Silk fibroin (SF) is a natural material with good biocompatibility and excellent mechanical properties, which are complementary to graphene with ultrahigh electrical conductivity. In this study, to maximally combine graphene and silk fibroin, a well-dispersed silk fibroin/graphene suspension was successfully prepared in a simple and effective way. Then we prepared a flexible conductive SF/graphene film with a minimum resistance of 72.1 ± 4.7 Ω/sq by the casting method. It was found that the electrical conductivity of the SF/graphene film was related to the water content of the film, and the variation was more than 200 times. Therefore, it will play an important role in the field of humidity sensors. It also has excellent mechanical properties in both wet and dry states. These unique features make this material a promising future in the fields of biomedical applications, wearable sensors, and implantable internal sensors. Meanwhile, SF/graphene composite materials also have good biocompatibility. Vera-Sanchez et al. [Flexible and wearable electronics have great potential to be applied in human health monitoring systems . They wez et al.  culturedz et al.  preparedBombyx mori larval silkworms with graphene. Secondly, it is well known that graphene is highly hydrophobic and chemical inert, and cannot be dispersed in silk fibroin solution, but graphene oxide (GO) can be dispersed. Therefore, GO is mixed with the silk fibroin (SF) and then reduced to obtain the graphene/SF composite material. Wang et al. [2+ suspensions, which are suitable for the fabricateion of polymorphic materials, such as fibers, films, and coatings. On Ling’s basis, Wang et al. [Accordingly, we intend to combine graphene and silk fibroin to prepare flexible and wearable electronics with good skin comfort. However, it is challenging to disperse graphene in silk fibroin ,26 and tg et al.  preparedg et al.  just disg et al.  used a kg et al.  to prepag et al.  made an Therefore, a safe, mild, and non-toxic dispersant is urgently needed, and poly  (PVA) is a good choice ,32,33. PIn this work, butanediol was used to improve the flexibility of the silk fibroin, and an appropriate concentration of graphene/PVA suspension was prepared. Then, the two suspensions were mixed, and the silk fibroin/graphene composite film was obtained by the casting method. This film material made by the method we proposed has excellent electrical, and mechanical properties and it is expected to be applied in flexible and wearable electronics, humidity sensing, and biomedical field. Bombyx mori) cocoons were purchased from Suzhou Siruibao Biotechnology Co., Ltd. . Graphene/N-methyl-2-pyrrolidone (NMP)  solution was purchased from Shanghai Najiu Intelligent Technology Co., Ltd. . Cellulose dialysis membrane  was bought from Union Carbide Corporation . PVA-103 was provided by Aladdin Industrial Corporation . Sodium carbonate was purchased from Sinopharm Chemical Reagent Co., Ltd. . 1,4-Butanediol was supplied by Shanghai Lingfeng Chemical Reagent Co., Ltd. . Lithium bromide was purchased from Tiancheng Chemical Co., Ltd. . All the chemicals were of analytical reagent grade and used without further purification. DC power supply (MT-1520) was bought from Maisheng Power Technology Co., Ltd. .Silkworm  with frequent water changes.The silkworm cocoons were degummed in a 0.02 mol/L Naw/v) solution of butanediol (BD) was added to the silk fibroin solution at a mass ratio of SF: Butanediol = 5:1 to obtain a SF/BD mixed solution.A 10% (w/v) PVA aqueous solution was added according to the mass ratio Graphene: PVA = 4:1. The dispersion was then dialyzed with deionized water for 48 h to obtain Graphene/PVA dispersion. The concentration of the graphene dispersion is 2 mg/mL.PVA was dissolved in deionized water at 95 °C for 3 h, and then the solution was cooled to room temperature. The NMP solution of graphene was diluted with a certain amount of deionized water, and a certain amount of 10% (The prepared SF (or SF/BD) solution and the Graphene/PVA dispersion were uniformly mixed according to the mass ratio, and the Graphene/SF/PVA mixed solution was dropped on the PDMS template, and air-dried at 20 °C and 65% relative humidity (RH). Thus the SF/Graphene film was obtained. The Hitachi S-4800 scanning electron microscope (SEM) was used to observe the cross-sectional morphology of SF/GrP/BD film (SF:GrP:BD = 10:2:1) at an acceleration voltage of 3 kV.For sample preparation, SF, SF/BD, SF/GrP, SF/GrP/BD films  were cut into powders. The crystal image of the sample was analyzed by the X’pert-Pro MRD X-ray diffractometer (XRD) . Test condition: tube current 35 mA, tube voltage 40 kV, scanning speed 5 °/min and using Cu–Kα rays. XRD patterns were recorded in the 2θ region from 5° to 40°.−1 using the Nicolet iS5 spectrometer, equipped with an iD7 ATR accessory . Each spectrum was acquired by the accumulation of 32 scans with a resolution of 4 cm−1.Attenuated total reflectance Fourier transformed infrared spectroscopy (ATR-FTIR) was used to analyze the structural changes of the SF in four kinds of films (the same as XRD). All infrared spectra were recorded in the range of 4000–550 cmM as the weight after drying. The film was then placed in 20 mL of deionized water and shaken in a 37 °C water bath for 1 h. The absorbance was measured for the solution using an ultraviolet-visible spectrophotometer at 278 nm to evaluate the concentration (C) of silk fibroin dissolution in deionized water, and the film loss rate was calculated according to the following formula [D is the dissolution rate, M is the weight after drying, C is the silk fibroin concentration obtained from the absorbance comparison standard curve, K is the percentage of silk fibroin content in dry weight of the film, and V is the total volume of the solution.Different SF/GrP/BD film samples were taken and placed in an oven at 90 °C for 3 h to test M1, and this film was placed in an oven at 90 °C for 3 h to be taken out and weighed, M2. Then the real-time water content of the film at that moment is E. The film’s mass swelling rate (water) is S.The water content of the SF/GrP/BD film was tested as follows. The film was firstly immersed in deionized water for 24 hours. After taking out, the water droplet on the film surface was absorbed with absorbent paper. The saturated water content and mass swelling rate (water) were calculated and the sheet resistance was measured. Then, during the film drying process, the water content, and the sheet resistance were tested at different times. When the quality of the film remained unchanged, it was placed in an oven at 90 °C and dried for 3 h, which was recorded as moisture content of 0%. In this process, the water content and the sheet resistance were tested separately at various times. Repeat the above process three times for the same film. At a certain moment, weigh the film mass to The conductivity of the films was compared by measuring the sheet resistance of the film. The sheet resistance of the sample was tested with the ST-2258C multi-function digital four-probe tester, and five points for each film were tested to calculate the average value and variance.The mechanical properties of the films were tested by a BOSE ELF3220 machine  in tensile mode at 20 °C and 65% relative humidity with a tensile speed of 0.008 mm/s. The SF, SF/BD, SF/GrP, and SF/GrP/BD films were cut with a dimension of 5 mm × 10 mm × 50 μm and placed in a constant temperature and humidity (20 °C and 65% (RH)) room for 24 hours for dry mechanical testing. For wet testing, films were soaked in deionized water for 24 h at room temperature. Subsequently, samples for wet testing were loaded into the tester and the surface dried with absorbent paper. The clamping distance and tensile speed were 8 mm and 20 mm/min, respectively. Each sample was repeated six times.A simple series circuit was built with 3 W Light-Emitting Diode (LED) lamp bead, the SF/GrP/BD film (cut with a dimension of 5 mm × 10 mm × 50 μm and placed at a constant temperature and humidity (20 °C and 65% relative humidity) for 24 hours), DC power supply, and wire. Once the power switch is turned on, and the small bulb starts to light, then 3 mL deionized water is dropped onto the SF/GrP/BD film with a dropper. The brightness change of the small bulb is then observed within the next three minutes .Graphene cannot form a stable aqueous solution alone due to its strong hydrophobicity. Many reports mentioned that PVA could solve the problem of graphene water solubility . On the In order to investigate the structural changes of SF after loading GrP and BD into the film, XRD and ATR-FTIR tests were performed . From th−1), amide II (1600–1500 cm−1), and amide III (1350–1200 cm−1). From the ATR-FTIR figure, it can be seen that SF and SF/GrP both have absorption peaks at 1640 cm−1, corresponding to the characteristic peaks of the random-coil . Since SF without BD mainly exists in the film in the form of a random-coil, the dissolution rate is as high as 76.5%. When the BD/SF (%) is 5%, the dissolution rate drops to 9.7%. With the increase of BD content, the dissolution rate of the film decreases gradually. When the BD/SF (%) is 20%, the dissolution rate of the film decreases slightly, and it was basically stable at about 3%. Therefore, the addition of BD could solve the water solubility problem of SF/GrP film materials. The properties of many polymer materials are strongly related to their water content . TherefoThe dry and wet mechanical properties of SF, SF/BD, SF/GrP, SF/GrP/BD films are shown in According to the above result of the sheet resistance changes with water content, the lower the water content, the lower the sheet resistance. Therefore, when a droplet was dropped onto the film, the film resistance became large, and the LED lamp bead gradually darkened. It can be seen from In this study, a flexible conductive SF/Graphene composite film with high conductivity (minimum resistance of 72.1 ± 4.7 Ω/sq) was successfully prepared by a simple combination of SF and graphene. The addition of BD solved the problem of water solubility of the film and improved the toughness. The structure of the film was characterized by SEM, XRD, and ATR-FTIR. The results showed the obvious and relatively neat orientation of the layer structure of graphene sheets, and the addition of BD changed the secondary structure of the silk fibroin, which reduced the dissolution rate of the film. The dissolution rate of the SF/GrP/BD films with different BD content was determined, which laid a foundation for the subsequent wet state experiment. It was found that the resistance of the film had a great relationship with the water content, which was caused by the increase of the distance between the graphene sheets derived from the swelling of SF. The change variation was more than 200 times, and the repeatability is good, far exceeding the ordinary humidity sensor. The SF/Graphene composite film had excellent mechanical properties in both wet and dry states, which can meet the needs of various situations. For demonstration purposes, we utilized SF/GrP/BD films for LED lamp bead test and flexible display. These attractive performances encourage further exploration of their applications in the fields of biomedical materials, wearable sensors, and implantable internal sensors.


The influences of glass film thickness and chemical potential/doping level of graphene on spontaneous emission enhancement are also studied in this paper. A comparison is made between graphene and other coating materials such as gold and silver to see their performances in SER enhancement.We propose an ultra-thin glass film coated with graphene as a new kind of surrounding material which can greatly enhance spontaneous emission rate (SER) of dipole emitter embedded in it. With properly designed parameters, numerical results show that SER-enhanced factors as high as 1.286 × 10 The chemical potential μc can be tuned by varying chemical doping levels during the fabrication, i.e., ng is the carrier concentration of graphene and μ is set to 10,000 cm2/(V s) which is a moderate value obtained in experiments [For MIR wavelengths considered here, the conductivity of graphene is dominated by intraband transition and can be simplified to a Drude-like expression as , 19:4\doeriments . It is rentclass1pt{minimagraphene , 22. Thed = 20 nm, dipole moment oriented in the z direction, chemical potential of graphene μc = 0.64 eV. Hx components in graphene monolayer 1 and monolayer 2 have the same field pattern , an SER-enhanced factor of 1.197 × 106 can be achieved in the structure. So in reality, the SER-enhanced factor will be lower than that obtained without taking mobility degradation into consideration, but still on the order of 106.However, there exist some factors that limit the SER-enhanced factor. Chemical doping will inevitably introduce scattering source and thus degrade the mobility of intrinsic graphene . MeanwhiL2 is the in-plane quantization area, k is the in-plane wave vector of plasmon. The dispersion relationships of TM SPs on silica–silver and silica–graphene interfaces are plotted in Fig. Finally, we compare graphene with other coating materials such as gold and silver. Two graphene monolayers shown in Fig. itten as .Fig. 6a6. The influences of glass film thickness and chemical potential/doping level of graphene on SER-enhanced factor were also studied. A comparison of graphene and other coating materials including gold and silver was made in terms of SER enhancement. Explanations and quantitative analysis were given to the observed phenomena. Different from previously reported works, the metamaterial we proposed benefits from the nature of graphene SPs. The radiated fields are better confined in the ultra-thin glass film coated with graphene, which gives rise to stronger light-matter interactions and higher SER-enhanced factors. It has great potential to work as the surrounding material of highly efficient quantum emitters for novel single-plasmon devices and quantum plasmonic applications in the near future.In conclusion, we proposed an ultra-thin glass film coated with graphene where the SERs of embedded quantum emitter were multiplied by a factor typically on the order of 10


It is important to note that the availability of F-P resonant probe with a defective clamped circular graphene membrane can be improved notably by fabricating the defected circular membrane to a double-end clamped beam, which gives a broader perspective to characterize the resonance performance of opto-thermally excited F-P resonators.An opto-thermally excited optical fiber Fabry-Perot (F-P) resonant probe with suspended clamped circular graphene diaphragm is presented in this paper. Then, the dependence of resonance frequency behaviors of graphene diaphragm upon opto-mechanical factors including membrane properties, laser excitation parameters and film boundary conditions are investigated via COMSOL Multiphysics simulation. The results show that the radius and thickness of membrane will linearly affect the optical fiber light-induced temperature distribution, thus resulting in rapidly decreasing resonance frequency changes with the radius-to-thickness ratio. Moreover, the prestress can be regulated in the range of 10 Q) of suspended single-layer graphene sheet over trenches, which indicated the feasibility of resonant graphene sensors [Graphene is a single atomic plane of graphite, which is the thinnest and strongest two-dimensional material in the universe known up to now ,2,3,4. D sensors . Then, C sensors . In addi sensors . Recentl sensors ,15. Alth sensors ,14,15,162 wafer actuated by a laser with a wavelength of 488 nm, therefore suggesting graphene’s application as thermal management material in nanoelectromechanical system (NEMS) [In contrast, opto-thermal excitation is a more efficient actuation mechanism for suspended resonant structures utilizing thermomechanical coupled ,18, becam (NEMS) . Additiom (NEMS) . Additiom (NEMS) . Althoug2 resonators at room temperature and then performed the thermomechanical resonance measurement, which offered a support for development of opto-thermally excited graphene resonator [2 nanomembrane resonator in air by using optical F-P interferometric excitation and detection method [In reality, in 2013, Lee et al. observed the intrinsic thermomechanical modes of the MoSesonator . Recentln method . They aln method . Then, Dn method . Unfortu8 Pa to 109 Pa, wherein the prestress was available to be tuned by changing the temperature ranging from 293.25 K (room temperature) to 393.15 K. Compared to those with asymmetrical defects at the edge of membrane, the F-P resonator probes under bilateral symmetry boundary conditions obtained better vibration modes when an unstable clamped boundary was turned into a stable double-end fixed one, thereby significantly enhancing the performance of F-P resonators.Therefore, in order to advance opto-thermal applications in graphene F-P resonators, in combination with the fabricated resonant probe with clamped circular graphene membrane, the relations between the membrane structural properties, optical excitation parameter and membrane boundary condition the and resonance frequency response were investigated by COMSOL Multiphysics simulation in this paper. The simulation results illustrated that the prestressed F-P resonator exhibited an excellent linearity in a prestress range of 104 V/W for observing and processing of useful F-P resonance signals. Q = 4.81) compared to the measured Q value of 3.37 in our previous work [With the use of the presented F-P resonant probe, the obtained natural frequency of 50-μm diameter circular graphene membrane with a thickness of 3.35 nm was confirmed as 1.7 MHz, which is much higher than the resonance frequency of 17 kHz in previously reported F-P resonator with a 125-μm diameter graphene film . Unfortuous work . Among thQ is the absorbed heat; n is the layer of graphene membrane; I is the intensity of the actuated laser; Regarding the graphene membrane as the resonant element of the F-P probe, the capillary substrate effect of the probe is ignored in the following simulation, which is similar to that in Reference . In thismembrane . It is wI) can be defined as:r is the radius of the membrane; r0 is the radius of laser beam; I0 is the peak laser power per unit area at the center of beam spot, which is given by [P is the output power of laser. In this case, a simulated “heat source” was assigned in the module “Solid heat transfer”, and then, the absorbed heat was determined by Equations (1)–(3). After that, the modules “Steady state study” and “Eigen frequency study” were chosen to achieve the resonance characteristics of graphene membrane imposed on by opto-thermal actuation.The laser beam generated by the laser S follows Gaussian light distribution so that the intensity (given by :(3)I0=Pπr) in the range of 10~30 μm and thickness (t) ranging from 0.335 nm to 3.35 nm (1–10 layers) in COMSOL simulation. As illustrated in the inset of r) of graphene membrane was fitted in c) of 99.86%, which demonstrates that the temperature in the center point of graphene membrane will increase with radius linearly under the opto-thermal actuation. Additionally, it can be clearly seen from n) varies from monolayer to 10 layers under fixed radii. However, due to the difference in absorption energy and attraction force among different layers, the thickness (or layer number) of graphene has a vital impact on the temperature distribution and resonance frequency of the membrane [r/t) was built to further estimate the effect of radius and thickness comprehensively. From r/t); however, the dependence on membrane thickness is dominated. In other words, when the ratio is constant, the temperature will increase sharply with an increase of layer number. This is mainly due to more heats accumulated in a thicker membrane. Furthermore, thicker graphene membrane may strengthen the opacity and absorbance of light on basis of Equation (1) [In view of the aspect ratio of graphene, an investigation of the radius and thickness of circular membrane as dominant structural parameters was introduced to evaluate the vibration performance of graphene membrane. We increasingly swept the radius  ,43, suchr/t, the thickness of membrane should be a trade-off for achieving high-efficient opto-thermal energy conversion and high natural frequency. Thus, regarding the 50-μm diameter capillary used in the presented F-P resonant probe, the radius of membrane was confirmed as 25 μm to match the capillary, and its thickness was set as 3.35 nm (10 layers) to ensure relative high natural frequency for the subsequent resonant characteristics simulation on F-P resonators with clamped circular graphene membrane.Referring to the inset of 3 Pa to 1 × 1010 Pa, which covers the measured values that may appear in graphene [7 Pa. By contrast, the natural frequency changes from 0.354 × 107 Hz to 1.878 × 107 Hz with a difference of 15.24 MHz when the prestress is larger than 1 × 107 Pa. In consideration of the actual prestress of graphene lower than 109 Pa for this type of resonant probe whose endface is adhered with graphene by van der Waals force [7 Pa and 109 Pa. Moreover, in terms of Ref. [f of circular graphene membrane can be determined as:8 Pa to 109 Pa. As depicted in the inset of Due to the ultrathin thickness of graphene, the van der Waals force between the film and the substrate will cause strain stretch, which is closely related to the natural frequency-dependent initial prestress of graphene. As a result, the prestress of clamped circular graphene membrane is simulated in the range of 1 × 10graphene . In accols force , the pre of Ref. , the nat8 Pa to 1.54 × 109 Pa  with a prestress-to-temperature scale factor (tk) of 14.2 MPa/K, which turns out that the prestress of graphene membrane can be tuned by adjusting environmental temperatures. Furthermore, in combination with the aforementioned results in fk) was calculated as 3 × 104 Hz/K. Thus, it could be concluded that the natural frequency of graphene membrane enhanced nearly 3 × 106 Hz if the environmental temperature increased to 393.15 K from 293.15 K, which is an extremely remarkable improvement for this type of F-P resonators.Currently, the typical ways to improve the prestress of graphene relate to reducing the surface roughness of substrate by polishing the endface , employiP) and light spot radius (r0), which reflects the total energy absorbed by graphene membrane. However, it is important to point out that the suspended graphene diaphragm will be partially burn out when the temperature at the center of the membrane is too high. This will definitely disable the F-P resonant probe and then lead to the absence of resonance effect. Generally speaking, the damage threshold of graphene membrane is in connection with the fabrication and transformation process due to the presence of the pretension of transferred graphene membrane. In view of our previous experiments, the output power was kept below 10 mW to avoid the physical damage of graphene membrane mentioned above. Hence, herein we set P and r0 in the range of 1-10 mW and 5–25 μm, respectively. As shown in P spanning from 1 mW to 10 mW, which had a same variation tendency as the influence of r/t on the temperature with a correlation coefficient of 99.86%. Particularly, it should be mentioned that r0 exhibited a more remarkable effect on the surface temperature of graphene membrane under a large laser output power, and the temperature decreased gradually as the radius of the laser became large. In light of the relation between r0 and I0 in Equation (3), the higher the light spot radius is, the weaker the light that approaches the membrane becomes. Furthermore, more surface areas of the suspended membrane will be covered if the light radius gets larger, leading to more temperatures distributed dispersedly and uniformly in the membrane. Therefore, the temperature at the center point may be regulated by controlling the laser output power and light spot radius to generate the enhanced resonance effect while avoiding the damage of suspended graphene diaphragm.Based on Equations (1)–(3), the light intensity excited by laser is strongly concerned with the laser output power (fR is the resonance frequency (MHz); f0 is the natural frequency (f0 = 2.3094 MHz). It is generally assumed that the fitted result possesses a good linearity when the relative error is below than 1%. Referring to r0 and the cross term f-P and f-r0) were also projected in f-r0 projection especially, the frequency verse laser radius curve showed a bigger nonlinearity than the f-P curve; however, the nonlinearity reduced gradually with the used smaller laser output power. In this way, the linearity can be promoted by limiting the laser output power range. f0 = 2.55 MHz. The maximum relative error between the simulated data and the calculated ones by Equation (6) was 0.85%, which is lower than the allowable error of 1% mentioned above. In consequence, Equation (6) is available to approximate the initial output power and radius of laser beam for this type of F-P resonators at room temperature to make the detected signals display a preferable linearity. As mentioned above, the change of laser parameters (output power and light spot radius) has an outstanding impact on the resonance frequency induced by temperature. As far as we know, previously reported studies mainly focused on the influences of the laser power on intensity. Nevertheless, the influence of laser output power and laser spot radius on the resonance frequency has not been involved. Thus, by setting the laser output power and light spot radius, respectively, spanning from 1 mW to 10 mW and from 5 μm to 25 μm, the simulated frequency response was achieved as labeled by black sphere in w) at the center point of film by the laser. However, during the process of transferring graphene to capillary endface, due to the imperfect attaching and clamping of circular membrane, certain boundary defects will appear as displayed in l1) of the unilateral defective membrane should be at least equivalent to 30 μm so as to ensure the integrity of detected signal irradiated on the membrane central area. As such, when l1 decreases from 50 μm to 30 μm, meaning a gradual increasing boundary defect as presented in l2), as well as the deflection of membrane. However, the first order vibration mode occurs at the central area of film instead of the edge of membrane until the length to width ratio is larger than 5. The phenomenon can be explained by the fact that the circular membrane becomes a clamped beam, which enables a higher-amplitude deflection than previous defective circular membrane, corresponding to an outstanding Q factor despite of lower natural frequency. That is to say, the circular graphene membrane can be fabricated to a graphene beam by photoetching or ion etching to achieve a remarkable detected signal and relatively high Q factor for F-P resonators. To sum up, the utilization and reliability of the resonant probe with a graphene membrane can be improved to a great extent, despite the lower successful rate of well transferring and attaching graphene film on a capillary endface with a hole no less than 50 μm in diameter.When an intact graphene film a is succA photo-thermally excited Fabry-Perot resonant probe with suspended clamped circular graphene membrane of 50 μm in diameter was proposed, and its opto-mechanical vibration behaviors were investigated by COMSOL simulation in this paper. The influence of the radius-to-thickness ratio of film on the optical fiber light-induced temperature absorbed by suspended graphene membrane exhibited an excellent fitted linearity with a correlation coefficient of 99.86%. Moreover, in order to achieve an enhanced linear vibration response, the film prestress could be adjusted in the range of 0.1–1 GPa by tuning environmental temperature based on the interaction between film thermal strain and substrate adhesion. In contrast to previously reported studies that primarily focused on the effect of laser output power on light intensity, this paper evaluated the dependence of the laser parameters (output power and light spot radius) on the temperature distribution and natural frequency of graphene membrane. More importantly, adverse clamped boundary conditions, such as edge defect and asymmetry clamped prestressed membrane, were improved by fabricating unstable defective circular membrane to stable double-ended clamped beam. Therefore, further research on the preferable trade-off between the opto-mechanical characteristic parameters and boundary conditions of suspended film acting as a reflective surface is needed to optimize opto-thermally actuated graphene resonators with an F-P micro cavity.


Toxoplasma gondii is an intracellular protozoan parasite widely distributed in animals and humans. Infection of host cells and parasite proliferation are essential steps in Toxoplasma pathology. The objective of this study was to develop and validate a novel automatic High Content Imaging (HCI) assay to study T. gondii infection and proliferation. We tested various fluorescent markers and strategies of image analysis to obtain an automated method providing results comparable to those from gold standard infection and proliferation assays. No significant difference was observed between the results obtained from the HCI assay and the standard assays . We developed here a robust and time-saving assay. This automated technology was then used to screen a library of compounds belonging to four classes of either natural compounds or synthetic derivatives. Inhibition of parasite proliferation and host cell toxicity were measured in the same assay and led to the identification of one hit, a thiosemicarbazone that allows important inhibition of Toxoplasma proliferation while being relatively safe for the host cells. Toxoplasma gondii is an obligate intracellular protozoan parasite responsible for toxoplasmosis, an infection which is asymptomatic in more than 80% of immune-competent subjects -uracil, quantitative PCR. Some of these assays present the disadvantages of being time-consuming and costly, especially when large series of samples have to be screened -uracil. Our method was then validated with different parasite strains described as presenting a failure in the infection or proliferation process. Finally, in the context of the discovery of novel and effective molecules against T. gondii infections, we screened in-house chemical libraries belonging to four chemical categories: two classes of natural products derivatives belonging to polyphenols -uracil was used to analyze parasite proliferation.A pyrimethamine (Sigma-Aldrich) stock solution was prepared at 10 mM in DMSO and was used at the final concentration of at 20 μM as a positive control for anti-3H]-uracil, as previously described -uracil per well. Monolayers were washed 3 times in phosphate buffered saline (PBS), disrupted with 500 μL of lysis/scintillation solution  and their radioactivity was measured by liquid scintillation counting using a Wallac MicroBeta TriLux (Perkin Elmer) calibrated for [3H]-uracil. Two experimental replicates were performed in each of the 3 separate experiments.The intracellular growth of RH-YFP2 parasites in HFF was monitored by selective incorporation of  was the average proliferation rate of Toxoplasma in the presence of 20 μM pyrimethamine, P/V[DMSO], the average proliferation rate of Toxoplasma in the presence of DMSO only and P/V[mol], the average proliferation rate of Toxoplasma in the presence of the tested molecule;the parasite proliferation inhibition (PPI), calculated with the following formula:[mol] / C[DMSO])x100, where C[mol] was the number of host cell nuclei per well containing the tested molecule and C[DMSO], the number of host cell nuclei per well containing the DMSO control.the cell viability (CV): (C 50) and the 50% of cell viability (CV50) of each tested molecule were determined by fitting their dose-response curve using the GraphPad Prism software. A selectivity index activity (SI) was calculated as SI = CV50/IC50.Both the 50% of parasite proliferation inhibition .The data generated by gold standard methods and compared to those obtained from the HCI assay were expressed as means ± SD and the significance of differences found between groups was determined using the two-tailed Student’s Toxoplasma infection in vitro. We optimized our cell culture and parasitophorous vacuoles’ labeling conditions to obtain efficient segmentation of both the vacuoles and the parasites in order to develop a High-Content Imaging assay (HCI) for the evaluation of Toxoplasma infection and proliferation. We optimized the HFF cell seeding density (1.104 per well) in 96 well plates to obtain a sub-confluent monolayer after 48 h of culture, which allowed satisfying segmentation of the host cells: the number of cells containing at least one Toxoplasma parasitophorous vacuole was > 15% following the inoculation of 4.104 parasites let to invade for 2 hours.Human foreskin fibroblasts are currently used as a host model to study Both the host cells and the parasites were identified and counted using the nucleic acid marker Hoechst-33342. However, when directly counted, it was sometimes difficult to discriminate parasites’ nuclei from host cells’ nuclei. Counting the parasites in each cell was facilitated by a first segmentation of the vacuoles.Toxoplasma proliferation was performed as described in the material and methods  or [3H]-uracil incorporation assay (p = 0.599)  .Toxoplasma strains (RH and PRU) described in the literature to have different infection- and proliferation rates. The infection rates of RH versus PRU were respectively 28.3% versus 10.5% (versus 1.6 ± 0.08 (PRU)  . Unlike arasites .Toxoplasma infection and treatment with DMSO or pyrimethamine. First we analyzed the number of HFF on 20 image fields per well within a 96-well plate and across multiple plates. The intra-plate variation coefficients for DMSO and pyrimethamine-treated wells were respectively 12.1% and 13.5%, while the inter-plate variation coefficients were 1.9% and 4.7%, respectively. These results showed an acceptable intra- and inter-plate repeatability of cell numbers  demonstrated the robustness of the HCI assay , 3-chalcones , 1-dipehyloxadiazol (DO3) and 1-aurone (AU11), respectively, led to a high proliferation inhibition activity. Following the primary screening, these 6 compounds were tested for dose-response studies as well as cell viability assays , showed characteristics  comparable to those of the pyrimethamine reference drug .Six compounds, y assays . Their Iand > 90 . The mosToxoplasma parasites. This technology has already been developed for other protozoan parasites such as Plasmodium, Leishmania and Trypanosoma -uracil incorporation assay because HCI provides the measure of several parameters at the same time. Inhibition of parasite proliferation in host cells can be due to the absence of parasite multiplication or to the loss of host cells. To avoid this potential bias, the HCI method takes both these parameters in consideration: it is possible to count the number of intracellular parasites in parallel to the number of host cells, and further proceed with normalization of the number of intracellular parasites to the number of host cells per well. Such normalization cannot be easily performed using standard proliferation assays such as the [3H]-uracil incorporation assay.Even if HCI has already been used to understand the process of nfection , this me3H]-uracil incorporation or spectrofluorimetry, the HCI assay allows the analysis, in a single test, of both the anti-Toxoplasma activity and the host cell cytotoxicity of tested compounds. This latter is particularly important for compounds that may induce necrosis, apoptosis, or which may interfere with the adherence of host cells. We would thus recommend to perform a first screen using [3H]-uracil incorporation or spectrofluorimetry to quickly identify the hits, followed by an HCI assay to check the absence of bias and understand the mechanism of action.In this study, we also demonstrated that HCI is an interesting assay to screen potential therapeutic compounds. Even if the time necessary to obtain data is longer than that necessary when using classical assays such as [T. gondii while having low host cell toxicity. These drugs will be further chemically modified in pursuing the pharmacomodulation and development processes. The compounds tested here were chosen because they offer high chemical diversity, and they possess a high potential of pharmacomodulation. We obtained six interesting hits, namely: 1 thiosemicarbazone (TS2), 3 chalcones , 1-dipehyloxadiazol (DO3), and 1-aurone (AU11). From the observed activities, some structure-activities relationships may already be highlighted in the chalcones series. One structural element that seems determinant for the inhibition activity is the presence of a halogen atom at their B-ring. Interestingly, pyrimethamine, the reference drug, possesses a chlorine atom. It should be noted that the methoxylation pattern of the chalcones is important for inhibition versus cytotoxicity. The latter behavior has been observed for chalcones in other pharmacological areas [TS2, which exhibits a profile comparable to that of the pyrimethamine reference drug. For further investigation, it will be straightforward to investigate TS2 analogs as well as chalcones derivatives.Our goal here was to provide hit compounds effective against al areas ,20. BaseThe synthesis easiness  and the availability of diverse starting blocks required to build both pharmacophores should allow the synthesis of large libraries of analogs, which will ultimately lead to the discovery of more active and less toxic compounds, suited for further development.Toxoplasma gondii infections constitute a challenging and life-threatening health problem in immunocompromised individuals. The emergence of parasite resistance against the few available molecules and the intolerance to the commonly used anti-T.gondii drugs during long-term therapy prompt the search for novel anti-Toxoplasma gondii agents. In this perspective, the development and the validation of this HCI assay to analyze Toxoplasma proliferation is a relevant and effective method to investigate new anti-Toxoplasma molecules.


Our metabolomic studies demonstrated that MTDH reduced intracellular cysteine, but increased glutamate levels, ultimately decreasing levels of glutathione and setting the stage for increased vulnerability to ferroptosis. Finally, we observed an enhanced antitumor effect when we combined various ferroptosis inducers both in vitro and in vivo; the level of MTDH correlated with the ferroptotic effect. We have demonstrated for the first time that MTDH enhances the vulnerability of cancer cells to ferroptosis and may serve as a therapeutic biomarker for future ferroptosis-centered cancer therapy.Ferroptosis is an iron-dependent, non-apoptotic form of regulated cell death driven by lipid hydroperoxides within biological membranes. Although therapy-resistant mesenchymal-high cancers are particularly vulnerable to ferroptosis inducers, especially phospholipid glutathione peroxidase 4 (GPx4) inhibitors, the underlying mechanism is yet to be deciphered. As such, the full application of GPx4 inhibitors in cancer therapy remains challenging. Here we demonstrate that metadherin (MTDH) confers a therapy-resistant mesenchymal-high cell state and enhanced sensitivity to inducers of ferroptosis. Mechanistically, MTDH inhibited GPx4, as well as the solute carrier family 3 member 2 (SLC3A2, a system X As a cellular process, ferroptosis acts in pivotal roles in both normal development and various diseases, including cancer1. The removal of phospholipid hydroperoxides relies largely on phospholipid glutathione (GSH) peroxidase 4 (GPx4), a GSH-dependent enzyme. Importantly, GPx4 is the only known enzyme that reduces lipid hydroperoxides within biological membranes2, converting polyunsaturated phospholipid lipid hydroperoxide into corresponding lipid alcohol3. Therefore, GPx4 is considered as a central regulator of ferroptotic cell death3. In order to enable the function of GPx4, the production and regulation of the metabolite GSH is necessary. GSH is synthesized from cysteine and glutamate, whose intracellular concentration is fine-tuned by system xc−, an amino acid antiporter that mediates the exchange of extracellular cystine and intracellular glutamate across the cellular plasma membrane3. Cystine is reduced to cysteine in order to act as a substrate to synthesize GSH by glutamate cysteine ligase. System xc− is a disulfide-linked heterodimer composed of solute carrier family 7 member 11 (SLC7A11) and SLC3A24.Ferroptosis is a non-apoptotic form of regulated cell death driven by lipid peroxidation, which requires abundant and accessible cellular ironc− inhibitors, compounds that deplete GSH1, and inhibitors of GPx4, along with other less reported mechanisms6. The inhibition or rescue from ferroptosis can be induced by iron chelators, reducing agents, and inhibitors of lipid peroxidation7. Accumulating evidence suggests that resistant, mesenchymal-high cancers are particularly vulnerable to ferroptosis inducers, especially GPx4 inhibitors6. However, the underlying mechanism is yet to be deciphered, and the full application of GPx4 inhibitors as cancer therapy remains unrealized.Ferroptosis can be induced through various modalities such as system x9. MTDH overexpression was documented in many types of cancer and correlates clinically to poor overall survival12. Several studies have established a bona fide role of MTDH in several hallmarks of cancer, including transformation, proliferation, evasion of apoptosis, and therapeutic resistance14. MTDH overexpression causes broad drug resistance to 5-fluorouracil, doxorubicin, cisplatin, mitomycin C, paclitaxel, histone deacetylase inhibitors, and other agents17, as well as resistance to radiation therapy18.Metadherin (MTDH), also known as astrocyte elevated gene-1 protein (AEG-1), is located on chromosome 8q22, a region frequently amplified in breast and hepatocellular cancersMTDH expression underscores the pleotropic interactions of MTDH with other signaling modules and networks. MTDH may act in the nucleus as a transcription co-factor, for example, MTDH can physically interact with p65 and therefore activate nuclear factor-κB19. MTDH can also interact with CREB-binding protein, preventing its ubiquitin-mediated degradation and thereby facilitating the epigenetic activation of Twist-related protein 1 (TWIST1)20. In addition, MTDH can function as an effector of multiple epithelial–mesenchymal transition (EMT)-related microRNAs, and incorporate oncogenic signaling pathways such as phosphoinositide 3-kinase-AKT and Wnt/β-catenin to promote EMT, cancer stemness, and metastasis24. While conferring resistance to chemotherapy agents and radiotherapy, MTDH was found to promote the EMT, invasion, and metastasis in various types of cancers including breast cancer27.These diverse findings of resistance associated with 18.As MTDH promotes a therapy-resistant, mesenchymal-high cell state, we therefore focused on whether MTDH enhances the vulnerability of cancer cells to ferroptosis inducers and the mechanism of the underlying vulnerability. This study provides evidence to support ferroptosis induction by GPx4 inhibitors can overcome MTDH-overexpression-mediated drug resistance to conventional chemotherapy and radiation therapyhttps://xenabrowser.net]. The messenger RNA (mRNA) expression data were sorted per MTDH expression and samples were split into tertiles. The high and low MTDH tertiles were subjected to Gene Set Enrichment Analysis (GSEA) using the C2, C5, and Hallmark libraries examining EMT and metastasis gene sets29. A false discovery rate (FDR) cutoff of <0.25 was considered to be significant. In order to correlate the mesenchymal score with the sensitivity to ferroptosis inducers, we used previously published mesenchymal scores6, and drug sensitivity data from the Cancer Therapeutics Response Portal (CTRP) [http://portals.broadinstitute.org/ctrp.v2.2/]. Data and visualizations utilized R (v.3.5.1) with the base functions and ggplots2 package (v.3.1.0) were used to visualize results.The Cancer Genome Atlas (TCGA) RNA-sequencing level 3 processed data were downloaded from UCSC Xena Browser [S, 3R-RSL3  and ML162 were dissolved in DMSO.For in vitro experiments, sorafenib , erastin , sulfasalazine , M162 , M210 , and ferrostatin-1 (Fer-1)  were prepared in dimethyl sulfoxide (DMSO). For in vivo studies, sorafenib was dissolved in Cremophor EL/95% ethanol . The 1MTDH knockout (KO) cells of Hec50 and MDA-MB-231 as described previously30. The single guide RNA (sgRNA) CAAAACAGTTCACGCCATGA targeted the coding region of the MTDH gene at 97,686,713 to 97,686,733 . The sgRNA was cloned into lentiCRISPRv1 . The viral vectors were produced in HEK293T cells following the manufacturer’s protocol. Cells were infected with the lentivirus and cultured in the presence of puromycin. Single-cell clones were selected by limiting dilution. MTDH deletion was confirmed by quantitative PCR (qPCR) and by Western blotting. The cells were grown in RPMI-1640/Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum  cell lines A549 and H1975; small-cell lung cancer (SCLC) cell lines DMS53 and DMS273; endometrial cancer cell lines KLE, AN3CA, RL95, Hec1A, and Ishikawa; and breast cancer cell lines MDA-MB-231 and MCF-7 were purchased from the American Type Culture Collection. Hec50 uterine serous carcinoma cells were kindly provided by Dr. Erlio Gurpide in 1991 (New York University), and their identities were verified by genotyping service at Bio-Synthesis—CRO/CMO Life Sciences Company . The CRISPR approach was used to generate isogenic 31. Briefly, 10,000 cells per well were seeded into 96-well plates and treated with individual drug for 72 h. Cell viability was evaluated using the cell proliferation reagent WST-1  according to the manufacturer’s protocol. The absorbance of wells was measured with a micro-plate reader .Cytotoxic effects were determined using the WST-1 method as previously describedTotal RNA was isolated using RNeasy Plus Mini kit (Qiagen) and reverse transcribed into complementary DNA (cDNA) using the SuperScript First-Strand Synthesis SuperMix kit (Invitrogen). Quantitative real-time RT-PCR analysis was performed using SYBR Green. The following primers were used:MTDH: 5′-GTAAACGTGATAAGGTGCTGACT-3′5′-CGGTGGTAACTGTGATGGTATTT-3′GPX4: 5′-ACAAGAACGGCTGCGTGGTGAA-3′5′-GCCACACACTTGTGGAGCTAGA-3′SLC3A2: 5′-CCAAGGTGAAGGATGCTCTG-3′5′-TGTGTGACTAGGGATTTTGTATGC-3′SLC7A11: 5′-ATGCAGTGGCAGTGACCTTT-3′5′-GGCAACAAAGATCGGAACTG-3′18S: 5′-AACTTTCGATGGTAGTCGCCG-3′5′-CCTTGGATGTGGTAGCCGTTT-3′.5 cells were grown in 24-well plates and exposed to drugs for 48 h. After the medium was removed, cells were washed in cold phosphate-buffered saline (PBS), fixed with 100% methanol, and stained with 0.5% crystal violet solution.A total of 1 × 10Cells were collected and lysed with RIPA buffer , protease inhibitors). Equal amounts of proteins (40 μg) were separated on 10% SDS-polyacrylamide gel electrophoresis gels and then transferred to a nitrocellulose blotting membrane . After blocking with 5% non-fat milk, the membrane was incubated overnight at 4 °C with the respective primary antibody. The following antibodies were used: anti-c-MYC , anti-SLC3A2 , anti-E-cadherin , and anti-SLC7A11  from Cell Signaling Technology ; anti-β-actin , anti-MTDH , and anti-GPx4  from Santa Cruz Biotechnology; anti-vimentin  from Sigma-Aldrich; anti-ZEB1  from Novus Biologicals USA. Membranes were further incubated with secondary antibody  at room temperature for 2 h. Signal bands were detected using the Bio-Rad ChemiDoc system and densitometry were analyzed with Bio-Rad Image Lab Software (Bio-Rad Laboratories).TM (RNA-binding protein immunoprecipitation) kit  and real-time PCR were used to confirm the association between MTDH with SLC3A2 and SLC7A11 as per the manufacturer’s protocol as previously described13. Magna RIPTM kit and microarray were used to pull down MTDH-associated RNAs by a rabbit antibody against MTDH  and to identify mRNAs that associate with MTDH. Microarray data have been deposited at GEO (www.ncbi.nlm.nih.gov/geo) under accession number GSE30588. SLC3A2 was detected by quantitative real-time RT-PCR.Magna RIPCells were seeded in 6-well plates in DMEM or RPMI-1640, supplemented with 10% FBS, and treated with defined drugs for 16 h. Cells were then incubated with 10 μM BODIPY 581/591 C11 (Life Technologies) in the dark for 30 min at 37 °C. After incubation, cells were trypsinized, washed twice with PBS, and re-suspended in 300 μL phenol red-free DMEM with 0.2% bovine serum albumin. Flow cytometry was performed using the BD FACS Canto II equipped with a 488 nm laser for excitation; data were collected using the 530/30 nm band-pass filter.MTDH KO cells were plated in 100-mm dishes and allowed to grow for 24 h. Cells were then washed with cold PBS and scraped into 300 µL 5% 5-sulfosalicylic acid (Sigma) in water and stored at −20 °C for a maximum of 72 h. Total GSH content was determined as described previously33. GSH disulfide (GSSG) was determined by adding 35 μL of a 1:1 mixture of 2-vinylpyridine and ethanol to 175 μL of sample and incubating for 2 h before assaying. The rates of the reaction were compared with similarly prepared GSH and GSSG standard curves. GSH determinations were normalized to the protein content of the insoluble pellet from the 5-sulfosalicylic acid dissolved in 2.5% SDS in 0.1 N bicarbonate using the BCA Protein Assay kit (Thermo Scientific).Parental cells and 34. The activity was measured by a coupled enzymatic assay using GSH, GSH reductase, phosphatidylcholine hydroperoxides (PCOOH), and NADPH. The substrate (PCOOH) was prepared by enzymatic hydroperoxidation of phosphatidylcholine using soybean lipoxidase type IV. The nonspecific NADPH oxidation rate was recorded for 4 min at 340 nm; then, the enzyme reaction was started by the addition of PCOOH (10–30 μM). The rate of specific NADPH oxidation was recorded every 20 s for 4 min at 340 nm using a Beckman DU-70 spectrophotometer. The activity was calculated by subtracting the nonspecific NADPH oxidation rate from the observed NADPH oxidation rate after the substrate addition. The specific activity is expressed as milliunits per milligram total cell protein; one unit of enzyme activity catalyzes the oxidation of 1 μM of NADPH per minute.Cells were grown in DMEM media with 10% FBS, and then in 300 ng/mL of sodium selenite for 24 h. Cells were treated with the compound at the indicated concentration in media for 16 h, then harvested by scratch, and finally pelleted by centrifugation. GPx4 activity was determined as described previouslyMTDH KO MDA-MB-231 cells were analyzed. Whole cells were washed twice on plates with ice-coldPBS, and then twice with ice-cold water. Cells were frozen by floating the plate on liquid nitrogen.The metabolomics assay was performed with the mass spectrometer through our Metabolomics Core Facility . The Metabolomics Core of the Fraternal Order of Eagles Diabetes Research Center provides metabolite profiling using high-resolution mass spectrometry (MS) following gas chromatography (GC). The semi-targeted high-resolution GC-MS protocol can identify and measure more than 100 metabolites, including glycolysis and TCA cycle, neurotransmitters, amino acids, carbohydrates, and fatty acids. One well of a nearly confluent 6-well plate provides enough sample to obtain full metabolite coverage. Six replicates of parental and All animal studies were performed under animal protocols #7051085 approved by the University of Iowa Institutional Animal Care and Use Committee .6MTDH WT and KO MDA-MB-231 cells were injected into the second and fifth mammary fat pads  of the NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ  immunodeficient female mice. To study the metastasis from this orthotopic mouse model, tumor volumes were allowed to grow to ~1000 mm3, after which livers were resected to examine incidence as well as tumor burden of liver metastasis. To study the expression level of SLC3A2 and GPX4 under alternative MTDH status (WT vs. KO), xenografted tumors were resected upon sacrifice, followed by tissue fixation, sectioning, and immunohistochemistry (IHC).To generate tumor xenograft models, 5 × 10c− inhibitor sorafenib, 5 × 106 MDA-MB-231 cells were implanted on the right flanks of the mice. Once the tumors became palpable, mice were treated with either solvent  or intratumoral RSL3 , or sorafenib via oral gavage , or the combination of RSL3 and sorafenib (n = 5) for 20 days. Tumor volume and body weight were measured periodically twice per week. Tumor volume was calculated using the formula length x width2/2.To test the antitumor effect of GPx4 inhibitor RSL3, and its combination with system xc− inhibitors under alternative MTDH status (WT vs. KO), NSG mice were injected with 5 × 106 MDA-MB-231 cells expressing either WT or KO MTDH into one of the fifth mammary fat pads (left). Mice were then started on treatment with 50 mg/kg ML162 at the same site one day later. The ML162 was administered locally twice per week for 2 weeks. Sorafenib was given by gavage daily (20 mg/kg) for 2 weeks. Mice were sacrificed after 3 weeks and tumors were collected.To test the combination of GPx4 and system xImmunostaining was performed using 5-μm-thick sections of 4% paraformaldehyde-fixed paraffin-embedded tissue samples. Immunohistochemical staining for GPx4, MTDH, and SLC3A2 expression were performed on paraffin sections using a rabbit anti-GPx4 antibody . For secondary antibodies, a biotinylated rabbit anti-rat immunoglobulin G (IgG)  or a biotinylated goat anti-rabbit IgG  was used. Staining was performed using the Vector ABC kit and Vector DAB kit (Vector Laboratories).t tests and presented using box-and-whisker plots. All P values were two-sided and <0.05 were considered statistically significant.Two-way analysis of variance was used for comparisons between control and treatment over a range of doses or times. Median weights of the resected tumors were compared using two-sided paired 6, we determined the half-maximal inhibitory concentration (IC50) of ML162 in different cell lines, including the NSCLC A549, SCLC DMS53 and DMS273, breast cancer MCF-7 and MDA-MB-231, and endometrial cancer KLE, AN3CA, RL95, Hec50, Hec1A, and Ishikawa cell lines. When we used E-cadherin/β-actin ratio to correlate the IC50s, we observed that mesenchymal-high  cancer cells exhibited significantly enhanced sensitivity  to GPx4 inhibitor ML162  concentration) to GPx4 inhibitors ML162, ML210, and RSL3  significantly enhanced the sensitivity to the chemotherapeutic agent camptothecin, a topoisomerase inhibitor . We found that MTDH depletion decreased the tumor burden of liver metastasis by both IHC . We observed that MTDH KO dramatically increased cysteine levels , which could theoretically improve cell viability under glucose-deficient/glutamine-replete conditions due to enhanced ability to use intracellular glutamate to maintain respiratory chain activity38.As the downregulation of SLC3A2 by MTDH could potentially impair the function of system xTDH Fig. . In contate Fig. , a produtly Fig. . These dc− inhibitors erastin, sulfasalazine, and sorafenib; buthionine sulfoximine, which depletes GSH; and statins that are indirect inhibitors of GPx4; as well as direct GPx4 inhibitors RSL3, ML162, and ML210. Overall, GPx4 inhibitors exhibit the lowest half-maximal effective concentration (EC50)   Fig. . In addiration EC0 , such advantages are not gained without a price: due to the attenuation of system xc− function, MTDH-high cancers exhibit enhanced vulnerability to ferroptosis—a potential “Achilles” heel.” These observations also suggest a role for SLC3A2 in lipid peroxidation and ferroptosis, which is less known compared to its heterodimerization partner SLC7A11. We therefore established the connection between MTDH and lipid peroxidation , we observed MTDH-high (WT) cells exhibited enhanced vulnerability to GPx4 inhibitors ML162 and ML210. Mechanistically, we found that MTDH downregulated both GPx4 and SLC3A2. Interestingly, its regulation of SLC3A2 was found at both the transcriptional and post-transcriptional levels: aside from reducing the mRNA level of SLC3A2, MTDH also negatively affected its protein stability. In addition, the mRNA species of key members of the lipid hydroperoxide removal system have putative MTDH binding sites, including SLC3A2, SLC7A11, and GPX436. We have previously demonstrated that MTDH may act as an RNA-binding protein;13 thus, we hypothesized this as an additional mechanism for how MTDH regulates lipid peroxidation. Using RIP assay, we observed that MTDH antibody was able to pull down significantly more mRNA of SLC3A2 compared to control IgG. In addition, through metabolomics study, we found that MTDH reduced the intracellular concentration of cysteine, but increased glutamate, suggesting a negative regulation of system xc−, the cystine/glutamate antiporter. This was consistent with our observation of reduced levels of GSH, setting the stage for enhanced ferroptosis7, which also suggests a potential role of SLC3A2 in regulating lipid peroxidation.The role of MTDH in treatment resistance, EMT, and metastasis has been documented in previous studies, including oursion Fig. . Our hypc− inhibitors. Our in vitro assay suggested a clear combinational effect only in MTDH WT, but not MTDH KO isogenic cells, again confirming the role of MTDH in ferroptosis.As GPx4 activity relies on GSH levels, we also tested the combination of GPx4 inhibitors with various other ferroptosis inducers, especially system x39. We therefore tested intratumoral administration of both RSL3 and ML162 as an approach to bypass this application hurdle. Consistent with previous studies3, intratumoral RSL3 was effective, and enhanced the antitumor effect of the system xc− inhibitor sorafenib, confirming the combinational effect observed in vitro. In addition, intratumoral injection of ML162 also prevented tumor growth alone or combined with sorafenib, suggesting that intratumoral administration could be a feasible delivery approach for direct GPx4 inhibitors. This is clinically important as intratumoral injection is a viable treatment approach for certain cancers at certain locations . In the era of cancer immunotherapy, intratumoral injections of chemotherapy or biological agents are often explored in combination with systemic anti-PD-1/PD-L1 agents42. Since antigens released upon cancer cell death may potentiate the immune checkpoint blockade , and ferroptosis is an effective form of cell death in apoptosis-resistant cancers6, intratumoral injection of GPx4 inhibitors might offer a novel strategy to enhance cancer immunotherapy. In fact, this hypothesis is currently being tested in our laboratory. In addition, our in vivo study showed that ferroptosis inducers and their combination were effective only in MTDH WT, but not MTDH KO tumors, again confirming the value of MTDH in modulating lipid peroxidation.Although GPx4 inhibitors are the most potent inducers of ferroptosis, all currently reported direct GPx4 inhibitors  have biostability issues that make them unsuitable for systemic use in vivoIn summary, the present study demonstrates that MTDH enhances the vulnerability of cancer cells to ferroptosis, and suggests that MTDH may be a therapeutic biomarker for future ferroptosis inducing agents to treat cancer.Supplemental figure 1Supplemental figure 2Supplemental figure 3Supplemental figure 3Supplemental figure 4Supplemental figure 5Supplemental figure 6Supplemental table 1Supplemental table 2Supplemental table 3


Li favors incorporating into the interlayer region of G/Sb rather than the outside surfaces of graphene and antimonene of G/Sb heterostructure, which is caused by the synergistic effect. The in-plane lattice constants of G/Sb heterostructure expand only around 4.5%, and the interlayer distance of G/Sb increases slightly (0.22 Å) at the case of fully lithiation, which indicates that the capping of graphene on antimonene can effectively suppress the volumetric expansion during the charging process. Additionally, the hybrid G/Sb heterostructure has little influence on the migration behaviors of Li on the outside of graphene and Sb surfaces compared with their free-standing monolayers. However, the migration energy barrier for Li diffusion in the interlayer region (about 0.59 eV) is significantly affected by the geometry structure, which can be reduced to 0.34 eV simply by increasing the interlayer distance. The higher theoretical specific capacity (369.03 mAh g−1 vs 208 mAh g−1 for antimonene monolayer) and suitable open circuit voltage (from 0.11 V to 0.89 V) of G/Sb heterostructure are beneficial for anode materials of lithium-ion batteries. The above results reveal that G/Sb heterostructure may be an ideal candidate of anode for high recycling–rate and portable lithium-ion batteries.To suppress the volume expansion and thus improve the performance of antimonene as a promising anode for lithium-ion batteries, we have systematically studied the stability, structural and electronic properties of the antimonene capped with graphene (G/Sb heterostructure) upon the intercalation and diffusion of Li atoms by first-principles calculations based on van der Waals (vdW) corrected density functional theory. G/Sb exhibits higher Young’s modulus (armchair: 145.20, zigzag: 144.36 N m In 2 , black o2 ,7, SnP3 2 [3 , and MXe2 [3 , have at2 [−1 after 100 charging/discharging cycles, and the capacity fading during 100 cycles was only 3.8%, which is superior to those of Sb bulk and Sb nanoparticles [Recently, antimonene (Sb) possessing a buckled hexagonal structure has been successfully fabricated by mechanical exfoliation , van der2 , as well2 . Due to 2 ,14,15,162 [−1 [2 and WS2, the introduction of graphene could effectively enhance the electron transport properties of hybrid composites as anode materials, which is an important influence factor for high-performance LIBs. For instance, hybrid black-P/graphene (P/G) exhibits a small bandgap of 9 meV, which mainly arise from bandgap opening of graphene owing to the breaking of structural symmetry caused by the black-phosphorene interaction [2 can be improved [2, respectively.The vertical heterostructure stacked by different 2D materials is a promising way to construct new materials  that int2 , black p2 ,20, and 2 , as well2 . For exa2 . An obvi2 ,25. A hi2 [−1  has alreeraction ,27, and improved  by cappi2 pillared structure of Ti3C2O2 intercalated by quinine molecules [Inspired by the graphene/blue-P, graphene/MXenes, graphene/MoSolecules , we introlecules . Meanwhiolecules . Therefo2 method of Grimme [All calculations were performed based on the densityfunctional theory (DFT) as implemented in the Vienna ab initio simulation package (VASP) ,32. The f Grimme . The k-pf Grimme  were usef Grimme . To redu−5 eV and forces on each atom smaller than 0.01 eV/Å, respectively.To investigate the diffusion properties of Li in the G/Sb heterostructure, the nudged elastic band (NEB) method was adopted to calculate the transition state and migration barrier ,38. All The geometrical structures of monolayer graphene (G) and antimonene (Sb) are optimized for references. Graphene exhibits hexagonal honeycomb structure with lattice constant of 2.47 Å and its C–C bond length and bond angle are 1.43 Å and 120°, respectively. However, in order to increase the stability of layered structure, antimonene adopts buckled honeycomb structures with lattice parameter and layer thickness of 4.12 Å and 1.65 Å, which is similar to those of buckled arsenene and silicene ,40. The G, for G/Sb heterostructure as illustrated in N is the total number of atoms in the system. The results for bilayer graphene and bilayer antimonene are also calculated as references  [−1) are greatly enhanced compared with those of monolayer antimonene , which can be explained by the synergy effect between individual monolayers. This improved stiffness of G/Sb is rather vital as anode materials for LIBs due to the large stresses and deformations resistance caused by the lithium intercalation.In order to find out the most stable stacking pattern for the graphene on antimonene, here, we considered four typical stacking configurations, H-H, H-C, Sb-C, and Sb-Hshown in a with lo(3.61 Å) , while l2 or metallic characters  heterostructure) [The band structures and density of states (PDOS) of monolayer graphene, antimonene, and G/Sb are shown in ructure)  are of gTo further give insight into how the coating of graphene affects the electronic properties of the G/Sb system, we also investigated the charge transfer and charge density difference distributions. The planar averaged charge density difference along the z direction is plotted in analysis ,45 was cHC  and TC (the top site of C atom), as shown in TSb(the top site of lower Sb atom), T’Sb(the top site of upper Sb atom), and HSb, as observed in HSb site (IH1), the two-fold site of HC and HSb (IH2), the two-fold site of TC and HSb (Ic), and the top site of lower Sb atom, which is near the HC(ISb), as illustrated in To examine the properties of Li incorporating into the G/Sb heterostructure, several possible adsorption and intercalation sites were considered in our study. We have explored three different regions—(1) Li adsorbed on the outside graphene surface (Li/G/Sb), (2) Li adsorbed on the outside antimonene surface (G/Sb/Li), and (3) Li intercalated in the interlayer of graphene and antimonene of G/Sb heterostructure (G/Li/Sb). Nine adsorption and intercalation sites of Li adatom are considered to search for the most stable geometries. For the cases of Li adsorbed on the outside graphene surface (Li/G/Sb), two adsorption sites considered are n is the number of Li atoms adsorbed or intercalated in the G/Sb heterostructures. According to the definition, a larger value of Eb represents a stronger bonding of Li to the G/Sb heterostructures. The calculated results for the corresponding structural and energetic parameters are summarized in HC and TSb are the most favorable adsorption sites for Li/G/Sb and G/Sb/Li system, respectively, which are identical to the adsorption cases of Li adatom on the pristine monolayers (Li/G and Li/Sb). However, the adsorption behavior of Li adatoms on the antimonene surface can be remarkably enhanced by increasing the binding energy from 1.96 eV for pristine antimonene to 2.23 eV for Li adsorption at TSb sites, while the change of bE for Li/G/Sb is only 0.10 eV compared with the case of Li/G. Second, the binding energies for Li occupying different sites of the interlayer of G/Sb are in the order of IC(2.67 eV) < IH1(2.80 eV) < IH2(2.88 eV) < ISb(2.92 eV). It is obvious that SbI is the most stable site for Li intercalated in the interlayer site of G/Sb, since it is very close to the most stable sites of Li adsorbed on the outside graphene surface (cH) and the antimonene surface (SbT), indicating that the stability of the intercalation of Li in G/Sb depends on both graphene and antimonene. Finally, it can be seen that the G/Li/Sb system is energetically more stable than Li/G/Sb and G/Sb/Li systems by 0.89 eV and 0.69 eV, respectively, which is ascribed to the interaction of Li atom with both graphene and antimonene at the interlayer sites. These calculated results indicate that the Li adatom prefers to reside in the interlayer space of G/Sb heterostructure rather than be incorporate at the outside surfaces of graphene and antimonene during the lithiation process. In other words, Li will first intercalate into the interlayer of the G/Sb during the lithiation process and then occupy the outside surfaces of graphene and antimonene in G/Sb, which is similar to the case of G/black-P [2  heterostructure [To better compare the adsorption behaviors of Li at different adsorption and intercalation sites, we calculate the binding energy, which is defined astructure . FurtherQ) for G/Li/Sb and G/Sb/Li are similar to those on ML antimonene (Li/Sb), which indicates that graphene as capping layer does not influence the electronic properties of antimonene. For the case of G/Li/Sb and G/Sb/Li, a significant charge transfer from Li atom to the neighboring C and Sb atoms, which is due to the atomic electronegativity in the order of C > Sb > Li. While the Li atom adsorbed at the outside surface of graphene, the C atoms are accepters gaining about 1|e| from both Li and Sb, which verify the strong ionic interaction of Li with graphene and antimonene. Furthermore, the large amount of electron transfer listed in xC50Sb18  in the process of Li intercalation were explored. For each concentration, we calculated several different configurations. After full geometry optimization, the variation of the averaged binding energy (bE) as a function of the number of Li ions for the corresponding most stable configurations are shown in bE decreases gradually from 2.73 eV to 2.43 eV with the number of inserted Li ions increasing from three to 28, which indicated that the G/Li/Sb structures are stable even at high Li concentrations. We found that the charge transfer from the Li atoms to the G/Sb at higher concentrations was reduced compared to that of lower concentrations. When the number of embedded Li increases, the interatomic distances between positively charged Li ions is also reduced. The reduction trend on the binding energy can be attributed to two main factors—(1) the weak electrostatic attraction between the G/Sb heterostructure and the Li cations, which is closely related to the reduction of charge transfer from Li to G/Sb at high concentrations, and (2) the enhanced Li–Li repulsion at high Li concentrations, which is resulted from the shorter Li–Li distance.It is known that the performanceof LIBs relies greatly on the Li capacity of the anode materials. Therefore, it is of great importance to understand the electrochemical properties of different lithiated phases during the Li intercalation process by adding more Li atoms to the G/Sb heterostructure. The most stable structures of Lid = 3.44–3.45Å) was slightly reduced when an isolated Li adsorbed on outside antimonene surfaces of G/Sb, while the d values (3.50–3.54 Å) change little except for SbI configuration with Li intercalated into the interlayer of G/Sb. Taking the case of fully lithiation as an example, we found that the in-plane lattice constant of G/Li/Sb systems present a slight lattice expansion of around 4.5% along the a/b-axis direction as the number of Li ions is increased from 0 to 28, which is significantly smaller than the 1.22-fold expansion in-plane channel reported by experiments for few layer antimonene [x ≤ 3) and then gradually increases relatively significantly when additional Li atoms constantly insert into the interlayer (3< x ≤ 9). This fact can be explained by the new interaction mechanism of C–Li–Sb at the low concentration instead of the weak vdW interaction in G/Sb heterostructure, but the enhanced Li-Li repulsion and increasing number of the Li adatoms will lead to the separation of two monolayers at a high concertation. The calculated results on interlayer separation suggest that Li intercalation could cause a slight expansion of 0.22 Å in the vertical direction of the G/Sb heterostructure, which is quite a bit smaller than expansion of few layer antimonene along vertical direction (1.14 Å) during the sodiation process reported in experiments [It is reported that the performance of Sb anode is seriously restricted by its volume expansion due to Li intercalation , so we stimonene . Besideseriments . Therefo2 and graphene/black-P heterostructures [To get insight into the interaction between intercalated Li and the G/Sb heterostructure, we investigate the partial density of state (PDOS) of G/Sb heterostructure with 1, 9, 18, and 28 Li incorporated as depicted in ructures ,20. It sx1 and x2 Li atoms intercalated, respectively, and e is the unit of charge. The calculated OCV of G/Sb as a function of the Li concentrations and the corresponding optimized configurations are shown in Open circuit voltage (OCV) and specific capacity are commonly used to estimate the performance of batteries. Under the premise that the small change in volume and entropy are ignored, the OCV can be approximately computed from the total energy difference based on the following equation:x is the maximum number of Li adatoms intercalated, F is the Faraday constant (26.081 Ah mol−1), and −1, which is higher than that (208 mAh g−1) of pristine antimonene in reference [Li storage capacity is another factor to determine the performance of LIBs, and the theoretical specific capacity can be given byeference . The abo−1, which is close to the experimental value for few layer antimonene [The theoretical specific capacity of G/Sb obtained is smaller than the experimental values for a few layers of antimonene and Sb nanoparticles, but antimonene rapidly expands about 200% in volume . With thtimonene  by interThe rate capacity is the essential issue for high-performance LIBs, which is closely associated with the high electrical conductivity and Li mobility. It has been discussed that the presence of graphene could significantly reduce the bandgap of antimonene, and the incorporation of Li atoms further improves the electrical conductivity of G/Sb. Since the fast Li diffusion is vital for the battery to rapidly charge/discharge, we next turn to investigate the mobility of Li atoms at G/Sb heterostructure. As listed in CH site to the nearest-neighboring CH site in TSb site to the nearby TSb site passing through the intermediate stable HSb site as shown in On the outside surfaces of graphene, it is noted that the energy barrier is about 0.35 eV for diffusion of Li adatom from HSb site to IC site through the Sb–Sb ring is about 1.26 eV, which is compatible to the case of Li migrate cross Sb layers in few layer antimonene (1.14 eV) [As listed in 1.14 eV)  and in t1.14 eV) . These c1.14 eV) , which cSbI and H2I) were considered. The diffusion of the Li adatoms is mainly through two possible routes, namely path-I and path-II shown in SbI sites across the neighboring CI site as an intermediate state, which is quite similar to the case of the pristine ML antimonene. For path-II  are much lower than that in the interlayer space of bulk Sb (1.73 eV) [2CO2(0.60 eV) [3N/phosphorene (0.24 eV) [For the cases of Li adatom diffusion in the interlayer space of G/Sb, two possible adsorption stable sites (1.73 eV)  and are 1.73 eV) , and gra0.60 eV) , suggest0.60 eV)  and C3N/0.24 eV)  are very3C2O2 intercalated by quinone molecules displays fast kinetics and enhanced Li storage capacity [ISb and IC are 2.70 eV and 2.41 eV, which are smaller than those for G/Sb with equilibrium interlayer distance. The diffusion energy barrier for Li diffusion along the path-I reduces from 0.59 eV to about 0.34 eV. These calculated results support the energetic stability of Li embedded in the G/Sb interface, and we also find that the increasing of the G–Sb vertical distance could remarkably reduce the energy barrier of Li diffusion, due to the weakening of the interactions between adjacent sheets. Meanwhile, more Li atoms can be intercalated in the interlayer region and the theoretical specific capacity of G/Sb can be estimated to be 369.03 mAh·g−1 when interlayer distance is increased to 4.20 Å, which is very close to that of the commercial graphite anode (372 mAh g−1) [Previous experimental studies confirmed that weakening the interaction of Li ions with the constituent layers can effectively reduce the diffusion barriers of Li atom ,50. Thiscapacity . In ordemAh g−1) .−1) compared with free standing antimonene . It is noted that the existence of graphene could effectively reduce the bandgap of the G/Sb heterostructure to 0.03 eV, indicating the electrical conductivity of hybrid heterostructure is significantly enhanced compared to pristine ML antimonene, which is beneficial for G/Sb heterostructure–based LIBs. It is also found that the Li adatom prefers to intercalate into the interlayer region with larger adsorption energy (2.92 eV) instead of incorporating at the outside surface of graphene (2.02 eV) or antimonene (2.23 eV), which indicated that the interlayer region of the G/Sb heterostructure could provide a unique space for the accommodation of Li atoms due to the synergistic effect. Furthermore, Li adatoms maintain the high diffusion mobility of Li on the outside surfaces of antimonene or graphene of G/Sb heterostructure, while the diffusion barrier of Li in the interlayer region increases to 0.59 eV from 0.35 eV for pristine ML antimonene. It results from the limited interlayer distance and common modulation of individual monolayers on the Li bonding strength and can be reduced to 0.34 eV simply by increasing the interlayer distance. Importantly, the in-plane lattice constant of G/Sb heterostructure is slightly increased by around 4.5% along the a/b-axis direction in the case of full lithiation, which is significantly smaller than that of few layers of antimonene. The interlayer distance of G/Sb also increases relatively slightly (0.22 Å) during the full lithiation process, which is much less than that of a few layers of antimonene (1.14 Å) reported in experiments. Therefore, the capping of graphene can effectively minimize the volumetric expansion during charging process, which is desirable for anode materials of LIBs. The theoretical specific capacity of G/Sb heterostructure (369.03 mAh g−1) is also improved compared to pristine antimonene (about 208 mAh g−1) after full lithiation. The calculated open circuit voltage (OCV) is in the range of 0.89 V to 0.11 V, which is within the desired potential range (0.10–1.00 V) for anode materials. Overall, our findings show that G/Sb exhibits good mechanical properties, good electrical conductivity, very small volume expansion, relatively high capacity, and suitable open circuit voltage, which demonstrates that G/Sb holds promise to be a good candidate as an anode material in LIBs. Moreover, these studies could enhance the understanding of 2D antimonene-based heterostructures, which is important for the rational design of high-performance electrode materials for LIBs.In summary, we have systematically investigated the G/Sb heterostructure as an anode material for LIBs based on first-principles calculations. The calculated results reveal the G/Sb heterostructure not only exhibits high structural stability with formation energy of about –30.43 meV per atom, which is higher  than that of the stacked graphene bilayer (–24.95 meV per atom), but also possesses improved stiffness accompanied by greatly enhanced Young’s modulus (armchair: 145.20, zigzag: 144.36 N m


Different local atomic arrangements have been considered to model the Moiré patterns expected in these heterostructures. The magnetic exchange interactions between both ferromagnets  spacers in the design of synthetic graphene-based antiferromagnets. In the case of h–BN, d(Fe/Co) coupling. However, there is a larger contribution of local ferromagnetic interactions. Magnetic anisotropy energies were also calculated using a fully relativistic description, and show out–of–plane easy axis for all the configurations, with remarkable net values in the range from 1 to 4 meV.Following the recent synthesis of graphene–based antiferromagnetic ultrathin heterostructures made of Co and Fe, we analyse the effect of the spacer between both ferromagnetic materials. Using density functional calculations, we carried out an exhaustive study of the geometric, electronic and magnetic properties for intercalated single Co MLs on top of Ir(111) coupled to monolayered Fe through  Overall the interlayer distances are in reasonable agreement with previous theoretical works that include van der Waals corrections and report distances between graphene and Co in the range 2.0–3.3Å  and Ir(111)/Co/h–BN/Fe heterostructures. Due to the mismatch between graphene and Ir, the minimum size of the unit cell of these ultra-thin films would require at least ~600 atoms. Instead, we have taken a simplified model structure where a commensurable minimal unit cell is considered, and explored the properties of a selected subset of the possible vertical stacking geometries for Gr/h-BN over an intercalated Co ML that follows the epitaxial structure of Ir(111). Different adsorption sites of Fe atoms over the honeycomb lattice for graphene/h-BN have been considered  and antiparallel (AFM) alignments between Fe and Co magnetizations.We have carried out a theoretical study of the geometric, electronic and magnetic structures of Ir(111)/Co/ered See . In totah-BN) materials. However, in the ultrathin limit (one ML), the hybridization between ferromagnets and the spacer heavily distorts the electronic properties of the latter, and both spacers behave as spin–polarized metals. We explored the effect of thicker spacers with bilayer and trilayer graphene, and saw a oscillatory behaviour (antiferro/ferro) and a fast decay in the strength of the magnetic coupling, as expected for metallic spacers in a RKKY model. In terms of designing new synthetic magnetic heterostructures based on 2D materials, we conclude that only ML spacers provide strong magnetic couplings, of hundreds of meV.The two different spacers between the magnetic films were taken in order to search for different type of magnetic couplings, coming from semimetallic (graphene) or insulating , and non–collinear alignments between Fe and Co layers could be obtained.For all the configurations considered, we obtain a PMA (MAE positive and in the range of ~3 meV). It is mainly dominated by the strong contribution from the underlying cobalt ML, which is enhanced by the presence of the graphene/Although our calculations are based on a very simplified model based on commensurate lattices and not the real Moiré pattern, we believe that our simulations can provide some general guidelines for the design of synthetic magnetic heterostructures based on bidimensional materials, and give some clues on the expected properties that these could have. We expect that the final properties of the Moiré superstructure will be an averaging of the local properties here obtained, which are robust on well defined regions that will have similar stacking orderings as the one considered in the model. The agreement with the available experimental results obtained for Ir(111)/Co/Gr/Fe is remarkable, and points in this direction.