Graphitic carbon nitride (g−CN), a promising visible-light-responsive semiconductor material, is regarded as a fascinating photocatalyst and heterogeneous catalyst for various reactions due to its non-toxicity, high thermal durability and chemical durability, and “earth-abundant” nature. However, practical applications of g−CN in photoelectrochemical (PEC) and photoelectronic devices are still in the early stages of development due to the difficulties in fabricating high-quality g−CN layers on substrates, wide band gaps, high charge-recombination rates, and low electronic conductivity. Various fabrication and modification strategies of g−CN-based films have been reported. This review summarizes the latest progress related to the growth and modification of high-quality g−CN-based films. Furthermore, (1) the classification of synthetic pathways for the preparation of g−CN films, (2) functionalization of g−CN films at an atomic level  and molecular level (copolymerization), (3) modification of g−CN films with a co-catalyst, and (4) composite films fabricating, will be discussed in detail. Last but not least, this review will conclude with a summary and some invigorating viewpoints on the key challenges and future developments. H. H3N3  to the lowest unoccupied molecular orbitals (LUMOs). The HOMO and LUMO levels originated from nitrogen pectively . This baectively ,39.Until now, thermal pyrolysis of nitrogen-rich precursors such as cyanamide, dicyanamide, urea, melamine, thiourea, guanidine hydrochloride, trithiocyanuric acid, etc. has been employed to synthesize g−CN. This type of nitrogen-rich precursor has a slight effect on the band-gap structure of g−CN . The flaNumerous synthesis strategies have been developed to prepare g−CN films on substrates, which are classified as thermal vapor condensation (TVC), solvothermal route, liquid-mediated growth, seed growth, electrophoretic deposition, disperse coating, and other deposition methods. Detailed descriptions of the various types of synthesis methods for the construction of g−CN films are provided below. The main advantages and disadvantages of the different synthetic methods are summarized in −2 at the bias of 1.23 V versus RHE without a sacrificial reagent in a two-step TVC procedure  glass, fluorine-doped tin oxide (FTO) glass, silicon slice, quartz glass, and glass. Bian et al. discovered that the morphology of the g−CN film is substrate-dependent, grown on ITO glass and silica are similar to that of on FTO glass; they are continuous films with many nanoparticles on the surface. However, inhomogeneous particles and nanorods formed on glass a–c 48]..48]. The μA cm−2 e [48]. B2, HfO2, and c-plane sapphire through TVC at 600 °C. A crystalline g−CN thin film can be deposited on both non-crystalline and crystalline surfaces by employing an amorphous-like buffer layer. The bonding states and stoichiometric chemical composition (N/C ratio was 1.36–1.39) are close to those of ideal g−CN. From the dependence of the photoluminescence (PL) intensity and peak shift on the excitation–power–density, electron injection from the sp3 C–N σ to the sp2 C–N π conduction band was strengthened under the above excitation condition (Eex > Eg σ)  a,b.2 groups play an essential role in nucleation. However, a lack of one NH2 group in each precursor molecule is proven to fail to realize high-quality g−CN thin film ..−2 compaIn spite of the fact that g−CN powder has been extensively and deeply researched, the preparation knowledge of g−CN film is still in its infancy. Recently, disperse coating strategy from colloid sources has also been reported extensively owing to its extraordinary features, including facile and cost-effectiveness. It has been applied to the deposition of carbon nitride thin film on various substrates via conventional filming technologies, such as spin coating , silk-sc−2 ..−2 at 1.−2) for 4000 s with no degradation (2 to generate C–N compound on the FTO substrate. The resulting g−CN nanorod (NR) arrays eliminated the intralayer hydrogen bonds owing to the utilization of carbon and nitrogen sources alone. The change of the graphite target current allowed different lengths of the g−CN nanorod arrays from 550 nm to 1.1 µm. The gas-based approach via the VMFAIP system enabled the g−CN to form heterostructures easily with, including but not limited to, TiO2 nanorod arrays (2 nanotube array in Ar and N2 plasma media. The PEC studies summarized that the g−CN/TiO2 formed under Ar plasma (~290 μA/cm2 at 0.9 V) obtained a higher current density as compared to that under N2 plasma (~160 μA/cm2 at 0.9 V).Electrospinning as a practicably proven method is also used to fabricate the PEC photoanode, in which the ITO substrate is coated with prepared polyvinylpyrrolidone (PVP)/g−CN sol and annealed at 500 °C. The thickness of the films can be modulated by varying the electrospinning time and actually controlling the PEC performance. Furthermore, the photoelectrode demonstrates a stable photocurrent performance (6.64 μA cmradation f–h 77]..−2) for d arrays i–k 93]..−2) for −2), which can be ascribed to the uneven coverage and the weak adhesion of g−CN on the substrates [−2) [−2 at 1.23 V versus RHE under AM 1.5G in 0.1 M KOH aqueous solution. This is the best value reported for pure g−CN electrodes without a sacrificial agent. Enormous space still exists for achieving a theoretical photocurrent density of 14.5 mA cm−2 and a maximum solar-to−hydrogen (STH) efficiency of 17.9% under AM 1.5G (100 mW cm−2) [Although various techniques have been employed to prepare g−CN electrodes, g−CN films prepared by ex situ methods mostly show poor photocurrent density  . Shalom tes [−2)  grew higmW cm−2) ,25,26. TWhen pure g−CN films are employed as photocatalysts, their practical applications are still hindered by limited theoretical photocurrents. The p–p* transitions in the conjugated aromatic system of g−CN result in a band gap of 2.7 eV, suppressing the absorption above 460 nm . Doping 3 as the Ag dopant. The AgNO3 strongly affects the PEC efficiency of the Ag/g−CN films. The optimized photocurrent density was as high as 6.40 μA mm−2 for the Ag/g−CN films (molar ratio of 1: 10), which was approximately 6.8−fold higher than that of a pristine g−CN film electrode. This is due to the efficient charge transfer and improved light-harvesting property of Ag-doped g−CN film. Apart from Ag, Karimi-Nazarabad and co-workers [−2, which is notably higher than those of Ag/g−CN and pristine g−CN films. Additionally, the photoelectrocatalytic oxygen evolution performance is improved due to the facilitated transfer efficiency of photo-generated charge carriers, which can be ascribed to the deeper band bending and the remarkable trapping capability for electrons. Recently, doping engineering with Thorium Nitrate (ThO2) during g−CN polymerization altered the chemical bonding, with the improved PEC activity  as the B source. It was also noted that the pyrolysis temperature had an intimate connection with the compositional ratios, and the preferable temperature for B doping ranged from 618 °C to 650 °C  and improved the diffusion length, resulting in the enhancement of PEC activity. The photocurrent density of C-doped g−CN film (5 wt %) was 0.1 mA cm−2 at 1.23 V versus RHE, which was over four-fold higher than that of pure g−CN. Dang et al. [−2, which was five-fold higher than that of pure g−CN. Similar methods were also prepared for the p-doped g−CN with enhanced PEC activity [Similarly to B, other non-metallic atoms such as C, P, and S were also examined. Bian and co-workers  fabricatg et al.  preparedactivity .−2, which was mainly ascribed to the S-doping and particularly the modification of surface morphology, crystalline, and band structure by the polymerization of the precursors.Other non-metallic atom-doped g−CN photoelectrodes have also attracted extensive attention in PEC applications. Ye et al., 2016 , grew a 2 at 1.23 V vs. reversible hydrogen electrode. This manifestation acts as a platform to form the gradual variation of the band-gap employing metal-free films to enhance the performance of PEC for other extended applications [In addition, Fang et al.  modifiedications ,119,120.2 as the starting materials. It was found that the K&I co-doping transforms the from amphoteric to n-type semiconductor property. The photocurrent density of K&I co-doped g−CN is approximately 50.3 μA cm−2 at 0.5 V versus Ag/AgCl. Luan et al. [Lately, g−CN film via co-doping of nonmetal and metal atoms has gained wide attention because this strategy shows a positive impact on the structural and optical properties. Jing et al.  reportedn et al.  reported2 nanorod/g−CN not only narrows the band gap into a visible-light range from 2.70 to 2.13 eV but also forms a metal (Cu and Fe) phosphide water oxidation cocatalyst via one-step phosphide annealing process. As a result, the metal phosphide-modified sample yields an outstanding visible light (>420 nm) photocurrent response of approximately 0.3 mA cm−2 at 1.23 V versus RHE .The π−conjugated structure of g−CN is another factor that influences the PEC activity owing to the spoperties ,127,128.operties . Bian etoperties  copolyme−2 at 1.23 V (vs. RHE) under visible light irradiation without sacrificial reagents and cocatalysts, which is 7.5, 176.9 and 67.6 times higher than that of the CN-Me (melamine as a precursor), CN-CC (cyanuric chloride as a precursor), and bulk g−CN powders, respectively. The improved PEC performance is ascribed to the enhanced charge transport, increased number of terminal amino groups, and the efficient visible light harvesting caused by the intimate contact  and inorganic monomer (hydrazine hydrate) to generate intermediate melem hydrazine (MH) through hydrothermal reaction, following annealing at 450 °C for 2 h. In the C3N5 framework, two s-heptazine units are bridged together by azo linkage (−N≡N−), which is a completely different bonding mode compared to C3N4, where three heptazine units are connected with tertiary nitrogen. Owing to overlap between the π orbitals of azo-bridged units and the π system of heptazine motif, the band-gap of C3N5 was markedly reduced to 1.76 eV, which enables the optical light absorption to expand up to 670 nm. The photocurrent response for the C3N5 and g−C3N4 blended TiO2 electrode was proved to be 152 and 100 μA cm−2 at 1.23 V vs. NHE (−2 (nearly 13 times) owing to the more extended conjugation to promote electron transport and expand light-harvesting range  in the copolymerization of nitrogen-rich precursors is an effective method to adjust their band structure at the molecular level of g−CN.2 at 1.23 V vs. RHE, which is nearly three-fold higher than that of S-g−CN/BiOBr [−2 to 0.12 mA cm−2 after loading FeOOH on the g−CN film surface [−2 at 0.6 V vs. SCE, which was 2.8−fold higher than that of pristine g−CN  or alloCN/BiOBr . In addiCN/BiOBr . This reCN/BiOBr  and oxyhCN/BiOBr ,138 are CN/BiOBr ,140. The surface . In anot surface  modifiedine g−CN a,b.2−bonded carbon atom, carbon-based materials, e.g., carbon dots, carbon nanotubes (CNT), and graphene oxide, etc., are promising substrates and excellent reductive cocatalyst to integrate with g−CN, inhibiting the recombination of charge carriers [−2 at 1.23 V versus RHE (electrode thickness up to 36 µm) and subsequently decreased for a thicker photoelectrode. Photocurrent measurements with holes scavenger (TEOA) display a photocurrent response of 660 µA cm−2 at 1.23 V versus RHE and the external quantum yield of 60% at 400 nm  are realized  have been integrated with g−CN to form a heterojunction photoelectrode. In particular, various types of dopants have triggered keen interest in the preparation of N-doped graphene as it can ameliorate the optical, electronic, and chemical reactivity of graphene by making use of the lone-pair electrons of nitrogen atoms and the π−conjugated electrons of 2D graphene. Hou and coworkers  employedrealized e,f. The 2 (nearly 3.7 times) via the recombination of CNTs to promote electron transport. The enhanced PEC performance was accredited to the excellent electrons transport capability by CNTs, fundamentally resolves the poor electronic conductivity of the conglomerated g−CN nanosheets structure and allows valid electrons separation and collection  and conduction band (CB) potentials, which results in a new electronic structure after thermal equilibration. In other words, band bending forms at the heterojunction interface on account of potential differences between two sides of composite films . This trBased on the above discussions, Type II and Z-scheme pathways that effectively separate the electron-hole pairs are ideal in the preparation of the g−CN-based film. In view of these two types of heterojunctions, selecting an appropriate semiconductor material to couple with g−CN is of utmost significance not only to enhance the optical light absorption but also to facilitate the charge migration and separation to suppress the recombination rate. In the following section, we will critically exemplify recently published studies on these two types of g−CN-based composite films for PEC water splitting.The flexibility, as well as their 2D flat morphology, favors g−CN films as an accessible platform for the construction of heterojunction with various components. Based on the reported literature, a series of categories of g−CN-based heterojunctions have been successfully fabricated: (i) metal oxides/g−CN heterojunction, (ii) metal sulfides/g−CN heterojunction, and (iii) complex compound/g−CN heterojunction. The detailed studies for g−CN-based heterojunctions, efficiency, and applications will be discussed.2 is one of the most suitable candidates, and the triumphant formation of TiO2/g−CN heterojunctions has been reported to improve the PEC activity.Until now, various metal oxide semiconductors have been coupled with g−CN to form type II heterojunction for PEC applications. Among those metal oxides, TiO2 using laser transient absorption spectroscopy (L-TAS)  precursor. As expected, its photocurrent reached 1.4 mA cm−2, which was a 30−fold enhancement compared to pristine TiO2 (0.045 mA cm−2). They also studied the impact of barbituric acid molecules on the mixture precursor on the performance of g−CN/TiO2. A relatively high carbon doping content augments the defect levels and causes higher recombination of charge carriers, resulting in a decreased photocurrent to 0.7 mA cm−2.Albero et al.  analyzed path 2, a, sugges path 2, a was foua et al.  fabricat-workers  coated g2. In addition, the direct Ti–O–C bond creates supererogatory molecular orbitals at the interface, leading to a strong red-shift of the optical visible-light absorption  TiO2 nanostructures such as nanowires and nanotube arrays have drawn comprehensive interest owing to their high specific surface area and pore volume [2 nanotube array heterojunction by the vapor-pyrolysis of anodized Ti sheets over melamine. The obtained g−CN/TiO2 nanotube heterojunction exhibited strong optical absorption and reduced impedance compared to the pure TiO2 nanotube. In addition, it also demonstrated enhanced PEC activity and stability. In another work, Liu and co-workers [2 nanotube array (TNTAs) heterojunction with a g−CN shell and TNTAs core through TVC employing melamine and cyanuric acid supramolecular complex as the precursors. A noticeably enhanced photocurrent density of 0.86 mA cm−2 was realized, which was 4.5−fold and 2−fold higher than those of g−CN/amorphous TiO2 heterojunction and pure TiO2 nanotubes. It is worth emphasizing that the crystalline TiO2 nanotubes offer a more distinct photo-response due to their unique one-dimensional nanostructure in comparison with the amorphous TiO2 film.The improved PEC efficiency was in virtue of the prominent interface-bound energy transition of g−CN/TiOsorption b–e. The e volume ,171,172.e volume  reported-workers  reported2 nanorods using a dip-coating process with cyanuric acid-melamine–thiourea (CMT) complexes as starting materials. Using CMT complexes for the fabrication of TiO2/g−CN heterojunction is more advantageous than the individual materials. The photocurrent density can reach up to 2.74 mA cm−2 at 1.23 V (vs. RHE)  was added as a cheap phosphorus source. P-CN/TiO2 NTs exhibited the photocurrent response (1.98 mA cm−2 at 0 V vs. Ag/AgCl), which was higher than that of pristine TiO2 NTs. This promotion was because of the synergic mechanism between the introduction of P heteroatoms and the improvement of π−electron delocalization by 2−aminobenzonitrile (ABN). A previous study revealed that hydrogenation can improve the visible-light absorption range, structure matching, and stability [2 NTAs (g−CN/H-S−TiO2 NTAs) photoelectrode using a dip-coating approach. The results exhibited a higher transient photocurrent response of 1.64 mA cm−2 with good stability compared to TiO2 NTAs (0.29 mA cm−2). Hydrogenation reduces the energy gap of TiO2 due to the introduction of Ti3+ and oxygen vacancies. S-doping further forms a new impurity level that upshifts the TiO2 VB edge. The introduction of g−CN can form type II heterojunction with H-S−TiO2 NTAs, realizing an effective separation of charge and extended visible-light absorption  a–c. In atability . Inspiretability  reportedsorption d,e. Recesorption f. Additiactivity . Improveexplored .2 nanotube arrays (NTAs) with CNQDs via a delicate organic molecular linkage. The process included immersing TiO2NT in CNQDs aqueous solution with 1 v% 3−mercaptopropionic acid (MPA) at 80 °C for 12 h. The resulting products were rinsed and dried in air. The MPA-modified TiO2NT/CNQDs achieved a photocurrent response of 1.34 mA cm−2 at 0.3 V vs. Ag/AgCl, which was mainly ascribed to the effective heterojunction structure and particularly the strong affinity of TiO2 NTAs.Quantum dot (0D) semiconductors help to widen band structures and enhance redox capability and open-circuit voltage compared with their bulk counterparts (3D) ,182. Rec2/g−CN photoelectrode, ZnO is another pivotal metal oxide with a wide band gap (3.2eV), which can be valid coupled with g−CN [−2, over 50−fold that of pure g−CN films achieved by powder. The result reveals the photocurrent retention capability can be preserved at 95% for 160 min. The charge collector layer in type II alignment remarkably reduces the traveling distance of the carrier and minimizes the defect-trapping effect, thereby suppressing the recombination effect  sample was verified by SEM image analysis (xNy (1.0) photoelectrode possesses a photocurrent density of approximately 0.25 mA cm−2, more than 3.5−fold that of the pure ZnO. Mahala et al. [−2) compared to bare ZnO at 0.5994 V versus RHE , which lies in the visible-light range, thus increasing the potential for wider utilize in the PEC applications [Other than the recombination with UV-active TiOications ,193,194.3/g−CN nanosheet arrays (NSAs) on an FTO substrate through a hydrothermal growth and post-annealing process. It drastically suppressed the recombination of carriers and prolonged the lifetime of electrons manifested by EIS analyses for the WO3/g−CN heterojunction. The photocurrent density reached 0.73 mA cm−2 at the bias of 1.23 V vs. RHE , which was three-fold higher than that of pure WO3 under illumination. Li et al. [3 nanorod surface through the dip-coating method, which can achieve efficient visible-light−governed PEC water splitting. The photocurrent response of the g−CN/WO3 photoanode achieved 1.92 mA/cm2 at 1.23 V vs. RHE. The heterojunction was approximately stable, but distinct degradation (only decreases to 1.85 mA cm−2) was observed after 400 min of illumination. The stability performance promotion was realized via dipping WO3 nanorod into g−CN nanosheet dispersion for various impregnation times without intermediary drying steps. The enhanced PEC performance is attributed to the synergic effect between g−CN and WO3 originating from their well-matched overlapping band structures, which can availably boost the charge separation and the improvement of electrons’ lifetime.Li et al.  deposite vs. RHE a–c. Zhani et al.  uniforml2O3 has also recently been studied for PEC application. Liu et al. [2O3 photocatalysts via electrodeposition followed by chemical vapor deposition. By altering the filling degrees of the melamine during the annealing process, the morphology of the g−CN/Fe2O3 changes from regular nanosheets toward porous cross-linked nanostructures. The photocurrent density of the g−CN/Fe2O3 is almost 70−fold higher than that of individual Fe2O3  tends to be self-oxidized under strong radiation, and their corresponding photo-generated electron–hole pairs tend to be recombined [−2, which was approximately 2.5 times that of pure CdS NRs. After 3600 s continuous illumination, there was no obvious loss observed in the photocurrent density  has recently become an ideal co-catalyst on account of its high mobility, low cost, and narrow band gap (1.2–1.9 eV) [2 above the g−CN surface is realized due to the well-matched lattice and the similar layered structure [2/S-doped g−CN heterojunction film is fabricated through CVD deposition of S-doped g−CN film on ITO glass employing thiourea and melamine as the raw materials and subsequent growth of a MoS2 layer by hydrothermal treatment. The obtained film showed evidently improved PEC property with the anodic photocurrent density of 1.2 × 10−4 A/cm2 at an applied potential of +0.5 V (vs. Ag/AgCl), which is approximately twice the S-doped g−CN film [2) on PCN films by one-pot synthesis, resulting in remarkably promoted PEC performances  ,213. Platructure ,215. The−CN film . In addi−CN film . Recentl−CN film  reportedormances e,f.4 is an n-type semiconductor with a narrow band gap (2.4 eV), nontoxicity, low cost, and being more active in visible light [As we know, a complex compound consists of at least three elements. Generally, the incorporation of a complex compound into g−CN is deemed to be a promising technique for promoting photocatalytic activity, especially for the PEC, because of the efficient carrier separation and transfer ,219,220.le light ,222,223,4 composite films, employing facile electrospinning with a post-annealing technique. The exfoliated g−CN nanosheets prevented the agglomeration of BiVO4, forming heterojunctions with BiVO4 as well as profiting the formation of nanostructures. The obtained BiVO4/g−CN composite film displayed a photocurrent density of 0.44 mA cm−2 at 0.56 V, which was higher than that of the pristine g−CN and BiVO4 photoanodes. Inspired by this, Feng et al. [4 photoanodes via a facile ethylene glycol dispersion and impregnation. The photocurrent density of the ultrathin BiVO4/g−CN−NS photoanode was up to 3.12 mA/cm2 at 1.23 V (vs. RHE), which was approximately 7−fold and 4−fold higher than those of BiVO4 and BiVO4/bulk g−CN, respectively. The improved PEC performance was ascribed to the fact that the g−CN−NS serves as an efficient cocatalyst due to its enlarged band-gap (3.03 eV). Moreover, the intimate contact of ultrathin g−CN−NS and BiVO4 in core shell structure facilitated the migration and separation of the charge and restrained the interfacial charge recombination. This work accentuates the importance of geometry engineering of heterojunction film, which can be outspread to other ternary oxide semiconductor such as LaTiO3.Wang et al.  preparedg et al.  synthesi4 heterojunction. Jiang et al. [4 heterojunction photoelectrode through modified sol-gel tactics via varying the weight ratio of g−CN. The obtained g−CN/BiVO4 (30%) sample exhibited improved photocurrent density compared to pure BiVO4. Such augment can be ascribed to the effective charge separation and fast interfacial charge transfer of the type-II hetero-interface. The corresponding electronic structures and charge transfer properties were also discussed based on systematic experimental studies and density functional theory (DFT) calculations  in different stoichiometric proportions, Yang et al. [xFe1–xVO4 heterostructures through a one-pot hydrothermal synthesis. In addition, the impregnated BixFe1–xVO4 on the sulfur-doped g−CN (SCN) enhances the charge separation on account of the formation of heterojunction and attains a wide range of solar absorption due to the decrease of band gap  is also well matched with g−CN to form g−CN/LaTiO3 heterostructure with improves the PEC activity [2). Furthermore, the enhancement could be because of the formation of heterojunction , which was 8−fold higher than that of g−CN/TiO2 material . The obviously enhanced activity was accredited to the construction of Z-scheme heterostructure with an interfacial oxygen vacancy layer between TiO2 Nanotube arrays and g−CN nanoparticles. Furthermore, the presence of an interfacial oxygen vacancy layer provides sufficient charge carrier transfer to heterostructure.Vacancy defects are reported to play a critical role in the modification of the electronic structure. Inspired by this, Xiao et al.  fabricat2 nanotube array containing oxygen vacancies (OV−TiO2). Additionally, the protonation pretreatment was undertaken by ultrasonic bulk g−CN in the presence of hydrochloric acid, realizing larger contact areas and a close-knit contact interface between two semiconductors. The optimal photocurrent density of the obtained p-CN/OV−TiO2 Z-scheme heterostructure reached 96 μA cm−2, which was approximately 1.4 times that of CN/OV−TiO2 samples  heterojunction with different wt. % of g−CN by a simple hydrothermal approach. It can be seen from 4 (1.11 eV). This structure provides a strong driving force for the photo-generated electrons to transfer from the CB of BiVO4 to the VB of g−CN easily, leaving the photo-generated electrons and holes in the CB of g−CN and the VB of BiVO4, respectively. Then, the heterojunction restricts the electron–hole pair recombination, which was testified by EIS and PL spectra  at 1.23 V versus Ag/AgCl, which was higher than that of the γ irradiated g−CN or non-radiated heterojunction. Fast electron migration took place on the g−CN/BiVO4 heterojunction interface, which resulted from the effects of γ radiation.Tailored exposure of semiconductor materials to ionizing radiation  can adjust the bond structure and turn the optical, electrical, and structural properties so that a higher portion of the solar spectra is captured . Compare 2.76 eV a. The γ 3, a typical ABO3 perovskite oxide, presents a narrow band gap (Eg = 2.2 eV), high stability in oxidizing environments, and a large absorption coefficient [3 possesses appropriate band edges , which match well with g−CN  to form a direct Z-scheme heterojunction system [3/g−CN under ultrasonication and subsequent calcination treatment at 400 °C for 2 h. Benefiting from a unique nanostructure and g−CN-based direct Z-scheme charge transfer mechanism, such NiTiO3/g−CN composite films exhibit enhanced PEC performance (0.25 mA cm−2) compared with the pure NiTiO3 and g−CN. This study provides meaningful guidance for the research of inorganic perovskite materials for water splitting, and it is essential to further inquiries into this type of photoelectrodes.NiTiOfficient ,247,248.n system ,249,250.n system  synthesi−2 at 0.5 V vs. Ag/AgCl) was higher than that of g−CN/ZnO due to the cocatalyst promotion. In addition, the Pt/g−CN/ZnO photoanode showed a much lower photocurrent response than that of g−CN/Pt/ZnO, manifesting the charge carrier transportation and separation by the solid-state−mediators. Inspired by this, the 3D urchin-like ZnO/Au/g−CN Z-scheme heterostructure was prepared and applied as an excellent photocathode for PEC water splitting by Wen et al. [2/g−CN/Ag-AgBr (TCNAAB) multi-heterojunction by depositing Ag-AgBr nanoparticles onto 3D spherical TiO2/g−CN  incorporated into the interface of two components in the Z-scheme structure act as electron mediators to improve electron migration efficiency. For example, Xiao and co-workers  fabricatn et al. . Under sn et al.  designediO2/g−CN f–g.The key discrepancy between Z-scheme and type II heterojunction is the electronically regulated medium for photo-carriers transfer. Compared with the traditional type II heterojunction system, the Z-scheme heterojunction maintains a strong redox capacity, achieves a wide spectral response, and possesses high stability. The main advantages of Z-scheme heterojunction are presented as follows: Firstly, there is no intermediate, it is low cost, there are fewer components, and it is easy to prepare. Secondly, the contact between the two kinds of semiconductors forms a strong interaction, which can decrease the carrier transfer resistance and improve the PEC activity. Finally, in addition to noble metal, cheaper carbon-based materials can also be used as charge modulators. By optimizing the electronic medium, electronic interface resistance, carrier transfer efficiency, and PEC stability can be enhanced. In particular, graphene and its derivatives stand out in the Z-scheme heterojunction system due to their excellent electrical conductivity and electron transport in recent years. However, Z-scheme g−CN-based heterojunction photoelectrodes are still in their elementary stage of development, and they are beneficial for future experimentation.As mentioned previously, the coupling of g−CN with appropriate semiconductors enables an efficient amelioration in the separation and migration of charge carriers via a heterojunction-transfer mechanism. Therefore, ternary g−CN-based heterojunction films will possess more meaningful applications owing to their synergistic effects.3O4 nanocube (g−CN/CdS–Fe3O4) hybrid material electrode driven by a two-stage hydrothermal method and photocurrent response (~0.0235 mA/cm2) was achieved using the g−CN/CdS−Fe3O4 electrode as the small band-gap (2.17 eV) led to effective interfacial charge transfer, which was higher than those of individual single and binary catalysts under visible-light irradiation  photoanode by combining the hydrothermal strategy and successive chemical bath deposition process to enhance PEC performance. The ternary photoanode led to the rapid separation of charge to suppress the recombination rate  with g−CN nanosheets (NSs) as the interfacial layer. The hybrid heterojunction as photocathode accomplished the higher photocurrent density of 28 mA cm−2 at 1.23 V (vs. RHE) with ABPE 5.4% at 0.8 V versus RHE. Moreover, EIS measurements suggest that the hybrid photocathode holds a reduced interfacial charge transfer resistance with a prolonged lifetime of photon-excited electrons due to the well-aligned photocathode/electrolyte interface . Taking advantage of CoFeOx as a cocatalyst, Bhandary and coworkers [x was used to modify the HD–CN through a one-step electrodeposition process. The photocurrent density could be availably improved from approximately 27 to 600 μA/cm2 (nearly 22 times) via the recombination of the CoFeOx layer to promote electron transport. A plausible illustration for such a remarkable increment in PEC activity can be given in terms of the construction of a p–n junction between the HD–CN and CoFeOx layer. The as-prepared heterojunction can help facilitate photo-generated charge separation and inhibit the electron/hole recombination.In another work, Xu et al.  employedoworkers  employed+); (2) excellent PEC activity for HER. To meet the first requirement, the Fermi level of the cocatalyst must be lower than the conduction band bottom of the photocatalyst, resulting in the smooth flow of photoexcited electrons and facilitating the separation of electron–hole pairs. For the second requirement, the Gibbs free energy of hydrogen adsorption on the cocatalyst should be close to zero so that proton adsorption and rapid release of hydrogen molecules with the lowest activation energy occur at neutral thermal conditions. In order to achieve efficient PEC performance, a variety of modifications have been taken to improve g−CN-based materials, for example: adjusting morphologies, metal/non-metal element doping, copolymerization, loading cocatalyst, heterojunction, etc. Ternary g−CN heterojunction film will have a wider application prospect due to its synergistic effect. However, the reasonable construction of the ternary g−CN heterojunction system needs to be optimized from the aspects of energy level structure, electronic structure and interface structure. A performance comparison of g−CN-based heterojunction composite film photoelectrodes is shown in 2 carbon groups into the g−CN film enhances the stability of photoelectrodes. The photocurrent density of the modified g−CN film is approximately 46% of that at the initial stage after 1 h and maintains 42% in the second cycle. Huang et al. [−2 under illumination for 6000 s [The stability of photoelectrode, namely, the PEC activity does not decrease significantly during repeated recycles, is a key factor for practical PEC applications. Time-dependent current (I-t) curves under fixed bias are commonly used to evaluate stability. For semiconductor photocatalyst with poor stability, the photocurrent decreases with the increasing illumination time, and the photocatalytic properties disappear gradually . By doping with the elemental atoms, the stability of the semiconductor photocatalyst can be improved. Bian et al.  grew g−Cg et al.  successfr 6000 s . To achiThe low price, high physicochemical stability, and attractive electronic band structure of g−CN materials, together with the recent developments in their controlled growth on various substrates, contribute to expectations of an infusive future for g−CN materials in PEC applications. The overview of the topics involved in this review is illustrated in Improvement in the deposition methods:Currently, it is still difficult to achieve a final film that is both homogenous and with good contact with the conductive substrate while concurrently controlling morphology, electronic structure, as well as optical and PEC properties. The deposition methods should be further modified in the aspect of the starting materials design, interfaces, and reactor parameters optimization for achieving the g−CN layers in a more controllable manner. Morphology regulation is the most direct, simple and effective method to improve the electronic structure, surface characteristics, and photoelectrical properties of g−CN. The three-dimensional (3D) sphere or cage structure of g−CN has a larger specific surface area and sufficient reactive sites but has not been explored in the field of PEC water splitting. Compared with other morphologies , 0D semiconductor is generally more helpful in broadening the band structure, enhancing the redox ability and open-circuit voltage.2.Tuning the band gap of g−CN:Decreasing the band gap of g−CN would result in increased light absorption and photocurrent density. In general, it can be acquired by optimizing the C/N ratio or the spatial organization of the N atoms or by doping with heteroatoms and halogens. The electronic structure of g−CN materials can be modified by inducing vacancy defects or tailoring monomer design.3.Improvement of photo-excited charge separation and suppression of the recombination of the carrier:+ adsorption capacity and accelerates the adsorption and desorption of hydrogen.The low electronic conductivity of the g−CN layer leads to a high photo-excited charge recombination rate and to a low diffusion length. The latter restricts the thickness of the active layer and hence limits light absorption. While increasing the thickness of the active harvesting layer results in enhanced light absorption, this also results in higher charge recombination rates. This trade-off is crucial in the PEC efficiency and is restricted by light absorption and charge extraction properties. Replacing some C=N with C=C bonds or doping with heteroatoms/metals could highly improve the conductivity of electrons. Plenty of theoretical and experimental research has proved that the formation of well-designed g−CN-based heterostructures can improve its charge separation and transfer within the electrode. Constructing multi-heterojunction photocatalysts with multi-sources for carrier generation is one of the effective methods for enhancing the PEC activity. Cocatalysts also play an important role in enhancing charge carrier transfer. Two-dimensional transition metal sulfide has a unique electronic structure and large work function, which is considered a potential cocatalyst to replace precious metals. It has the following advantages: (1) a large number of active sites located at both edge and substrate; (2) strong conductivity to promote the rapid transfer of photogenerated electrons; (3) the edge position has higher H4.Enhancing the stability of g−CN:In a photoanode, water oxidation on the semiconductor surface is still the bottleneck that needs to obtain more attention in future studies. The introduction at the g−CN/electrolyte interface of a co-catalyst that can both serve as the catalytic sites for oxygen evolution and act as an extract for holes from the g−CN was demonstrated to be a successful tactic. There are several factors that should be taken into account when incorporating a co-catalyst onto a g−CN layer. One problem is the nature of the contact, in which different types of co-catalysts change the transport of photo-induced charges across the g−CN/electrolyte interface. On the other hand, the electronic properties of the co-catalyst need to match the VB potentials of the CN for promoting hole transfer. To achieve a long-term stability, the modified technologies should be further studied, including protecting g−CN from photo− or chemical-corrosion by protective layers and using both a co-catalyst and a protective layer is possible.To summarize, although there is a mass of obstacles hindering and taking advantage of carbon nitride materials in PEC devices, we believe that their excellent and unique physicochemical properties will enable us to conquer the current limitations. We believe that enhancements in the synthesis and deposition tactics, as well as a better understanding of their photoelectrochemical and charge separation and transfer processes by the study of structure-activity relationships, will result in their exploitation in many optoelectronic devices.


Species composition and diversity were investigated as the assessment indices. The results showed that the importance value of grasses significantly increased with the increase of the N addition rate, while that of forbs significantly decreased. A high N addition rate (72 kg N ha−1 y−1) induced species composition change, making Leymus secalinus become the most dominant species within the entire plant community. Compared with the control (without N addition), species richness, Shannon–Weiner diversity, Simpson dominance and Pielou Evenness were significantly reduced under a high N addition rate. The changes of plant diversity in the alpine steppe were closely correlated with dynamics of soil nutrients, especially total carbon (TC), total phosphorus (TP) and ammonia nitrogen (NH4-N). Our findings suggested that a high N deposition rate (72 kg N ha−1 y−1) could significantly change plant composition and reduce the diversity of the alpine steppe, though they were less affected by low N deposition rates at present. With the increase of the N deposition rate, plant composition and diversity of the alpine steppe may be negatively affected in the future. In addition, Leymus secalinus is more competitive than other species with an N deposition rate increase. Soil C, soil P and soil NH4-N variation induced by N deposition might play a key role in regulating changes in plant composition and diversity in the alpine steppe. In addition, longer term field investigation needs to be carried out to testify to this phenomenon with the increase of N deposition in the future.The N deposition rate is notably increased in China, especially in the Qinghai-Tibetan Plateau (QTP). How plants respond to the projected N deposition on the alpine steppe is still in debate. In this study, to investigate the effects of N deposition on the plant community of the alpine steppe, we simulated N deposition at six different N addition rate levels (0, 8, 24, 40, 56, 72 kg N ha World in 2005 . It is e century . In ChinIn recent years, due to the differences of study areas and test methods, the results about the impacts of N deposition on the grassland ecosystem are often inconsistent. Some studies showed that adequate N deposition was beneficial to ecosystems in N-constrained environments ,10,11. O8 km2 and the average altitude is more than 4000 m [−1 yr−1 on average, with a minimum of 1.08 kg N ha−1 yr−1 and a maximum of 17.81 kg N ha−1 yr−1 [The Qinghai-Tibetan Plateau (QTP), located in southwestern China, is well known as “The Third Pole” of the earth . The totn 4000 m . This una−1 yr−1 ,28,29, ka−1 yr−1 . On this2  decreased the importance value of Aster alpinus (forb plant). N addition had no significant effect on the importance value of Carex capillifolia and Carex melanantha (sedge species). The importance value of Leymus secalinus (grass species) significantly (p < 0.05) increased with the increase of the N addition rate. From the perspective of functional groups, the importance value of grasses significantly increased with the increase of N addition rate, while that of forbs significantly decreased  higher than that under the control, while the NⅤ level led to the lowest richness among all the levels (p < 0.05). Compared with CK, the Shannon–Wiener diversity index was significantly (p < 0.05) lower than under the NⅤ level. The Simpson dominance index under the NⅤ level was significantly (p < 0.05) lower than that under other levels. The Pielou index was the lowest (p < 0.05) under NⅤ level.The richness index, Shannon–Wiener diversity index, Simpson dominance index, and Pielou evenness index significant decreased with the increase of N addition rate . The effp < 0.05) and NH4-N . There was a strong negatively correlation between Shannon–Wiener diversity and soil TC , TP , and NH4-N . There was a strong negatively correlation between Simpson dominance and soil TC , TP , and NH4-N . Pielou evenness had a significantly  negative relation with soil TP. There was no strong correlation (p > 0.05) between the plant diversity index and other soil factors. No significant (p > 0.05) correlations were found between soil pH and plant diversity index. 4-N, TN, TC, TP, AP, AK, and Ca under N addition (p < 0.05), while N addition had no significant effect on other soil properties such as, soil NO3-N, pH, Mg, and S  may be an effective way to maintain plant biodiversity under the background of enhanced atmospheric N deposition in the future ,47,48.Stipa capillata L. and Poa crymophila Keng. The soil is mostly sandy loam [This study was conducted in Tiebujia Village, Gonghe County , which is located at the west sides of Qinghai Lake in Qinghai Province, China . The stundy loam .−1·y−1 as the control (CK), 8 kg N ha−1·y−1 (NⅠ), 24 kg N ha−1·y−1 (NⅡ), 40 kg N ha−1·y−1 (NⅢ), 56 kg N ha−1·y−1 (NⅣ), 72 kg N ha−1·y−1 (NⅤ), with three replicates for each N treatment  in early May and July every year since 2014. Until 2019, we have fertilized for 5 years continuously. In May 2014, 18 plots (replicates) of 2 m × 5 m with 1 m the buffering area were randomly placed in an alpine steppe in Tiebujia Village. The plots were randomly treated by six levels of N deposition, 0 kg N hareatment . AccordiIn July 2019, we randomly placed one 1 m × 1 m quadrat in each plot for investigating plant species composition including name, abundance, coverage, and frequency of each species. Species abundance is the number of species in the community. Species coverage refers to the percentage of the vertical projected area of the above-ground part of the species to the surveyed area. Species frequency refers to the frequency of occurrence of the species within the survey range. After recording the plant species composition, we collected the soil samples at a depth of 0–20 cm depth soil cores from each plot. The collected soil samples were sealed in Polyethylene bags and transported to the lab for analysis after being air-dried, ground, and sieved (a 100 mesh sieve was used for soil nutrients measurement and 18 mesh for pH measurement).4-N) and nitrate nitrogen (NO3-N) were measured using a flow injection auto-analyzer . We used ICP-MS  to measure the total phosphorus (TP) according to the following procedures. Soil samples were first digested in sulfuric acid and then quantified using the ICP Elemental Analyzer [2) solution, and the supernatant was collected with a pipette and stored at 6 °C until analysis [An elemental analyzer  was applied to measure the total nitrogen (TN) and total carbon (TC) content of the soil. Ammonium nitrogen  of the plant species using the following formula :IV = , Shannon–Weiner diversity index (H), Simpson dominance index (D), Pielou Evenness index (J), which were calculated based on the following formulas ,43,51:R Our data were presented as means ± standard error (SE) in figures and tables. The ‘vegan’ and ‘ggplot’ packages in R (4.1.2) were used for Nonmetric multidimensional scaling (NMDS) of species abundance among different N addition levels. Duncan’s Multiple Range Test in analysis of variance (ANOVA) was used to test the effects of different N addition on the plant community composition, diversity indices, and soil nutrients in SPSS 23.0. We employed the simple linear regression model to determine the relationship between functional group importance value, plant diversity, and N addition rate. We used SPSS 23.0 to perform Normality Test and assessed the relationship between plant diversity and soil nutrients by using Pearson‘s correlation. GraphPad Prism 8.0 was used for drawing.Leymus secalinus was the most adaptive species within the entire community. The high level of N addition (72 kg N ha−1·y−1) has a greater impact on the diversity of alpine steppe ecosystems. Additionally, under the continuous N addition for 5 years, the changes of plant diversity in the alpine steppe had a close relation with soil nutrients, especially total carbon (TC), total phosphorus (TP), and ammonia nitrogen (NH4-N). Soil nutrients imbalance induced by long term N addition may be the main cause that leads to the species diversity loss and composition shift in alpine steppe.From this 5-years N addition study, we found that importance values of different functional groups and species responded differently to N addition. It is clear that the importance value of the grass functional group significantly increased, while that of forbs significantly decreased with the increase of N addition.


Knowing the leading patterns will help timely predict that addictive behavior is being formed.To identify the leading patterns of addictive behavior in adolescents in Central Siberia according to the results of the CIAS test.200 adolescents aged 11-18, 69 males and 131 females, with Internet addiction living in an urban area (Krasnoyarsk) were surveyed. The patterns of addictive behavior were assessed using the CIAS test, which includes scales of key symptoms: “Com” (compulsive symptoms), “Wit” , “Tol” (tolerance symptoms); and negative consequences scales: “IH” , “TM” (problems with time management).The mean results (M) of key symptoms were obtained at 14.56 on the “Com” scale, 15.27 on the “Wit” scale, 12.23 on the “Tol”. The mean indices of negative manifestations were obtained at 17.00 on the “IH” scale and 13.94 on the “TM” scale. When comparing the mean results of the scales of addicted behavior by the method of one-way analysis of variance (ANOVA), statistically significant differences between representatives of different sex and age groups were not revealed (p> 0.05).The leading key symptoms of Internet-addicted behavior in Central Siberia adolescents include withdrawal symptoms: decreased mood, anxiety and irritation in the absence of access to the Internet. Symptoms of negative consequences include decreased social contacts, reduced communication with family members, and problems at school. When such symptoms emerge, one should suspect the formation of Internet addiction and carry out the necessary diagnostics for timely intervention. The study was funded by RFBR project № 18-29-22032\18.The study was funded by RFBR project № 18-29-22032\18.


Granulosa cell tumors (GCT) are rare malignant ovarian tumors. The two subtypes, adult and juvenile granulosa cell tumors, differ in clinical and molecular characteristics. GCT are low-malignant tumors and are generally associated with favorable prognosis. However, relapses are common even years and decades after diagnosis. Prognostic and predictive factors are difficult to assess in this rare tumor entity. The purpose of this review is to provide a comprehensive overview of the current state of knowledge on prognostic markers of GCT to identify patients with a high risk of recurrence.n = 409 English full text results from 1965 to 2021. Of these articles, n = 35 were considered for this review after title and abstract screening and topic-specific matching. A specific search for pathologic markers with prognostic relevance for GCT identified n = 19 articles that were added to this review.Systematic research for adult ovarian granulosa cell tumors and prognosis revealed FOXL2 mRNA were inverse and immunohistochemical (IHC) expression of CD56, GATA-4 and SMAD3 was associated with reduced prognosis. IHC analysis of estrogen receptor, Anti-Mullerian hormone (AMH) and inhibin was not associated with prognosis for GCT. Analyses of mitotic rate, Ki-67, p53, β-catenin and HER2 revealed inconsistent results.FOXL2 mutation and  Granulosa cell tumors (GCT) are a rare malignant subtype of ovarian tumors. They comprise about 1–2% of all ovarian neoplasms and 5% of malignant ovarian tumors . Furthern = 1 new article for Ki-67; n = 2 for p53, n = 3 for CD56, n = 3 for inhibin, n = 2 for catenin, n = 1 for cadherin, n = 3 for GATA4, n = 3 for HER 2 and n = 6 for FOXL2, respectively  AND (ovary OR ovarian)) AND (prognosis OR prognostic)’ added up to 564 results from 1952 to 2021. Filtering only articles with available English full text left 409 results from 1965 to 2021. Three hundred seventy-four articles missed the topic of this review after title and abstract screening, 35 articles referring to pathological markers were considered in this review. The prognostic markers were chosen based on these selected articles. Specific search for the GCT markers ‘((granulosa cell tumor) AND (ovary OR ovarian)) AND (xxx)’; xxx representing ‘mitosis OR mitotic’, ‘Ki-67’, ‘p53’, ‘CD56’, ‘estrogen receptor’, ‘inhibin’, ‘AMH’, ‘catenin’, ‘cadherin’, ‘GATA4’, ‘HER 2’, ‘FOXL2’ and ‘SMAD3’, respectively, confirmed that no articles were missed. Specific search revealed 2 is advocated by the WHO rather than using HPF due to different microscopes and field diameters [The mitotic rate is the number of mitoses traditionally counted in an area of 10 high power fields (HPF). The area with the highest density of mitotic figures is chosen and a light microscope using a 10x ocular and 40x objective magnification is used . Currentiameters . The exaiameters , 14–20. iameters –26. Afteiameters   had positive staining for p53m, which did not correlate with stage of disease, recurrence risk or survival [n = 30) were positive for p53m. p53 was more common in patients with stage II or higher compared to stage I. Furthermore, the overall survival (OS) of p53-negative tumors was approximately 10 times higher than the median survival of p53-positive patients  [Historically, p53 has been thought to be an oncogene and mutations of p53 occur frequently and transform p53 into an oncogene   and accusurvival . Other s= 0.037) . In this= 0.037) . In the = 0.037) , which w= 0.037) , 38, 39.TP53 mutation in ovarian cancer [TP53 mutation. These tumors were characterized by numerous alterations and increased mitotic activity [Currently, immunoexpression of p53 is evaluated as follows: staining of 1–80% is regarded as normal (wild type) activity, strong staining of > 80–100% as well as absence of staining in tumor nuclei are regarded as abnormal (mutant). Mutant expression of p53 is considered as a surrogate for n cancer . Roze etactivity .TP53 mutations, however the prognostic impact is unclear. Also, the previous studies revealed inconsistent results on the influence of p53 as a prognostic marker in AGCT and the relative number of positive tumors throughout the studies varied widely.To summarize, in older publications, analysis of p53 expression was based on outdated knowledge. Currently, a molecular approach is used to analyze n = 32 GCT to be positive for CD56, helping to distinguish between different entities of ovarian tumors [n = 30 AGCT (16 primaries and 14 relapses). They were able to show an increased staining intensity of the high molecular CD56 isoforms in relapses and relapsing primaries compared with CD56pan in unrelapsed primaries. They concluded this molecular isoform to be a possible sign for a more aggressive behavior of the tumors [p = 0.001) [CD56 (NCAM) is an immunoglobulin participating in organogenesis . Its ison tumors . Volker e tumors . In the = 0.001) .n = 30 GCT (19 AGCT and 11 JGCT), Staibano et al. examined the expression of ER-β. In five cases, expression of ER-β was scored negative, eight cases showed low expression, in 10 cases medium expression was found and seven cases revealed high expression of ER-β. These results were compared with follow-up data. Loss of ER-β was significantly associated with worse prognosis [n = 149 AGCT of a multicenter study. They did not find a correlation between the expression of ER and prognosis. However, PR expression showed to be a predictor of recurrence free survival (RFS) and OS. In their study, a high PR expression score was significantly associated with worse RFS and OS [Studies have shown that estrogen plays an important role in carcinogenesis of ovarian neoplasms . Two typrognosis . ContradS and OS . Balan eS and OS . AnotherS and OS . The samS and OS .n = 80) except for three stained positive for inhibin-α, but data failed to correlate with recurrence risk, stage or prognosis [Inhibin is a glycoprotein hormone that is produced in granulosa cells of the ovary. It is a heterodimer consisting of α and β dimers. The β dimer is divided in two subunits βA and βB, differentiating between inhibin-A and inhibin-B. It is responsible for suppressing the secretion of follicle-stimulating hormone (FSH) by the pituitary gland via a feedback system . As showrognosis .Anti-Mullerian hormone (AMH), also known as Mullerian inhibiting substance (MIS), is a growth factor produced in the gonads and is responsible for folliculogenesis and sexual differentiation . It was n = 1 of 6) and equine (n = 14 of 18) GCT, but not in normal ovarian tissue samples. These results were refuted by Ohishi et al. who did not find nuclear expression of β-catenin (n = 0 of 30 AGCT), which contradicts the hypothesis that nuclear β-catenin supports tumor progression in AGCT. Rather they found nuclear expression of E-cadherin (n = 27 of 30 AGCT), which is usually located at the cell membrane. However, nuclear E-cadherin expression was not associated with prognosis [n = 62), and its influence on prognosis. They detected β-catenin expression in all AGCT samples and E-cadherin expression in 85%. E-cadherin staining was mostly restricted to sex cord-like components of the tumors and was in general weaker in extent and intensity than β-catenin. In cells with strong E-cadherin expression, staining was prevailing in the membrane, whereas cells with weaker staining showed more cytoplasmic staining activity. Consistent to Ohishi et al., Stewart et al. did not find nuclear β-catenin expression. In correlation with patients clinical outcome, they proved that less extensive β-catenin staining was associated with a higher rate of AGCT recurrence and shorter DFS compared to a more extensive staining. No clinical correlation was found to cytoplasmic β-catenin staining intensity as well as both, E-cadherin extent and intensity [E-cadherin is a transmembrane protein responsible for cell-cell adhesion. Through a cytoplasmic binding site, the catenin binding domain (CBD) β-catenin controls and modulates E-cadherin function . When acrognosis . Stewartntensity .n = 11 with high expression, n = 3 with intermediate expression). In the same tumor samples, opposite results were shown for GATA-6. Expression of GATA-6 was shown to be reduced in GCT. Consistently with AMH, reduced GATA-6 expression correlated with larger tumor size, but not with prognosis [GATA-4 is a zinc-finger transcription factor that is responsible for various genes in the steroidogenesis and normal granulosa cell function –71. It hrognosis . Likewisrognosis . In contrognosis .n = 40 GCT tumor samples [n = 31 cases of AGCT and found positive staining of HER1/EGFR1 in 23 cases (74.2%), but negative staining results for HER2 in all samples [Human epidermal growth factor receptor (HER2) is a member of the epidermal growth factor receptor (EGFR) family . It is a samples . These r samples . Menczer samples . In cont samples , 82, 83. samples . This wa samples .FOXL2 results in an alteration of its pro-apoptotic function [FOXL2 gene is also associated with blepharophimosis-ptosis-epicanthus-inversus syndrome (BPES) which manifests in two forms, BPES type II resulting in isolated craniofacial abnormalities and BPES type I additionally being accompanied by premature ovarian failure [FOXL2 on prognosis. They showed that FOXL2 mutation (402C > G), which was detected in 70% of cases, correlated with a poor prognosis (DFS). DFS was also reduced in patients with increased FOXL2 mRNA expression. In IHC staining, reactivity for FOXL2 was higher in tumor samples expressing mutant FOXL2, but was not associated with prognosis (DFS or OS) in AGCT. Contrarily, in JGCT, where FOXL2 mutation is rare, strong FOXL2 immunoreactivity correlated with decreased DFS and OS [FOXL2 (402C > G) mutation in 38 of 40 AGCT. Three of the recurrent tumors exhibited homozygous genotype. The authors concluded that FOXL2 homozygous genotype is more likely to relapse than heterozygous genotype [n = 26 patients with AGCT, Rosario et al. were also not able to find significant correlation between FOXL2 mutation and tumor size or prognosis [FOXL2 is a member of the forkhead transcription factors and is involved in embryogenesis and ovarian differentiation as well as granulosa cell differentiation and follicle development , 85. In function , and thefunction , 89, 90. failure , 92. D’AS and OS . Kraus egenotype . In a sarognosis .n = 88 primary GCT cases, Sakr et al. showed that increased expression of SMAD3 was significantly associated with increased recurrence and a shorter DFI . SMAD3 was also revealed as a predictor of recurrence in GCT  [SMAD3 is a mediator of transforming growth factor beta (TGFβ)-function. It is responsible for cell viability in AGCT . SMAD3 w= 0.001) .This review gives an overview about the multiple pathways and molecular factors and their prognostic role in AGCT using IHC staining. Numerous studies pointed out different factors and mutations that were associated with proliferation or tumorigenesis of AGCT and analyzed their IHC expression. The studies were confronted with various challenges. The two different entities, AGCT and JGCT, vary widely in characteristics. AGCT, the more common form of GCT, can occur at all ages with a peak in perimenopausal women, whereas JGCTs commonly occur before the age of 30. Clinical behavior also differs between these tumor types. Both tumors are associated with a good prognosis, but relapses are common. AGCT tend to recur late, even later than 10 to 20 years after diagnosis, JGCT generally within a few years , 99. As n = 10) and an index between 5 and 25% in 45% (n = 9) [The greatest challenge in the study of GCT is its low incidence. As AGCTs, and even more JGCT, are rare tumors, statistically significant results are difficult to obtain due to the low number of cases. Differentiation between the individual tumor stages is often not possible. Many of the reviewed studies in this article did not differentiate between AGCT and JGCT. Regarding the molecular and prognostic differences of these subtypes, results for prognostic IHC markers are difficult to obtain. No significant correlation between pathological markers and prognosis was found concerning ER- and inhibin-expression. In regard to the mitotic rate, Ki-67, p53, β-catenin, and HER2, the results of the individual studies were contradictory (Table  (n = 9) . FurtherFOXL2 mutation and increased FOXL2 mRNA were associated with reduced DFS in AGCT.The prognostic relevance of AMH-IHC remains unclear as in only one study, AMH expression correlated with larger tumor size, but not with prognosis. FOXL2-IHC correlated with decreased DFS and OS in JGCT and TP53 and FOXL2 mutation. In further studies a new approach including both, immunohistochemical and molecular data might improve assessment of prognosis [In recent studies whole genome sequencing was performed and yielded new aspects, such as rognosis .Of all examined markers, this review only revealed a prognostic value for worse outcome of CD56, GATA-4 and SMAD3. To gain more knowledge about this rare tumor entity and its prognosis, large multi-center studies with higher case numbers and clear distinction between AGCT and JGCT are needed. The implementation of national and international tumor registries represents a great opportunity for further evaluation.


Frameshift mutations have been considered of significant importance for the molecular evolution of proteins and their coding genes, while frameshift protein sequences encoded in the alternative reading frames of coding genes have been considered to be meaningless. However, functional frameshifts have been found widely existing. It was puzzling how a frameshift protein kept its structure and functionality while substantial changes occurred in its primary amino-acid sequence. This study shows that the similarities among frameshifts and wild types are higher than random similarities and are determined at different levels. Frameshift substitutions are more conservative than random substitutions in the standard genetic code (SGC). The frameshift substitutions score of SGC ranks in the top 2.0-3.5% of alternative genetic codes, showing that SGC is nearly optimal for frameshift tolerance. In many genes and certain genomes, frameshift-resistant codons and codon pairs appear more frequently than expected, suggesting that frameshift tolerance is achieved through not only the optimality of the genetic code but, more importantly, the further optimization of a specific gene or genome through the usages of codons/codon pairs, which sheds light on the role of frameshift mutations in molecular and genomic evolution.The online version contains supplementary material available at 10.1186/s12864-022-08435-6. All these natural genetic codes share several important properties: (1) the genetic code is universal, with only a few variations found in some organelles or organisms, such as mitochondrion, archaea, yeast, and ciliates [The genetic code was deciphered in the early 1960s . As showciliates ; (2) theIt has been reported that the natural genetic code was optimized for translational error minimization, which is being extremely efficient at minimizing the effect of point mutation or mistranslation errors and is optimal for kinetic energy conservation in polypeptide chains –7. MoreoA frameshift mutation alters the reading frame of a coding gene and may produce frameshift proteins (frameshifts). Frameshifts have been considered mostly meaningless since they look completely different from the wild type and are often interrupted by many stop signals. A frameshifted gene yields truncated, non-functional, and potentially cytotoxic peptides . Until tMoreover, there have been a handful of cases of functional frameshifts that retain their function. For example, Hahn and Lee identified nine frameshift homologs between humans and chimpanzees by collecting human coding exons bearing InDels compared with the chimpanzee genome, some of which seem to be functional in both species . By blasAs is well known, a protein can become dysfunctional by changing even one residue, so it is puzzling how a frameshift protein can maintain the integrity of its tertiary structure and function while substantial changes occur in its primary sequence. Based on ClustalW alignments, we have observed high similarities among frameshifts and wild-type protein sequences ; recentlEscherichia coli, Saccharomyces cerevisiae, Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, Xenopus tropicalis, Mus musculus, Pan troglodytes, and Homo sapiens, were retrieved from GenBank Genome Database. Program RandomCDSs.java produced ten thousand sets of CDSs, each containing three CDSs and each CDS containing 300 or 500 random sense codons.All reference coding sequences (CDSs) in ten model species, including δij be the pairwise similarities of its three translations, i, j = 1, 2, 3, i ≠ j, δij = δji. The average similarity among the frameshifts and the wild type is defined as the shiftability of protein-coding genes (δ),For a given CDS, let δ12, δ13, and δ23 are all considered in the formula.Shiftability is a quantitative measurement of frameshift tolerability. As frameshifting occurs between any two of the three reading frames, Program Similarity.java batch translates CDSs and computes the pairwise similarities among the three translations, in which CDSs are translated using the SGC in the different reading frames of the sense strand, and the three different translations are aligned by different methods, including ClustalW2, MSA, and FrameAlign. To calculate pairwise similarity, a pair of matched AAs in a pairwise alignment is considered conserved if their substitution score is ≥0 in the scoring matrix GON250, i.e., gaps and negative scores are considered different. The percent of conserved sites gives the pairwise similarity between a pair of protein sequences.E. coli to humans, while the readthrough rules may vary among different species [E. coli, nonsense suppression tRNAs reported includes amber suppressors , ochre pF [supG ), and oppG [supU , su9 [33n triplet codons is written as,Bk ∈{A, G, U, C}; i = 1… n; k = 1…3n. Each pair of neighboring codons are separated by a bar to show the native reading frame. Its encoded wild-type protein sequence (WT), consisting of n amino acids, can be written as,A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y}, represents the amino acid encoded by the ith codon (B3i-2B3i-1B3i). If a frameshift is caused by deleting or inserting one or two bases in the start codon, there are only four cases:: B2B3B4| B5B6B7| … | B3i-1B3iB3i + 1 |B3i + 2B3i + 3B3i + 4| …Delete one (− 1): B3B4B5| B6B7B8| … | B3iB3i + 1B3i + 2 |B3i + 3B3i + 4B3i + 5| …Delete two (− 2): B0B1B2| B3B4B5| B6B7B8| … | B3i-3B3i-2B3i-1| B3iB3i + 1B3i + 2| …Insert one (+ 1): B− 1B0B1| B2B3B4| B5B6B7| … | B3i-4B3i-3B3i-2| B3i-1B3iB3i + 1| …Insert two (+ 2)A wild-type protein-coding sequence consisting of ith codon, the (i + 1)th codon (B3i + 1B3i + 2B3i + 3) has only two possible changes:FF): Forward frameshifting (RF): Reverse frameshifting  has two bases overlapping with the (i + 1)th codon of WT (B3i + 1B3i + 2B3i + 3); the encoded amino acids (The last codon of ac (N) and act (T) are both small amino acids, while gtt (V) and ttg (L) are both aliphatic (with the shared bases underlined), and the corresponding amino acid substitution scores are positive .WTvs.RF: insert one gap at the beginning of RF.(3).FFvs.RF: no gaps are needed.. Based on the above analysis in Random substitutions (RCSs) are produced by randomly changing all three bases of the codons; there are 64 × 64 = 4096 possible RCSs.Interchangeable substitutions (ICSs) are produced by randomly changing only the third position of the codons; there are 64 × 4 = 256 possible ICSs.Frameshift substitutions (FCSs) are produced by forward or reverse shifting. Each codon has four forward and four reverse FCSs, and there are 64 × 8 = 512 possible FCSs.According to whether the encoded AA is changed or not, codon substitutions have been classified into synonymous substitutions (SSs) and nonsynonymous substitutions (NSSs)In most cases, all three bases in the frameshifted codon are changed compared with the original codon, except for triplet monomers . The AA substitution scores of FCSs and RCSs are defined as frameshift substitution scores (FSSs) and random substitution scores (RSSs), respectively. The sum FSS of all possible FCSs is considered a measure of the frameshift tolerability of the genetic code. Program ShiftCodons.java computes the substitution scores for each type of codon substitutions using a scoring matrix, BLOSSUM62 , PAM250 18) genetic codes randomly using a random-number-based sampling algorithm, in which the probability of an AA being swapped is proportional to its proportion in the code table. The sampling was repeated 100 times independently. AlternativeCodes.java gives all (13824) possible compatible codon tables by permuting the nucleotide in each codon position independently . One miB1B2B3|B4B5B6, its encoded AA pair is Forward frameshifting: Reverse frameshifting: where B0 and B7 each have four choices. There are 4096 × 8 = 32,768 different codon pair frameshift substitutions (CPFSs). For each CPFSs, FrameshiftCodonPair.java computes the FSSs for all possible codon pairs. For a given codon pair, E) and observed (O) number of occurrences, frequency, together with a value for the χ2 statistics. A codon or codon pair was identified as over-represented if O > E (or under-represented if O < E), and the average FSSs were calculated for each genome weighted by their codon or codon pair usages.The number of occurrences was counted for each codon/codon pair for each genome. The observed and expected frequencies were calculated using the Gutman and Hatfield method for each codon or codon pair, resulting in a list of 64 codons and 4096 codon pairs, each with an expected . In order to distinguish the two different implications of the term frameshift, hereafter, we refer to the two frameshifted protein sequences (FF and RF) as frameshift translations. Frameshift translations usually do not exist in nature, and they have been considered mostly meaningless since they look like random sequences.Usually, a frameshift refers to an organism that has a frameshift mutation, i.e., having a protein-coding gene with an altered reading frame compared to the wild type; sometimes, it also refers to a putative protein sequence artificially translated from an alternative reading frame of a CDS, e.g., the second and third translations of zebrafish faa Fig. A. As desWT, FF, and RF). Each set of them was aligned using ClustalW and FrameAlign, and their similarities were calculated as real frame similarities.All available reference CDSs  for ten model species were translated, each producing a set of real frame translations . Each set of them was aligned using ClustalW, MSA, and FrameAlign, and their similarities were calculated as random frame similarities.Ten thousand random CDSs were translated, each producing a set of random frame translations (Ten thousand sets of CDSs (each containing three random CDSs) were translated in the native frame of the sense strand, each producing a set of random native translations; each set of them was aligned using ClustalW, MSA, and FrameAlign, and their similarities were calculated as random similarities.In this section, three groups of CDSs were translated, the translations were aligned, and their similarities were calculated:When the frame translations were aligned using ClustalW, the estimated (hereafter est) means of real and random frame similarities are respectively 0.456 ± 0.033 and 0.452 ± 0.013 . As well, the overall average of the real frame similarities is 0.450 ± 0.030 (Table As described in 30 Table a and S1bE. coli) to 0.466 (human), but the standard deviations are generally as low as 0.030 in all tested species, i.e., the shiftability is species-dependent and is concentrated at a particular value for most genes in a specific species.As described in vegfaa look different from the wild type, but ClustalW aligns them well  and 2.896 × 10− 9 (reverse frameshifting vs. random substitutions), respectively.Besides, no matter which AA substitution scoring matrix is used, the average FSSs are always significantly higher than random substitutions. Using GON250, e.g., the average FSS (− 1.78) is significantly higher than the average RSS (− 10.81). As shown in Table SIn the most common scoring matrices, such as BLOSSUM62, PAM250, and GON250, most scores are negative, and the percentage of positive scores is about 35%, i.e., in random codon substitutions, the percent of positive substitutions is about 35%, which is consistent with the observed mean random similarity, 0.383 (Table 18) random codon tables, 100 independent samples, each with 1 million codon tables, were sampled using a simple random sampling algorithm. As shown in Fig. Random codon tables are produced by swapping the amino acids assigned to sense codons while keeping all degenerative codons synonymous . From al3  compatible codon tables were produced, and their FSSs were computed  and the human glutenin gene (δ = 0.660). As shown in Table E. coli, A. thaliana, and C. elegans are lower than expected , showing that frameshift-resistant codons (FRCs) are not overrepresented in these genomes. The weighted mean FSSs are significantly higher than expected in humans, mice, Xenopus, and yeast, suggesting that FRCs are overrepresented in these genomes. In other words, the shiftability of certain genes or genomes can be adjusted through the biased usage of codons.As abovementioned, shiftability is species-dependent Table . For somne δ = 0.60. As shE. coli, C. elegans, and A. thaliana, showing that frameshift-resistant codon pairs (FRCPs) are not overrepresented in these genomes; in humans, mice, Xenopus, and yeast, however, the weighted mean FSSs are significantly higher than expected, indicating thatFRCPs are overrepresented in these species. In these higher species, genome-level shiftability is also higher than those in the lower species  [We calculated frameshift substitution scores and showed that they are significantly higher than random substitution scores. Recentlys (PCPs) . From a s (PCPs)  pointed This study incorporates computational frameshifting and readthrough into the analysis. It is important to note that such computational operations are conceptually different from biological frameshifting and translational readthrough. They do not require that they truly occur in an organism because these operations are used only for calculating similarities. So, in the present study, they are not taken as biological laws but computational methods borrowed from biology. However, the expected proportion of hidden stop codons (HSCs) in the frameshifted CDS s is 3/64 = 4.69%, and the proportion of HSCs in real genes may even be higher than expected [Therefore, the differences between readthrough and non-readthrough translations are not negligible. All these data suggest that the readthrough rules are probably adapted to the genetic code and explain part of its optimality. As the presentation of these results depends on the present study, we will present these data in another article.Based on the above analysis, we conclude that the genetic code, many genes and certain genomes were optimized for frameshift tolerance. Shiftability ensures high similarities between frameshifts and their wild-type counterparts, endowing coding genes the inherent tolerability to frameshift mutations in either forward or reverse direction. Thanks to this unique property, the natural genetic code obtained excellent fitness better than its competitors, thus winning the competition in the early evolution. The shiftability serves as an innate mechanism by which coding genes and genomes tolerate frameshift mutations, and thus, deleterious frameshift mutations could have been utilized as a driving force for molecular evolution. However, the impacts of frameshift tolerance on molecular or genomic evolution remain to be characterized across the tree of life.Additional file 1: Table S1a. Frame similarities aligned by ClustalW or MSA. (1) The summary of the similarities of natural or simulated proteins and their frameshifts aligned by ClustalW or MSA . (5) The similarities of yeast frame translations . (6) The similarities of human frame translations . (7) The similarities of chimpanzee frame translations . (8) The similarities of mouse frame translations . (9) The similarities of Xenopus frame translations . (10) The similarities of zebrafish frame translations . (11) The similarities of fruit fly frame translations . (12) The similarities of nematode frame translations . (13) The similarities of Arabidopsis frame translations . Table S1b. Frame similarities aligned by FrameAlign. (1) The summary of the similarities of natural or simulated proteins and their frameshifts aligned by FrameAlign . (4) The similarities of yeast frame translations . (5) The similarities of human frame translations . (6) The similarities of chimpanzee frame translations . (7) The similarities of mouse frame translations . (8) The similarities of Xenopus frame translations . (9) The similarities of zebrafish frame translations . (10) The similarities of fruit fly frame translations . (11) The similarities of nematode frame translations . (12) The similarities of Arabidopsis frame translations . Table S1c. Computing frame similarities of zebrafish vegfaa. (1) The three frame translations are aligned by ClustalW. (2) The three frame translations are aligned by FrameAlign. (3) The Gon250 scoring matrix used in (1) and (2).SA Table . (2) ThestalW). 1 The simiAdditional file 2: Table S2. FSSs of the natural genetic code. (1) The FSSs of the natural genetic code using Scoring Matrix Gon250. (2) The FSSs of the natural genetic code using Scoring Matrix Blossum62. (3) The FSSs of the natural genetic code using Scoring Matrix PAM250.Additional file 3: Table S3. FSSs of the alternative genetic codes. (1) The summary of the FSSs of the natural and alternative genetic codes (Table es Table . (2) ComAdditional file 4: Table S4. FSSs of different codon usages. (1) The summary of codon usages and their weighted mean FSSs (Table E. coli. (11) The 64 triplet codons and their FSSs.Ss Table . (2) TheAdditional file 5: Table S5. FSSs of different usages of codon pairs. (1) The summary of codon pair usages and their weighted mean FSSs (Table E. coli. (11) The equal codon pair usages and their weighted mean FSSs.Ss Table . (2) The


Identifying these mechanism-independent interactions could improve drug safety and support drug repurposing. Here, we develop iDTPnd (integrated Drug Target Predictor with negative dataset), a computational approach for large-scale discovery of novel targets for known drugs. For a given drug, we construct a positive structural signature as well as a negative structural signature that captures the weakly conserved structural features of drug-binding sites. To facilitate assessment of unintended targets, iDTPnd also provides a docking-based interaction score and its statistical significance. We confirm the interactions of sorafenib, imatinib, dasatinib, sunitinib, and pazopanib with their known targets at a sensitivity of 52% and a specificity of 55%. We also validate 10 predicted novel targets by using in vitro experiments. Our results suggest that proteins other than kinases, such as nuclear receptors, cytochrome P450, and MHC class I molecules, can also be physiologically relevant targets of kinase inhibitors. Our method is general and broadly applicable for the identification of protein–small molecule interactions, when sufficient drug–target 3D data are available. The code for constructing the structural signatures is available at https://sfb.kaust.edu.sa/Documents/iDTP.zip.Current FDA-approved  Proteins that contain kinase domains are involved in numerous cellular processes including signaling, proliferation, apoptosis, and survival Recent studies have estimated that many unintended targets of approved drugs are yet to be discovered Computational methods have increasingly been used for hit identification and lead optimization i.e., a conserved structural signature in the kinases that is known not to interact with the respective drug). We now also provide a docking-based interaction score along with its statistical significance. In a blind test of five FDA-approved kinase inhibitors, we predict the known targets with 52% sensitivity and 55% specificity. This is a significant improvement compared to a baseline model based on sequence similarity and to a recently published study in vitro experiments. It is important to note that our predictions are not limited to kinases.In this study, we propose a computational method for large-scale discovery of new drug targets, named iDTPnd, which markedly improves our previous methodology https://www.rcsb.org/). This means that the number of structures has almost doubled in 7 years. Similarly, in case of the membrane proteins which are the targets of more than 60% marketed drugs, we had 328 structures in 2011, while this number increased to 876 on April 4, 2019 (https://blanco.biomol.uci.edu/mpstruc/). Therefore, we expect our method to be applicable for broader set of studies going forward.We extracted the positive and negative datasets from the kinome scan assay of Davis et al.’s work The sequence similarity baseline model used nearest neighbor algorithm to allocate a protein to the interacting or non-interacting cluster. Using leave-one-out cross validation, global pairwise sequence similarity (not identity) was calculated between the left-out protein and all other proteins. The left-out protein was assigned to the cluster that contained the protein with the maximum pairwise global sequence similarity to the left-out protein. If none of the protein pairs had a global sequence similarity >0.6, a label was not assigned to the left-out protein.The flowchart of our method is shown in α as long as it is close to 1. The α can be adjusted according to the empirical insight from the data. In this study, we used α = 1.2. RMSD is the root mean square distance, N is the number of positions aligned, i, i, and the summation is over all aligned positions. Every position in the signature is present in at least 50% of the structures. To achieve a minimalistic structural signature, preservation ratio cut-off is increased if the number of atoms in the signature is more than 100. While combining the positive and the negative structural signatures, predicted targets are those that have a better positive score than the negative one (Scorepositive – Scorenegative < 0).Our method is not sensitive to the exact value of The predicted protein targets, human leukocyte antigen A , human leukocyte antigen B , mitogen-activated protein kinase-activated protein kinase 2 , cAMP-specific 3ʹ,5ʹ-cyclic phosphodiesterase 4B , protein kinase C eta type , estrogen receptor α , cyclin-dependent kinase 2 , and tyrosine-protein kinase ITK/TSK  were labeled by using NHS chemistry with the help of an NT647-labeling kit . In an initial step, the Tris-containing storage buffers were exchanged by the MicroScale thermophoresis (MST) labeling buffer as indicated by the manufacturers in order to avoid labeling primary amines in Tris. After addition of a two-molar excess of reactive NT647 dye to the respective target protein, the reaction was incubated in the dark for 30 min. After this, the unbound dye was removed using a size exclusion column as indicated by the manufacturers. Using the buffer containing 1× PBS pH 7.5, 0.1% pluronic F127, and 2% DMSO did not result in aggregation or sticking effects for HLA-A, HLA-B, MAPKAPK2, CDK2, PKCη, ITK, and ERα. Standard, premium, and hydrophobic capillary types were tested for non-specific sticking of the proteins to the glass surface. HLA-A, HLA-B, and ERα showed sticking in standard capillaries but no sticking in premium capillaries. Hence, premium capillaries were used for the further experiments for these proteins. The other proteins remained in solution in standard capillaries. PDE4B showed aggregation in all conditions and hence was not tested further. The LED power was set to 10%–25% to obtain optimal signal intensities. The laser power was identified being optimal at 40% or 80%. Changes in amplitudes between the lower and upper binding curve plateau of more than 4 units and a signal-to-noise ratio of more than 6 were considered significant for binding events. Each experiment had two replicates.positive – Scorenegative < 0). Combining the positive and negative structural signatures helped improve the performance of the methodology significantly across all the kinase inhibitors  of structures deposited in PDB using CASTp in vitro (see Method for details). The results showed that PKCη and MAPKAPK2 interacted with sorafenib with dissociation constant (Kd) values of 1.1 ± 0.4 μM and 3.7 ± 0.1 μM, respectively  of sorafenib to test, considering the target’s ranking in our results as well as the availability and cost of the purified protein. We used MST experiments to test the predicted interactions ectively . Considein vitro. We also included sorafenib and imatinib in our experiments to test our false negative rate.ERα is a nuclear receptor that is activated by estrogen and is important for hormone/DNA binding and transcription activation +/HER2− breast cancer tumors to pazopanib after the tumors had developed resistance to endocrine therapy Sunitinib, dasatinib, and pazopanib were found to interact with ERα with Kd values of 14.7 ± 5.7 nM, 1.2 ± 0.5 μM, and 3.2 ± 1.0 μM, respectively . While sin silico prediction. In our next round of MST experiments, we also included sorafenib as the positive control and used two other kinase inhibitors (sunitinib and pazopanib) to test our false negative rate.Cyclin-dependent kinases (CDKs) perform important roles in cell division cycle, transcription, differentiation, neuronal functions, and apoptosis Dasatinib, imatinib, and sorafenib were found to interact with CDK2 with Kd values of 2.2 ± 0.9 μM, 6.6 ± 2.9 μM, 9.1 ± 2.7 μM, respectively . We founWe observed that MHC class I (HLA-A/HLA-B) proteins were predicted as potential targets for all kinase inhibitors used in this study except pazopanib. The cell surface of all nucleated cells contains MHC class I proteins in jawed vertebrates Cytochrome p450 enzymes (CYPs) are the most important enzymes involved in drug metabolism. They account for about 75% of the total metabolism. Most drugs are deactivated by CYPs, either directly or indirectly Due to the importance of protein–drug interactions, several computational studies have addressed the problem of identifying novel targets of drugs from different angles. However, most of these studies do not benchmark their performance on known targets and use different datasets, thus making it hard to compare among these studies. Other studies reported low precision values  in vitro binding experiments that validated 10 predicted interactions. Moreover, we also compared our model with recently published studies of Zhou and colleagues Herein, we proposed iDTPnd, a computational method for large-scale discovery of novel targets of known drugs. Our method had the following advantages: 1) it incorporated a negative structural image into the probabilistic scoring function, increasing the sensitivity from 31% to 52%  that appeared in the top 10 predicted target list of more than one kinase inhibitors. In both cases, our predicted interactions were verified by in vitro experiments. Beyond validating our predictions, experimental results also suggest that our method can serve as a platform for kinase inhibitor combination studies. The experimental validation showed that our false positive rate is very low compared with other studies. The false negative rate can be improved in future studies by incorporating structure-independent information like expression data from GTEx and ENCODE projects. Our method is generic and can be used broadly for all types of small molecule drugs for which sufficient 3D structures of known targets (∼30) have been solved.In this study, we developed a computational model iDTPnd to discover the novel targets of known drugs. For the five kinase inhibitors in our dataset, we identifed the known targets with 52% sensitivity and 55% specificity. The predictive capability of iDTPnd was supported by the validation of top predicted targets using https://sfb.kaust.edu.sa/Documents/iDTP.zip.The code for constructing the structural signature is available at The authors have declared no competing interests.Hammad Naveed: Conceptualization, Methodology, Investigation, Software, Writing – review & editing, Writing – review & editing. Corinna Reglin: Validation. Thomas Schubert: Validation. Xin Gao: Supervision, Investigation, Writing – review & editing. Stefan T. Arold: Investigation, Writing – review & editing. Michael L. Maitland: Investigation, Writing – review & editing.