It was found that the dependence of the current in the graphene film on the frequency of the surface acoustic wave (SAW) excitation repeats the amplitude-frequency response of the SAW delay time line. It is shown that increasing the SAW amplitude leads to an increase in the current in the graphene film, and the current in the graphene film depends linearly on the amplitude of the high-frequency input signal supplied to the interdigital transducer . It is demonstrated that at a positive bias potential on the graphene film, the SAW propagation allows to change the direction of the current in the graphene film by changing the amplitude of the SAW. It is also shown that in the frequency range of the amplitude-frequency response of the SAW delay time line, the current in the graphene film can vary from positive to negative values depending on the frequency. The capability to control the SAW excitation frequency or the SAW amplitude makes it possible to control the value and direction of the current in the graphene film. The SAW propagation lets to collect and transport the photo-stimulated charges in the graphene film on the crystal surface.The process of acoustically stimulated charge transport in the graphene film on the surface of the  Modern acoustoelectronics is based on the use of surface and bulk acoustic waves for the transmission and processing of information in a real time mode . Also, aRecently, the development of gas sensor acoustoelectronic devices is associated with the use of 1D (ZnO and AlN nanorods) and 2D crystals (graphene and graphene oxide) ,8,9,10. An interesting proposal is to use the surface acoustic waves in solar energy for acoustically stimulated transport of charge carriers generated in semiconductor structures under the influence of light ,12,13,143 ferroelectric crystal depending on the SAW amplitude and the bias potential on the graphene film. The process of charge transport in a graphene film under the conditions of generation of electron-hole pairs under the action of optical radiation is also studied.The purpose of these investigations is to study the process of the SAW propagation in the graphene film on the surface of a LiNbO3 crystal was used to study the process of the SAW propagation in the graphene film. This crystal cut has large values of piezoelectric moduli and a large value of the electromechanical coupling coefficient. The process of SAW propagation in the 3 crystal is characterized by the autocollimation of the acoustic beam. The velocity of SAW propagation in the 3 crystal is 3 crystal is 3. The substrate surface has been polished. The substrate roughness does not exceed 3 Å at the damaged layer thickness of 24 Å.The 3 crystal. The IDTs (input and output) were fabricated by photolithography and consisted of 25 pairs of Al electrodes. The period of the IDT electrodes was 15 μm, which corresponds to the SAW wavelength Λ = 30 μm. The aperture of the IDT was W = 50 × Λ = 1500 µm.A SAW delay time line consisting of two identical interdigital transducers (IDTs) was fabricated on the surface of the 3 crystal. In the left part of the spectrum, strong peaks of the LiNbO3 crystal are visible. The inset shows a zoomed-in part of the spectrum, in which the peaks of graphene  are visible. The spectrum demonstrates features characteristic of single-layer graphene, namely, a symmetric 2D peak with a full width at half-maximum FWHM = 28 cm−1 and intensity ratio I2D/IG = 2.1 [3 substrate are marked by asterisks.IG = 2.1 ,27. In tTo study the acoustically stimulated charge transport on the graphene surface, four gold electrodes were fabricated . The disUsually, to study the operation of acoustoelectronic devices (in our case the SAW delay time line), the amplitude-frequency characteristics are measured, which compare the electrical signals at the input and output interdigital transducers. To study the process of the SAW propagation on the surface of a piezoelectric substrate with graphene, a scanning electron microscopy method was used to visualize the SAW in real time ,29,30. TTo study the process of charge transport in the graphene film by the surface acoustic wave, the scheme shown in 3 crystal allows to change the direction of charge transport in the graphene film. In endences  and on tendences  are deteU=−80 mV a in the   leads to an increase in the current in the graphene film. The dependence of the current in the graphene film depends linearly on the amplitude of the input signal on the IDT in dB.The process of acoustically stimulated charge transport in the graphene film on the surface of the At a positive bias potential 3 crystal, has been demonstrated. The SAW propagation allows the collecting of the photo-stimulated charges and transferring them in the graphene film on the substrate surface. This method of charge collection and transfer will significantly improve the efficiency of solar cells in the future.The possibility of the transport of the photo-stimulated charges by the surface acoustic wave, i.e., the charges generated by the optical radiation in the ferroelectric substrate of the


MXene/graphene composites possess high potential in future biomedical applications.The hybridization and surface functionalization of MXene-graphene composites should be further explored to improve the biocompatibility, high stability, and multifunctionality.The synthesis methods, performances, potential toxicologies, as well as future perspectives of MXene/graphene composites are discussed. MXenes, transition metal carbides and nitrides with graphene-like structures, have received considerable attention since their first discovery. On the other hand, Graphene has been extensively used in biomedical and medicinal applications. MXene and graphene, both as promising candidates of two-dimensional materials, have shown to possess high potential in future biomedical applications due to their unique physicochemical properties such as superior electrical conductivity, high biocompatibility, large surface area, optical and magnetic features, and extraordinary thermal and mechanical properties. These special structural, functional, and biological characteristics suggest that the hybrid/composite structure of MXene and graphene would be able to meet many unmet needs in different fields; particularly in medicine and biomedical engineering, where high-performance mechanical, electrical, thermal, magnetic, and optical requirements are necessary. However, the hybridization and surface functionalization should be further explored to obtain biocompatible composites/platforms with unique physicochemical properties, high stability, and multifunctionality. In addition, toxicological and long-term biosafety assessments and clinical translation evaluations should be given high priority in research. Although very limited studies have revealed the excellent potentials of MXene/graphene in biomedicine, the next steps should be toward the extensive research and detailed analysis in optimizing the properties and improving their functionality with a clinical and industrial outlook. Herein, different synthesis/fabrication methods and performances of MXene/graphene composites are discussed for potential biomedical applications. The potential toxicological effects of these composites on human cells and tissues are also covered, and future perspectives toward more successful translational applications are presented. The current state-of-the-art biotechnological advances in the use of MXene-Graphene composites, as well as their developmental challenges and future prospects are also deliberated. Due to the superior properties and multifunctionality of MXene-graphene composites, these hybrid structures can open up considerable new horizons in future of healthcare and medicine. For iactivity . In addi devices .Fig. 1MX3C2Tx)-graphene hybrid aerogels with aligned cellular microstructures were prepared through hydrothermal assembly followed by directional-freezing and freeze-drying processes [–1) exhibited superb electrical conductivity (695.9 S m–1) and EMI-shielding effectiveness (more than 50 dB in the X-band at a low MXene content of 0.74 vol.%) [3C2Tx)-graphene composite aerogel electrodes with high conductivity and large specific surface area were constructed utilizing self-healing polyurethane (outer shell) [MXene-graphene hybrids with high conductivity, thermal stability, and excellent EMI have found their applications in designing novel supercapacitors and multifunctional sensors , 23. 3D rocesses . These p4 vol.%) . Additior shell) . Likewisr shell) . Since, via the selective removal of “A” layer from their MAX or non-MAX phase parents by acid etching, where A is generally group 13 or group 14 elements in the periodic table [via heating [MXenes have been typically prepared ic table , 28. Addic table , chemicaic table , molten ic table , hydrothic table , and eleic table . Chemicaic table . Notablyic table . On the  heating .3C2Tx)/reduced graphene oxide structures were prepared at low temperature (65 °C) followed by a freeze-drying process. In the hydrothermal reaction, ascorbic acid was utilized to prevent MXene structures from being oxidized /reduced graphene oxide aerogels were synthesized via a hydrothermal technique under a temperature of 95 °C. The designed electrodes from these hybrid aerogels exhibited high electrochemical activities, including significant capacity (~1270 mAh g−1 at 0.1C), enhanced cycling life (~500 cycles), and low capacity decay rate (~0.07% per cycle), with excellent areal capacity (5.27 mAh cm−2). Such composites with unique properties should be further explored for constructing intelligent devices with biomedical potentials [Various physical and chemical approaches have been reported for the synthesis of MXene/graphene composites, including mechanical mixing, self-assembly method, hydrothermal technique, heat treatment, and reagent reduction treatment. Among them, hydrothermal techniques have been widely applied to prepare composites. For instance, MXene Ti3CTx/reducetentials .Fig. 2Th−1) [Several methods have been reported for synthesizing MXenes and graphene based on green chemistry principles to avoid the utilization of toxic/harmful agents and laborious processes. However, greener methods for synthesis and functionalization are still in the infancy stages, and more elaborative studies should be planned to find low-cost, simple, up-scalable, and environmentally benign techniques for the synthesis of these structures. In one study, MXene nanosheets were synthesized using an electrochemical method without using dangerous acid/alkali etchants; although the prepared MXenes exhibited high stability and battery performance, it should be further explored for a variety of applications . Greener−1) .Fig. 3Th2N) quantum dot-based systems with high stability and targeting/selectivity properties were introduced with cancer photoacoustic imaging and photothermal therapy potentials [3C2Tx) structures with cellular uptake features exhibited high transferring potentials from vascular endothelial cells with localization, stability, and auto-fluorescence advantages at different emission-excitation wavelengths allowing post-transport examination and tracking [e.g., face masks) [3C2) could be applied for recognition of human papillomavirus (HPV) with high selectivity and fluorescence quenching ability to dye-labeled single-stranded DNA (ssDNA) as well as significant affinity for ssDNA and double-stranded DNA (dsDNA) [MXenes and MXene-based (nano)structures possess abundant functional groups on their surfaces, offering modification/functionalization opportunities with flexibility. In addition to their high stability and hydrophilicity, they also contain complete metal atomic layers and tunable composition which make them attractive candidates for clinical and biomedical purposes . For instentials . Furthertracking . On the e masks) , 48. Groe masks) . It was e masks) . However (dsDNA) . Under t (dsDNA) .3C2)-based structures to repair bone defects; MXene structures were incorporated into composite scaffolds constructed from hydroxyapatite and sodium alginate through extrusion-based 3D printing for bone regeneration. These scaffolds with uniform structures and macropore morphologies had significant mechanical strength with improved alkaline phosphatase performance, upregulated osteogenic gene expression, suitable biocompatibility, and stimulated mineralized-nodule generation/cell proliferation. They could efficiently promote the regeneration of bone (in vivo), providing great opportunities for bone healing [3C2) quantum dots with immunomodulatory effects have been explored for improving tissue repairing after injury. They selectively reduced the human CD4+IFN‐γ+ T‐lymphocytes activation and stimulated the expansion of immunosuppressive CD4+CD25+FoxP3+ regulatory T‐cells in a triggered lymphocyte population [Different types of MXene and graphene-based (nano)composites have been deployed for tissue engineering and regenerative medicine purposes, with efficient multifunctionality and good biocompatibility. Mi et al.  introduc healing . Notably healing . They ha healing . MXene -graphene hybrid films were synthesized using a mixing-drying technique to produce biosensors for glucose detection /graphene/polydimethylsiloxane layered structures fabricated via vacuum filtration and pre-polymerization mainly contained two layers of MXenes (upper layer) and flexible graphene/polydimethylsiloxane composites (bottom layer). These composite films could be deployed in designing wearable strain sensors  with a large range of linear response, as well as high sensitivity (low limit of detection was ~0.025%), linearity (R2 > 0.98), and cycling stability  [The designed MXene-graphene hybrids have been applied as flexible supercapacitors, electrodes, ion batteries, and EMI-shielding –58. Howe protein . Additio protein . In one ion Fig. . Consequ cycles) .Fig. 4A 3C2TX)-graphene oxide film-based sensors were designed with flexibility, conductivity, and cycling stability advantages [3C2Tx) composites. The designed temperature sensor exhibited high sensitivity/accuracy and competitive thermos-power output (~53.6 μV/°C) with great flexibility/stability (negligible degradations after 1000 bending cycles), opening many opportunities for manufacturing multifunctional devices with biomedical potentials [e.g., respiratory monitoring sensors) [−1 from 0–500 Pa, 126 kPa−1 from 500 Pa–7.5 kPa) and high conductivity were designed utilizing MXene-reduced graphene oxide aerogels [Layer-structured homogenous MXene (Tivantages . One stusensors) . Furtheraerogels . These saerogels .Graphene-based materials have been extensively used in various biomedical applications such as bioimaging, biosensing, theranostics, drug/gene delivery, antibacterials/antivirals, and tissue engineering applications. Therefore, for any effective and successful translation of these materials and becoming commercialized products, there should be a significant exposure of the human body to graphene-based materials, which makes it essential to evaluate the degree of biocompatibility and cytotoxicity of these materials to human cells. The potential cytotoxic effects of graphene-based materials on human cells mainly depend on their physicochemical characteristics, the nature of their interaction with cells, and their accumulation in specific tissues/organs . The tox3C2Tx) was analyzed for possible toxicity in a zebrafish embryo model (in vivo assessments) [−1, the lethal concentration 50 ≈257.46 μg mL−1, and LOEC (lowest observed effect concentration) ≈100 μg mL−1. Notably, no meaningful teratogenic influences could be detected in the examined model at 100 μg mL−1. After locomotion and neurotoxicity assessments, MXenes (50 μg mL−1) demonstrated no harmful influences on neuromuscular performances. Based on the results at concentrations below 100 μg mL−1, these MXene structures could be categorized as practically nontoxic based on the Acute Toxicity Rating Scale (ATRS) by the Fish and Wildlife Service [For clinical translation of MXene- and graphene-based (nano)structures, future studies should be comprehensively focused on (eco)toxicological and cytotoxicity properties of these materials –72. For ssments) . Accordi Service . By deve Service –78. Also Service . Another Service , 81.e.g., cellulose or chitosan nanofibers) can be combined with MXenes and graphene structures to improve their biomedical applicability. Additionally, the commercialization and eco-friendly manufacturing of these structures should be comprehensively explored to find inexpensive and up-scalable strategies with higher safety profile. Optimization of reaction conditions, environmental stability, surface chemistry characterizations, nanotoxicological studies, systematic biocompatibility analyses (both in vitro and in vivo), and pre-/clinical assessments still need to be addressed. MXene structures with single-layer, fewer defects, and larger size had higher conductivity, illustrating that the synthesis conditions and their properties can significantly affect their quality and future applications; the intrinsic features of MXenes and graphene can be improved by controlling reaction mixture conditions , surface functional groups/terminations, and interlayer spacing.MXene-graphene hybrids have been explored due to their fascinating physicochemical properties, which make them attractive candidates for future biomedical explorations. Although MXenes-graphene nanomaterials have attracted wide attention in bio- and nanomedicine, their possible applications for drug delivery, cancer therapy, and theranostics are still uncertain concerning their biocompatibility and toxicity, lack of clinical assessments, and enough specificity/selectivity. Their biological sensing and imaging properties are typically restricted by their non-specific adsorption. MXenes with a larger interlayer spacing had higher specific surface area and additional exposed active sites. In this context, hybridization or surface modifications can remarkably improve multifunctionality and reduce the possible toxicity of these structures. Natural polymers (


The graphene–silicon junction is one of the simplestconceivableinterfaces in graphene-integrated semiconductor technology that canlead to the development of future generation of electronic and optoelectronicdevices. However, graphene’s integration is currently expensiveand time-consuming and shows several challenges in terms of large-scaledevice fabrication, effectively preventing the possibility of implementingthis technology into industrial processes. Here, we show a simpleand cost-effective fabrication technique, based on inkjet printing,for the realization of printed graphene–silicon rectifyingdevices. The printed graphene–silicon diodes show an ON/OFFratio higher than 3 orders of magnitude and a significant photovoltaiceffect, resulting in a fill factor of ∼40% and a photocurrentefficiency of ∼2%, making the devices suitable for both electronicand optoelectronic applications. Finally, we demonstrate large-areapixeled photodetectors and compatibility with back-end-of-line fabricationprocesses. However,the future generation of electronic devices does require the use ofthinner materials with higher electronic performance, which cannotbe achieved with traditional semiconducting bulk materials.4 In addition to the unique propertiesof these materials, their dimensionality and low-temperature integrationenable easy integration in a standard Si CMOS process flow.4 Indeed, back-end-of-line (BEOL) process techniques,where graphene is used as last layer of the process, have been usedto demonstrate graphene–Si prototypes, such as graphene gassensors,5 optical receivers,6 and image sensor arrays.7The family of two-dimensional (2D) materials,in particular, graphene,represents a very attractive platform for the future generation ofelectronics and optoelectronics.8 For this reason, in the past few years, severalstudies have reported the fabrication of graphene–silicon-basedrectifying or barrier-variable devices,10 photovoltaiccells,11 bias-tunable photodetectors,13 chemical sensors,14 and Schottky-barrier-basedfield-effect transistors.15 Despite thesepromising results, there are still several challenges associated withoptimizing the material integration and device performance.4 In particular, previous works did focus on graphene produced bychemical vapor deposition (CVD), whose integration into a siliconchip requires to finely control the material growth (grain-free graphenewill provide better performance) and to minimize wrinkle formationand residue associated with the transfer process,17 hence enabling device reproducibility. These challenges not onlymake the integration process expensive in terms of time and money,but it also considerably limits the possibility of extending the fabricationprocess on a large scale, effectively preventing the possibility ofimplementing this technology in an industrial process.The graphene–silicon junction is one of the simplestconceivabledevices in a graphene-integrated semiconductor technology. Not onlydoes it offers a great opportunity to study the physics occurringat the interface between a 2D and a 3D material, but it also providesthe possibility to realize electronic devices with outstanding properties.18 It also enables reductionof materials and energy consumption, limiting the number of processingsteps and the waste production during the fabrication.18 Graphene and 2D materials can be easily formulatedin inkjet printable inks, either by using liquid phase exfoliation(LPE)20 or electrochemical exfoliation.21 Many works have reported hybrid or fully printedelectronic devices made of 2D materials,26 but to date, a printed graphene–silicon diode has never beenreported in the literature.In thisframework, inkjet printing represents a very attractivetechnique for device fabrication because of its scalability and costeffectiveness.2 from the silicon substrate. Then grapheneis inkjet-printed on the exposed area to allow the formation of thegraphene–silicon junction. The obtained devices show a typicaldiode behavior with an ON/OFF ratio (measured in air and at room temperature)of about 3 orders of magnitude. Several theoretical models have beenapplied to study the transport mechanisms of the junction and to estimatethe figures of merit of the diode, such as the Schottky barrier heightand the ideality factor. Furthermore, the behavior of the devicesat different temperatures and under light irradiation is investigated.A significant photovoltaic effect is observed, making the diode suitablefor advanced optoelectronic applications, such as photodetectors ableto work in the long-wavelength range. In this framework, we demonstratea device made of a network of printed diodes made on the same substrate,which acts as a spatially selective photodetector; i.e., the deviceallows one to identify which area of the chip has been irradiatedwith white light through the measurement of the variation of currentmeasured by different diodes. Finally, to demonstrate the ease oflarge-scale production through this technique and compatibility withSi technology in a BEOL process, we fabricated the same diode on aprepatterned silicon substrate, where the oxide layers were selectivelyremoved with standard clean-room-based etching techniques.In this work, we present a simpleand cost-effective fabricationtechnique for the realization of printed graphene–silicon rectifyingdevices. Mechanical scratching is used to remove a small region ofthermally grown SiOInconclusion, we demonstrate that inkjet printing offers a simpleway to integrate graphene into silicon-based electronics, which canbe used to fabricate diodes with a high ON/OFF ratio and a strongphotovoltaic response that make them suitable for optoelectronic applications.2  of ∼103 at V = ±2 V. The polarity of the diodeis consistent with the formation of a n–n heterojunction betweenthe slightly doped graphene27 and n-typesilicon.28 Measurements have been performedon 12 samples, showing reproducible results. The I–V characteristics of all of the devicestested are reported in section II in the Supporting Information. Note that no rectifying behavior was observedwhen graphene was replaced by metallic electrodes (section II of the Supporting Information).Devices wereprepared on doped n-Si (100) wafers covered with 300 nm thermallygrown SiO2 see . An area surface 1A. A wat surface  and was 28I0 is thereverse saturation current, q is the electron charge, n is the ideality factor, k is the Boltzmannconstant, and T is the temperature. In the case ofpure thermionic transport, n should be 1, but interfacialdefects as well as unwanted insulating layers can lead to Schottkybarrier inhomogeneities increasing the ideality factor.30 The fit of V = −0.2 V. This is expectedsince in this range any series resistance can be neglected. The bestfit is obtained with I0 = −1.4× 10–10 A and n = 2.1. Accordingto the thermionic emission theory, the reverse saturation currentcan be written as:28S is the area of thejunction, A* is the Richardson constant (112 cm–2 K–2 for n-Si31), and φb0 is the Schottky barrierat zero voltage. By using I0 = −1.4× 10–10 A, we obtain φb0 = 0.89 eV. This value seems too high and is likely overestimateddue to the arbitrary choice of the fitting region and the A* value that can be quite different from the theoreticalone in real devices. To improve the fit, we consider a more realisticmodel that takes into account the possible presence of a series resistance Rs, which describes the diode current with thefollowing equation:3233 to obtain the diode parameters: for V – RsI ≫ nkT/q, Rs and n. Then defining thefollowing function:33Supporting Information, we obtain Rs = 35 kΩ, n = 2.7, and φb0 = 0.28. The blue dashed curve in V – RsI ≃ 0, where the diode equation . This discrepancy islikely due both to graphene being doped (n-doping would raise theFermi level reducing its work function) and to a probable Fermi levelpinning caused by the presence of defects or impurities at the junctioninterface, in agreement with what was observed in other works basedon CVD graphene.38To furtherinvestigate the conduction mechanisms of the diode,we study how the current in the devices is affected by the temperature. I–V characteristicsat different temperatures have been used to construct the Richardson’splot and to estimate the values of A* and φb0 through a different method . Although the originof a lower effective Richardson constant has been attributed to differentphenomena, such as the massless Dirac fermion nature of carriers ingraphene,39 a Landauer transport mechanism40 or the presence of a native oxide layer at theGr–Si interface,30 it can be seenthe A* and φb0 obtainedthrough Richardson’s method are very unrealistic as they areaveraged on I–V curves inwhich the thermionic emission is not the only conduction mechanism,such as those at low temperatures. Hence, we can state that Cheung’smethod gives a better estimate of the diode parameters among the modelsused.The I–V characteristics of the Gr–Si diodein the dark and under illumination by a supercontinuum white laser.The measurement carried out under light exposure shows that the I–V curve is shifted to the left,compared to the one obtained under dark conditions, indicating anevident photovoltaic effect. When the Gr–Si junction is irradiatedby light, the Si substrate absorbs photons with energy higher thanthe band gap, and electron–hole pairs are generated. Thesepairs are quickly separated by the built-in field of the Gr–Sijunction formed in the depletion zone, and electrons and holes arecollected at the electrodes, generating the observed photocurrent.Moreover, electrons from graphene can also be excited over the Schottkybarrier and contribute to the photocurrent.13 The parameters commonly used to evaluate the performance of a photovoltaicdevice are the open-circuit voltage (Voc), the short-circuit current (Isc), thefill factor (FF), and the power conversion efficiency (PCE). Voc corresponds to the maximum voltage availablefrom the photovoltaic device, and it occurs when the current is zero.It matches the amount of direct polarization on the junction due tothe illumination, and it has a logarithmic dependence on the intensityof light. In our case, we obtain Voc ∼0.2 V, comparable to values reported from graphene–siliconheterojunction-based devices.42Isc is the current measured when the voltage across the deviceis zero. It is entirely due to generation and collection of carriersgenerated by the light, and it corresponds to the maximum currentachievable by the photovoltaic device. We observe a significant currentof a few nA, confirming the good photovoltaic performance of our diodes.FF is defined as the ratio between the maximum power of the device(Imax·Vmax) and that of the one of the ideal device (FF = (Imax·Vmax)/(Isc·Voc)), and in ourcase, it results in FF ∼ 40%. PCE is defined as the ratio betweenthe energy output from the device and the intensity of the incidentlight (Imax·Vmax/Plight). Despite being stronglyinfluenced by several factors, such as the intensity of light andthe temperature of the device, the PCE is the most commonly used parameterto compare the performance of different photovoltaic devices. In ourcase, we obtain a PCE of ∼2%, which is a good achievement comparedto values commonly reported in the literature for CVD graphene withno further processing (see section III in the Supporting Information), considering the low intensity ofthe incident light and that our devices have not been subjected toany engineering process.43 In particular, ourresults indicate that Si–Gr devices with good performance canbe achieved using printed graphene films; i.e., parameters such assheet resistance and mobility have little effect on the device performance,as also theoretically shown in ref /Idark) calculated at V = 2 V, as shown in in section III in the Supporting Information, follows a linear relationship which ensures thata maximum light effect is reached at the maximum laser power. Therefore,we further investigated the optical response of the devices settinga power of 100 μW/cm2 to test the Gr–Si devicein photocurrent and photovoltage modes. V =0 V and I = 0 A, respectively. It is observed that,under illumination, the device generates both a current and a voltage;i.e., it can be operated in a self-powered mode for over 15 min ofcontinuous cycles. Dark–light–dark sequences have beenused to estimate the characteristic time for the electron–holepairs’ photoexcitation and recombination, respectively. In Supporting Information section III, we show thatthe electron–hole couple generation and recombination timeare 0.38 and 0.25 s, respectively.I–V characteristics of the fourdiodes simultaneously measured under dark conditions: all four diodesshow the same electrical behavior, with slightly different currentvalues mainly due to different series resistances. To test the deviceas a photodetector, the current in the four diodes was measured whilesetting a common bias of V = 0 V on all devices andalternating 20 s in the dark and 20 s of light irradiation only ondiode 1. 45 so the least resistive pathfor the holes is the one through the graphene and not the one throughthe substrate. This conduction mechanism is confirmed when testingthe photodetector using only two or three diodes, as shown in sectionIII of the Supporting Information. Theobserved feature allows the photodetector to be used in self-poweredmode. Indeed, it is possible to identify the irradiated area of thechip simply by looking at the current sign. Furthermore, by illuminatinga single junction, it is possible to generate current in all of theother diodes, considerably improving the photovoltaic efficiency ofthe device as a consequence of an improved charge collection. Theresolution of our detector is in the range of few millimeters  but is limited by the size of thelaser spot. Indeed, the resolution of the photodetector could easilybe further improved by using a focused laser beam.The potential of the “scratchand print” approachproposed in this work enables a network of diodes to be made in avery simple and fast way. To demonstrate this idea, we realized adevice with four parallel diodes . Doped n-Si (100) wafers with a resistivityof ∼10 Ω·cm, corresponding to a phosphorus dopantdensity of ∼4.5 × 1046 Note that the nanosheets are exfoliated withnoncovalent functionalization in water, so the thickness of the nanosheetalso includes a contribution from the stabilizer molecules adsorbedon both sides of the flake.46 Previouscharacterization shows that the nanosheets have high crystallinity,and they are C–O free.47 A VarianCary 5000 UV–vis spectrometer was used to measure the absorptionspectrum of the ink. The Lambert–Beer law was used to obtainthe concentration of graphene by using an absorption coefficient of2460 measured at 660 nm.48 Immediatelybefore printing, the Si substrates were cleaned with acetone, isopropylalcohol, and DI water. A Dimatix Materials Printer 2850 (Fujifilm)was used to print 2 mg mL–1 graphene ink on topof the scratched area. The spatial resolution of the printer is ∼50μm on silicon. The platen temperature was set at 42 °C,and cartridges with a droplet volume of 10 pL were used, setting adrop spacing of 35 μm and 80 printing passes.The water-based inks wereprepared as described in ref . The graSupporting Information). Electrical measurements were performed with aJanis probe station (Janis ST-500 probe station) equipped with fournanoprobes connected to a Keithley 4200 SCS (semiconductor characterizationsystem), working as source-measurement unit with current sensitivitybetter than 1 pA. The electrical measurements were performed in apressure range from 1 bar (room pressure) to 10–5 mbar, obtained by evacuating the air in the chamber by a rough anda turbo pump, and at different temperatures from 220 to 400 K. Thephotoconductivity was tested by irradiating the devices with a supercontinuouswhite laser source .A Bruker Dektak XT system wasused to measure the roughness of the substrate and the thickness ofthe printed graphene (section I in the


It was discovered that the stimulation of M1 macrophages promoted fibroblast proliferation and fibrosis formation in vitro, accompanied with a disorder of the APN/CPT1A pathway, an overproduction of lipid peroxides, and a low level of autophagy in HFL-1 cells. Thereafter, APN treatment or CPT1A overexpression greatly suppressed above lipid peroxide accumulation, fibroblast proliferation, and fibrosis but activated autophagy in vitro. Furthermore, an in vivo IPF rat model was established by injection of bleomycin (BLM). Consistently, CPT1A overexpression exerted a protective role against pulmonary fibrosis in vivo; however, the antifibrosis property of CPT1A was partly abolished by 3-methyladenine (an autophagy inhibitor). In summary, APN/CPT1A-mediated fatty acid metabolism exerted its protective role in IPF partly through activating autophagy, shedding a new prospective for the treatment of IPF.Idiopathic pulmonary fibrosis (IPF) is a chronic progressive interstitial lung disease that leads rapidly to death. The present study is aimed at discovering the in-depth pathogenesis of IPF, exploring the role of adiponectin/carnitine palmityl transferase 1A- (APN/CPT1A-) mediated fatty acid metabolism during the development of IPF, and excavating its potential mechanism. Here, THP-1 cells were differentiated into M0 macrophages, followed by polarization to M1 macrophages upon hypoxia. Subsequently, lung fibroblast HFL-1 cells were stimulated by M1 macrophages to simulate hypoxia-related IPF condition  The rats were randomly assigned into 5 groups  solution. The pathological morphology changes were observed under a light microscope .After fixing with 4% paraformaldehyde for 24 h, the tissue samples were dehydrated in ethanol, embedded in paraffin, cut into 4 t-test, and the difference among more than two groups was evaluated by one-way ANOVA analysis, followed by Tukey's post hoc test. A p value less than 0.05 was considered to be statistically significant.Data were expressed as mean ± standard deviation (SD) from at least three independent experiments or groups of 6 rats each. The normal distribution of variables was assessed by the Shapiro-Wilk test. The differences between two groups were determined by Student's α and mammalian target of rapamycin (mTOR)  . mTOR isn (mTOR) . Furthern (mTOR) . All theβ, α-SMA, and Collagen I for the M1-treated group  was also introduced to HFL-1 cells. The subsequent western blot assay illustrated that addition of APN or overexpression of CPT1A could greatly elevate the protein expression of AdipoR1 and CPT1A in HFL-1 cells (μg/ml) and CPT1A overexpression remarkably lowered HFL-1 cell viability cocultured with M1 macrophages  (Figures Next, to further explore the regulatory role of fatty acid metabolism during the progression of pulmonary fibrosis, particularly to the induction of macrophages, APN/CPT1A signaling was activated to modulate fatty acid metabolism in HFL-1 cells with the coculture of M1 macrophages. As shown in -1 cells , suggestrophages . In addi Figures . Further Figures . These aβ, α-SMA, and Collagen I in pulmonary tissues, whereas C75 treatment lowered this rise, indicating that C75 protected rats against fibrosis; however, this antifibrosis effect of C75 was then partly weakened by the simultaneous treatment of 3-MA (Finally, to validate the critical regulation of APN/CPT1A during the progression of IPF, we successfully established an IPF rat model using BLM injection. In addition, the BLM-induced rats were injected with C75 and/or 3-MA for treatment. The histological analysis presented in  of 3-MA . In addi of 3-MA . What is of 3-MA . The fol of 3-MA . Further of 3-MA . Taken tin vitro, accompanied with a disorder of the APN/CPT1A pathway which is critical for lipid acid metabolism, an overproduction of lipid peroxides, and an activation of autophagy. Thereafter, APN treatment or CPT1A overexpression greatly suppressed lipid peroxide accumulation, fibroblast proliferation, and profibrotic protein expression but activated autophagy in vitro. The following in vivo experiments further validated the protection of CPT1A overexpression in pulmonary fibrosis; however, the antifibrosis property of CPT1A was partly abolished by 3-MA, indicating that APN/CPT1A-mediated fatty acid metabolism exerted its protective role in IPF partly through activating autophagy.IPF is a chronic progressive interstitial lung disease that leads rapidly to death. The incidence of pulmonary fibrosis keeps increasing and brings great burden to global healthcare. Thus, exploring the pathogenesis of IPF and discovering novel effective targets for IPF treatment are essential. Currently, accumulating evidence discloses that the alternations of fatty acid metabolism are commonly detected in fibrosis diseases. In the present study, we uncovered a critical function for APN/CPT1A-mediated lipid acid metabolism in the development of pulmonary fibrosis. Here, we disclosed that hypoxia induced macrophage polarization to M1, which was beneficial to fibroblast proliferation and fibrosis formation β [in vivo [β, α-SMA, and Collagen I, revealing that hypoxia induced the generation of M1 macrophages, which subsequently promoted the fibroblast proliferation and fibrosis. Nevertheless, in this study, in addition to verifying the influences of macrophages on fibroblast proliferation and fibrosis, more attention was focused on the influences on autophagy, oxidative stress, and fatty acid metabolism following hypoxia.Fibroblasts are effector cells driving the progress of fibrotic diseases including IPF featured as unrestrained deposition and stiffening of the extracellular matrix. Despite diverse and varied etiologies of fibrotic diseases, it is pointed out that macrophages play a critical role in the initiation and development of fibrosis . Macrophβ . In addi[in vivo . Thus, min vitro and in vivo, accompanied with the elevated expression of profibrotic proteins, including TGF-β, α-SMA, and Collagen I, but also demonstrated that strengthening of APN/CPT1A signaling was beneficial to restore the balance of lipid peroxides and profibrotic proteins, so as to suppress oxidative stress and alleviate pulmonary fibrosis.Emerging evidence uncovered that alterations in fatty acid metabolism are core pathways and common mechanisms which play a crucial role in multiple fibrotic disorders, including kidney fibrosis, liver fibrosis, and pulmonary fibrosis . Excessiβ-oxidation and accumulation of excessive lipids, suggesting that fatty acid oxidation may be mediated through an autophagy-dependent mechanism [in vivo and in vitro, and APN exerts as a novel therapeutic target of diabetic retinopathy through inhibiting autophagy [Last, but not least, fatty acid metabolism is closely associated with autophagy. It has been proposed that cells may be unable to deal with lipid droplets via acid lipases in the absence of autophagy, leading to the reduction of mitochondrial echanism , 46. Coiutophagy ; howeverutophagy , emphasiutophagy ; Qi et autophagy . ConsistIn summary, we demonstrated not only that hypoxia-induced macrophage polarization to M1 could induce pulmonary fibrosis but also that the activation of APN/CPT1A-mediated fatty acid metabolism suppressed lipid peroxide accumulation and fibroblast proliferation but activated autophagy in IPF. Mechanically speaking, the protective role of APN/CPT1A-mediated fatty acid metabolism was autophagy-dependent. Therefore, targeting fatty acid metabolism is an effective approach for the treatment of IPF.


Here, we demonstrate a physical interaction between 5-HT2A and TrkB in vitro and in vivo using co-immunoprecipitation and biophysical and biochemical approaches. Heterodimerization decreased TrkB autophosphorylation, preventing its activation with agonist 7,8-DHF, even with low 5-HT2A receptor expression. A blockade of 5-HT2A receptor with the preferential antagonist ketanserin prevented the receptor-mediated downregulation of TrkB phosphorylation without restoring the TrkB response to its agonist 7,8-DHF in vitro. In adult mice, intraperitoneal ketanserin injection increased basal TrkB phosphorylation in the frontal cortex and hippocampus, which is in accordance with our findings demonstrating the prevalence of 5-HT2A–TrkB heteroreceptor complexes in these brain regions. An expression analysis revealed strong developmental regulation of 5-HT2A and TrkB expressions in the cortex, hippocampus, and especially the striatum, demonstrating that the balance between TrkB and 5-HT2A may shift in certain brain regions during postnatal development. Our data reveal the functional role of 5-HT2A–TrkB receptor heterodimerization and suggest that the regulated expression of 5-HT2A and TrkB is a molecular mechanism for the brain-region-specific modulation of TrkB functions during development and under pathophysiological conditions.Serotonin receptor 5-HT Tropomyosin receptor kinase B (TrkB) is a major transducer of intracellular signals from mature brain-derived neurotrophic factor (BDNF). TrkB is predominantly expressed within the CNS, especially in the frontal cortex, hippocampus, cerebellar cortex, visual system, hypothalamus, striatum, substantia nigra, and dorsal raphe nucleus ,3,4,5,6.2A) belongs to the G-protein-coupled receptor (GPCR) family and is broadly distributed within the brain, showing high expression in the olfactory tubercle, cortical areas, and dentate gyrus [2A is involved in neurogenesis and can modulate synaptic plasticity via long-lasting increases in the excitability and firing rate of glutamatergic and GABAergic neurons [2A receptor is implicated in numerous CNS disorders, including bipolar disorder, depression, and schizophrenia [2A-blocking antipsychotic drugs elicit better clinical responses in bipolar or treatment-resistant depressive patients [Serotonin receptor 2A , leading to increased Ca2+ release from the endoplasmic reticulum. In neurons, Ca2+ influx can activate cyclic AMP (cAMP) response element-binding protein (CREB), which, in turn, boosts the transcription of BDNF [2A stimulation [2A and TrkB signaling pathways. Although the underlying mechanisms are not completely understood, a direct interaction between 5-HT2A and TrkB may be an intriguing explanation. Research groups have demonstrated that 5-HT2A can form heterodimers with other GPCRs, including the 5-HT1A receptor [4 and 5-HT7 subtypes, can form heterodimers with adhesion molecule L1 [2A interacts with non-GPCR proteins.The coupling of 5-HT of BDNF . Interesmulation ,27,28,29receptor , dopaminreceptor , and metreceptor . Moreoveecule L1 , CDK5 [3ecule L1 , or CD44ecule L1 , respect2A and TrkB form heteroreceptor complexes both in vitro and in vivo. We further demonstrated the functional effects of 5-HT2A and TrkB receptor heterodimerization.In the present study, we found that 5-HT2A antagonist ketanserin  or the selective 5-HT2A agonist 25CN-NBOH  was dissolved in saline, and each was administered i.p. at a dose of 1 mg/kg of body weight (eight mice in each group). The control group (eight mice) received an equivalent saline injection. At 10 min (for 25CN-NBOH) or 30 min (for ketanserin) after the injection, the animals were euthanized, the brains were removed on ice, and the frontal cortex, hippocampus, and striatum were dissected.Adult (P60) male C57BL/6J mice  were used for acute treatment with ketanserin or 25CN-NBOH. The mice were housed under standard laboratory conditions in the natural light–dark cycle (16 h of light and 8 h of dark) with free access to water and food. The preferential 5-HT2 in DMEM  that contained 10% of fetal bovine serum  and penicillin/streptomycin . Transient transfection was performed with Lipofectamine 2000 (Invitrogen) according to manufacturer’s protocol, and after transfection cells were incubated in the serum-free medium. For treatments, we applied 500 nM selective TrkB agonist 7,8-DHF , 1 μM 25CN-NBOH , and/or 1 μM ketanserin . The doses of all drugs were chosen based on literature data. In one work, it was shown that a 7,8-DHF concentration of 500 nM is maximally effective for TrkB activation [2 sites [2A receptor [Mouse N1E-115 neuroblastoma cells from the American Type Culture Collection  were grown at 37 °C and 5% COtivation . 25CN-NBreceptor .2A cDNA was cloned into the pcDNA3.1(+) donor vector  carrying an HA-tag. TrkB_mEGFP was kindly gifted by Ryohei Yasuda ; RRID: Addgene_83952).Murine 5-HT2A and GFP-tagged TrkB was performed as described previously [Co-immunoprecipitation in lysates of N1E-115 cells that co-expressed HA-tagged 5-HTeviously  with an 2A  followed by incubation with Protein A-Sepharose and Western blotting with an anti-TrkB antibody .Co-immunoprecipitation from hippocampal, cortical, and striatal homogenates was performed according to a protocol described elsewhere . BrieflyAdult (P90) male and female C57BL/6J mice were subjected to this assay. The mice were bred and housed at the animal facility of the University of Hohenheim at a controlled temperature (25 °C) and photoperiod (12/12 h light/dark cycle) and were allowed unrestricted access to standard food and tap water.2 asphyxiation, the brains were removed, postfixed overnight at 4 °C, processed routinely, and embedded in paraffin. Next, 5 µm-thick sections were prepared on a Leica RM2255 microtome  and transferred to SuperFrost®Plus glass slides . The sections were air-dried at least overnight at 37 °C and then subjected to the immunofluorescence assay.Mice were euthanized by CO2A receptor  and goat polyclonal anti-TrkB  antibodies diluted in PBS was carried out overnight at 4 °C. After a wash in PBS, appropriate secondary antibodies were applied for a 1 h incubation at room temperature: a donkey anti-rabbit IgG antibody conjugated with Alexa Fluor® 488  and a donkey anti-goat IgG antibody conjugated with Alexa Fluor® 594  both diluted 1:800 in PBS. After a subsequent wash in PBS, cell nuclei were visualized with a bis-benzimide solution . Finally, the sections were mounted in an antiquenching medium and examined under a fluorescence microscope Zeiss Axiovert 200M  with 20× air objectives.The brain sections were deparaffinized according to a standard protocol and washed with PBS. Antigen retrieval was performed by immersion in 0.01 M sodium citrate buffer (pH 9.0) heated at 80 °C in a water bath for 30 min. The blocking of nonspecific binding sites was performed for 1 h at room temperature in PBS containing 5% normal donkey serum , 0.1% Triton X-100 , and 0.02% sodium azide . Incubation with rabbit polyclonal anti-5-HT2A-mTurquoise2 and TrkB-YPet served as the donor and acceptor, respectively. As a negative control, cells were co-transfected with mTurquoise2-tagged CD86 and YPet-tagged CD86. After 16 h, the cells were imaged with a Zeiss LSM 780 microscope equipped with a C-Apochromat 40×/1.2 W Korr water immersion objective via the excitation of the fluorescent proteins at 440 and 514 nm according to the protocol. For the image analysis and evaluation, custom-written MATLAB scripts were employed. We calculated the predicted apparent FRET efficiency as EfDA = ½ EfD/(1 − xD) = ½ EfA/xD, assuming a standard dimerization model [These measurements were performed on live N1E-115 neuroblastoma cells as described elsewhere . 5-HT2A-on model ,41.2A-mTurquoise2, or both. We acquired time series data under the Zeiss LSM 780 microscope for 10 min per recording (5 s per frame). Ca2+ activity was assessed by means of GCaMP6f fluorescence signals (F) and changes calculated as F/Fmax. To raise Ca2+ levels to saturated, 10 µM ionomycin was applied after 6 min. This saturated Ca2+ signal was used as Fmax for the calculation of basal Ca2+ levels.This assay was performed on N1E-115 cells expressing TrkB-YPet, 5-HTA PLA assay was performed with the Duolink in situ PLA Probes and Duolink in situ Detection Reagents Red Kit (cat. # DUO92008). Brain sections of 20 μm thickness were utilized in the PLA. Antibodies, the same as those used for immunohistochemistry, were diluted 1:100 with the Antibody Diluent provided with the kit. For imaging, the slides were dried and mounted with a cover slip by means of ~7 μL of the Duolink in situ Mounting Medium with 4′,6-diamidino-2-phenylindole (DAPI). The imaging and analysis of the stained brain sections were carried out under the fluorescence confocal microscope (Zeiss LSM 780) using a 40× objective and excitation wavelengths of 561 nm for PLA and 405 nm for DAPI. In each brain section, 3–4 measurements in the frontal cortex, hippocampus, and striatum were performed. Each image was analyzed in ImageJ  to count individual fluorescent spots. The data are presented as the number of spots per cell, normalized to the number of cells.Polr2a) cDNA, which served as an internal standard [Total RNA was extracted from the brain tissue of C57BL/6J mice using ExtractRNA , treated with RNA-free DNase , and diluted to 0.125 µg/µL with diethyl pyrocarbonate-treated water. One microgram of total RNA was subjected to cDNA synthesis with a random hexanucleotide mixture ,43,44. Tstandard ,43,44. A3VO4, 2 mM phenylmethylsulfonyl fluoride, and a protease and phosphatase inhibitor cocktail. The protein concentration was estimated spectrophotometrically using the Pierce BCA Protein Assay Kit  and a NanoDrop 2000C spectrophotometer , followed by the adjustment of samples to equal concentrations with 2× Laemmli sample buffer. After denaturation by boiling for 10 min at 95 °C, the cell extracts  were resolved on 10% SDS-PAGE and blotted onto a nitrocellulose membrane . The membrane was incubated with a primary antibody , anti-pTrkB , or anti-5-HT2A antibody ) at 4 °C overnight, then the membrane was washed in Tris-buffered saline supplemented with 0.05% Tween 20 (TBS-T) and incubated with an HRP-conjugated secondary antibody. After protein detection, the blot was stripped and then re-probed with an anti-β-tubulin antibody  as a loading control. Immunoreactive bands were detected by means of the Clarity Western ECL Substrate . Protein bands were documented on a C-DiGit Blot Scanner  and quantified in the Image Studio software . Target protein levels were assessed in chemiluminescence relative units and normalized to β-tubulin chemiluminescence relative units.The extraction of total protein from N1E-115 cells was performed in RIPA buffer  with the addition of 1 mM Nat test after a Gaussian distribution evaluation by the D’Agostino–Pearson normality test. Groupwise comparisons were made by one-way ANOVA followed by Fisher’s post hoc test. Cell groups co-expressing different amounts of 5-HT2A receptors and treated with 7,8-DHF were compared by two-way ANOVA followed by Fisher’s post hoc test. In figures, significance is displayed as p < 0.05 (*), p < 0.01 (**), and/or p < 0.001 (***).The data are presented as means ± the standard error of the mean (SEM). Unless stated otherwise, the significance of pairwise differences was assessed by Student’s 2A and TrkB, we performed co-immunoprecipitation experiments with neuroblastoma N1E-115 cells co-expressing HA-tagged 5-HT2A and GFP-tagged TrkB. It is noteworthy that non-transfected N1E-115 cells express neither 5-HT2A nor TrkB (data not shown). After immunoprecipitation with an antibody against the HA tag, GFP-tagged TrkB was detectable only in cells co-expressing both HA- and GFP-tagged receptors (2A or GFP-TrkB) were mixed prior to lysis and analyzed in parallel. While individual receptors could be detected by the same antibody, co-immunoprecipitation did not occur -based approach. We measured the apparent FRET efficiency (EfDA) between mTurquoise2-labeled 5-HT2A  and YPet-labeled TrkB  in living N1E-115 cells using the linear unmixing FRET (lux-FRET) method combined with confocal microscopy (DA = 14.6%) for 5-HT2A-mTurquoise2 and TrkB-YPet. In contrast, cells expressing a monomeric fluorophore-tagged CD86 protein [DA values  . In all and TrkB C. The qustriatum D.2A heterodimerization, we first assessed Ca2+ activity in N1E-115 cells expressing TrkB-YPet, 5-HT2A-mTurquoise2, or both. To this end, we used the Ca2+ indicator GCaMP6f [2+ dynamics with the multi-threshold event detection approach [2+ signal (Fmax) indicated that basal Ca2+ levels were significantly higher following heterodimerization compared to control cells  protein in N1E-115 cells, using a phospho-specific antibody that specifically recognizes the Y515 site [2A , either alone or together with an increasing concentration of HA-tagged 5-HT2A C. Basal f 5-HT2A B.2A–TrkB oligomerization impacted TrkB function in a manner depending on the receptors’ expression ratio. Therefore, we next determined the expression profiles of 5-HT2A and TrkB in mouse hippocampus, striatum, and cortex at different stages of postnatal development using real-time quantitative RT-PCR. An analysis of the Ntrk2 transcript encoding TrkB revealed that the Ntrk2 mRNA level was continuously elevated from P1 to P30 in the hippocampus and frontal cortex and that this increase was sustained in the striatum until P60  concentrations of HA-tagged 5-HT2A. These experiments revealed significantly reduced basal phosphorylation of TrkB in the cells expressing 5-HT2A at concentrations ≥0.05 μg  C57BL/6J mice with either ketanserin (1 mg/kg) or the selective 5-HT2A agonist 25CN-NBOH (1 mg/kg). TrkB phosphorylation was significantly increased in the hippocampus and frontal cortex of mice treated with ketanserin but not those treated with 25CN-NBOH  in the mouse hippocampus [2A-TrkB heteroreceptor complexes both in vitro and in vivo.Several research articles have reported the modulation of BDNF expression by the 5-HTcircuits ,28,29, bcultures . We recese brain , indicatecule L1 , CDK5 [3ecule L1 , or CD44ecule L1 . The exiecule L1 ,58. Morepocampus . In the 2A receptor functions. Importantly, our data demonstrated that the degree of heterodimerization played a pivotal role in this process. This suggests that changes in the relative amounts of both receptors and the corresponding changes in heterodimerization rates can provide an intriguing mechanism for the differential regulation of TrkB functions in health and disease. Several studies have described substantially decreased TrkB expression and lower TrkB phosphorylation in the hippocampus of depressive suicidal victims [2A levels in some brain structures, including the frontal cortex and hippocampus, in post-mortem samples from patients with major depressive disorder and suicide victims [2A expression is reportedly sensitive to basal 5-HT concentration, with the 5-HT2A receptor becoming more abundant in response to a diminished synaptic 5-HT concentration and vice versa [2A expression in the brain of suicide victims may be explained by an adaptive response to deficient 5-HT2A signaling due to a reduced 5-HT level in depression. Based on the above-mentioned data, it might be hypothesized that under pathological conditions , the balance between 5-HT2A and TrkB becomes shifted toward increased levels of 5-HT2A–TrkB heteroreceptor complexes, with a subsequent loss of TrkB function. The prevalence of 5-HT2A–TrkB heteroreceptor complexes in the hippocampus, even under the basal conditions obtained in this study, suggests that this brain structure is among the most vulnerable to the above scenario.From a functional perspective, this heterodimerization suppressed basal TrkB autophosphorylation and prevented agonist-mediated TrkB activation without affecting 5-HT victims ,61,62,63 victims ,65,66. Mce versa ,68,69. I2A expression was drastically increased (up to five-fold) in all analyzed brain regions, while TrkB expression exhibited only a moderate change (up to a two-fold increase). Considering that even a very low relative amount of 5-HT2A receptor elicited inhibitory effects on TrkB, such a shift in the receptor ratio could result in the decreased ability of BDNF to activate TrkB.Notably, our findings may also explain why the ability of BDNF to activate TrkB gradually declines during early postnatal development in mice . Startin2A–TrkB interaction could also play a role in the action of antidepressants. It is generally accepted that BDNF–TrkB signaling is a critical component of an antidepressant response [2A receptor inactivation may also facilitate antidepressant action [2A receptor antagonist ketanserin increased neurogenesis in the adult rat hippocampus [2A receptors because a combined treatment with fluoxetine and ketanserin applied at lower dosage  failed to produce a neurogenic effect while it boosted the expression of Bdnf mRNA in the rat hippocampus [2A antagonism can stimulate BDNF expression and/or secretion [2A receptor activation can suppress Bdnf transcription in the hippocampus [2A and BDNF could thus explain the functional crosstalk between these receptors at the molecular level. Indeed, we found that an acute blockade of 5-HT2A with ketanserin reversed basal TrkB phosphorylation in vitro. We also demonstrated that acute treatment with ketanserin led to increased TrkB phosphorylation in the mouse hippocampus and frontal cortex in vivo, which is in accordance with our findings regarding the prevalence of 5-HT2A–TrkB heteroreceptor complexes in these brain regions.The 5-HTresponse ,71,72,73t action ,75,76,77pocampus . Howeverpocampus . Moreoveecretion ,88,89,90pocampus . Our pre2A receptor, a member of the GPCR family, could form heterodimers with the non-GPCR receptor TrkB, both in vitro and in vivo. Heterodimerization considerably decreased TrkB autophosphorylation and prevented TrkB activation by its ligand, and these effects could be reversed by a pharmacological blockade of 5-HT2A with ketanserin. Importantly, our findings suggest that the regulated and balanced ratio of heterodimerization in different brain structures may be crucially involved in both the onset and treatment responsiveness of psychiatric diseases such as depression and anxiety.Overall, our present results demonstrated that the 5-HT2A receptor, a member of the GPCR family, and the non-GPCR receptor TrkB, both in vitro and in vivo.In the present study, we provide a multilevel analysis demonstrating, for the first time, the physical interaction between the 5-HT2A receptor functions. Importantly, these detrimental effects on TrkB functions were reversed by a pharmacological blockade of the 5-HT2A receptor with ketanserin, a drug widely used to treat hypertension. Moreover, our data demonstrated that the stoichiometry of heterodimerization played a pivotal role in this process. This suggests that changes in the relative expression of both receptors and the corresponding changes in heterodimerization rates can provide an intriguing mechanism for the brain-region-specific regulation of TrkB functions in health and disease.From a functional perspective, heterodimerization suppressed basal TrkB autophosphorylation and prevented agonist-mediated TrkB activation without affecting 5-HT


A subgroup of IPF patients can meet IPAF criteria (features suggesting an underlying autoimmune process without fulfilling established criteria for a CTD). This study was aimed to evaluate whether IPAF/IPF patients compared to IPF patients differ in clinical profile, prognosis and disease course.This is a retrospective, single center, case–control study. We evaluated 360 consecutive IPF patients  and compared characteristics and outcome of IPAF/IPF to IPF.more frequently females , suffered more frequently from gastroesophageal reflux , and showed a higher prevalence of arthralgias , myalgias  and fever . The serologic domain was detected in all cases (the most frequent were ANA in 17 and RF in nine cases) and morphologic domain (histology features) was positive in 6 out of 10 lung biopsies (lymphoid aggregates). Only patients with IPAF/IPF evolved to CTD at follow-up . The presence of IPAF was a positive prognostic determinant , whereas the isolated presence of circulating autoantibody did not impact prognosis .Twenty-two (6%) patients met IPAF criteria. IPAF/IPF patients compared to IPF were The presence of IPAF criteria in IPF has a major clinical impact correlating with the risk of evolution to full blown-CTD during follow-up and identifying a subgroup of patients with a better prognosis. In 2015, ATS/ERS international task force defined the criteria describing a new research entity named IPAF  , 4. DespIPF can be reclassified as IPAF when, in addition to the usual interstitial pneumonia (UIP) pattern, have a combination of one feature from at least two of three different domains; clinical, serologic or morphologic [either pathological  or related to a multi-compartment involvement ] , 5.The paucity of studies investigating the impact of IPAF features on IPF natural history provides a strong rationale for the present study that was conducted in a large and well-defined IPF cohort using rigorous inclusion criteria for IPAF and was aimed to evaluate whether IPAF/IPF patients compared to IPF patients have a different prognosis and disease course.In this single-center, retrospective, investigator initiated comparative study, we evaluated consecutive patients presenting to the pulmonary unit of the GB Morgagni Hospital  with suspected interstitial lung disease who received a multidisciplinary diagnosis of IPF . Patients with incomplete clinical data, less than 3 months of follow-up and those without a complete autoimmune clinical and serological evaluation performed at our center were excluded. Baseline and follow-up data were collected as detailed in the This study was approved by the Comitato Etico di area vasta ROMagna, Italy .The primary endpoint was the prognostic significance of the presence of IPAF among IPF patients. This was measured by comparing transplant-free survival for IPF with IPAF to that of IPF without IPAF.Secondary endpoints included:(1)The prognostic significance of the presence of positive autoimmune serology alone  in IPF patients, compared to IPAF/IPF and to IPF only . This was measured comparing transplant-free survival for IPF with positive autoimmune serology to that of IPF with IPAF and to that of IPF only.(2)Evaluation of natural history: development of full-blown CTD at follow-up. We described the baseline characteristics and compared the prevalence of CTD at follow-up between three patients subgroups: IPF with IPAF, IPF with positive autoimmune serology alone  and IPF only .t-test or Chi2 test, as appropriate. We used exact probability values  and an alpha level of 0.05.For baseline data, the summary descriptive statistics were be generated with categorical data displayed as absolute numbers and relative frequencies. Continuous data were shown as mean (SD) for normally distributed data or as median (interquartile range) for skewed distribution. Comparisons between groups was performed using a p-values ≥0.2 and excluding covariates with significant collinearity (r > 0.8) at univariate analysis. The proportional hazard assumption for each predictor was tested using approximate score statistic of linear correlation between the rank order of failure times in the sample and Schoenfeld partial residuals. We calculated hazard ratios (HRs) for overall mortality analyses. Patients were censored at death, lung transplant, or date of last known follow-up. Data cut-off was December 28, 2016. Results are reported as HRs, 95% CIs and p values, and are shown graphically as Kaplan–Meier survival curves. All statistical analyses were performed using STATA 15.For regression analysis, sample size calculation met the rule of thumb of at least 10 observation per variable, with 170 observed events we could evaluate 17 variables without overfitting. The small size of missing data allowed an analysis restricted to individuals with complete information on all variables of the main analysis . The fundamental method of univariable/multivariable analysis was Cox regression. Causal model based on previous literature was used to identify confounders: age, gender, smoking status, comorbidities and disease severity. The models were formulated by systematically removing predictors that were not significant using a backward selection procedure removing variables with N = 274) was excluded due to the lack of rheumatologic and/or serologic evaluation performed at our center. The flow chart of cases inclusion is presented in the p = 0.003), as detailed in the We extracted from the Forlì database 703 consecutive IPF patients, between January 1, 2002 and December 28, 2016. Among those 360 met protocol requirements and were included in the study. A vast minority , p = 0.02. No significant differences were noted in age, smoking history, family history and comorbidities profile with the notable exception of GERD that was significantly more prevalent in IPAF/IPF compared to non-IPAF/IPF . The pulmonary function profile showed a slight but significantly higher DLco in the IPAF/IPF patients  and a marginally higher FVC not statistically significant  without significant differences in the GAP stage distribution. Similarly to non-IPAF/IPF patients, IPAF/IPF patients presented in the vast majority with a chronic onset (75%) of dyspnea (86.4%) and/or cough (59.1%). However, in contrast to what observed in non-IPAF/IPF patients, all IPAF/IPF patients presented with at least one systemic symptom, with a strikingly higher prevalence of arthralgias , myalgias  and fever  compared to non-IPAF/IPF. Further rheumatologic evaluation of arthralgias revealed inflammatory arthritis only in IPAF/IPF cases, as shown in Comparison between IPAF/IPF patients and non-IPAF/IPF patients is shown in Among the 338 IPF patients that did not meet IPAF criteria, 43 (12.7%) showed a positive autoimmune serology.All cases showed isolated positivity for a single class of autoantibodies. Four patients  showed ANCA positivity and one of them developed a full-blown vasculitis after 10 years of follow-up . Seventeen (39%) patients had an isolated anti-thyroid positive autoimmunity, all were clinically identified as autoimmune thyroiditis without evidence of systemic autoimmune disease. 11  patients showed isolated ANA positivity, two ENA, two anti-CCP, and 7 RF positivity. None of them developed CTD at follow-up.Comparison of clinical characteristics of non-IPAF/IPF cases with and without positive autoimmune serology are reported in p values >0.05, not shown) with two notable exceptions: (1) higher prevalence of arthralgias in the non-IPAF subgroup with positive autoantibodies  compared to non-IPAF with negative autoantibodies , p < 0.0001. Those patients were not classified as IPAF because arthralgias was interpreted by the rheumatologist as non-specific. (2) higher prevalence of digital clubbing in the non-IPAF subgroup with positive autoantibodies  compared to non-IPAF with negative autoantibodies , p = 0.003.N = 22), the difference was robust both by univariate analysis HR 0.17  and after adjusting for age, sex, lung cancer, pulmonary function variables (%pred FVC and %pred DLco) and diagnosis before or after 2011, HR 0.22 . Univariate and multivariate analyses are reported in The presence of IPAF criteria in patients who received a multidisciplinary diagnosis of IPF was associated with significantly lower overall mortality compared to IPF patients lacking IPAF criteria. Despite the small number of cases (N = 338) the presence of positive autoimmune serology (N = 43) did not impact patients’ prognosis, unadjusted HR 1.01 (95% CI 0.69–1.48), p = 0.96 and HR 1.00 (95% CI 0.67–1.49), p = 0.99 after adjusting for age, sex, lung cancer and pulmonary function variables (%pred FVC and %pred DLco). Overall mortality rate per 100 person-year and survival at 1, 3, and 5 years were all similar between the two groups, data are reported in When analysis was confined to IPF patients not meeting IPAF criteria  developed CTD: rheumatoid arthritis , scleroderma  and one Sjogren. Mean latency time from IPAF diagnosis to CTD diagnosis was 21.5 months (range 6–60 months).The characteristics of the 22 IPAF cases are reported in To the best of our knowledge this is the first study evaluating IPAF features and clinical meaning in a well-defined cohort of IPF patients, showing that IPF patients can present IPAF features in a minority of cases (6%) with a clinical profile and natural history strikingly divergent from that of IPF. IPAF/IPF compared to IPF is characterized by:(1)a significantly better prognosis;(2)a high risk of evolution to full blown CTD (45.5%), that among IPF patients seems exclusive of those presenting with IPAF features;(3)a specific clinical profile with a higher prevalence of females and gastroesophageal reflux. Most notably, systemic signs and symptoms, rare in IPF, are universally present in IPAF/IPF  and are associated with at least one positive autoimmune finding on serology (most commonly elevated levels of ANA and/or RF).To define IPAF in this cohort of IPF patients we have meticulously applied the current IPAF ERS/ATS criteria to the clinical and serological features. The morphologic domain was defined by histopathology patterns  of UIP with interstitial lymphoid aggregates with germinal centers and/or diffuse lymphoplasmacytic infiltration (with or without lymphoid follicles), and these features were present in one third of IPF/IPAF cases (6/22). The multi-compartment involvement was defined using more stringent criteria: unexplained pleural or pericardial effusion or thickening, or by the presence of unexplained airway disease as seen by histopathology (follicular bronchiolitis or constrictive bronchiolitis). Interestingly none of our IPF/IPAF patients met the multi-compartment features following these restricted criteria. This punctilious approach led to a prevalence of IPAF among IPF cases lower than previously described, but with a strikingly prognostic divergence that previous studies could not detect .p = 0.003). Age, lung cancer, pulmonary function (% of pred FVC and % of pred DLco), diagnostic time period (before/after 2011) and IPAF were all significant prognostic factors at both univariate and multivariate survival analysis. IPAF/IPF patients compared to IPF had a significantly lower overall mortality rate per 100 persons year (3.13 vs. 16.26) and a significantly better prognosis . Previous studies have compared the prognosis of IPAF to that of historical IPF patients cohorts, showing that IPAF carries a better prognosis compared to IPF  (The Oldham study  is the m= 0.009) .In the absence of IPAF criteria the sole presence of autoantibodies in IPF did not influence survival. It is difficult to compare our results with the existing literature on this topic because our study was not powered to detect the prognostic significance of specific antibodies. In the literature the effect of ANA and RF on all causes mortality does not seem significant.  The subsN = 22) imbalanced compared to the controls groups . Biases related to the observational retrospective design of this study spanning over a wide time period (2002–2016) are alleviated by the consecutive enrollment of patients, the reassessment of all IPF diagnosis based on ERS/ATS 2011 guidelines and the introduction in the multivariate survival analysis of the diagnostic time period (before/after the year 2011) as a dummy variable. However, concerns about the high dropout rate, a price we had to pay to achieve a highly accurately selected IPAF population with serology and rheumatology evaluation completed at our center, are only partially mitigated by the observation that the excluded cases compared to included cases had homogeneous clinical profile, although a slightly divergent prognosis.This study has several limitations: the retrospective and monocentric study design, the high rate of cases exclusion , the very small sample size of IPAF cases  and identifying a subgroup of patients with a clearly better prognosis .Future prospective and larger studies will help to better define IPAF diagnostic criteria and their utility to identify in IPF specific subgroups with different prognosis and treatment response.The original contributions presented in the study are included in the article/This study was approved by the Comitato Etico di area vasta ROMagna, Italy . Written informed consent [from the patients/participants OR patients/participants legal guardian/next of kin] was not required to participate in this study in accordance with the national legislation and the institutional requirements.ST, CR, SPu, SPi, AW, JR, MB, and VP: conception and design. ST, SPu, CR, MB, AW, JR, AD, SPi, FG, FL, ER, VL, MC, and VP: acquisition, analysis or interpretation, and drafting the manuscript for important intellectual content. VP had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. All authors contributed to the article and approved the submitted version.ST declares speaker’s fee from Boehringer-Ingelheim, Roche, Erbe, PulmoniX; and VP declares speaker’s fees from Boehringer-inghelhem, Erbe, Ambu, and Roche.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The three-dimensional full-field morphologies at different compressive loads were obtained. The evolution of flaws, including pores, transverse cracks, and vertical cracks, during compressive loading was observed. The number and volume of pores, transverse cracks, and vertical cracks were extracted. It was found that most pores and transverse cracks tend to be compacted. However, high compressive loads cause vertical cracks near the upper surface to expand, leading to the failure of the specimen. Real flaws with morphological and positional information were introduced into the finite element models. The influence of different types of flaws on the mechanical behavior is discussed. It was found that vertical cracks have a greater influence on mechanical behavior than do transverse cracks under compression. The presence of transverse cracks contributes to the evolution of vertical cracks. This study may be helpful for process optimization and performance enhancement of additively manufactured ceramic materials.The additive manufacturing process may create flaws inside ceramic materials. The flaws have a significant influence on the macroscopic mechanical behavior of ceramic materials. In order to reveal the influence of flaws on the mechanical behavior of additively manufactured ceramic, flaw evolution under mechanical loads was studied by in situ X-ray computed tomography (XCT) in this work. In situ compression XCT tests were conducted on stereolithographic additively manufactured Al The higiC, etc. ,8,9.2O3 depends on the relation between the printing and loading directions. The microstructural characteristics and fracture strength of yttria-stabilized tetragonal zirconia polycrystal fabricated by stereolithographic additive manufacturing and traditional subtractive manufacturing were studied. Although both materials showed similar microstructures considering grain size and phase composition, significant differences in critical defects were observed [Stereolithography additive manufacturing technology for ceramics generally comprises shaping, debinding (binder burnout), and sintering processes ,8,9. Durobserved . Anotherobserved . The infSEM, as a commonly used method for microstructural characterization, provides only two-dimensional information of outer surfaces. To obtain three-dimensional information of the whole body, successive sectioning and subsequent microscopy are adopted. However, the sectioning process may cause damage to materials. X-ray computed tomography (XCT) technology can obtain internal information of materials and has become an established technique for nondestructive analysis ,17,18. I2O3 ceramics. The flaw evolution behaviors under compressive loads were investigated. The influence of different types of flaws on the mechanical behavior is discussed. It is believed that this study may provide guidance for the process optimization and performance enhancement of additively manufactured ceramic materials.In this paper, compression tests with in situ XCT were conducted on additively manufactured Al2O3 ceramics is presented in 2O3 green bodies was reported in a previous work [2O3 coarse grain particles of 50 vol.%  were used as the raw material to prepare Al2O3 ceramic slurry. Al2O3 fine grain particles  were added as fine grains. Al2O3 slurry was dispersed in a pre-photosensitive resin suspension that consisted of photosensitive resin monomers (HDDA and TMPTA), photoinitiator (1 wt% TPO), dispersant (2 wt% KOS110), and sintering additives (3 wt% TiO2 and 1 wt% MgO). The photosensitive slurry was mixed via ball milling at 400 rpm for 24 h. Al2O3 green bodies were then fabricated using stereolithographic additive manufacturing equipment with a 405 nm ultraviolet light projector . The light intensity, exposure time, and slicing thickness were set to 14,000 μw/cm2, 4 s, and 100 μm, respectively. After that, the Al2O3 green bodies were debound at 550 °C for 2 h (a heating rate of 0.5 °C/min) and pre-sintered at 1000 °C for 2 h in a furnace . Finally, the debound bodies were sintered at 1650 °C for 3 h in air in a furnace . Based on the above process, Φ 5 mm × 12.5 mm (diameter × height) sintered specimens that were built with layers in the height direction were prepared for in situ XCT tests. The microstructures of the specimens were observed using a scanning electron microscope .A schematic for the preparation of additively manufactured Alous work . Al2O3 c2O3 ceramics using an in situ X-ray computed tomography apparatus assembled in our laboratory, as shown in In situ compression tests were conducted on additively manufactured Al2O3. The relative density of the sintered specimen was 96.63 ± 0.52%. The layer-by-layer characteristic was observed on the surface of the material in layer-by-layer processing of stereolithography additive manufacturing. It can be seen that the material had a relatively fine-grained microstructure. The grain size in the specimen ranged from 1 to 6 μm. The crystallization of Al2O3 particles was also observed.2O3 specimens.Load and displacement data were recorded during the compression tests. A typical load–displacement curve is shown in 2O3 ceramics, and the flaws evolve with increasing compressive load. At 10,000 N, flaws near the upper surface evolve into large flaws, resulting in the failure of the specimen. To quantitatively analyze the evolution behavior of the flaws during compressive loading, the number and volume of the total flaws at different compressive loads were also obtained, as shown in Three-dimensional volume rendering of total flaws at different compressive loads was obtained by stacking two-dimensional images, as shown in To gain insight into the evolution behavior of flaws with the load, the flaws were classified and analyzed. As mentioned before, flaws mainly include pores and cracks. The sphericities of pores were calculated to distinguish between pores and cracks. Flaws with a sphericity value less than 2.9 were classified as pores, and those with a sphericity value larger than 2.9 were classified as cracks. Three-dimensional volume renderings of the pores at different compressive loads were individually extracted and are shown in z-axis was located along the height direction of the specimen and parallel to the loading direction. Cracks with a maximum size greater than 200 μm in the z-direction were defined as vertical cracks, and those with a maximum size less than 200 μm in the z-direction were defined as transverse cracks. As is widely known, the relationship between crack orientation and loading direction may have an important influence on the crack evolution. Thus, cracks were further subdivided according to crack orientation. A coordinate system was created and is shown in Three-dimensional volume renderings and the number and volume of transverse cracks at different compressive loads were also obtained, as shown in To explain the change in volume with the load, some transverse cracks with a large volume were chosen and extracted separately for analysis, as shown in 2O3 ceramic specimens. Tensile stresses on the tip of vertical cracks induced by compressive loads can lead to specimen failure.Under compressive loads, most pores and transverse cracks tend to be compacted. However, the vertical cracks have an important influence on the failure of additively manufactured Al2O3 ceramic specimens. Four three-dimensional (3D) finite element models, as shown in Finite element analysis was adopted to further investigate the influence of different types of flaws on the mechanical behavior of the additively manufactured AlAlthough the displacement data could be obtained from the sensor of the material testing machine, the measured displacement was the displacement of the crossbeam rather than that of the specimen. Two-dimensional XCT images were used to calculate the true displacement of the specimens, as shown in Load–displacement curves corresponding to the four models were obtained from the finite element analysis, as shown in 2O3 ceramic. We conclude the following:(1)2O3 ceramic specimens. In situ XCT compression tests were conducted on additively manufactured Al2O3 ceramic specimens. Details on the three-dimensional full-field morphology of the specimens at 0, 3000, 6000, and 10,000 N were obtained by stacking two-dimensional XCT images.Stereolithography additive manufacturing technology was used to prepare Al(2)Different types of flaws, including pores, transverse cracks, and vertical cracks, were studied. The number and volume of the pores, transverse cracks, and vertical cracks were analyzed. It was found that the number of flaws tended to decrease with increasing compressive load, resulting from the compaction effect. At 10,000 N, vertical cracks near the upper surface expanded and formed large vertical cracks, leading to the failure of the specimen.(3)Real flaws with morphological and positional information were introduced into the finite element models to study the influence of different types of flaws on the mechanical behavior. The true displacements were obtained by analyzing XCT images. The failure mode obtained from the model including two vertical cracks and one transverse crack was similar to the experimental result. It was found that the influence of vertical cracks on the mechanical behavior was greater than that of transverse cracks. The presence of transverse cracks contributes to the evolution of vertical cracks.In this paper, we investigated the influence of manufacturing flaws on the macroscopic mechanical behavior of additively manufactured Al