did not yield a consolidatedgraphene hydrogel. Instead, distorted and broken pieces were producedfrom aGO precursors using the same hydrothermal synthesis parameters(rameters1. Some nWe have found that this issue could be overcomeby exposing themto oxygen plasma. The plasma-treated aGO (pGO) materials have formeda compact and stable rGO hydrogel using hydrothermal synthesis again. –1, which are associated with the D and G bands of graphene oxide.The ID/IG ratioof GO and aGO is 0.87 and 0.81, respectively. No major noticeablechange can be observed in the Raman spectra of GO compared to aGO.The FTIR absorbance spectra of GO and aGO (–1), hydroxyl , epoxide (C–O–C at 1200–1385cm–1), sp2-hybridized (C=C at1550–1650 cm–1), and ketonic species (C=Oat 1725–1810 cm–1).43 The main difference between the FTIR spectra of the GO and aGO samplesis observed in the relative intensities of the peaks. This observationshows a quantitative difference between the total content of oxygenand oxygen functional groups in the new and aged GO samples. However,due to the overlap of the peaks, quantitative analysis of the chemicalchanges is difficult and inaccurate from FTIR.43 Therefore, quantitative chemical analysis was done usingXPS.To understand the effects of the GOaging, a thorough examination of the structure and chemical compositionof GO before and after the aging was performed in  and aGO 2b revealTables S2–S4) confirmsthat the aged samples are slightly chemically different from the freshGO. The C 1s spectrum of the fresh GO  are located at 287.1 eV, and the carboxylspecies (COOH) are at 288.5 eV.46 The corresponding O 1s spectra with the deconvoluted C–O,C=O, and COOH peaks of the GO are shown in XPS analysis of GO and aGO (fresh GO 2c after Tables S3 and S4) reveal that the aging causes a loss of the oxygen-functionalizedcarbon, while the relative percentage of the sp2 + sp3 carbon content increases. A 7% decrease is observed relatedto the C–OH groups and an ∼1% decrease in the contentof C=O in aGO. Overall, the aging results in the loss of oxygenand modification of the oxygen-related functional groups in GO.After the aging, the C 1s and O 1s spectra 2c,d depiID/IG ratio down to 0.65 compared to theaGO and GO samples and improvement of the crystallinity of the sample.This observation can be explained by the partial removal of defectsrelated to oxygen functional groups with the plasma.40 There is no major difference in the FTIR spectrum of pGOwith respect to aGO. On the other hand, XPS exhibits notable chemicalchanges in pGO compared to aGO (Tables S2–S4). The deconvoluted XPS peaks in the C 1s and O 1s spectra show thatthe absolute oxygen content was decreased by 4% after the plasma treatment.This is mainly reflected in the decrease of hydroxyl (C–OH),epoxide (C–O), and ketonic (C=O) functional groups.However, the relative content of carboxyl (COOH) was increased from6.1 to 8.3% in pGO compared to aGO (see Table S3). The amount of sp2-bonded carbon is also increasedby the plasma treatment. Interestingly, the amount of defect-relatedsp3 carbon remains unchanged in the pGO after plasma treatment,which is in line with the Raman observation.The structural and chemical compositionanalysis of the plasma-treated aged graphene oxide (pGO) in 2 plasma treatment.Most of the oxygen species are diminished in the GO samples, exceptfor COOH, which is increased. As the carboxyl groups are negativelycharged, this observation suggests that a small increase in the COOHspecies by plasma is sufficient to recover the highly negative chargeof aGO and improve the solubility in water, as demonstrated below.However, it needs to be highlighted that it is challenging to drawany concrete conclusion from the structural, vibrational, and compositionalanalysis due to the insufficient sensitivity of the spectroscopictechniques to the carbon–oxygen bonds.3 This is because, in reduced GO, all carbon atoms in the defectiveregions are bonded to three neighbors that maintain a planar sp2 configuration, making them undetectable by spectroscopictechniques.32 Therefore, the GO properties,such as the surface charge and flake size distribution, influencedby the aging and plasma treatment are analyzed in water solutionsin the next section.From XPS analysis,the following conclusion can be derived. There are minor compositionalchanges in graphene oxide caused by the OAs the hydrothermalsynthesis of GA is started with an aqueous solution of GO, it is necessaryto have good colloidal stability of GO suspensions in water for thesuccessful preparation of 3D graphene hydrogels. 47Dynamic light scattering (DLS) and zeta potentialmeasurements3b–dreve48 The fresh GO solution exhibits a broad distributionof zeta potentials with peak boundaries from −66 to −7mV and a peak center at −39 mV  are inherited from the starting GO material because it containsa high density of sp3-hybridized carbon bonds due to theadsorbed oxygen species.31 Some intrinsicdefects, including lattice/topological and edge defects, are thenproduced during the ultrasonication and reduction of GO during thehydrothermal synthesis of rGA due to the loss of oxygen functionalgroups.56 3D porous graphene structuresmade of interconnected graphene sheets also contain other types oflonger-range defects, such as pore defects, cracks, and lack-of-fusionpore structures . The high-temperatureannealing can significantly improve the electrical conductivity (σ)of the aerogels (58 At lower temperatures (≤1000 °C), thechange in σ is low due to the presence of numerous oxygen-relatedfunctional groups. The electrical conductivity increases significantlyat high temperatures, demonstrating σ of ∼390 S/m at2700 °C. This electrical conductivity is one of the highest thatwas reported in the literature for graphene aerogels.17In 60 The ratioof the intensity of the D to G peaks can be used for the determinationof the defect density61 (nD) and crystallite size (La) using the equation defined by Cancado et al.62 For the nonannealed rGA, the crystallite size is foundto be around 10 nm, and the density of defects is roughly 4.3 ×1011 cm–2  and C=C(sp3) hybridized carbon atoms along with several carbon–oxygenfunctional groups, such as C–O , C=O , and O–C=O .66 The rGA sample is composed of 89% of carbon and 11% of oxygen. Annealingof the GA at 400 °C shows almost no change in the composition.Once the annealing temperature is increased to 750 °C, the relativecontent of carbon is increased to 96% and oxygen is decreased to 4%.When the aerogel is further annealed at 1000 °C, the carbon andoxygen content remains almost the same as in the GA annealed at 750°C. A significant reduction of oxygen is observed after 1300°C annealing. The GA sample annealed at 1300 °C has >99.4%of carbon and <0.6% of oxygen content. The oxygen is completelyremoved from the sample when the GA is annealed at 2700 °C (Table S5). After this high-temperature annealing,a highly mechanically stable and intact graphene aerogel is obtainedat 9.1 eV. The experimentally obtained values from our XPS measurementsare in line with this sequence of the theoretical binding energies.The amounts of different oxygen species at a specific annealing temperaturein the GA samples determined from XPS are summarized in Tables S6 and S7. The experiments show that hydroxylgroups are removed first. As a result, the GA sample annealed to 1000°C contains mainly carboxyl and ketonic species.69 After 1300 °C annealing, all the remaining oxygenspecies are removed from the sample.72 As no more oxygen functionalgroups are bound to the GA samples at temperatures above 1300 °C,the observed increase of the sp2 carbon content in GA between1300 and 2700 °C  annealingwas able to remove only hydroxyl species from the aerogels. The completeremoval of oxygen species was achieved at temperatures above 1300°C. Furthermore, it is observed that even after the completeremoval of oxygen from GA, the electrical conductivity is still limiteddue to the presence of intrinsic structural defects. The crystallographicdefects can be up to a large extent repaired by heating the aerogelsat extremely high temperatures (≥2700 °C). The high-temperatureannealing is found as an effective strategy to heal defects and improvethe electrical properties of complex 3D graphene structures withoutaffecting their morphology.In this work, we investigated the effectsof the aging and deoxidationof graphene oxide in the synthesis of complex 3D graphene aerogelstructures. We also demonstrated a universal strategy to reverse theaging and remove defects using O


Idiopathic pulmonary fibrosis (IPF) refers to chronic progressive fibrotic interstitial pneumonia. It is called a “tumor-like disease” and cannot be cured using existing clinical drugs. Therefore, new treatment options are urgently needed. Studies have proven that ferroptosis is closely related to the development of IPF, and ferroptosis inhibitors can slow down the occurrence of IPF by chelating iron or reducing lipid peroxidation. For example, the ferroptosis inhibitor deferoxamine (DFO) was used to treat a mouse model of pulmonary fibrosis, and DFO successfully reversed the IPF phenotype and increased the survival rate of mice from 50% to 90%. Given this, we perceive that the treatment of IPF by delivering ferroptosis inhibitors is a promising option. However, the delivery of ferroptosis inhibitors faces two bottlenecks: low solubility and targeting. For one thing, we consider preparing ferroptosis inhibitors into nanomedicines to improve solubility. For another thing, we propose to deliver nanomedicines through pulmonary drug-delivery system (PDDS) to improve targeting. Compared with oral or injection administration, PDDS can achieve better delivery and accumulation in the lung, while reducing the systemic exposure of the drug, and is an efficient and safe drug-delivery method. In this paper, three possible nanomedicines for PDDS and the preparation methods thereof are proposed to deliver ferroptosis inhibitors for the treatment of IPF. Proper administration devices and challenges in future applications are also discussed. In general, this perspective proposes a promising strategy for the treatment of IPF based on inhalable nanomedicines carrying ferroptosis inhibitors, which can inspire new ideas in the field of drug development and therapy of IPF. Idiopathic pulmonary fibrosis (IPF) is a specific form of chronic progressive fibrotic interstitial pneumonia having complicated causes .Its path® trials, nintedanib 150 mg twice daily , and promotion of lipid peroxidation . At presTherefore, inhibition of ferroptosis will possibly emerge as a promising approach to the treatment of IPF. Encouragingly, increasing studies have demonstrated the inhibitory efficacy of ferroptosis inactivation on IPF, as detailed below. 2-induced weight loss. Compared with the control group, the body weight of mice in the model group was sharply reduced by 10 g. When DHQ was treated, the body weight of mice was reduced by only about 5 g compared with the control group. Also, the expression of IPF biomarkers such as α-SMA, collagen I, and fibronectin was significantly reduced in the DHQ-treated mice. These results further demonstrated that by inhibiting ferroptosis, DHQ could greatly reduce the extent of SiO2-induced inflammation and fibrosis in lung tissue. This evidence suggests that ferroptosis inhibitors might provide potential targets for the treatment of IPF. Cheng et al.  found abFerroptosis inhibitors have emerged as a promising remedy to slow down the progression of IPF. Currently, classical ferroptosis inhibitors usually include the iron chelator DFO, the aromatic amine antioxidants Fer-1  and liprHowever, there are still two bottlenecks that restrict the delivery of these ferroptosis inhibitors to treat IPF. Firstly, the molecular structure of ferroptosis inhibitors often contains aromatic cycles, which leads to poor drug solubility and low cellular uptake. Moreover, conventional oral or injectable administration of ferroptosis inhibitors confronts the problems of focal accumulation and poor selectivity in lung area, due to the first-pass effect and off-target effects. Therefore, designing a drug-delivery system with high solubilization and targeting capacity is of great significance for the ferroptosis inhibition-based treatment of IPF.In recent years, the advancement of nanotechnology has opened up new avenues for the field of drug delivery. Various nanoplatforms have been reported to improve drug solubility via solubilization or accommodation effects, such as micelles , liposomPulmonary drug delivery system (PDDS)  refers tTaken together, engineering ferroptosis inhibitors as inhalable nanomedicines may provide a promising therapeutic regimen for IPF, and further considerations should be taken to promote such inhalable nanomedicines from bench to bedside.® , Arikayce® , and Theodur® (theophylline sustained-release tablets)]. The following content will briefly summarize the major preparation methods of these nanomedicines.Currently, there is a multitude of inhalable nanomedicines to load ferroptosis inhibitors. Considering the industrialization applicability, micelles, liposomes, and nanocrystals are the most promising nanomedicines, as relevant products have witnessed commercialization [such as CequaMicelles refers to surfactant molecules in an aqueous solution that self-assemble to form an orderly arrangement of thermodynamically-stable colloidal aggregates, when the surfactant reaches a certain concentration. The micelles that are mainly studied at present are formed by amphiphilic polymer materials, and thus they are also called polymeric micelles. Polymeric micelles have the advantages of small particle size, stable structure, and strong solubilization ability. The current methods for preparing polymeric micelles include solvent-evaporation method, solid-dispersion method, dialysis method, and so on. Solvent evaporation is a widely-used approach to prepare micelles, which typically includes emulsification-solvent evaporation and self-assembly-solvent evaporation. Of note, compared with the solid-dispersion technology, micelles prepared by solvent evaporation have better stability. Compared with the dialysis method, micelles prepared via solvent evaporation have a narrower particle-size distribution, fewer side reactions, and easier operation . The emuIn summary, we believe that the best method to prepare micelles is solvent evaporation. This method not only has a simple process, but also prepares nanomicelles with narrower particle-size distribution and better stability.® , Emend® (arepitant capsules), and Tricor® (fenofibrate tablets). Nanocrystals may also be used for respiratory drug delivery, which has been widely reported in the literature. Ming et al. [Nanodrug crystals are stable nano-colloidal dispersion systems formed by drugs with the help of a small amount of stabilizer , suspending nano-scale drug molecules in a dispersion medium. The usual methods for preparing nanocrystals are “bottom-up” and “top-down” techniques, also known as self-assembly techniques and comminution techniques. The “bottom-up” technology focuses on the process of drug precipitation and nanocrystal formation, including precipitation method, supercritical fluid technology, emulsification method, and microemulsion method. Compared with the “top-down” technology, the “bottom-up” technology has the advantages of simple operation and low cost. The nanocrystals prepared by “bottom-up” technology have poor physical stability, which affects their bioavailability. At the same time, the residue of the organic solvent used in the preparation process also seriously affects the safety of the preparation. Therefore, there are no products of this technology yet. “Top-down” technology can be utilized for producing nanocrystals  by reducg et al.  preparedg et al.  designedTo sum up, from our point of view, the best way to prepare nanocrystals is the “top-down” technique. The “top-down” technique has a simple process and strong applicability, and the particle size of the drug nanocrystal product prepared is controllable. Besides, it has good reproducibility, and is easy to scale up production.Liposomes are a type of ultrafine spherical particle with a bilayer structure formed by self-aggregation of phospholipids as a wall material in water. Due to the amphiphilic nature of the lipids in liposomes, they can be used as candidate carriers for delivering drugs. Methods for preparing liposomes include thin-film hydration, mechanical stirring, reverse-phase evaporation, solvent injection, etc. Thin-film hydration is a widely used method for preparing liposomes . It refeFrom the above discussion, we believe that the thin-film hydration method is the best solution for the preparation of liposomes. The thin-film hydration method is of simple operation. In addition, liposomes prepared by the thin-film hydration method have high stability, which allows safe and leak-free drug delivery to target organs.Herein, we presented three promising nanomedicines and the advantages and challenges of different preparation methods. The authors argued that liposomes might be the most promising and robust nanomedicines for carrying ferroptosis inhibitors. The reasons are as follows.In extensive studies of PDDS, it has been found that the vast majority of drug monomers are metabolized and cleared within four hours after direct PDDS administration, thus requiring frequent multiple doses to maintain efficacy . It was Compared to micelles, liposomes possess better physicochemical stability. Therefore, liposomes can be protected from environmental factors and protected from enzymatic degradation before reaching the lesion site, which can improve drug stability, reduce drug toxicity, and exert better therapeutic effects. Additionally, the components (mainly phospholipids and cholesterol) and structure (lipid bilayer) of liposomes are similar to cell membranes, which makes them highly biocompatible and biodegradable . CompareNumerous studies have been performed to achieve better lung targeting by modifying liposomes to target surface molecules to specific cells in the lung. Yan et al.  develope® . It is an inhalable liposome nanosuspension for the treatment of non-tuberculous mycobacterial (NTM) lung disease caused by Mycobacterium avium and was launched in 2018 [It was worth mentioning that there are already-marketed inhalable liposomes such as the abovementioned Arikayce in 2018 . This prAs mentioned above, thin-film hydration is the most suitable preparation method for liposomes in our opinion, and hence we suggest that researchers in the field adapt this method for inhaled liposome development.The next consideration is bench-to-bedside translation. To render inhalable liposomes applicable in clinical application, a PDDS device should be employed. Currently, the most commonly used PDDSs in clinical practice are metered-dose inhalers (MDIs), dry powder inhalers (DPIs), nasal spray, and nebulizers ,50,51,52MDIs mean that the drug and the propellants  are enclosed together in a pressurized container with a dosing valve, and are sprayed out by means of an external force, which have the advantage of being stable, cheap, and simple to install . HoweverDPIs refer to a preparation in which the dry powder of the drug itself or the dry powder of the drug mixed with a carrier , stored in a capsule or a multi-dose reservoir, is inhaled actively by the patient using a special device . CompareNasal sprays refer to nasal liquid formulations made from a clear solution, suspension, or emulsion of raw drug and suitable excipients  for atomization . Compare®) and aminotransol inhalation solution (Cayston®).Nebulizers are preparations in which a drug-containing solution is nebulized into small droplets by a nebulizer for inhalation . CompareTo conclude, nebulizers are the optimal PDDS device to deliver liposomes loaded with ferroptosis inhibitors for the treatment of IPF, bringing benefits to IPF patients. We recommend that relevant investigators pay more attention to this system.In summary, we propose that inhibiting ferroptosis could slow down the pathogenesis of IPF, and that formulating a ferroptosis inhibitor as an inhaled nanomedicine holds great promise for the treatment of IPF. In addition, we also described a series of classical nanoengineering technologies to construct the inhalable nanosized ferroptosis-inhibiting systems, such as micelles, liposomes, and nanocrystal preparations.Although engineering ferroptosis inhibitors as inhalable nanomedicines for the highly efficient treatment of IPF is promising, there are some concerns about the safety of ferroptosis inhibitors and inhalable nanomedicines. First of all, ferroptosis is a recently discovered type of cellular death that generally occurs in various types of cells, that is, ferroptosis can occur in both normal cells and IPF-related cells. In the case of IPF, inhibition of ferroptosis in diseased alveolar cells slowed the progression of IPF, but the consequences of inhibiting ferroptosis in normal cells could not be assessed. Therefore, the safety issues posed by the delivery of ferroptosis inhibitors still need to be further explored. Secondly, drug delivery systems such as micelles, nanocrystals, and liposomes raise some important issues. For example, after the micelles enter the human body, their stability is greatly weakened, which leads to the premature release of the drug and the loss of targeting effect, while the effective drug content has not yet accumulated in the lesion. In addition, premature drug-release will have toxic side effects on normal cells. For nanocrystals, there is a general problem of Ostwald ripening in the nanocrystal systems, which causes crystal growth of the product and formation of large-scale crystals, affecting the particle size and its distribution. At the same time, the residual organic solvents can bring serious toxic side effects. As to liposomes, it is not yet clear whether the membrane-fusion phenomenon occurs upon contact with IPF-related and normal cells provokes an unpredictable outcome. How to solve these problems will also be the subject of our long-term exploration. In the future, we will experimentally verify the therapeutic effect of inhalable ferroptosis inhibitor-loaded nanomedicines on IPF. With the in-depth study of ferroptosis and inhaled nanomedicines, especially the industrialization and translation aspects, it is foreseeable that inhaled ferroptosis inhibitors may provide alternative treatment options for IPF.


We read with interest the recent study by Gilson and colleagues , “How DoWe evaluated this issue of varying responses using all questions from the most recent quarterly, online, open-book American Board of Preventive Medicine (ABPM) pilot evaluation of a longitudinal assessment program for the maintenance of certification of its clinical informatics diplomates. We evaluated ChatGPT, version 3.5 (OpenAI), and Google Bard (Alphabet Inc) by copying and pasting each of the 12 questions and the corresponding 4-part multiple-choice options into the chatbots’ message boxes on March 30, 2023, and April 1, 2023, respectively. We added a request to provide citations for each question. Both chatbots supplied the option they considered best, with a justification, references, and an explanation as to why each option was either incorrect or inferior to the recommended answer.For ChatGPT, the series of 12 questions was performed 10 times in separate chat sessions to avoid memory effects from a previous search, with each session scored against the answer key provided by the ABPM. The results showed that out of the 12 questions, there were 9 sessions where 8 correct responses were achieved and 1 session where 9 correct responses were achieved. Although 8 questions had perfect (10/10) concordance with the answer key, there were 2 questions with 2 different answers and one with 3 different answers. There was a twelfth question where the same answer was provided for each session that disagreed with the answer key. These scores were at least as good as the average performance of the diplomates participating in the maintenance of certification process , which allows the use of online resources, and likely would have represented a passing score. We also evaluated the experimental ChatGPT, version 4.0, in 5 separate chat sessions, which produced sequential scores of 10, 8, 8, 6, and 7. For Google Bard, the process was performed 9 times, and the most common answer was selected as the best response. The modal responses were correct for 7 out of 12 questions . There were 5 questions for which 2 different answers were provided and 1 question for which all 4 answers were provided as correct answers during different sessions. Google Bard agreed with the ABPM answer key for only 4 questions in all sessions.The questions where the large language models consistently disagreed with the ABPM answer key were either based on low-level evidence or involved an opinion on a “best” approach. As implied by Gilson et al , these d


The significant association between the nadir SpO2 and IPF or non-IPF ILD grouping remained even after adjusting for gender, age, body mass index, lung function, 6MWD, and dyspnea . Conclusion: Even after adjusting for confounding factors, IPF patients had lower nadir SpO2 during 6MWT. Early assessment of exercise desaturation using the 6MWT may be more important in patients with IPF compared with patients with other ILDs.Objective: Interstitial lung disease (ILD) is classified into several disease groups. Among them, idiopathic pulmonary fibrosis (IPF) has higher incidence and poor prognosis; therefore, it is important to characterize specific IPF symptoms. Exercise desaturation is a strong factor related to mortality in patients with ILD. Thus, the purpose of this study was to compare the degree of oxygen desaturation between IPF and other ILD (non-IPF ILD) patients during exercise, using the 6-minute walk test (6MWT). Methods: This retrospective study included 126 stable patients with ILD who underwent 6MWT in our outpatient department. The 6MWT was used to assess desaturation during exercise, 6-minute walk distance (6MWD), and dyspnea at the end of exercise. In addition, patient characteristics and pulmonary function test results were recorded. Results: Study subjects were divided into 51 IPF patients and 75 non-IPF ILD patients. The IPF group had significantly lower nadir oxygen saturation determined by pulse oximetry (SpO Interstitial lung disease (ILD) comprises a group of diverse diseases that damage the lung parenchyma through inflammation and fibrosis. ILD is divided into several disease groups, with different treatment goals and assessment methods1). Idiopathic pulmonary fibrosis (IPF) is the most frequent form of ILD and has a very poor prognosis with a median survival time of 2–3 years from diagnosis2). In contrast, non-IPF ILDs, such as idiopathic nonspecific interstitial pneumonia (NSIP), connective tissue disease-associated ILD, and hypersensitivity pneumonitis, usually have a better prognosis than IPF3). Hence, it is important to characterize the specific symptoms in IPF patients compared with those in non-IPF ILD patients.5). The 6-minute walk test (6MWT) is an inexpensive and simple test that assesses desaturation during exercise and has been shown to be reproducible in patients with ILD7). Desaturation during exercise is a strong risk factor for mortality in ILD patients12) and is also associated with quality of life14). A few studies have assessed the difference in desaturation during exercise between IPF patients and non-IPF ILD patients, but no adjustment was made for some important confounding factors15). Moreover, several studies reported controversial results on whether desaturation during exercise is more severe in patients with IPF17); hence, this relationship remains unclear. Clarifying the differences in desaturation during exercise between patients with IPF and patients with non-IPF ILD will be useful in determining how to evaluate IPF patients and help determine the appropriate treatment strategy.Chronic hypoxemia influences prognosis in patients with ILD. Many patients with ILD present with severe desaturation during exercise even if they do not have hypoxemia at restTherefore, the purpose of this study was to compare the degree of oxygen desaturation in IPF and non-IPF ILD patients during exercise, using the 6MWT.1). Assessment items for the analyses were age, gender, the modified British Medical Research Council (mMRC) scale, comorbidities, treatment, smoking status, 6MWT, and pulmonary function tests.This retrospective study included 195 stable patients with ILD who underwent the 6MWT as part of an examination at our outpatient clinic at Kobe City Medical Center West Hospital (Japan) between April 2016 and March 2020. No respiratory rehabilitation was performed before or after 6MWT because it was performed to assess physiological status of the patients. Among these, patients with long-term oxygen therapy, active coronary artery disease, other severe comorbidities, and missing data were excluded. The classification of ILD, including the diagnosis of IPF, was made using the American Thoracic Society/European Respiratory Society (ATS/ERS) consensus statementThis study was approved by the Institutional Review Board  of Kobe City Medical Center West Hospital with waiver of informed consent because of its retrospective nature .18). Patients were instructed to walk as much as possible in 6 min. Oxygen saturation determined by pulse oximetry (SpO2) during 6MWT was continuously recorded using a pulse oximeter with a finger probe  and 6MWT analysis software . To minimize errors in SpO2 measurement, patients were instructed to hold the finger with the pulse oximeter attached to the front of their chest while walking. SpO2 was also measured at rest for 1 min prior to the test and for 3 min immediately after. Patients were asked to rate their dyspnea every minute during 6MWT, using the modified Borg scale scores by selecting a number from 0 to 10, with 0 being no appreciable dyspnea and 10 being maximal sustained dyspnea19). From the 6MWT, 6-minute walk distance (6MWD), rest SpO2, nadir SpO2, ΔSpO2 (rest SpO2 − nadir SpO2), maximum heart rate, and modified Borg scale scores during 6MWT were obtained.The subjects performed 6MWT on a flat, 25-m walking course according to the ATS statement20). Vital capacity (VC), forced vital capacity (FVC), and forced expiratory volume in 1 s (FEV1) were evaluated. Predicted normal values for the Japanese population were derived from reference values of the Japanese Respiratory Society21) for VC, FVC, and FEV1.All patients were subjected to pulmonary function tests by spirometry  according to the method described in the ATS 1994 update2 and IPF or non-IPF ILD groups. Additionally, multiple regression analysis was performed with the nadir SpO2 as the dependent variable and IPF diagnosis as the independent variable to evaluate the relationship after adjusting for potential covariates. Dummy variables were created with patients with IPF as 0 and non-IPF ILD as 1. These were entered into the regression analysis. Gender, age, body mass index (BMI), chronic obstructive pulmonary disease (COPD) or non-COPD, 6MWD, %VC, and mMRC scale scores were included as confounders because they are assumed to be involved in desaturation23). p-value <0.05 was considered significant.Statistical analysis was performed with EZR Ver. 1.38 . Subjects were categorized into the IPF group or the non-IPF ILD group. Continuous variables were presented as mean ± standard deviation and were compared using unpaired t, Welch’s t, and Mann–Whitney U tests between the two groups. A simple regression analysis was performed to examine the relationship between the nadir SpOAmong 195 patients with ILD who underwent 6MWT, 18 patients were on long-term oxygen therapy and 51 cases had missing data. Therefore, 126 cases were included in the analysis. Fifty-one patients (40%) were diagnosed with IPF and the other 75 patients (60%) had non-IPF ILD. Non-IPF ILD included collagen disease, n = 12 (16.0%); hypersensitivity pneumonitis, n = 10 (13.3%); idiopathic pleuroparenchymal fibroelastosis, n = 6 (8.0%); asbestosis, n = 6 (8.0%); idiopathic NSIP, n = 3 (4.0%); sarcoidosis, n = 2 (2.7%); cryptogenic organizing pneumonia, n = 1 (1.3%); and unclassifiable, n = 35 (46.7%). p = 0.50) and %FVC , between the IPF group and the non-IPF ILD group. In addition, no significant differences were observed in the other pulmonary function measures or patients characteristics between the two groups, with the exception of treatment modalities used for IPF and non-IPF ILD.2 during 6MWT than the non-IPF ILD group . The IPF group also had significantly higher ΔSpO2 during 6MWT than the non-IPF ILD group . In contrast, no significant difference was observed in the dyspnea scale at the end of 6MWT  and the 6MWD  between the two groups.2 over time during the 6MWT.p = 0.02) between nadir SpO2 and IPF or non-IPF ILD grouping (p <0.05) remained even after adjustments for gender, age, BMI, COPD or non-COPD, 6MWD, %VC, and mMRC scale score . Their finding supports our results, but the study did not adjust for any confounding factors. Another study comparing SpO2 during 6MWT between patients with IPF and patients with idiopathic NSIP reported that there was no significant difference5). Although there were only two cases of idiopathic NSIP in the present study, the results could not be simply compared, and it is necessary to classify non-IPF ILD patients in detail and compare the results using 6MWT in the future.The results of this study indicate that patients with IPF tended to have severe desaturation during exercise despite not having hypoxemia at rest. Holland et al. reported that nadir SpO17). Although these results were similar to our findings, 6MWT is a more clinically feasible and simpler exercise stress test than using a bicycle ergometer. In addition, they also did not adjust for confounding factors. Therefore, this study expanded on the results of these two studies and indicated that similar results can also be obtained with 6MWT, which is simple and easy to administer in the clinical settings.We performed the 6MWT to assess desaturation during exercise in patients with ILD. In two previous studies, wherein a bicycle ergometer was employed as an exercise stress test, IPF patients were shown to exhibit more severe desaturation during exercise than non-IPF ILD patientsCO) is more likely to decline earlier compared with %FVC. In fact, it was reported that patients with IPF had lower DLCO than patients with non-IPF ILD24). Ventilation–diffusion inequality and diffusion limitation play an important role in desaturation at rest and during exercise25). A number of studies showed that low DLCO is strongly associated with desaturation during exercise. For example, Du Plessis et al. reported that DLCO was a predictor of nadir SpO2 during 6MWT among ILD patients22). In another study of 300 patients with ILD, a simple regression analysis identified DLCO as the explanatory factor for decreased oxygen saturation during exercise26). Therefore, we speculated that early decline in DLCO in IPF patients could lead to more severe desaturation during exercise. However, this study did not have sufficient data on DLCO, which is a future challenge.More severe desaturation during exercise in patients with IPF might be explained by more severe diffusion limitation. IPF is a progressive disease, and diffusing capacity for carbon monoxide (DL9). Our results indicated that IPF patients have more severe exercise desaturation compared with non-IPF ILD patients. Therefore, early assessment of exercise desaturation using 6MWT may be useful, especially in patients with IPF who do not have hypoxemia at rest. In addition, the 6MWT does not require much time or complex equipment to implement; therefore, the test is easy to perform clinically.Desaturation during exercise is a strong prognostic indicator in patients with IPF28). For that reason, these can be confounding factors. However, in the present study, there was no difference in the maximum pulse rate during the 6MWT, which is related to the circulatory response during exercise, so the difference in circulatory dynamics may not have been significant between IPF and non-IPF ILD patients.Our study has some limitations. First, this was a single-center cross-sectional study and may not represent the general characteristics of IPF patients. Therefore, further multicenter studies with larger samples are needed. Second, we did not evaluate the presence of pulmonary hypertension, right heart pressure, or other circulatory dynamics. It has been reported that patients with pulmonary hypertension present with severe exercise hypoxia2 during 6MWT. Early assessment of exercise desaturation using the 6MWT might be more important in patients with IPF than in patients with non-IPF ILD. Even if the resting SpO2 is good, it may be important to perform 6MWT more aggressively in patients with IPF to detect a decrease in oxygen saturation during the test, which is a prognostic factor at an early stage.The results of this study showed that even after adjusting for confounding factors, IPF patients had lower nadir SpOwww.enago.jp) for the English language review.The authors would like to thank Kiyohiko Kado, Chisato Nagatani, Takashi Kadooka, Shoko Mitani, Takuya Okamoto, Kumi Matumura, Ryoya Fujisaki, Yuya Kawate, Suzuka Higashiyama, Takumi Yamaguchi, Yutaro Oki, Yukari Oki, and Haruko Yamamoto for cooperation in acquisition of data and constructive comments on this paper. The authors would also like to thank Enago (There is no conflict of interest to disclose.


Hypoxia, a state of insufficient oxygen availability, promotes cellular lactate production. Lactate levels are increased in lungs from patients with idiopathic pulmonary fibrosis (IPF), a disease characterized by excessive scar formation, and lactate is implicated in the pathobiology of lung fibrosis. However, the mechanisms underlying the effects of hypoxia and lactate on fibroblast phenotype are poorly understood. We exposed normal and IPF lung fibroblasts to persistent hypoxia and found that increased lactate generation by IPF fibroblasts was driven by the FoxM1‐dependent increase of lactate dehydrogenase A (LDHA) coupled with decreased LDHB that was not observed in normal lung fibroblasts. Importantly, hypoxia reduced α‐smooth muscle actin (α‐SMA) expression in normal fibroblasts but had no significant impact on this marker of differentiation in IPF fibroblasts. Treatment of control and IPF fibroblasts with TGF‐β under hypoxic conditions did not significantly change LDHA or LDHB expression. Surprisingly, lactate directly induced the differentiation of normal, but not IPF fibroblasts under hypoxic conditions. Moreover, while expression of GPR‐81, a G‐protein‐coupled receptor that binds extracellular lactate, was increased by hypoxia in both normal and IPF fibroblasts, its inhibition or silencing only suppressed lactate‐mediated differentiation in normal fibroblasts. These studies show that hypoxia differentially affects normal and fibrotic fibroblasts, promoting increased lactate generation by IPF fibroblasts through regulation of the LDHA/LDHB ratio and promoting normal lung fibroblast responsiveness to lactate through GPR‐81. This supports a novel paradigm in which lactate may serve as a paracrine intercellular signal in oxygen‐deficient microenvironments. These molecules serve as proton donors for the oxygen‐dependent electron transport chain and the efficient generation of ATP  is converted to lactate through anaerobic metabolism which generates ATP in a much less efficient manner. Lactate dehydrogenase (LDH), an NADIn some circumstances, best studied in the context of cancer as the “Warburg effect” or “aerobic glycolysis,” pyruvate is converted to lactate even in the presence of sufficient oxygen. Recently, this phenomenon has been characterized as “metabolic reprogramming” in lung fibroblasts stimulated with the pro‐fibrotic cytokine transforming growth factor beta 1 (TGF‐β1)  that is induced in control and IPF fibroblasts under hypoxic conditions in a TGF‐β‐independent manner and regulates lactate‐induced myofibroblast differentiation in control fibroblasts. Taken together, our studies support a novel paradigm in which hypoxic conditions induce IPF fibroblasts to produce extracellular lactate which may then function to directly propagate myofibroblast differentiation in “normal” fibroblasts within the same environment.22.1Lung tissues were removed at the time of transplantation or death from non‐IPF and IPF patients. The tissue samples were stripped of all identifiers and designated as waste (exemption 4). Written informed consent was obtained on all patients or their surrogates prior to the procedure being performed. Use of human lung tissues was approved by the Institutional Review Board (IRB) at the Ohio State University (protocols 2017H0309 and 2021H0180) and all identifiers other than the diagnosis  were removed from the specimens provided. Normal fibroblasts were isolated from the normal lungs of decedents with no evidence of pulmonary disease who were HIV, hepatitis C, and SARS‐CoV‐2 negative and had no evidence of significant trauma. For the preparation of IPF and control lung fibroblasts, the visceral pleura was dissected and removed. The parenchyma was washed with PBS to eliminate RBCs. Minced tissue was transferred to GentleMACs C‐Tubes containing the enzymatic cocktail  and incubated at 37°C. After 1 h, cold serum was added to reduce the enzymatic activity. The filtered lung cell suspension was resuspended in RBC lysis solution, incubated for 7 min, and plated in a 75 cm flask for 24 h. Nonadherent cells were removed, and fresh media was added every 2 days. Experiments were conducted with isolated control and IPF fibroblasts between passages 4 and 7.2.25 control and IPF fibroblasts cultured in the normoxic or hypoxic chambers for 4 days were treated with the GPR‐81 antagonist 3 hydroxy butyric acid  or TGF‐β (2 ng/mL) for an additional 24 h. For silencing, control and IPF fibroblasts were transfected with 50 nM FoxM1, LDHA, or GPR‐81 siRNA  using Lipofectamine 3000 . Scrambled siRNA was used as a negative control . Control and IPF fibroblasts (3 × 105 cells/30 mm cell culture dish) were lysed with 1 × cell lysis buffer  containing protease inhibitor  and phosphatase inhibitor , and western blotting was performed. Cell lysates were collected, sonicated on ice for 15 s, and then denatured using 3× Laemmli buffer at 95°C for 3 min. Protein quantification was conducted by DC™ Protein Assay Kit I . Protein samples (5 to 15 μg) were loaded and separated through the gradient polyacrylamide gels (Novex WedgeWell Tris‐Glycine Gel) having 4%–20% polyacrylamide concentration . Precision Plus Protein Kaleidoscope Prestained Protein Standards  were used as a protein marker. Proteins on the gel were then electrically transferred to a PVDF membrane  using a Protean III tank transfer system (Bio‐Rad). After blocking with 5% bovine serum albumin in 1× TBST  for 1 h at room temperature, the membrane was incubated with antibodies  as follows: FoxM1  for antigen retrieval. All slides were then transferred to a humid staining rack in distilled water. All tissue on slides were circled with an immunopen, and then two drops of TBS were added to each slide and left to sit for 5 min. Endogenous peroxidases were quenched with 3% hydrogen peroxide in PBS for 5 min and sections were incubated with diluted normal swine serum to block nonspecific binding of secondary antibodies for 15 min at room temperature. The slides were incubated overnight at 4°C with 1:200 dilution of LDHB  or 1:100 dilution of GPR‐81 . After incubation, the slides were washed with PBS and then incubated with goat anti‐rabbit biotin‐conjugated secondary antibody  for 60 min (1:1500 dilution). After washing with TBS, the slides were incubated in HRP‐Streptavidin solution (Vector Laboratories) for 45 min and subjected to the application of the chromogen substrate 3, 3′ Diaminobenzene for 3 min and counterstained with hematoxylin. Slides were then put into a water bath with running tap water and then were rinsed in PBS before being dehydrated through a graded series of ethanol. Slides were then put into xylene before being treated with permanent mounting medium and coverslips being applied. Slides were then left to dry overnight. Images were obtained using a EVOS M7000 microscope (Invitrogen) and processed with Celleste Image analysis version 5.0 software. Control specimens were processed under the same conditions without the primary antibody to produce negative control (NC) sections.2.32, 5% CO2, and 94% N2. Control and IPF fibroblasts (2 × 104 cells/well of a 96‐well plate) were cultured in serum containing DMEM medium under the normoxic and hypoxic conditions for 5 days. To minimize the potential for reoxygenation, the cells were cultured in the hypoxic conditions without interruption. To precisely measure extracellular lactate levels, control and IPF fibroblasts were incubated in the hypoxic chamber for 5 days without changing the medium. For the measurement of cell viability, control and IPF fibroblasts cultured under normoxic and hypoxic conditions as indicated were incubated with 20 μL of Cell Titer Blue reagent  for 3 h, and cell viability was measured at 560 nm (Ex)/590 nm (Em) of fluorescence using a 96‐well plate reader . Extracellular lactate levels were measured using a Lactate Assay kit . Briefly, extracellular medium derived from control and IPF fibroblasts cultured under the conditions as described above was collected. Lactate standard was prepared and the final concentration of lactate (ng/μl) was measured according to the manufacturer's protocol.Control and IPF fibroblasts were cultured at 37°C in a modular hypoxic chamber  flushed with a gas mixture containing 1% O2.4t test (two‐tailed), and two sample equal variance was used. Significance level was set at p < 0.05.Data are expressed as the means ± SD. Two‐dimensional column graphs were prepared using Microsoft Excel. Protein expression levels are also presented as a box‐whisker plot showing the lowest expression, lower quartile, median, upper quartile, and the highest expression using SPSS v.19. N equals the number of unique and independent experimental samples. The significance of the sample data was determined using student 33.1Prior studies have linked hypoxia to the pathogenesis of fibrotic diseases including lung fibrosis  regulate the interconversion between pyruvate and lactate  have been implicated in the pathogenesis of pulmonary fibrosis  is known to be activated leading to increased production of lactate despite an ample supply of oxygen Devic, . In lungIPF is a disease of aging and it has been demonstrated that IPF fibroblasts exhibit a senescent phenotype. However, it is not clear how senescent IPF fibroblasts contribute to the maintenance and propagation of the persistent wound‐repair response that leads to progressive lung fibrosis. It has been postulated that senescent fibroblasts influence the phenotype of other cells via paracrine mechanisms in fibrosis and cancer; these effects have been attributed to the senescence‐associated secretory phenotype  leading to phosphorylation of the common co‐SMAD, SMAD4, which traffics to the nucleus and serves as a transcription factor. Transcriptional responses to SMAD4 can be modulated by co‐activators and corepressors, including serum response factor (SRF) and myocardin‐related transcription factor‐A (MRTF‐A), both of which have been implicated in myofibroblast differentiation. We have shown that SMAD‐mediated activation of the non‐receptor tyrosine kinase, focal adhesion kinase (FAK), is essential for TGF‐β‐induced myofibroblast activation, that upregulation of α‐SMA is evident within 12 h of treatment, and that formation of organized alpha smooth muscle actin stress fibers, indicative of the myofibroblast phenotype, occurs within 24 h of treatment under the culture conditions used in this study , although the extent of α‐SMA induction was reduced compared to that from non‐pH adjusted medium. Thus, our findings suggest that lactate signaling through GPR‐81 functions as an additional mechanism, along with the potential effects of TGF‐β activation and direct regulation of pH sensors in the induction of myofibroblast differentiation. As lung tissues of patients with IPF have enhanced lactic acid concentrations, we suggest that each of these mechanisms may have a role in propagation of the myofibroblast phenotype and contribute to progressive pulmonary fibrosis in vivo.Intercellular communication and cellular reprogramming are key underlying mechanisms of lung fibrogenesis. Such paracrine signaling for the induction of myofibroblasts may account for the geographically distinct development of fibroblast foci described in IPF , NIH/NHLBI HL141195 (JCH), and 1 U01 HL14555‐01(MR).All authors declare no conflict of interest.This study was approved by the Ethics Committee of the Ohio State University. The use of human lung tissues was approved by the Institutional Review Board (IRB) at the Ohio State University (protocols 2017H0309 and 2021H0180) and all identifiers other than the diagnosis  were removed from the specimens provided.https://figshare.com/s/8b30179f7f1b085292efURL: https://doi.org/10.6084/m9.figshare.22189201DOI: Figure S1.Click here for additional data file.Figure S2.Click here for additional data file.Figure S3.Click here for additional data file.Figure S4.Click here for additional data file.Figure S5.Click here for additional data file.Figure S6.Click here for additional data file.


GRN). Modeling TDP-43 pathology has been challenging in vivo and in vitro. We present a three-dimensional induced pluripotent stem cell (iPSC)-derived paradigm—mature brain organoids (mbOrg)—composed of cortical-like-astrocytes (iA) and neurons. When devoid of GRN, mbOrgs spontaneously recapitulate TDP-43 mis-localization, hyperphosphorylation, and LoF phenotypes. Mixing and matching genotypes in mbOrgs showed that GRN−/− iA are drivers for TDP-43 pathology. Finally, we rescued TDP-43 LoF by adding exogenous progranulin, demonstrating a link between TDP-43 LoF and progranulin expression. In conclusion, we present an iPSC-derived platform that shows striking features of human TDP-43 proteinopathy and provides a tool for the mechanistic modeling of TDP-43 pathology and patient-tailored therapeutic screening for FTD and ALS.Loss of function (LoF) of TAR-DNA binding protein 43 (TDP-43) and mis-localization, together with TDP-43-positive and hyperphosphorylated inclusions, are found in post-mortem tissue of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients, including those carrying LoF variants in the progranulin gene (  •GRN−/− mbOrgs made of mature astrocytes and neurons show traits of TDP-43 pathology•GRN−/− astrocytes are necessary and sufficient for STMN2 mis-splicing in mbOrg•STMN2 mis-splicing is rescued, adding progranulin to neuron and astrocyte co-cultures GRN expression, these cultures present features of ALS- and FTD-associated pathology hardly ever observed in vitro. These phenotypes are shown to be primarily driven by diseased astrocytes and can be rescued by pro-granulin supplementation.In this article, de Majo et al. present a novel 3D iPSC-derived model to study neurodegenerative disorders such as ALS and FTD. When devoid of  TARDBP) gene encodes for the TDP-43 protein, which plays a pivotal role in these two devastating neurodegenerative disorders. TDP-43 was also recently implicated in limbic-predominant age-related TDP-43 encephalopathy (LATE) (STMN2), a neuron-specific gene important for neuronal survival  and amyotrophic lateral sclerosis (ALS) are two fatal neurodegenerative diseases. ALS is estimated to affect 2.1 cases per 100,000 people per year , while Fy (LATE) . Althougy (LATE) . Loss ofsurvival .GRN) gene. (GRN transcripts are composed of 13 exons and encode for the progranulin (PGRN) protein, a highly conserved, approximately 80-kDa protein involved in lysosomal function, neuronal survival, and inflammation  gene. . GRN traammation . The vasmination . The GRNdiseases in vitro or in vivo has been a challenge, with most in vitro models applying exogenous stress to replicate pathology akin to what is observed in post-mortem ALS/FTD central nervous system (CNS) tissue. Within these paradigms, induced pluripotent stem cells (iPSCs) hold great promise; they allow for patient- and tissue-specific human-derived in vitro models, which can be used for therapeutic screening and phenotype testing (GRN loss of function (LoF)  . GRN depignaling  and cellignaling . Interespatients . One poss manner ; 2016. Rs manner . Indeed,neration . The rolGRN LoF in FTD. When devoid of granulin expression (GRN−/−), our model develops features of TDP-43 pathology, including cryptic STMN2 (CrSTMN2) splicing, and extranuclear and hyperphosphorylated TDP-43 inclusions. This study presents the first in vitro model showing robust evidence of numerous FTD/ALS pathology markers spontaneously developing, overcoming the need of exogeneous chemical-induced stress or overexpression. Furthermore, we obtained partial phenotype rescue when GRN−/− cells were treated with exogenous full-length PGRN, demonstrating that the development of TDP-43 pathology depends on PGRN expression. We believe this model could provide insight into cell biological mechanisms, leading to TDP-43 pathology and offer a platform for patient-tailored phenotype and therapeutic screening for FTD and ALS patients with suspected or confirmed TDP-43 proteinopathy.To address this challenge, we applied our previously developed iPSC-derived 3D co-culture model  composedGRN LoF, we modified a previously described protocol to study glial-neuronal interactions (GRN+/+) iPSCs or isogenic GRN knockout (GRN−/−) iPSCs , an astrocyte specific gene well expressed by astrocytes  iPSCs G. Import of PGRN B and S1E neurons B and 1C.trocytes , was str− mbOrgs A. Simila− mbOrgs B. Both rGRN-associated FTD TDP-43 proteinopathy is characterized by increased levels of phospho-TDP-43 (pTDP-43) and abnormal TDP-43 cytoplasmic accumulation, seen in postmortem CNS tissue  has been considered a robust indicator of TDP-43 pathology and correlates with the level of pTDP-43 . Immunostaining for TDP-43 showed expected pathology in the both-cell-type GRN−/− mbOrgs. We also found TDP-43 pathology in the GRN+/+ neurons + GRN−/− astrocytes mbOrgs  cultures. The results point to a profound deficit in synaptosome phagocytosis in the GRN−/− astrocytes  and post-synaptic marker post-synaptic density protein 95 (PSD-95). Confocal microscopy analysis and IMARIS 3D reconstruction showed that in comparison with 4-week-old GRN+/+ mbOrgs. GRN−/− mbOrgs contain more SYP and PSD-95 puncta   B. When i(28 DIV) B. As pre neurons G. This rl bodies F. We nex neurons G. These in vitro human iPSC-derived neuro-glia 3D model of TDP-43 proteinopathy in a context of PGRN deficiency. While iPSC-induced neurons have provided numerous cell-autonomous biological insights into neurodegeneration, most models do not recapitulate multiple aspects of TDP-43-associated pathology. These phenotypic challenges have made therapeutic discovery difficult for TDP-43 proteinopathies Further resources and reagents inquiries should be directed to and will be fulfilled by the corresponding authors . If normally distributed, two-tailed unpaired Student’s t test was performed. If normally distributed, a one-way ANOVA was performed. A p value of less than 0.05 was considered significant. If non-normally distributed, a Mann-Whitney U-test was performed. A p value of less than 0.05 was considered significant. Using DESeq2, Wald tests were performed to evaluate genes for differential expression between conditions. The false discovery rate multiple testing correction method was then used for adjusted p values.−/− iPSC lines. M.dM, E.M.U., E.M., H.L., and K.S. conceived the hypothesis. E.M., J.R.K, M.Kurnellas, M.Kampmann, M.E.W., E.J.H., and E.M.U. provided resources. M.dM., M.Koontz, E.M., and E.M.U. wrote the manuscript. All authors reviewed and approved the manuscript.M.dM., M.Koontz, and E.M.U. designed experiments. M.dM., M.Koontz, E.M., N.S., A.R., Y.K., S.L.G., N.M.D., and K.L. performed experiments and/or analyzed data. Y.M. generated the GRN