Esophageal cancer is one of the deadliest cancers in the world, with high incidence and mortality rates ranking among the top ten in China. The efficacy of conventional treatments is limited and often accompanied by severe adverse reactions, which results in unsatisfactory outcomes. The mechanism of immune checkpoint inhibitors (ICIs) is to activate cytotoxic T cells to kill tumor cells expressing tumor antigens. The application of ICIs has profoundly changed the mode of cancer treatment. However, the use of ICIs also induces a series of adverse reactions similar to autoimmune reactions, called immune-related adverse events (irAEs). Some ICIs can cause manifestations similar to those in the development of sarcoidosis, which are called sarcoidosis-like reactions or granulomatosis.We report a 50-year-old Chinese male patient.The patient had been diagnosed with advanced esophageal squamous cell carcinoma , and was confirmed to have pulmonary sarcoidosis-like reactions associated with sintilimab, a human programmed cell death protein 1 (PD-1) inhibitor.The patient was administered corticosteroid treatment.After receiving steroid treatment, the patient’s systemic and pulmonary symptoms improved rapidly. To our knowledge, this is the first report of pulmonary sarcoidosis-like reaction in a patient with esophageal squamous cell carcinoma. The patient then continued to receive 1 year of follow-up antitumor treatment after the appearance of lung pulmonary sarcoidosis-like reactions. The prognosis was good and the patient’s condition is currently stable.The diagnosis of ICI-induced sarcoidosis often requires comprehensive evaluation through clinical, pathological, and radiological assessment. A subset of patients with sarcoidosis-like reactions may not require treatment unless there is organ dysfunction or severe clinical symptoms, and these reactions generally respond well to treatment. The occurrence of sarcoidosis-like reactions after immunotherapy is positively correlated with the long-term prognosis of cancer patients. However, this hypothesis requires larger prospective studies for validation. PD-1 inhibitor Sintilimab is a highly selective recombinant humanized anti-PD-1 antibody, which can specifically block the interaction between PD-1 and its ligands, and has stronger affinity for human PD-1 than other PD-1 inhibitor such as Nivolumab and Pembrolizumab.–4 Therefore it has been approved as a standard therapeutic drug for the treatment of programmed death ligand 1 positive or advanced esophageal squamous cell carcinoma. Unfortunately, immunotherapy can also cause some unexpected side effects called Immune-related adverse events (irAEs), which refers to a series of immune checkpoint inhibitors (ICI)-induced effects similar to autoimmune reactions. It has been proved that some ICIs can cause symptoms similar to those in the development of sarcoidosis, which are called “sarcoidosis-like reactions”. The etiology of sarcoidosis-like reactions is still unknown. Biopsy shows non-caseous granuloma, and patients may suffer from severe systemic manifestations.,6Esophageal cancer is one of the most lethal cancers in the world, and the morbidity and mortality rank among the top 10 in China. At present, the means of treatment are surgical resection, radiotherapy and chemotherapy. However, due to the limited efficacy of routine treatment and serious adverse reactions, the outcomes are still unsatisfactory. Immune checkpoint molecules are promising anticancer targets in recent years. Among them, the immune checkpoint antibodies which inhibit programmed cell death protein 1 (PD-1)/PD-Ligand1 pathway have shown activity against a variety of malignant tumors, and has been widely used in the treatment of unresectable or metastatic solid tumors.A 50-year-old man, with no previous medical history, was diagnosed in September 23, 2021 with lower thoracic esophageal carcinoma  with left supraclavicular, mediastinal, abdominal and retroperitoneal lymph node metastasis and fluorodeoxyglucose hypermetabolism  scan showed multiple enlarged lymph nodes in bilateral hilum, mediastinum and supraclavicular areas . Hazard ratios (95% CI) were estimated with a stratified Cox proportional hazards model where the stratification factors were PD-L1 CPS, ECOG performance status score, chemotherapy regimen, and hepatic metastasis. For programmed death ligand 1 positive population: 8.3 versus 6.4 months .Sintilimab has been used as a clinical therapeutic strategy of a variety of malignant tumors. According to the results of Shen et al, the overall median progression-free survival: 7.2 versus 5.7 months of unresectable locally advanced, recurrent or metastatic ESCC treated with sintilimab combined with chemotherapy , gastrointestinal manifestations (such as colitis) and endocrine disorders (including hypophysitis and hypothyroidism) Table The pathogenesis of sarcoidosis is very complex, and it is not clear whether there is a causal relationship between sarcoidosis-like reaction and primary tumor, or whether it interacts with primary tumor. However, the adverse events do not seem to hinder their curative effect. Therefore, we speculate that the sarcoidosis-like reaction may be the immune defense response against tumor cells. Das et al reviewed a large number of clinical ipilimumab studies and found that the emergence of irAEs usually indicates a better clinical response. In another retrospective study of 119 patients who received anti cytotoxic T-lymphocyte antigen 4 treatment, 20 patients with imaging manifestations of irAEs (including sarcoidosis-like reactions) had better responses than other 99 patients without irAEs: the disease control rate and complete remission rate in the irAE group were 55% and 25%, while those without irAEs were 10% and 3% respectively.There are several theories about the pathogenesis of local sarcoidosis-like reaction in the lung: Anti-PD-1 antibodies produce objective reactions in about 20% to 25% of patients with non-small cell lung cancer, melanoma or renal cancer. believe that sarcoidosis-like reaction is a T-cell-mediated immune response. Related studies have found that the sarcoidosis-like reaction after immunotherapy is mainly T-helper1 cell response mediated by interleukin-2 and IFN- γ, and the increase in the number and function of Th17 cells in tumor patients receiving anti-ICIs therapy. Th17 cells can promote the development of sarcoidosis fibrosis and play an indispensable role in sarcoidosis-like granuloma formation. Long-term antigen stimulation causes a series of T-lymphocyte immune response, which stimulates monocytes to form epithelioid granuloma.In addition, June et alSome studies suggest that the occurrence of sarcoidosis is related to age, environmental and genetic factors, and familial inheritance. In addition, some research supports that mycobacterium tuberculosis infection, acne, and even certain fungi may be the cause of sarcoidosis; certain occupations or exposure to toxins such as pesticides are also risk factors for sarcoidosis.There are both similarities and differences between ICI-induced sarcoidosis and typical sarcoidosis. The histopathology of the reported cases of sarcoidosis induced by ICI is exactly the same as that of sarcoidosis. Biopsy revealed focal infiltrative lesions of non-caseous epithelioid granuloma and giant cell granuloma, and these lesions can be integrated into micro-nodules. In addition, sarcoidosis-like reaction has the manifestation of organizing pneumonia which is different from sarcoidosis: cough, fever, bilateral diffuse shadow, ground glass shadow, nodular shadow, especially in the inferior field of the lung. About half of the patients with sarcoidosis-like reactions do not need treatment, and it is necessary to temporarily use corticosteroids for local or systemic treatment only when organ function is affected or when the patients have serious clinical symptoms. In reported cases, sarcoidosis-like reactions responded well to treatment (Table The staging and therapeutic strategies of sarcoidosis-like reactions depends on the severity of systemic symptoms and organ injury.nt Table .Most patients who develop sarcoid-like reactions after immunotherapy can be cured with timely treatment. However, reapplication of ICI may lead to recurrence of sarcoidosis, with different sites and severity than the initial presentation, but the treatment approach is usually similar. Therefore, clinicians need to carefully evaluate the benefits and risks to the patient before deciding whether to continue ICI therapy.Immune therapy has profoundly changed the landscape of cancer treatment and brought new hope to cancer patients. However, as the use of ICI in cancer therapy increases, the incidence of irAEs and sarcoid-like reactions will also increase accordingly. From this perspective, it is an urgent problem to achieve maximum therapeutic efficacy while avoiding the occurrence of irAEs as much as possible. This requires clinical physicians to have a clear understanding of the mechanisms of related diseases and to be conscious of irAEs during diagnosis and treatment. Moreover, more extensive and in-depth research on related diseases and treatment strategies is needed in the future.Conceptualization: Linlin Wang.Methodology: Haoqian Li, Fengchun Mu, Bing Zou.Project administration: Haoqian Li.Visualization: Fengchun Mu, Bing Zou, Linlin Wang.Writing – original draft: Haoqian Li.Writing – review & editing: Haoqian Li.


The intercalating of anions into cost‐effective graphite electrode provides a high operating voltage, therefore, the dual‐ion batteries (DIBs) as novel energy storage device has attracted much attention recently. The “graphene in graphite” has always existed in the graphite cathode of DIBs, but has rarely been researched. It is foreseeable that the graphene blisters with the intact lattice structure in the shell can utilize its ultra‐high elastic stiffness and reversible lattice expansion for increasing the storage capacity of anions in the batteries. This review proposes an expected “blister model” by introducing the high elasticity of graphene blisters and its possible formation mechanism. The unique blisters composed of multilayer graphene that do not fall off on the graphite surface may become indispensable in nanotechnology in the future development of cathode materials for DIBs. The intercalation of solvated anions enables a few graphitic layers to separate from the bulk graphite and form a nano‐blister. The number of graphite layers in the blister shell is often less than 10, and the physical properties of blister shell similar to those of graphene make it different from bulk graphite, so it can be referred as “graphene blister”. Additionally, the electrode materials in DIBs are predominantly carbon materials (such as graphite); when graphite is used for the anode and cathode, the DIBs can also be referred to as “dual‐graphite batteries” rather than transition metal‐based materials, presenting great advantages of DIBs such as long cycling‐life, environmental protection, inexpensiveness, and high energy density. A schematic illustration of a DIB is shown in FigureDIBs are considered as a promising energy storage device wherein anions and cations are intercalated/de‐intercalated into the cathodes and anodes, respectively.1 Comp researchers explored the structure and composition of various graphite anion intercalation compounds (GICs) primarily through graphite oxidation in the early stage. A brief development history of DIBs until the device has a unified name have been provided in an image  as the cathode and anode, respectively, for assembling the DIBs, and a solution of 1 m BF4− in PC was used as the organic electrolyte. Although traditional carbon‐based supercapacitors possess high power density, they often possess low energy density. However, the combination of graphite and AC electrodes could significantly improve the energy density of the devices and was known as “megalo‐capacitance” capacitor. Although the name of this energy storage device varied, the most significant feature of this battery type is the reversible intercalation/de‐intercalation reaction of anions into/out of the graphite cathode. The device did not have a unified name until 2012, when the name of “dual‐ion cell” was first mentioned by Tobias et al. Furthermore, Wang et al. attached significant importance to the research on the solvation effect, and proposed that the electrolyte significantly influences the performance of this type of capacitor. The charge‐storage behavior can be regarded as the competition between the electrode and electrolyte for acquiring anions; thus, anion solvation could play a key role in the anion intercalation into carbonaceous materials. For example, the interactions between the PF6− anion and EC or PC solvent were explored earlier, and the results demonstrated that the solvation effect grew stronger with decreasing anion size. Therefore, the strong interaction between PF6− and EC hinders the PF6− intercalation into graphite. In addition, the influences of EC, PC, and GBL solvents on the intercalation behavior of BF4− anion with a small size were also well summarized. Owing to the strong bonding between EC and BF4−, the EC solvent significantly shortened the discharge plateau and specific capacity.Although DIBs have been studied since 1938,4 resePrevious studies have focused on i) the GIC structure in graphite electrodes formed by electrochemical anion intercalation, and ii) the influence of the solvated anion composition on battery capacity and cycle life. However, these two research directions were relatively separated, and the electrolyte as the only source of anions and the graphite as the anion host should be simultaneously focused on. From this point of view, methods to identify hidden key research priorities must be explored to guide and promote the future development of DIBs. Therefore, the interface properties optimization is required to improve the electrochemical performances of DIBs. In recent years, the regulation of electrolyte components on the graphite cathode interface in DIBs has gradually attracted attention. This method, via the regulation of CEI or SEI composition in Li‐ion or Li‐metal batteries, has been demonstrated to be effective to an extent. For example, the oxidative decomposition of the electrolyte was suppressed, and the structural integrity of graphite was maintained by regulating the CEI composition at the graphite interface using fluorinated solvent. However, the special 2D structure of graphite differ significantly from those of other cathode materials. In‐depth research on the interface properties of graphite cathodes remains lacking, and the influence of anion intercalation on the graphite surface properties in DIBs also required to be further studied.As an important electrochemistry research topic, the electrode/electrolyte interface affects the properties of ion adsorption, redox reaction, and desorption.15 The Graphite surface blistering was considered to be the initial stage of graphite exfoliation and has attracted the attention of several researchers. Similarly, the graphite cathode of the DIBs underwent the same electrochemical oxidation process. However, significant information on graphite surface blistering remain uncovered. Once the blisters are formed, multi‐layer graphite that was originally closely connected to the bulk graphite, turns into bended graphene. The number of graphite layers in the blister shell is often less than 10 layers, and the physical properties of blister shell similar to those of multilayer graphene make it different from bulk graphite, so we can refer to it as “graphene blister.” It has been reported that this type of blister generated during the charge/discharge process of a graphite cathode is beneficial for improving the specific capacity of DIBs. However, research specifically focusing on the contribution of graphene blisters to the DIB performance remains lacking. It is foreseeable that the graphene blisters intact lattice structures in the shell can utilize ultra‐high elastic stiffness and reversible lattice expansion for increasing the anion storage capacity in the batteries.Blister formation on graphite surfaces was a relatively common phenomenon observed in the field of graphite electrochemical oxidation.20 GraIn this review, therefore, the combinations of reported results from many different research fields, including graphite oxidation, nano‐mechanics of graphene, condensed‐matter physics, electrowetting, and DIBs, are presented for sorting out which supports intercalation of anions and formation of graphene blister in the bulk graphite. Besides, we discussed the reversible deformation and anion storage capacity of graphene blisters from the elasticity of graphene blisters and their relationship with graphite grain boundaries. A “blister model” was proposed by linking the many characterizations and analyses in the literatures. The conclusions of the model were applied to explain many anomalous behaviors in DIBs. The revelation of the importance of “graphene in graphite” for anion intercalation can develop future research propositions that can extend the field of DIBs.22.1 Thought provokingly, Tatsumi et al. found that introduction of nano‐sized pore into graphene sheet could provide a large capacity of 147 mAh g−1 through intercalation of PF6−. Moreover, the blistering on the graphite surface was also regarded as a necessary pre‐step in electrochemical methods to prepare graphene. Therefore, many research studies on graphene blisters or bubbles have been conducted by scientists in a variety of fields, and the researches related to graphene blisters were growing in decades as shown in FigureAlthough graphene was discovered in 2004, graphene blisters with multilayer formed on the graphite surface were researched much earlier. The graphene blisters are a kind of microscopic 3D network nanostructures with a series of special properties. Benefiting from their ultrahigh adhesion, ultrahigh elasticity and excellent electrical conductivity, graphene blisters gather plenty of attractive physical properties. Dresselhaus and Kalish prepared carbon materials with specific properties through ion implantation, which induced modifications to graphite like blistering.24 Tho2.22, while the blister is not completely detached and keeps intact lattice. The structural representations of graphene blisters in many forms are shown in FigureThere are several physical and chemical environments among various forms in which the graphene blister might exist. A monolayer or multilayer graphene blister forms on graphite surface or other substrates, such as SiO2.3Elasticity, adhesion, flexility are particularly important physical parameters in graphene blisters. The excellent elasticity can facilitate the repeated or even reversible changes of the blister shell. The ultrahigh adhesion between graphene blister and substrate enable repairability or even recyclability after blister generation, and the blister is restricted to the bulk materials that adhere to the substrate surface. Graphene blisters with hollow space are more appropriate for storage of ions in electrolyte due to reversible changes of the blister shell with intact lattice. More importantly, the elastic structure of blisters can be tailored by electrochemically permeating the ion species or regulating the voltage between the graphite electrode and electrolyte to better match DIBs in terms of surface chemistry.3 Once the blisters are formed, the multi‐layer graphite that was originally closely connected to the bulk graphite transforms into bended graphene. Similar blisters that are beneficial for improving the capacity have also been observed on the graphite cathode surface. Therefore, this section aims to analyze the graphene blister as a special phenomenon that has been ignored in DIBs for a long time. Herein, we describe this graphene blister in detail and predict its role in the future development of DIBs. The evolution of important events in the field of graphite blistering since its discovery was organized into an image as shown in FigureIn the field of oxidation or electrochemical oxidation of graphite, anion/solvent intercalation was a crucial step in graphite exfoliation, during which nano/micro‐sized blisters were observed on the graphite basal plane.30 Onc3.1 In 1992, in a study on the electrochemical intercalation process of ClO4−, SO42−, and PO43− anions into graphite in an acidic aqueous solution and the corresponding Raman spectral changes during the process, McCreery et al. observed whether the graphite lattice was destroyed was correlated with the different types of anions and solvents. The results demonstrated that PO43− could not intercalate, and no lattice damage in graphite was observed. Anion intercalation was accompanied by lattice damage in both 1 m HClO4 and 1 m H2SO4 aqueous solutions, whereas in NaClO4/CH3CN electrolyte, ClO4− could intercalate into the graphite lattice without lattice damage. Particularly, the rapid electrochemical intercalation process occurred on the graphite basal plane rather than the edge plane. These results indicate that the intercalation process can be controlled by regulating the anion and solvent compositions, and the anion intercalation sites cannot increase merely by increasing the fracture exposure. In 1995, Erkang Wang et al. used STM to study the electrochemical oxidation of HOPG. They observed the blister formation on the graphite surface in the phosphate buffered solution under different voltages. The blisters were triangular in shape, and the graphitic structure remained intact, indicating that intercalated ions or solvents were present under the subsurface, as shown in the bright triangle in Figure This type of blister was characterized by its small size, and the STM image showed that the distance between the centers of the dark and light triangles was merely 3 nm. The size and shape of the blisters were uniformly distributed periodically. In addition, the authors believed that the intercalation sites were located one or two graphite layers below the surface layer, and the graphite defects were beneficial to the blister formation.The blistering phenomenon on the graphite surface was discovered in the 1990s, when researchers studied the electrochemical graphite oxidation in electrolytes.32 In  They performed electrochemical oxidation of graphite cathode in the 1 m LiClO4, 1 m (NH4)2SO4, 1 m HNO3, and 1 m H2SO4 aqueous solutions, respectively, and observed that the anion intercalation into graphite could lead to blistering, whereas no blistering was observed in the 1 m K2HPO4 and 1 m H3PO4 electrolytes owing to the lack of intercalation of phosphate ions. The energy‐dispersive X‐ray elemental microanalysis (EDX) results demonstrated that graphite oxide was present on the top surface of the inner blister, which was covered by 5–12 unoxidized graphite layers. The average size of semicircular blisters observed was larger than that of triangular blisters, as shown in Figure  as shown in Figure In the same year, Murray et al. continued studying the blistering phenomenon on the HOPG surface that they discovered in 1993 using an in situ atomic force microscopy‐electrochemistry (AFM‐EC) system and optical microscopy in aqueous electrolytes.36 The4− anion in HOPG. The results demonstrated that during the charge/discharge cycle (non‐first), two groups of blisters were distinctly observed at 0 V  The interlayer expansion caused by anion intercalation caused mechanical deformation of the graphite layer, and the energy required to overcome the deformation increased with the increasing number of graphite layers on both sides of the intercalation point. Therefore, the intercalation generally initiates near the surface and then proceeds into a deeper interlayer. The intercalation reaction for the solvated anion could be described by:x is the number of carbon atoms involved in the reaction in graphite; A− represents the anion; solv represents the solvent molecules in the solvation shell; y is the number of solvent molecules involved in the intercalation reaction.This same phenomenon was also observed by Alliata et al. in 1999 when they studied the intercalation behavior of the ClOV Figure, the smaV Figure, the dis6− in the graphite cathode in “dual‐graphite battery” and observed that a nano‐sized hollow structure was present below ≈8 surface layers of graphite, and named it “bubble” , and this bubble is the same as the aforementioned blister. Furthermore, the authors also observed that this nanobubble with a diameter <10 nm was beneficial for improving the specific capacity of the battery. A non‐aqueous solvent (electrolyte: 1 mn‐BPPF6 in PC/MEC) was used in this battery, which complements the speculation of the previous researchers that blistering is related to water molecules in an aqueous solution, indicating that the blistering phenomenon caused by the intercalation of solvated anions can appear in both aqueous and non‐aqueous systems. However, such bubbles (or blisters) in DIBs have not received significant attention and were only considered as lattice expansions due to anion intercalation.Because the electrochemical graphite oxidation occurs at the cathode of the DIB during charging, the blisters have been also observed in the study of DIBs. In 2010, Ishihara et al. studied the intercalation behavior of PF They observed that the graphite basal plane possessed excellent elasticity, and was morphologically stable in the electrolyte under low voltages. The anions cannot form the stage‐III structure in the graphite, while the graphite surface was not damaged. After further increasing the voltage, although the larger potential caused cracks on the graphite surface, the blisters predominantly remained stable. Furthermore, when the blisters were exposed to the high accelerating voltage probe of the scanning electron microscope (SEM), the blisters tended to disappear or “deflate”  is generally orders of magnitude higher than their interlayer shear stiffness and strength. The extreme mechanical anisotropy may enable interlayer sliding in the layered materials.41 Gra In the field of solid mechanics, numerous theoretical and experimental studies have been performed on the elastic strength of graphene bubbles and their adhesion energy to substrates. For example, in 2010, Levy et al. studied the magnetic field behavior in graphene nanobubbles affected by the strong stress. Moreover, the relationships between the adhesion mechanics, elastic mechanics, and the geometry of graphene bubbles have been theoretically studied in depth. For example, Koenig et al. studied the law of the change in the adhesion energy and shape of the graphene bubbles, wherein the gas is stored, on SiO2 substrate. The authors proposed that single‐layer graphene exhibited the strongest adhesion to the substrate, and the height of the single‐layer graphene bubble was larger. The nanoscale adhesions of graphene to substrates are higher than those measured in typical micromechanical structures, and are comparable to solid/liquid adhesion, which is attributable to the ability of graphene to conform to the topography of the smooth substrates, consequently facilitating its interaction with a substrate that is more like a liquid than a solid. Although the substrate surface is rough, the van der Waals forces across this contact area play an important role, which is greater than the adhesion of gold‐coated submicron beams by a factor of ≈5. Furthermore, Bunch et al. reviewed the reported results of the adhesion energy of graphene to the substrate. The authors claimed that the nano‐sized graphene materials were considerably affected by the surface forces, and the stiffness of the materials decreased with size, which exhibits various nonlinear mechanical relationships, such as a cubic relationship between bending stiffness and multilayer graphene thickness. When the graphene bubble is formed, its adhesion energy to the substrate can be expressed asγ is adhesion energy; E, stiffness; a, radius of the bubble; h, thickness of graphene bubble shell; w, height of graphene bubble. High adhesion allows a high flexibility of graphene bubbles. It can also be observed from this formula that graphene bubbles tend to form spherical bubbles with larger radii but lower heights because of the relatively weak interaction between the graphite layers, which is also lmost the same phenomenon observed in electrochemical graphite oxidation.Graphene bubble formation is a manifestation of the strong adhesion between graphene and its substrate, and the strong adhesion enables the graphene bubbles to exhibit a series of unique properties.42 In  however, sufficient experimental evidence to demonstrate the existence of the gas in the bubble and the its escape mechanism remains lacking. Past analyses of this hypothesis have relied on the possibility of generating gas via solvent oxidation. Thus, determining whether the bubble contains gas or liquid requires further research. In 2018, Sanchez et al. studied the morphology of a graphene bubble containing liquid and its adhesion energy to the substrate. Although no consensus exists on whether the blisters are filled with gas or liquid, adhesion is a well‐established controlling parameter for blister formation as aforementioned. The authors reported that the subtle nature of the content inside the blister may render invalid the previously reported assumption that bubbles contain gas. Compared to the well‐established adhesion measurements, the direct application of this model resulted in unrealistically small adhesion values for graphene. Blister formation is energetically favorable only when the adhesion between the blister and substrate is sufficiently strong. The results of the elastic membrane model established by Sanchez et al. demonstrated that if the liquid volume  remains unchanged, the elastic energy decreases, and the interface energy increases with increasing bubble diameter a. For a liquid‐filled (water) blister, the mathematical relationship of the height/diameter ratio can be expressed as:Γ is the work of adhesion (or adhesion energy) of the membrane‐substrate interface; γw is the surface tension of water (≈0.072 J m−2); θs and θm are the water contact angles of the substrate and membrane, respectively; h and a represent the height and diameter of the blister, respectively; E2D is the in‐plane elastic stiffness. The theoretical model showed that the weak shear limit (complete exfoliation of graphene owing to interlayer slip caused by the blister formation) predicts a larger aspect ratio h/a of the blister compared with that of a strong shear limit , particularly for graphene (Poisson's ratio ν = 0.165).Whether it is a bubble or blister, it refers to the same research object, that is, the bulge on the graphite surface in different fields. However, the elastic properties of the graphene bubble when the bubble contains a liquid are different from those when it contains gas. Although most studies in the field of electrochemical oxidation of graphite have repeatedly mentioned the existence of gas in the bubbles when describing the bubble formation mechanism , and it was believed that the appearance of graphene bubbles was related to gas evolution;47 howFigureh/a of the blisters was moderately large, and the blisters with excessive height might cause self‐rupture and surface damage, which is inconsistent with previous experimental results that the surface structures of graphene blisters caused by anion intercalation is maintained undamaged. However, in another case of the lower adhesion energy (<0.2 J m−2), the top of blister with low aspect ratio h/a was nearly flat. The adhesion energy of graphene on HOPG is 0.086 ± 0.016 J m−2, and thus, the top of the graphene blisters produced by the electrochemical graphite oxidation are predominantly nearly flat. This prediction is extremely similar to the microscopically observed blister morphology in the literature on the electrochemical oxidation of graphite in Section 3.1. In addition, according to the literature in the field of graphite oxidation, if the electrolyte solvent is water, the gas inside the blister is generated from the oxidation of carbon atoms, which is primarily based on the reaction formula C + 2H2O → CO2 + 4H+ + 4e−, indicating that 2 mol of water produces 1 mol of CO2. Then the volume of the blister increases by a factor greater than 100, which is inconsistent with the phenomenon observed via AFM. There are at least two reasons why it is contradictory: i) Only a significantly small quantity of gas in the blister, or ii) the gas is highly compressed owing to the high elastic stiffness of the graphene blister. However, no reported study is devoted to this topic. Based on the above analysis, the matter contained in the blister generated by the electrochemical oxidation of graphite is more likely to be liquid. Similarly, the graphene blister that appears on the graphite cathode surface in the DIB undergoing the same electrochemical process is also likely to be liquid, that is, intercalated solvated anions. We can now distinguish between the word “blister” and “bubble,” which contain liquid and gas, respectively.Further molecule dynamics (MD) simulation results Figure10a show Tripathi et al. reported a logical plausibility to this explanation. They characterized the local potential distribution on a single blister surface, as shown in Figure If no gas exists in the blister, then another important phenomenon should be considered. Research teams varying with countries or regions have reported the same phenomenon: After first charge/discharge cycle, graphene blisters grow larger under lower voltage, but decrease in size under higher voltage, which does not appear to be a logical electrochemical behavior. Because the solvated anions are more likely to intercalate to form graphene blisters under higher voltage, consequently leading to larger blisters, whereas, the experimental phenomenon is completely opposite. There were no ready‐made logical connections in the literature linking these contradicting results from those different fields; therefore, we provide a possible explanation: The blister serves the accommodation of reactants. Under higher voltage, solvated anions intercalate into the surrounding lattice connected to the inner blister, and then the loss of ions and solvent decreases the blister sizes; under lower voltage, the ions in the lattice are de‐intercalated and returned to the blister, so the surface structure is not damaged, and the electrochemical process is reasonable. Additionally, the solvated anions are de‐intercalated and returned into the graphene blisters rather than the electrolyte near the cathode surface possibly owing to the Marengoni effect caused by the high elastic stiffness of the graphene blisters; this has not been reported in the literature on micro‐ and nano‐sized liquid blisters or bubbles.50 Tri Therefore, for graphene blister formation, the elastic stiffness of graphene blisters also requires to be considered when calculating the theoretical operation voltage from the difference between the intercalation energy and desolvation energy. Moreover, as mentioned above, blistering caused by anion intercalation always occurs in the vicinity of the graphite subsurface at the beginning and subsequently proceeds to the deeper layers, which is explained in this section: The shell thickness of graphene blisters is positively related to the elastic stiffness of the multilayer graphene, so the maximum elastic energy and number of shell layers of graphene are determined by the interlayer force of graphite and intercalation ability of anions, which facilitates the appearance of blisters in the subsurface below 5–12 surface layers. After the formation of graphene blisters in the subsurface layer, the deeper graphene blisters continue to be generated. It can be inferred from the conclusions that a high interlayer force requires to be overcome during the 1st intercalation of anions to form graphene blisters, and in the subsequent intercalation/deintercalation cycles, only relatively low elastic moduli of graphene blisters require to be overcome. The DIB will predictably have a high charge/discharge voltage plateau in the first cycle, and will gradually decrease in the subsequent cycles, however, the specific capacity exhibits a significantly opposite trend to that of the voltage plateau, and the specific capacity gradually increases owing to blister formation and its excellent anion holding capacity. When the number of graphene blisters reaches a threshold value, the voltage platform and specific capacity tends to stabilize, which is almost a normal phenomenon in the long‐term stability tests of DIBs.The above analysis suggests that a core issue in increasing the specific capacity of DIBs is the controllable formation and reversible deformation of graphene blisters. The intercalation energy is related to the operation voltage of the DIB, however, previous research predominantly considered the simplest condition, that is, the intercalation of anions into the graphite interlayers.51 The3.342− anions were generated along the same line‐shaped trajectory as the graphite grain boundaries. The graphitic structure of the top layer on the blister remained intact, and this result was consistent with those reported by McCreery et al. in 1992. Furthermore, in some previous studies, the researchers claimed that pre‐existing defects on the graphite surface contribute to blister formation by serving as stable ion channels and supporting the reversible transport of ions in the blisters. Subsequent reports have indicated that this defect actually refers to the GBs. Graphite GBs, also known as “superlattices,” are produced by the contact of two graphite lattices at different angles in the same plane. Then, the entry mechanism of the solvated anions into the lattice through the graphite GBs to form graphene blisters is a topic that requires further exploration, and it can help to realize the controllable formation of graphene blisters.In 2018, Sinitsyna et al. observed an important phenomenon while preparing graphite oxide via the Hummers method. The blisters on the graphite surface caused by the intercalation of SO When HOPG was electrochemically treated in perchloric or sulfuric acid, the intensities of the characteristic ionic peaks of graphite, such as C2−, C3−, C4−, C5−, and C6−, were significantly reduced, and the mass spectrum in the negative‐ion mode showed a stronger oxygen ion peak compared with that of the pristine graphite surface. These results suggested that the carbon atoms in the topmost lattice of HOPG were oxidized by the aqueous electrolyte to form graphitic oxides. In addition, a few chemicals, including chloride Cl−, perchlorate ion ClO4−, sulphite ion SO3−, bisulfate ion HSO4−, and solvated ClO4−, related to the anion intercalation process were also detected. These ion signals were stronger at fractures on the graphite surface  Solvated ions are involved in the intercalation reaction. Moreover, through analyzing the signal distributions of the intercalation of various chemical species, the authors concluded that graphitic defects  were the preferred initiation sites for the electrochemical anion intercalation process. It has also been proposed in other studies that electrochemical oxidation at the edge sites and GBs subsequently leads to the expansion of the graphite layers, thereby facilitating anion intercalation within the graphitic layers in both ionic liquid and aqueous solution.Although this phenomenon has long been discovered, it was only in recent years that advanced characterization instruments were used to further analyze the formation process of graphene blisters grown on GBs and the specie compositions in the blisters. In 2019, Rosa et al. used time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS) to analyze the chemical element distribution and change in chemical compositions on the HOPG surface before and after electrochemical oxidation of HOPG in the electrolyte.55 Whe This unique periodic superstructure (or superlattice) affects the surface charge density distribution of graphite. Simulation results demonstrated that positive charges were concentrated on the heptagon, whereas negative charges were concentrated on the pentagon, consequently resulting in a dipole moment at the GBs  solvent on the surface morphology of HOPG. The experimental results verify the previous conclusion that the formation of SEI (contributing to the blister structure) on the graphite was triggered by the intercalation of Li(TFPC)n, and the SEI was formed rapidly at step edges before graphite layers began to exfoliate. Therefore, the stability of the graphite against solvent co‐intercalation is one of the key factors influencing the stability of the SEI formation. However, the “blister” obtained in TFPC was quite distinct from the blister on the graphite surface in EC‐based electrolytes. The STM image showed that the top of the blisters obtained in the EC‐based electrolyte exhibited the same bright spots at intervals of ≈0.25 nm as the graphite basal surface, indicating that the blister was an interior structure formed under the surface. In contrast, the decomposition products of Li(TFPC)n were exposed on the surface rather than the subsurface. Meanwhile, compared with EC‐based electrolytes, TFPC‐based electrolytes exhibited larger initial irreversible discharge capacity , as well as smaller reversible discharge capacity . Therefore, the subsurface blister with an intact graphite structure was more beneficial to improve the reversible capacity of the batteries.Inaba et al. have noticed the blistering phenomenon on HOPG surface in EC‐based electrolytes and studied its effect on electrode performances in ref. . When us of HOPG.68 The They studied the decomposition precipitation, swelling at step edges, and blistering that occurred on HOPG surface in the electrolyte of LiPF6 EC:EMC when lithium was intercalated. The authors believed that the swelling at step edges is the SEI played an important role in the intercalation/de‐intercalation of lithium ions. Subsequently, a layer of decomposition products was covered on the basal plane of HOPG after adding vinylene carbonate (VC) additive to the electrolyte, which could cause the isolating of the electrolyte and graphite, while maintaining the swelling at step edges and blistering. The phase image of AFM showed that there was a phase contrast between the “blister” and the basal plane in the electrolyte containing VC. In contrast, there was no significant phase contrast between the blister area and the basal plane in the EC‐based electrolyte  in PC:tetraglyme (20:1 v/v), and the anion of OTf− could perform stable reversible intercalation/de‐intercalation process in graphite. The discharge capacity remained ≈300 mAh g−1, and the discharge curves coincided well. Indeed, these blisters mentioned above all showed the characteristics of blistering along the step edges of the basal plane, consisting with the previous conclusion that the blister was preferentially formed at the defects on basal plane.The above conclusions were also verified by the results published by Matsuoka et al. in 2002.69 Thee Figure, and the The authors designed phosphorus‐doped and nitrogen‐doped graphene anodes to introduce “protrusion” (similar to blister) and “hole” structures . DFT calculation results showed that the enlarged (0002) spacing of graphene with protrusion could bring in more Na+ storing sites, which would greatly facilitate the mobility of both electrons and Na+ ions during the sodiation process . It was found that the new graphene electrode was compounded with 2‐naphthalene sulfonate that provide continuously anion SO32−. The self‐stand film exhibited good elasticity and made it possible to form blisters after swelling of the polymer film during charge/discharge process . Specifically, the specific capacitance only showed a sharp decline in the initial 100 cycles and then displayed insignificant changes in the following 900 cycles (97% is maintained from 242 to 234 F g−1), demonstrating the improved structural stability of PPy anchored partially exfoliated graphene electrodes. It is worth noting that the other sample prepared by the authors did not exhibit the blister structure. The difference between the two samples was that they contain different matters that continuously donated anions. As a result, the absence of the blister structure led to a poor rate performance of the electrode .The blistering on graphite can also enhance the electrochemical performances of other energy storage devices, for instance, the supercapacitors. Liu's group prepared partially exfoliated graphene through anion intercalation into multilayer graphene in phosphate buffer solution (pH = 6.68) containing 1 m KNO3.21b Sus Figure . The autIn conclusion, the graphene blisters on the graphite surface exert a great impact on the battery performance. That is, the ability of the electrode to accommodate ions can be enhanced, and a stable electrode/electrolyte interface by forming the SEI in the inside can be obtained through forming the blisters. However, currently little information is available concerning the role of blisters in the battery field. In view of this, the effect of the blisters on battery performance and its microscopic mechanism still need to be further studied.4.2In the field of DIBs, a few unique experimental phenomena related to blister formation have been neglected by researchers. These abnormal phenomena can easily relate to graphene blisters by reviewing the literature in several research fields including DIBs, electrowetting, and electrochemical oxidation of graphite, and considering the previous discussions.4− anions on graphite surfaces when the cyclic voltammetry (CV) curves of the intercalation/de‐intercalation of the anions in EC solvent were measured. The authors observed an abnormal oxidation current peak in the discharge segment of the CV curve  and referred to it as “countercurrent,” as shown in Figure Therefore, a preliminary explanation was provided by the authors: the EC solvent inevitably hinders BF4− intercalation owing to the strong interaction between BF4− and EC, leading to the partial intercalation of BF4−, and the rest of anions remain outside the graphite. Subsequently, the intercalated BF4− re‐solvates during de‐intercalation. If the re‐solvation occurs in the graphite lattices, the entry of BF4− immobilized outside the graphite electrode into the graphite lattice causes a backflow process, resulting in the abnormal counter oxidation current, because the BF4− immobilized outside the graphite electrode also requires to be re‐solvated by EC solvent molecules before appearing as the ionic current of de‐intercalated BF4−. After reviewing the literature, we observed that the countercurrent in the CV curve likely occurs when the anions and solvents that are easier to intercalate into the graphite lattice are used. For example, in the following work, Wang et al. used GBL solvent to replace the EC solvent. The co‐intercalation of BF4− and the EC solvent was suppressed owing to the weak interaction between GBL and BF4−, and thus, the de‐solvation of internal BF4− in the graphite lattice was reduced, resulting in the disappearance of the phenomenon of countercurrent. Therefore, the smaller BF4− anion sizes and its stronger interaction with the solvent are the factors that lead to the countercurrent phenomenon. Moreover, the authors also observed that the strong interaction between Li+ and BF4− could regulate the BF4− intercalation properties if the main salt of SBPBF4 was replaced by LiBF4. Although a countercurrent peak was also observed in this case, a higher cut‐off voltage was required to induce this phenomenon, which was more evident in the experiments conducted by Yazami et al. who also thought it was an “anomaly.” When they used the LiBF4 electrolyte in EC‐DMC to study the anion intercalation behavior of anions into single‐walled carbon nanotubes, they observed that a wide anomalous anodic reverse current appeared in the initial de‐intercalation segment of the CV curve  were more likely to lead to ion intercalation and de‐intercalation, consequently resulting in significant electrowetting. In contrast, the HOPG samples with a considerably higher step edge density (with multilayer step) exhibited considerably less electrowetting. It seems illogical that more edge planes cannot increase amount of intercalation sites. Furthermore, although the HOPG samples with higher multilayer step density underwent potential scans under the same test condition, no significant intercalation and de‐intercalation peaks appeared, indicating that the voltage was not the only factor affecting the intercalation/de‐intercalation of anions. However, the authors simply attributed this inability to the electrochemical intercalation of anions to the strong binding force imposed by the high density of multilayer step edges. Additionally, the authors conducted a series of experiments for analyzing the abnormal phenomenon, including the operando characterizations of contact angle (CA) and relative contact diameter (RCD) during the CV curve measurements. The droplet was gradually spreading with increasing voltage. The results of the CA and RCD tests showed that the RCD increased by 8% when the voltage increased from 0 to 1.13 V, and the CA decreased from 51° to 38°. When the voltage reverted to 0 V, the RCD curve, which should be immediately decreased, exhibited an abnormal increase in during the de‐intercalation process. A similar phenomenon was also observed in the CA curve, which was not synchronized with the reverse voltage. The authors referred to this phenomenon as “hysteresis” that was repeatable and reversible, and they proposed that hysteresis occurred because of the change in potential under a certain scan rate was faster than the wetting rate. Notably, the AFM images of the wetting area showed the formation of dense blisters on the HOPG surface after 30 intercalation/de‐intercalation cycles . When the lattice was not damaged by anion intercalation in the electrolyte of NaClO4 in acetonitrile, only G peak splitting occurred. Thus, the G peak can represent the degree of structural perfection of blister shell, while the G′ peak symbolises anion intercalation and possible formation of the blister. These results indicate that graphene blisters should be generated in the experiments performed by Balabajew, and that such blisters are reversible, and can revert to graphite with strong interlayer forces. This phenomenon that graphene blisters can be recovered has only recently been strongly corroborated by the experiments conducted by Xu's group. They reported that the solution‐enclosing blister could be reversibly modulated by the solution osmotic pressure.Another inexplicable anomaly discovered by Balabajew et al. in their study on DIBs can also be explained by the “blister model”. When they studied the behavior of the storage anions of graphite foam in the electrolyte of LiTFSI in Pyrstanding.78 Ram When the anions were intercalated into the graphite and stage‐III was reached, thin lines and blisters appeared on the graphite surface. The thin line represents the fracture, and numerous small blisters are present around it, so these so‐called fracture lines are likely to be lines composed of several tiny blisters and cracks generated on the graphite GBs, which does not conflict with the conclusions in Section 3.3. The authors tested the Raman spectra of these lines, as shown in Figure Referring to the study of Yivlialin et al. in 2017 investigating the relationship between graphene blisters and Raman spectra.39 Whe5The graphite cathodes in DIBs are inevitably associated with graphene blisters owing to the electrochemical oxidation process that graphite undergoes. Therefore, according to our analysis, a preparation method for graphene blisters with intact lattice and reversible deformation is a key factor for the structural design of graphite cathodes with large capacities. In addition to the electrochemical oxidation methods that have been widely reported in the literature, numerous other physical methods, such as electron irradiation, laser‐induced, and ultraviolet (UV) oxidation, that can be used to introduce the graphene blisters on graphite cathode are present.80 The hollow nanotube‐like defect could be observed in the graphite interlayer, and significant expansion in the c direction occurred between the basal planes. The authors referred to this lattice blister induced by high‐temperature electron‐irradiation as “localized swelling.” Additionally, the laser irradiation can be used to induce blistering. The interaction between the two neighboring graphene layers was governed by the balance between van der Waals forces and repulsive molecular orbital interactions. The laser pulse could supply the necessary energy required to overcome the interlayer cohesion without disrupting the in‐plane bonding. Therefore, the laser‐induced photoexfoliation technique was an effective method to form graphene blisters. Moreover, blister formation could be achieved via the combination of UV transparent anatase thin film and photocatalytic oxidation in the presence of water vapor. The gas‐assisted photo‐oxidation provided a novel photocatalytic method to uniformly oxidize the graphite surface. Oxidative gas species at reactive atomic carbon surface defects  caused graphite surface functionalization and the blistering of basal planes.Johns et al. used in situ electron‐irradiation to treat nuclear graphite on a 200 kV transmission electron microscope with a dose rate and observed that the defect domains induced by electron‐irradiation underwent atomic rearrangement, which subsequently resulted in nanostructure formation in nuclear graphite.81 The Therefore, the anion intercalation rate can be well controlled in the electrochemical oxidation experiment, which is still the most effective method to induce blistering. The influences of the operating conditions, such as scan rate of CV, that control blister formation require further exploration. Moreover, how to characterize and detect graphene blisters is also important, which will affect our understanding of the properties of graphene blisters and how they can be used. Combined with the results of many references in this paper, we can know that several characterization methods play an important role in the study of graphene blisters, as listed in FigureIt can be observed that graphite oxidation may induce blistering on the graphite surface, and in the meantime, the blister formation is often accompanied by intercalation of matters  into graphite.86 The6DIBs use graphite, in which anions can intercalate, as the cathode material, and use the reversible intercalation/de‐intercalation process for secondary energy storage. During battery charging, the graphite cathode undergoes an electrochemical oxidation process, which can be viewed as a competition between the accommodation of anion inside graphite lattice and structural exfoliation of graphite. The researchers focused more attention on the composition of the electrolyte and the structural optimization of graphite cathodes in previous studies on DIBs. However, a special interfacial phenomenon has been neglected in battery research. Anion intercalation induces an intermediate state that is neither fully exfoliated nor initial surface—graphene blister—which was first discovered in the field of electrochemical oxidation of graphite. The intercalation of solvated anions enables 5–12 graphitic layers on the surface to separate from the bulk graphite and form a micro/nano‐sized semi‐circular protrusion, that is, graphene blister. Once the blister is formed, the blister with the shell containing several graphene layers exhibits unusual membrane‐like elastic behavior, which allows the graphene blister to remain its shell structure when it undergoes elastic deformation through reversible expansion and contraction. Although graphene blisters in DIBs have received little attention, the results of the Raman spectroscopy of graphene blisters in the field of electrochemical oxidation are almost the same those of in situ Raman spectrum changes during the cyclic charge/discharge process in DIBs. Therefore, it is known that graphene blisters can have favorable reversibility by regulating the cut‐off voltage, anionic components, and solvation. Moreover, we applied the “blister model” of graphene blisters to explain numerous anomalies in DIBs, and learn how the results from many different research fields correlate with each other, including the experimental or theoretical results from the electrochemical oxidation of graphite, electrowetting, DIBs, condensed matter physics, graphene bending nano‐mechanics, etc. Moreover, by reviewing the literatures we provide an expected physico‐electrochemical process: When the anions intercalate into graphite, the anions preferentially enter the lattice through the graphite GBs, producing graphene blisters with super elastic stiffness (5–12 graphitic layers in the shell); during anion de‐intercalation, the graphene blister enlarge owing to the absorption of the surrounding de‐intercalated solvated anions. The de‐intercalated anions will be attracted into the blister owing to the strong blister elasticity instead of returning to the bulk electrolyte immediately, which may be affected by the Marangoni effect.In addition, numerous studies have shown that the formation of such graphene blisters can significantly increase the specific capacity of DIBs, but the formation and energy storage mechanism of graphene blisters in DIBs has rarely been researched. Therefore, we provide our expectations for graphene blisters: i) Highly reversible elastic deformation of graphene blisters for anion storage can be achieved by regulating the cut‐off voltage, anion, and solvent composition, or other physical oxidation methods; ii) Thin‐layer graphite with dense graphene blisters may significantly improve the specific capacity. However, several unresolved issues remain: i) After graphene blisters formation, the special physical and chemical properties of the blister shell, such as whether it has the properties of the selective permeation membrane, require to be further studied; ii) We have further predicted that a few solvated anions can be propelled via the Marangoni effect of graphene blisters, but this specific physical process requires to be characterized using advanced instruments; iii) The shape, pressure, cracking limit, and size of the graphene blisters are related to the electrolyte composition, so it is required to determine the appropriate solvent and the composition of the solvation shell of anion; iv) Self‐discharge rate of graphene blister should be studied in‐depth. Moreover, whether the inside of the graphene blister is equivalent to a minute electrolytic cell needs to be experimentally confirmed, which can hinder the outward diffusion of anions, if confirmed, significantly shortening the anion diffusion distance. We hope that graphene in graphite can bring fundamental changes and provide valuable insights into the research on cathode materials for DIBs in the future.The authors declare no conflict of interest.


The task of writing multiple choice question examinations for medical students is complex, timely and requires significant efforts from clinical staff and faculty. Applying artificial intelligence algorithms in this field of medical education may be advisable.During March to April 2023, we utilized GPT-4, an OpenAI application, to write a 210 multi choice questions-MCQs examination based on an existing exam template and thoroughly investigated the output by specialist physicians who were blinded to the source of the questions. Algorithm mistakes and inaccuracies, as identified by specialists were classified as stemming from age, gender or geographical insensitivities.After inputting a detailed prompt, GPT-4 produced the test rapidly and effectively. Only 1 question (0.5%) was defined as false; 15% of questions necessitated revisions. Errors in the AI-generated questions included: the use of outdated or inaccurate terminology, age-sensitive inaccuracies, gender-sensitive inaccuracies, and geographically sensitive inaccuracies. Questions that were disqualified due to flawed methodology basis included elimination-based questions and questions that did not include elements of integrating knowledge with clinical reasoning.GPT-4 can be used as an adjunctive tool in creating multi-choice question medical examinations yet rigorous inspection by specialist physicians remains pivotal. The global healthcare system faces a pressing challenge: increasing the number of healthcare professionals, particularly physicians, without impairing the quality of their education (1). Competency-based medical education (CBME) addresses this issue; however, written knowledge tests remain an essential component in evaluating the basic knowledge acquired by medical school graduates (2). As a result, the demand for creating multiple-choice questions (MCQs) for healthcare professionals' examinations, especially for medical students, is expected to rise, further complicating an already difficult task (3).Each manager and leader in systems engaged with medical education worldwide, should ask themselves “who are my examination writers and what are their qualifications? What tests are employed to ensure that their product is of high quality, not only in terms of content but also in terms of their examinations’ methodology?” The fact that a healthcare professional is experienced and talented in his realm of practice does not indicate his ability and experience in writing MCQs (4). This realm of competencies encompasses a wide range of skills and expertise such as: syllabus tracking, topics’ selection, adhering to the correct rules of medical case presentation, directing the question to a certain domain  and rightfully choosing the way and extent of discrimination between the correct answers and false ones.Since the realm of written tests in medicine is based on combining acquired knowledge and pre-determined methodology, it is only prudent to assume that in the future, the task of writing examinations in medicine would become another output of artificial intelligence applications . Recent advances in this technology have proven this assumption to be correct and reality is overriding imagination. The artificial intelligence application “GPT-4” developed by a private firm known as “Open-AI” – has successfully passed the USMLE, United States Medical Licensing Examination (7). It is only reasonable to hypothesize that this private firm application would be successful, no less, in writing such examinations – as modifications of existing ones and as de-novo MCQs writing.Recognizing the potential benefits of applying artificial intelligence algorithms in exam writing, we describe the results of utilizing GPT-4 to write a 210 MCQs medical examination. The number of questions was chosen according to the number of questions included in our national medical licensing examination. We provide a qualitive description of the test, evaluated by various domain experts and present the challenges of this method.This study did not include research in humans. All methods were carried out in accordance with relevant guidelines and regulations. We have tested GPT-4 abilities in the realm of medical written, MCQs tests—in the challenges of creating new questions on the basis of existing ones  and maintaining high quality of both content and examination-constructing methodology. The GPT-4 model used did not go through any specific training in the fields / clinical disciplines included in the generated examination.We inserted a request/prompt to GPT-4 asking it to rewrite a 210 MCQs test, and to create a new test based on the former one. The relevant, preliminary prompt given to the algorithm is shown in Table Since the preliminary prompt resulted in short, informative questions, without clinical background, we reiterated our request by giving the algorithm another prompt, as presented in Table After receiving the algorithm initial output, we did a quality assurance phase by introducing the GPT-4 generated MCQs to five specialist physicians in the different test domains: internal medicine, general surgery, obstetrics and gynecology, psychiatry and pediatrics. The five specialists were blinded to the source of questions: they did not know the purpose of the study but only knew that they are serving as researchers in a quality-assuring study for MCQs writing in medicine. Also, their feedbacks were presented separately and they were blinded to each other’s opinions on the MCQs quality and professional level. They were also prompted to assess the correctness and appropriateness of the examination MCQs relating to previous versions of this test.The researchers collected the specialists’ feedbacks and sorted them according to different potential errors: a). wrong questions (content mistakes included in the question stem), b). wrong answers , c). methodological faults in either questions or answers , d). interdisciplinary mistakes .All data generated or analyzed during this study are included in this published article. We included all quantitative data regarding the number of questions written and number of rejects presented by the specialists. Also, we present in this article all qualitative data presenting all verbal rejects presented by specialists regarding the AI-generated MCQs. Overall, GPT-4 performance was extremely rapid and efficient. All questions were generated according to the secondary prompt and were introduced to five, blinded specialists who were not aware to the research question and to the optional writing of questions by an artificial intelligence algorithm. Of note, currently there is no option to write image-based questions, which is a major limitation in the field of medicine. In addition, the algorithm had difficulty differentiating between close disciplines, e.g., distinguishing between general surgery versus gynecological pathologies requiring surgical treatment in clinical scenarios addressing the lower abdomen.Only one question (0.5%) out of 210 required replacements due to a completely mistaken answer. This question was in the domain of surgery. A total of 13 questions had more than one possible matching answer due to incomplete clinical information in the question stem, or optional answers that could not be definitively differentiated from each other. These questions and answers were not replaced but necessitated correction and re-writing in a better precision of the question stem or the answer options.In addition, 3 questions presented patients’ age that was unconcordant with the clinical description (categorized above either as wrong questions or wrong answers). Such mistakes were included in disciplines that included questions that could be “age sensitive”: gynecology and pediatrics. For example, a question presenting a 38-year-old woman with irregular menses as postmenopausal. Also, one mistake that could be classified as “gender sensitive”, once again, in gynecology, when an abdominal complaint of a male was questioned, and the optional answers included ectopic pregnancy and ovarian cyst rupture.In the chapter of internal medicine, two questions were considered, by specialist physicians, too easy and replaced, although considered qualifying.In 2 cases, our specialists defined terminology used by GPT- 4 as being outdated or inaccurate: using the term SIRS (Systemic Inflammatory Response Syndrome) in the field of surgery and the term amenorrhea instead of irregular menses. Both questions necessitated correction but otherwise were judged as qualified.Overall, 3 questions were identical / appeared twice in the same test and necessitated writing new ones. These questions were in the field of surgery. Additionally, three questions needed replacement as they presented a repeated topic that exceeded its appropriate weight within the exam syllabus. These questions were in the domain of internal medicine. Two questions were in the elimination format, which is considered methodologically flawed, despite the absence of such question types in the provided example questions.One of the questions had wrong spelling, writing “GI track” instead of “GI tract”.Overall, the majority of problematic questions were in the field of surgery, reaching up to 30% of questions in this subject. It is worth mentioning that this is the only subject in which the algorithm provided an incorrect answer. In the chapter of gynecology, 20% of the questions had inaccuracies, most commonly due to lack of relevant clinical descriptions. In pediatrics and internal medicine only 10% of the questions needed some kind of correction. All MCQs written by GPT-4 in psychiatry qualified and did not necessitate corrective measures. It should be emphasized that the original examination, serving as an example for GPT-4 had no such inaccuracies.Example 01. A). A question necessitating change of the preliminary prompt due to lack of clinical case description at the base of the question.Presented hereafter are examples for questions necessitating replacement or correction:A.HallucinationsB.DelusionsC.AnhedoniaD.disorganized speechExample 01. B). A question generated according to the second prompt, relying on a clinical case presentation as a basis for the knowledge question.Aschizoaffective disorderBSchizophreniaCMajor depressive disorder with psychotic featuresDbipolar disorder with psychotic featuresExample 02. A). A question presenting without a correct answer (content mistake).A.Surgical thrombectomyB.Anticoagulation therapyC.Compression stockingsD.Elevation of the affected limbExample 02. B). Corrected question .A.Surgical thrombectomyB.Non-Steroidal Anti-Inflammatory DrugC.Compression stockingsD.Elevation of the affected limbExample 02 shows a question with incorrect answer regarding the treatment of superficial vein thrombosis. The answer provided by the algorithm was anticoagulation therapy, which is the correct answer for deep vein thrombosis. The above question describes a patient with superficial vein thrombosis, which is treated using non-steroidal anti-inflammatory drugs.Example 03. A). A question using out-of-date terminology.A.It is activated mainly by the Innate Immune systemB.Vasoconstriction is caused mostly by Histamine and BradykininC.Anti-cytokine medications such as Anti-TNF were proven effective in decreasing mortalityD.The Cardiac Index is decreased due to an increase in the AfterloadExample 03 shows a question using the term SIRS, which is no longer used as part of sepsis diagnostic criteria that were replaced two years ago.Example 04. A). A question presenting with an inaccurate terminology.A.Premature ovarian failureB.Polycystic ovary syndrome (PCOS)C.HyperprolactinemiaD.HypothyroidismExample 04. B). A correction of the question, replacing the incorrect professional term.A.Premature ovarian failureB.Polycystic ovary syndrome (PCOS)C.HyperprolactinemiaD.HypothyroidismExample 04 shows a question mistakenly using the term amenorrhea when the correct description had to be irregular menses.Example 05. A). Presenting 38-year-old woman as postmenopausal (age sensitive question).A.Progesterone therapyB.HysterectomyC.Endometrial ablationD.Observation and repeat biopsy in 3 monthsExample 05. B). A question correction, replacing the age of the patient to 58 years old woman.A.Progesterone therapyB.HysterectomyC.Endometrial ablationD.Observation and repeat biopsy in 3 monthsExample 06. A). A question presenting a “pediatric patient” without specifying the fact that he should be an infant (age-sensitive question).A.Cytomegalovirus (CMV) infectionB.Congenital rubella infectionC.Congenital syphilisD.Congenital toxoplasmosisExample 06. B). The age of the patient was added for the appropriateness of optional answers.A.Cytomegalovirus (CMV) infectionB.Congenital rubella infectionC.Congenital syphilisD.Congenital toxoplasmosisExample 07. A question deemed as wrong, necessitating replacement.A.Meniere's diseaseB.Benign paroxysmal positional vertigoC.Migraine-associated vertigoD.Acoustic neuromaExample 07 shows a question in which the algorithm associated Meniere’s disease, a disease classically appearing between the age of 20 to 60, as a viable option (intended to be the correct answer) in children. This mistake should be classified as age-sensitive.Example 08. A). A question presenting a male patient, with two of the provided answers describing gynecological pathologies (gender sensitive mistake).A.Acute appendicitisB.DiverticulitisC.Ovarian cyst ruptureD.Ectopic pregnancyExample 08. B). The gynecological pathologies were replaced.A.Acute appendicitisB.DiverticulitisC.Acute cholecystitisD.Liver abscessExample 09. A question presenting a clinical case of Lyme disease, without mentioning traveling to high incident countries. A mistake potentially classified as geographically sensitive.A.Rocky Mountain spotted feverB.Lyme diseaseC.Erythema multiformeD.Stevens-Johnson syndromeExample 09 shows an example of a question aiming for a disease that has specific geographical distribution, without mentioning traveling to endemic areas.Example 10. A). A question lacked information, resulting in the incorrectness of the marked answer. While the question asks about the most common cause of anemia in children, the marked answer was physiological anemia, which is true for neonates but not for children. Once again, an age-sensitive question.A.Iron deficiencyB.Sickle cell diseaseC.ThalassemiaD.Physiologic anemiaExample 10. B). The question was changed to “neonates” instead of “children” to match the correct answer.A.Iron deficiencyB.Sickle cell diseaseC.ThalassemiaD.Physiologic anemiaExample 11. An elimination request in a too-short question .A.Deficits in social communicationB.Restricted, repetitive patterns of behaviorC.Sensory sensitivitiesD.Excessive worry and anxietyExample 12. Two questions showing the tendency of the algorithm to form too simplistic questions without need for clinical integration.A.SerotoninB.NorepinephrineC.DopamineD.GABAA.Respiratory syncytial virus (RSV)B.Influenza virusC.Parainfluenza virusD.AdenovirusIn this study we aimed to evaluate the performance of GPT-4, an artificial intelligence application, in generating multiple-choice questions (MCQs) for medical exams. Overall, when compared to the tedious process of gathering examination writers from five clinical disciplines, the performance of GPT-4 was rapid and efficient, with the majority of questions deemed suitable for the exam by a panel of specialists, blinded to the source of the questions. However, some errors were identified, including mistaken answers, age and gender inconsistencies, repeated questions, and methodological flaws.The healthcare system worldwide faces a critical dilemma: the need to enhance the quantity of healthcare professionals, particularly physicians, while ensuring the excellence of their education (8). Since written knowledge tests continue to play a vital role in assessing the core knowledge acquired by medical school graduates, the task of generating multiple-choice questions (MCQs) for healthcare professionals' examinations, becomes even more challenging due to the anticipated increase in demand. As written by Alexander Pope (9), more than 200 years ago, “to err is human” and indeed, expertise in a healthcare profession does not automatically translate to the ability to write effective multiple-choice questions (MCQs), which requires a separate set of capabilities. As a result, there is a constant need to reflect on the qualifications of examination writers and the methods used to ensure the quality of written exams.Recent advances in the subject of artificial intelligence (AI), specifically GPT-4 which is a large language model, offers valuable contributions in the field of education . It can be utilized for automated scoring of student papers, easing the burden of grading for teachers . In addition, it can be used as a teacher assistant, providing help in exercises and quizzes generation for both practice and assessment . Additionally, GPT-4 can enhance personalized study plans, further contributing to student understanding and facilitating tailored learning experiences based on individual needs and preferences. GPT-4 was the first algorithm to successfully pass the United States Medical Licensing Examination (USMLE), reaching a very high score of 87 (15). Lastly, it was also demonstrated that it was capable of generating USMLE-like exam questions that are challenging to distinguish from human-generated questions, showing the potential of GPT-4 to assist in exam preparation (16).The objective of our study was to propose the utilization of GPT-4 as an aid in exam preparation while identifying its limitations, helping reduce the workload of the physician-educator (17). In general, the process of generating tests with GPT-4 was fast and efficient in terms of time. However, approximately 15% of the questions generated from the detailed prompt required some correction, primarily due to inaccuracies in content or faulty methodology.The main revised questions in terms of wrong content basis were due to the lack of sensitivity in specific topics, ignoring the requirements to use specific age, gender or geographical location for either the question or the answers. Such lack of sensitivity was mentioned in a recent paper, describing a user getting the wrong answer generated by GPT-4 in chemistry, providing an answer with wrong units (18). This limitation seems to be due to GPT-4’s limitations in reasoning recalled knowledge (19).One of GPT-4’s major limitations is that its primary source of training data is the internet, which can be inaccurate and unreliable . This results in the necessity to validate GPT-4 output for tasks requiring high levels of credibility (20). A less concerning example is typos, e.g., “GI track” instead of “GI tract”. While more concerning inaccuracies can result in providing incorrect answers, e.g., treating superficial vein thrombosis using anticoagulation instead of NSAIDs, due to either inaccurate training data or “factualness error” which is caused by lack of training data in the specific, asked subject (19).Our findings of GPT-4 limited integration of knowledge and clinical reasoning, align with recent papers highlighting the challenge of logical reasoning (20), incapability of GPT-4 to generate novel findings based on existing knowledge and limited capacity to innovate (21). This is the result of GPT-4 architecture, which focuses on providing coherent responses from its vast knowledge base rather than extrapolating new insights or hypothesizing connections through identification of hidden patterns and relationships (18).Medical, MCQs-based examination writing by GPT-4 is feasible yet necessitates rigorous inspection by specialist physicians. Learning the characteristic flaws and mishaps of GPT-4 in this task is essential for faculty members intending to apply AI potential in this realm. We did not challenge artificial intelligence with questions containing figures, tables of graphs that are sometimes desirable.This study concentrated on the production of one, multidisciplinary MCQ examination. Also, we describe our experience with GPT-4 which will be updated in the future. Our findings lay the fundaments for further, similar studies.


IPF is a fatal lung disease characterized by intensive remodeling of lung tissue leading to respiratory failure. The remodeling in IPF lungs is largely characterized by uncontrolled fibrosis. Fibroblasts and their contractile phenotype the myofibroblast are the main cell types responsible for typical wound healing responses, however in IPF, these responses are aberrant and result in the overactivation of fibroblasts which contributes to the inelasticity of the lung leading to a decrease in lung function. The specific mechanisms behind IPF pathogenesis have been elusive, but recently the innate and adaptive immunity have been implicated in the fibrotic processes of the disease. In connection with this, several in vitro co-culture models have been used to investigate the specific interactions occurring between fibroblasts and immune cells and how this contributes to the pathobiology of IPF. In this review, we discuss the in vitro models that have been used to examine the abnormal interactions between fibroblasts and cells of the innate and adaptive immune system, and how these contribute to the fibrotic processes in the lungs of IPF patients. Idiopathic pulmonary fibrosis (IPF) is a progressive, debilitating lung disease that has risen dramatically in incidence, with a 78% increase between the years 2000 and 2012 . CurrentThe pathologic mechanisms that underline IPF have been shown to involve an exaggerated and abnormal response to wound healing and tissue repair , 5, 10. To assess and understand immune-fibroblast crosstalk in IPF, different in vitro models have been established. These diverse in vitro models that range from 3-dimensional (3D) hydrogel transwell co-cultures to decellularized lung scaffolds, incorporate immune cells and fibroblasts derived from IPF patients and healthy control individuals as well as continuous lung cell-lines and mimic the spatial configuration of cells in the in vivo environment to allow for the study of abnormal cellular communication , 22. TheIn this review, we will summarize different studies that have used 2D and 3D in vitro models to examine the mediators and mechanisms involved in the crosstalk between pulmonary fibroblasts and immune cells in the pathogenesis of IPF. We will assess these studies in the context of the fibrotic mechanisms of IPF  as well as how this interaction may lead to the production of inflammatory mediators that contribute to lung tissue remodeling in IPF. We then place these in the context of (future) therapeutic studies in IPF.To examine the role of fibroblast-immune cell interactions in IPF, various in vitro models have been utilized to mimic the microenvironment of the lung . The simIPF is characterized by chronic remodeling of the lung tissue that involves excessive fibrosis which results in the destruction of the lung parenchyma, subsequently disrupting gas exchange and concluding in respiratory failure , 33, 34.In line with the role of defective fibroblast-immune cell interactions in aberrant matrix turnover , 39, BagFurther, Novak et al., isolated IPF and control-derived primary alveolar macrophages (AMs) and lung fibroblasts. Various combinations of CM experiments, direct co-cultures in tissue culture plates, transwell and 3D collagen hydrogel cocultures were then set up to assess the interaction between different combinations of IPF- and control- derived lung fibroblasts and AMs . After eIn addition to macrophages, neutrophils are also important innate immune cells with elevated numbers in the lungs of IPF patients –46. AlthThe adaptive immunity has also been identified as an important contributor to fibrotic mechanisms in IPF , 49, 50.Taken together, the studies presented in this section demonstrate the importance of the innate and adaptive immunity in regulating mechanisms of immune cell-fibroblast crosstalk that may drive fibrotic changes in IPF. Innate immune cells such as mast cells, macrophages and neutrophils all interact with fibroblasts through the release of growth factors and enzymes  that leads to increased ECM and structural proteins such as collagen, fibronectin, and α-SMA , 48, 54.The fibrotic mechanisms that characterise IPF have been shown to be impacted by the survival, proliferation, and migration of lung (myo)fibroblasts , 56. In To corroborate the role of lung fibroblast-immune cell crosstalk in mechanisms of fibroblast proliferation in IPF, Wygrecka et al., isolated IPF- and control -derived PHLFs and mast cells and co-cultured them directly . The dirDue to the difficulty in culturing neutrophils in vitro, Gregory et al., used a neutrophil derivative to assess how human lung neutrophil-fibroblast crosstalk may influence defective fibroblast proliferation in IPF . Here, tThe interaction between adaptive immune cells such as T and B cells with fibroblasts have also been shown to influence fibroblast proliferation and migration in IPF. In a study by Chavez-Galan et al., human T cells enriched via negative selection from PBMCs isolated from the peripheral blood of healthy volunteers was exposed to CM collected from control- or IPF-derived primary lung fibroblasts . IPF-derTaken together, the studies in this section suggest that lung immune cell-fibroblast interactions are crucial for aberrant rates of proliferation and migration of lung fibroblasts that add to remodeling mechanisms in IPF. The interaction between fibroblasts and innate immune cells lead to the release of mediators  that increase lung fibroblast proliferation and migration , 54, 61.In IPF, it is believed that repeated epithelial injury leads to chronic inflammation and the activation of various immune cells as well as fibrosis via the activation of (myo)fibroblasts , 64. In To analyze the contribution of macrophage-fibroblast interactions in IPF, Prasse and colleagues isolated PHLFs and alveolar macrophages (AMs) from patients with IPF and healthy controls and used them to establish both direct and indirect co-cultures . AMs useMast cell-fibroblast interactions have also been implicated in inflammatory mechanisms that contribute to tissue remodeling in IPF . Bagher Taken together, these data demonstrate a distinct role for fibroblast-immune cell crosstalk in inflammatory mechanisms that may contribute to remodeling in the IPF lung. Here, lung fibroblasts may interact with macrophages to produce classical inflammatory mediators  that attract other immune cells to the site of injury in the lungs. The interaction between mast cells, neutrophils and fibroblasts lead to the release of IL-8 and IL-6 which are classical chemoattractants for neutrophils  co-culture and CM exposure models to assess the role of immune cell-lung fibroblast interactions in various mechanisms such as fibrosis, fibroblast migration and proliferation as well as inflammatory processes associated with the chronic remodeling of lung tissue in IPF  co-culture models and how they contribute to the various mechanisms of chronic remodeling in IPF. A common significant finding was that the interaction between lung fibroblasts and different lung immune cells in both the innate and adaptive immunity such as mast cells, neutrophils, macrophages, B cells and T cells result in an increase in fibroblast proliferation and migration as well as alterations in lung fibroblast activation and ECM protein deposition which are crucial mechanisms involved in excessive fibrosis and lung tissue remodeling in IPF. Specifically, immune cell-fibroblast crosstalk contributes to fibroproliferative mechanisms in IPF because of interactions through mediators, growth factors and enzymes such as HGF, PGE, tryptase and NE. Interestingly, immune cell-fibroblast interactions through mediators such as CCL18, CCL2, CX3CL1, CXCL1, IL-6 and IL-8 also contribute to inflammatory mechanisms that may add to tissue remodeling in IPF and need further investigation to ascertain the role of inflammatory mechanisms in IPF therapeutics. In line with this, through these studies, a protective role of T-cells on fibroblast activation and fibrotic phenotype has been uncovered that may serve as a basis for further (therapeutic) studies. These and other disease processes which have also been shown to be targeted as part of the mechanism of action for the antifibrotic drug Nintedanib, points to how crucial multicellular co-culture model studies are in understanding nuanced and complex disease mechanisms and eventually discovering a cure for IPF.


Introduction: Holt–Oram syndrome (HOS) is a rare genetic disorder characterized by upper limb abnormalities, congenital heart defects, and/or conduction abnormalities. Sequence alteration of T-box transcription factor 5 (TBX5) is correlated with the incidence of HOS.Case description: We present the case of a 24-year-old female with upper limb alterations  and an anomalous left main trunk arising from the right coronary sinus. The patient inherited a base T (reference C) at rs883079 from her mother and base C (reference T) at rs10850326 from her father, both of which belong to the 3′-untranslated region (UTR) of the TBX5 gene; no alterations in TBX5 expression or single-nucleotide polymorphisms (SNPs) in other exon areas were found. We explored the effects of TBX5 on cardiomyocytes using the HL-1 cell line and TBX5-knockdown cells.Discussion: Quantitative polymerase chain reaction analysis demonstrated that TEKT2, TEKT4, and SPTB expression decreased after TBX5 knockdown, while chromatin immunoprecipitation analysis further revealed that TBX5 binds to the TEKT2, TEKT4, and SPTB promoter regions to promote gene transcription. Our findings support a novel TBX5-related pathogenic mechanism in HOS. Holt–Oram syndrome  is a rare genetic disorder characterized by upper limb abnormalities, congenital heart defects, and/or conduction abnormalities . The uncHOS is caused by a mutation in the T-box transcription factor 5 (TBX5) gene (located on chromosome 12q24.1), a protein-coding gene consisting of nine coding exons and belonging to the T-box domain . The NkxBecause of its rare occurrence and consistent phenotypes and symptoms, only a few studies have been conducted on HOS development and little is known of the functions of its molecular components. In this study, we aimed to describe a novel TBX5-related pathogenic mechanism of HOS in which there was no mutation in TBX5 or single-nucleotide polymorphisms (SNPs) in other exon areas except the 3′-untranslated region (UTR). Our study findings could promote the development of more effective prognostic and treatment tools and enable early identification of individuals at risk of transferring the disease.In December 2021, a 24-year-old female was admitted to the hospital because of intermittent chest tightness and suffocation for 6 years, followed by 3 months with no medical history. The results of the medical examination were as follows: temperature 36.8°C, pulse 72 times/min, respiratory rate 16 times/min, and blood pressure 113/76 mmHg. More results included clear, stable breathing, coarse breath sounds in both lungs, no rale or wet rale, no uplift in the precardiac area, strong heart sound, consistent heart rhythm (HR 72 beats/min), no murmur in the auscultation area of each valve, flat and soft abdomen, no tenderness, internal rotation malformation of both upper limbs, no external rotation, and no edema in either of the lower limbs.A bilateral elbow wrist joint examination demonstrated the partial fusion of the bones at the proximal end of both the ulna and radius, widening of the radial ulnar joint at the distal end, and poor alignment, suggesting congenital dysplasia in the wrist and elbow joints . CardiacThe transient heart rate increased and chest tightness appeared after admission. An electrocardiogram examination suggested a high sinus heart rate, HR 123 beats/min, and ST segment depression. A three-dimensional printed model was used to determine the appropriate surgical approach . Midlinehttps://ncbi.nlm.nih.gov/clinvar/), which indicated that the patient inherited a base T (reference C) at dbSNP ID rs883079 from her mother and base C (reference T) at dbSNP ID rs10850326 from her father, both of which belong to the 3′-UTR of the TBX5 gene . The screened mutations and SNPs were correlated using ClinVar  and it plays a fundamental role in tissue and organ formation during embryonic development and in maintaining the normal development of the upper limbs and heart (structure and electric system) through the activation of different genes . VariantHand deformities sometimes lead to a diagnosis of congenital heart disease; however, a genetic investigation is more appropriate to diagnose and prevent complications in subsequent generations. TBX5 variants, such as those with non-sense, splicing, and missense mutations, were detected in two previous studies of 78 HOS patients and their family members as well as three other families . TBX5 vamiR-98-5p regulated the myocardial differentiation of mesenchymal stem cells by targeting TBX5 in rat bone marrow samples, and miR-10a and miR-10b targeted the 3′-UTR of TBX5 to repress its expression in human embryonic kidney 293T cells . miR-182The mechanisms mediated by miRNAs and TBX5 may be spatiotemporally regulated, which may explain why the patient’s father and mother had SNPs and mutations but not HOS. The patient also inherited the SALL4 SNP in the exon area from both her father and mother. In zebrafish, SALL4 acts downstream of TBX5 and is required for pectoral fin outgrowth; mutations at the SALL4 locus on chromosome 20 resulted in a range of clinically overlapping phenotypes, including Okihiro syndrome, HOS, acro-renal-ocular syndrome, and thalidomide embryopathy . KoshibaWe identified 578 exons, along with 498 3′-UTRs and 346 5′-UTR mutations, in the patient using whole-exome sequencing, including the female urogenital genes PKD1, SON, and BMPR1B, as well as the reproduction-related genes TEK, COL11A1, ANKRD11, LEMD3, and SPTB, which was consistent with the findings of This study had some limitations. To further explore the effects of TBX5 on cardiomyocytes, the HL-1 cell line was used with TBX5 knockdown. Quantitative polymerase chain reaction and chromatin immunoprecipitation analyses revealed that TBX5 binds to the TEKT2, TEKT4, and SPTB promoter regions to promote gene transcription. Further studies on mouse models and human cohorts are required. In addition, we only carried out sequence alignments between the TBX5 3′-UTR regions and previously reported miRNAs, and further validation is still needed. The effects of other genes on HOS formation and progression still need to be investigated.We provide a novel gene signature of HOS, including TBX5 3′-UTR and downstream genes, such as SALL4 SNP, which may be a potential therapeutic target. Our findings support a novel TBX5-related pathogenic mechanism in HOS, though further experimental validation is needed. If this mechanism is correct, novel approaches can be devised for gene expression correction using pharmacological agents.