Graphene is a popular material with outstanding properties due to its single layer. Graphene and its oxide have been put to the test as nano-sized building components for separation membranes with distinctive structures and adjustable physicochemical attributes. Graphene-based membranes have exhibited excellent water and gas purification abilities, which have garnered the spotlight over the past decade. This work aims to examine the most recent science and engineering cutting-edge advances of graphene-based membranes in regard to design, production and use. Additional effort will be directed towards the breakthroughs in synthesizing graphene and its composites to create various forms of membranes, such as nanoporous layers, laminates and graphene-based compounds. Their efficiency in separating and decontaminating water via different techniques such as cross-linking, layer by layer and coating will also be explored. This review intends to offer comprehensive, up-to-date information that will be useful to scientists of multiple disciplines interested in graphene-based membranes. Over the last twenty years, separation via membranes has been deemed as an innovative technology suitable to address massive challenges, such as lack of resources and the environmental impact of human activities . ContrarA membrane is essentially a selective boundary with pores or channels. They are more cost-efficient and effective, and their use is fundamental in eco-friendly technologies . An ideaGraphene and graphene oxide (GO) have demonstrated marked potential in separation processes using membranes, owing to being accessible and because of their strength and notable resistance to various types of solvents . The inc2 hybridization and thicker atomic layers expedite the formulation of ultrathin selective barriers with expected separation capacity [In this context, additional effort has been devoted to comprehending their behavior and exploring the uses of materials based on graphene . Graphencapacity . The intcapacity ,12. The capacity . It shoucapacity ,13.The pristine graphene layer was found to be impenetrable to liquids and gases because of the very small gaps in its structure . GraphenConsidering the issues discussed, graphene-based membranes produced through diverse processes possess specific structures and transport pathways, allowing their application in multiple membrane processes such as water desalination ,19,20, rThis work delves into the most recent (past four years) research findings on the topic of GO-based membranes, going over the vast and scattered literature to obtain and organize the information on the synthesis approaches, the potential challenges related to them and the recorded data on the separation and decontamination efficiency. The novelty of this article is its mission to review the very recent literature and curate up-to-date (and easily comparable) information on the synthesis methods of graphene-based membranes, their applications and efficiency in water purification, bringing attention to the factors affecting the separation/decontamination processes. Appropriate information regarding the pollutants susceptible to each process is further collected and assessed, along with details on the rejection rates achieved.Graphene quantum dots (GQD) is a term used for single or few-layer graphene nanosheets, often during studies of the photoluminescence property. GQDs have generally extremely small size, usually less than ten nm (and an average of five nm), which places them at the lower end of the graphene nanosheets’ range, whose upper end starts at less than 100 nm .The reduction of graphene oxide can be conducted via chemical, thermal, electrochemical and photochemical approaches, resulting in the production of graphene-like materials, such as reduced graphene oxide (rGO) and highly reduced GO (HRG), etc. In spite of the considerable differences between pristine graphene and HRG, they still possess remarkable similarities. Their physicochemical attributes define this step as one of the most significant processes of GO, especially for bulk applications. The attributes of HRG produced via GO-reduction, are highly dependent on the reduction processes .Turbostratic carbon is a 3D sp2-bonded carbon material, with layers whose carbon atoms don’t have a spatial relationship with the atoms in other nearby layers .Graphene-based materials, which are also mentioned as graphene or graphene-family nanomaterials—refer to a group of 2D materials containing the name ‘‘graphene’’, multilayered materials (less than ten layers) included. They may be chemically modified forms , or materials produced using graphene, its oxide, or other graphene material as a component .Multi-layer graphene (MLG)—a two dimensional material in sheet form, comprising of a limited number (ranging between two and ten) of precisely-defined, stacked layers . They maFew-layer graphene (FLG)—a subgroup of MLG with an average of two to five layers. Examples include graphite nanoplates, nanosheets and nanoflakes; They are two dimensional with ABA or ABCA stacking, whose thickness does not exceed 100 nm .Exfoliated graphite—a multilayer version, produced through partial graphite exfoliation , creating thin multilayer products that maintain the three-dimensional structure of graphite .Graphene nanosheet—a single layer of hexagonally placed, sp2-bonded carbon atoms that is not the main part of a carbon material, but is either free or adhered on an external substrate, whose lateral dimension does not exceed 100 nm ,32.Metal oxides: In order to further improve and expand the applications of graphene, a wide range of metal and metal oxide nanoparticles have been combined with graphene. The CVD method requires the dissolution of carbon in metal surfaces, such as nickel and copper that serve as catalysts and then by cooling the metal to forcing it to separate. Metal and metal oxide nanocompounds are produced by chemically reducing metal precursors such as chloroauric acid, silver nitrate and potassium tetrachloroplatinate, using hydrazine hydrate, amines, and sodium borohydride. The combined effect of graphene and the metal-based materials in these nanocompounds have a new research pathway, including biomedical applications .Polymer Nanocomposites: The binding or loading of metal and metal oxide nanoparticles on graphene for the preparation of graphene-based nanocomposites is commonly achieved in two different ways: post immobilization or in-situ binding. Post immobilization requires combining different graphene nanosheets options and pre-produced nanoparticles. Prior to that, the nanoparticles and/or the surface of graphene sheets are functionalized to improve the ability to process the end results, which greatly improves their solubility and thus offers additional alternatives for the creation of graphene-based compounds. Nevertheless, post immobilization might be affected negatively if the structure of the graphene sheets is not dense enough or if the coverage by the graphene sheets is irregular .Before discussing the actual membranes, the inherent attributes and the creation process of graphene-based components will be summarized, because they heavily impact the preparation process and general efficiency.There is a plethora of articles describing the simple yet perfect structure of graphene, with its atoms placed in a honeycomb-looking lattice , that leThe functional groups that can be found on the surface of component based on graphene offers the potential of formulating bonds with proteins, whereas the structure of pristine graphene allows for hydrophobic π–π interaction to lead any hydrophobic leftovers to the graphene surface . FurtherLi et al. demonstrated that graphene is a very effective additive . Using aThere are two main synthetic pathways, top-down and bottom-up. Graphene can be synthesized via various approaches , such asMechanical exfoliation was the first successful method of producing graphene by consecutive peelings via scotch tape . SeparatThe growth of thin graphene films has proven to be a successful approach for the synthesis of graphene . Xu et aCVD is an effective approach for mass manufacturing of graphene-based membranes. The technique’s advantage is the ability to continuously prepare graphene films using an ever growing list of potential substrates, such as copper, platinum and nickel . The cheThe reduction of GO is a different option for the fabrication of graphene sheets. GO is created according to the Brodie, Staudenmaier and Hummers’ methods . HummersHummer expanded on Brodie’s and Staudenmaier’s approaches, by establishing an oxidation phase for the production of GO  by combiVia reduction, the GO partially reverts to its initial state, but with improved properties, such as better electrical conductivity ,60,61. TLiquid-based exfoliation of graphite is another promising method for manufacturing graphene at a larger scale. It refers to the exfoliation of bulk graphite into thin graphene while being in a liquid medium, excluding the chemical oxidation phase . This apDespite the fact that bottom-up approaches are able to produce single-layer, flawless graphene, they are not currently applicable for large-scale synthesis of graphene and graphene-based materials. As a result, and taking into account the growing demand for the mass production of graphene, top-down solutions have garnered the spotlight. Such methods traditionally require the repeated oxidation and reduction of graphite, which is an inexpensive resource . AdditioNevertheless, there are still some gaps in our knowledge regarding cost-effective top-down synthesis on a large scale, without harming the environment in the process of producing defect-free graphene. The majority of the known synthetic approaches cannot consistently regulate properties such as the size, shape, edge and layer number of the produced graphene, because of the randomness of the exfoliation, growth and assembly steps. In spite of the significant progress in the synthesis of graphene-based inorganic nanomaterials, there are still certain barriers to their large-scale application. For instance, advanced uses of graphene-based metal and metal oxide nanomaterials call for a comprehensive analysis, before understanding the interactions between the surface of graphene and the nanomaterials, since they directly affect the attributes of these resulting composites. This would undoubtedly improve the potential of the produced nanomaterials in various areas, such as biosensors and drug delivery. Multiple techniques have been employed for the synthesis of homogeneously dispersed nanomaterials, using diverse reduction and functionalization approaches. It should be noted however that there are numerous reduction and surfacing materials with adverse impact on their possible uses. It is thus crucial to examine how biocompatible and/or toxic these reductants and surfactants are, to ensure the safety of the produced materials for use in biomedical applications . More spCVD and epitaxial growth are not appropriate for the production of the graphene that is required for the resulting graphene-based nanomaterials, since they typically require large quantities of graphene sheets with a modified surface architecture. Furthermore, mechanical exfoliation via Scotch-tape is a painstaking process and does not usually result in adequate quality graphene monolayers, whereas epitaxial growth requires high-vacuum and a generally high-priced production system. The production of graphene monolayers with large surface areas is now feasible owing to the quite recent advances in chemical vapor deposition techniques; however, the growth of graphene monolayers remains a challenge and appropriate techniques have not yet been fully implemented .Regardless, graphene is an innovative two-dimensional building block for the growth and assembly of metal/metal oxide nanoparticles, and the integration and collaborative effects with graphene significantly improves the performance of the resulting nanocomposite. Graphene-based metal and metal oxide composites with specific or mixed structures have thus great potential for multiple large-scale applications, and they are commercially viable. The regulated production of these nanocomposites, with specified attributes, stops the restacking of graphene nanosheets, but also offers great templates for the creation of three-dimensional porous networks with improved electrical and electronic properties. Additionally, enhancement of the produced quantity will result in the synthesis of nanocompounds, which can be calibrated for numerous possible applications. A multidisciplinary approach should thus be used to establish better protocols for the large-scale synthesis of graphene, and to achieve a higher technological maturity level .Guidelines are required for the creation of graphene-based membranes that offer the optimal transport of water, while still being able to be reused in separation processes. This requires the minimization of the energy barriers upon entering the membrane. Rejection capacity may also be adjusted in a similar manner by ensuring that the inter-sheet spacing only allows a single monolayer of water. To summarize, through the optimization of the process at a molecular level, the viability of graphene oxide as a suitable material for separations  can be maximized .Despite the fact that bottom-up approaches are able to produce single-layer, flawless graphene, they are not currently applicable for mass production of graphene and graphene-based materials. As a result, and taking into account the growing demand for the mass production of graphene, top-down solutions have garnered the spotlight. Such methods traditionally require the repeated oxidation and reduction of graphite, which is an inexpensive resource .Additionally, top-down techniques have become more popular owing to the progress achieved in the area of direct exfoliation of graphite while using different types of solvents without any reducing or stabilizing agents. Graphite-based top-down approaches are thus more financially appropriate for large-scale applications. Nevertheless, there are still some gaps in our knowledge regarding cost-effective top-down synthesis on a large scale, without harming the environment in the process of producing defect-free graphene. The majority of the known synthetic approaches cannot consistently regulate properties such as the size, shape and layer number of the produced graphene, because of the randomness of the exfoliation, growth and assembly steps. In spite of the significant progress in the synthesis of graphene-based inorganic nanomaterials, there are still certain barriers to their large-scale application. For example, advanced uses of graphene-based metal and metal oxide nanomaterials call for a comprehensive analysis, before understanding the interactions between the surface of graphene and the nanomaterials, since they directly affect the attributes of these resulting composites. This would undoubtedly improve the potential of the produced nanomaterials in various areas, such as biosensors and drug delivery. Multiple techniques have been employed for the synthesis of homogeneously dispersed nanomaterials, using diverse reduction and functionalization approaches. It should be noted however that there are numerous reduction and surfacing materials with adverse impact on their possible uses. It is thus crucial to examine how biocompatible and/or toxic these reductants and surfactants are, to ensure the safety of the produced materials for use in biomedical applications Therefore, the graphene-based nanocomposite synthesis primarily revolves around the oxidation and exfoliation of graphite oxide, which is then chemically reduced to create highly reduced graphene oxide or chemically modified graphene .The green reduction of graphene oxide (GO) requires the use of biocompatible components under physiological conditions of temperature and pressure . Most ofSalvadora persica L. roots (miswak) extract. Multiple results have verified that the biomolecules in the roots also functionalize the surface of SP-highly reduced graphene oxide, serving as a capping ligand, stabilizing it in various solvents. This method could offer a better substitute for the large-scale manufacturing of graphene and graphene-based materials for diverse applications such as electronics, nanomedicine, and bionic materials [A previous work by Mujeeb Khan et al. in 2015 described an easy and eco-friendly approach for the bio-reduction of GO using aterials .Abdulhadi H. Al-Marri et al. produced similar results: a green, single-step method for the creation of graphene/silver nanocomposites through the concurrent reduction of both graphene oxide and silver ions, using a plant extract, which functionalizes the surfaces of highly reduced graphene oxide, linking it to the silver nanoparticles. Higher amounts of silver improved the density of silver nanoparticles linked to the highly reduced graphene oxide .The main categories, based on their structure are: (1) porous layers, (2) laminates and (3) compounds that shall be further explored as follows:Porous layers. By limiting the number of defects introduced via physicochemical approaches, uniform nanosized pores can be created on the graphene membrane surface, achieving outstanding selectivity in separation of gases, water desalination, and other applications. Moreover, ultra-thin, porous graphene membranes exhibit much higher permeability than other compounds. The size of the pores is a pivotal factor for use in desalination projects .Their production methods are related to specific types of manufacturing approaches. For example, coating techniques, such as spin-, spray- or dip-coating and membrane casting are widely used for the preparation of membrane layers on the substrates a. Due toAssembled graphene laminates. Laminated membranes based on graphene use GO nanosheets as basic stackable components. There are lot of research works on the subject of the optimal production of laminated membranes with minimum defects and enhanced efficiency. Kim et al.  used a G2. Tsou et al. [2·h.Comparable behavior can also be achieved through a casting process. Akbari et al.  stated tu et al.  examinedGraphene-based composite. GO-modified polymeric membranes have demonstrated enhanced permeability, selectivity, and anti-microbial outcomes . Two mai2/bar/h, and better rejection rates for sodium sulfate (95%), magnesium sulfate (91%), magnesium chloride (62%), and sodium chloride (60%). Zhang et al. [The first method has been tested by various scientists, including Lee et al. , who man sulfate %, magnesPerreault et al.  implemenZhang et al.  developeWater purification through membranes is based on the filtration, the adsorption, the rejection, and the degradation of pollutants. The efficiency of such membranes is evaluated by checking if they are water permeable, using contaminated organic solutions and deionized water. The efficiency of the prepared membrane can be impacted by the concentration of cations, the pore size, spacing between layers, chemistry of the surface, dosage, pressure and other parameters .The various applications such as desalination, rejection of ions, and pollutant removal are depicted in The desalination of seawater guided by a vapor pressure gradient is a relatively new treatment approach. Despite its promising experimental results, the purification of a combination of different pollutants and keeping the temperature stable are significant setbacks that have not yet been addressed . NanofilXu et al.  suggesteRomaniak et al.  created Dong Han Seo et al. proposed a water desalination process via membrane distillation. Graphene films procured from renewable oil achieved notably better water vapor flux retention and rejection rates, and an improved antifouling capacity under a mixture of saline water that contained diverse pollutants ,12.- radicals as indicated in Photocatalysis refers to the process of accelerating a photo-generated electron during catalysis. Recently, Chen et al.  examinedYu et al.  describeMaterials based on graphene play a significant role for the elimination of environmental contaminants via adsorption, which relies on the bonds betwixt the pollutant and the adsorbent surfaces. Its mechanism may be based on adsorption, charge transfer or dipole interactions, electrostatic or dispersion forces, and quadrupole interactions ,111. TheZhan et al.  evaluatePollutant rejection from wastewater through membranes is based on the size of the pores and the interaction between the surface and the ions ,117. GO-Attempts to prevent fouling and swelling have relied on electrokinetic effects, intercalation of ions, physical confinement of organic material molecules, pore size or channel heigh tuning, cross-linking blending with nanocarbons, and reduction of GO. The adjustment of nanochannels and nanowrinkles properties may enhance the rejection rates, because of the existence of extra water transport channels . Yuan etSeunghyun Hong et al. demonstrated the very high charge selectivity for graphene oxide membranes. By estimating the diffusion of a wide range of ions, sizes and charges, they were able to comprehend the corresponding mechanisms and the factors affecting the ionic sieving in GO membranes, namely electrostatic repulsion and the compression of the ionic hydration shell within the membrane’s nanochannels .The pollution of heavy metal ions in water sources and wastewater is an issue of global concern due to their high toxicity even at very low concentrations . The eliVarious approaches have been suggested to eliminate them from water; out of which, membranes based on graphene were deemed to be low cost, effortless and more effective. Wang et al.  demonstrEmerging pollutants such as herbicides, pharmaceuticals and personal care products commonly detected in both potable and waste water, posing a global concern. The inability of traditional approaches of treatment is an important concern for the extraction of these emerging pollutants from wastewater. Graphene-based nanomaterials demonstrated promising potential to eliminate them from wastewater via adsorption, filtration, and photocatalysis .Khalil et al.  producedVarious approaches were established for the manufacturing of GO membranes, such as LbL, vacuum or pressure-assisted, cross linking, intercalation, annealing and coating. Some of the recent works and their findings are summarized in Vacuum/Pressure-Assisted Method. Such techniques are the most frequently used and clear-cut approaches for the mass production of graphene oxide nano-, micro, and ultra-filtration membranes .Cheng et al. used vacuum filtration to fabricate hybrid tungsten disulphide/GO 2D membranes to accomplish fine molecular sieving. The nanochannel size of the tungsten disulphide membrane became smaller and more regular after introducing the GO nanosheets. The hydrated hybrid membrane with 15 wt% GO showed high rejection rate of more than 90% versus dyes and ions. In addition, the nanochannels in the fabricated membrane exhibited strong rigidity under up to 0.5 MPa pressure, while the separation effectiveness slightly changed during 120 h of cross-flow filtration .Sun et al. fabricated new membranes comprising of large-area, high-strength metallurgical graphene, GO, hydrazine and polyamide on porous polysulfone scaffold. The GO layer was created from GO suspensions via vacuum filtration. The effectiveness of the membranes was assessed via forward osmosis tests. The membranes showed promising results in ion blocking (>95%). The results indicated that the manufactured membranes can be useful in treating water .Zhao et al. successfully prepared from GO quantum dots and sheets via a simple vacuum filtration process. The study revealed that the resulting composite displayed improved markedly improved permeability. When more quantum dots were used, the structure was not affected, while the average spacing between layers increased. Furthermore, the intercalation of the quantum dots enhanced the hydrophilicity of the membranes to a degree. More specifically, the permeability of the membranes in all cases was two to four times better without affecting their retention for direct yellow, bovine serum albumin, humic acid, and gold nanoparticles (higher than 99%). This work proves that the homo-structure of zero dimensional GO quantum dots in two-dimensional GO sheets can efficiently enhance the separation performance of GO-based nanofiltration membranes .−2h−1, suggesting that the fabricated membranes have good application potential [Recently Hou et al. created a GO/Methylene Blue (MB) membrane through vacuum filtration, in order to enhance the stability and rejection of dyes. The rejection rate of the GO/MB membrane reached approximately 93% for methyl orange, 99.9% for disperse black 9 and 82% for rhodamine B, whereas the pure water flux was approximately 7.7 Lmotential .The same year Kunimatsu applied vacuum filtration to produce niobate nanosheet (NbN)- GO membranes. The impact of the weight ratio of the nanosheet versus GO on the structure and efficiency were evaluated, finding that the fabricated membranes are more structurally stable in wet conditions and were comparable with GO-rich membranes. NbN55-GO45 particularly exhibits excellent water permeability, practically six times better than the plain niobate one and two times better than a plain GO membrane, without affecting the rejection abilities of anionic dyes and salt .Liu also applied vacuum filtration to manufacture a novel titanium dioxide nanorod inserted in a graphene-based membrane on a cellulose acetate mat. The nanorods extended the spacing between the graphene sheets and thus improved the separation performance of the membranes, achieving a very high flux and very high rejection rates versus dyes, exceeding 99.3 for the examined dyes .Hung studied a series of graphene-modified GO membranes for nanofiltration by pressure-assisted self-assembly. The fabricated membrane exhibited high water flux, salt rejection at approximately 88%, sodium chloride at 91%, magnesium chloride at 97% and magnesium sulphate at 98% .Layer by layer. Layer by layer provides a more accurate regulation of the concentration levels, the deposited film, the monolayer elasticity and the surface state. This technique, able to create nanoscale multilayer films, has thus been at the forefront of the research in various fields .Arenas et al. utilized the technique to produce rGO membranes for desalination purposes and assessed the performance versus sodium and magnesium cations .Slot dye coating. This is a technique that uses GO with reduced viscosity levels, allowing for the production of thin membranes without requiring the GO solution to be pre-treated. The thin wet layer facilitates spontaneous drying, which minimizes the production time needed. Minimal waste of stock solution occurs; thus, this approach could be further examined for membrane production using various materials .Kim et al. also fabricated GO membranes using a slot die coater. The membrane thickness is controllable, according to the quantity of the GO solution and morphology of the substrate. Furthermore, the coating layer and spacing are not affected, as the GO is deoxygenated (dGO). An efficiency test indicates that the manufactured membrane is effective for filtering sub-nanosized dye molecules, with a rejection percentage that reaches 99% for brilliant blue G. The fabricated membrane is stable in the case of cross-flow and high-pressure levels. The process is scalable .Cross-linking methods. Cross-linking has the ability to prevent membrane swelling and dissociation, whereas the resulting membrane is also more structurally stable. Cross-linking refers to both covalent and non-covalent approaches. The covalent option improves the stability and sieving capacity of membranes based on graphene, whereas the non-covalent one assists in the dissolution of the materials .Thakur et al. produced a set of cross-linked laser-induced graphene-poly  LIG−PVA membranes. These membranes demonstrated improved selectivity and permeability and their performance was of the same levels as polymeric ultrafiltration membranes. The fabricated membranes significantly improve the strength of the LIG. Increasing the PVA levels had a positive effect on the rejection rates. The produced membranes were fouling resistant and had good flux recovery ratio .Annealing process. Graphene’s properties can be impacted by either substrate interactions or the processing steps needed to manufacture contacts and devices. Annealing is used when cleaning graphene devices, which might result in doping, defect changes and strain effects . The cleA review conducted by Li et al. verified the applicability of mild annealing for creating partially reduced GO membranes and 2D laminar channels for nanofiltration. The membrane changes were methodically characterized, and results show the process not only made GO membranes more stable but also facilitated the customizable tunable salt rejection of the resulting membranes. Mild annealing rGO membrane (MRGO) accomplished better water permeance and sodium sulfate selectivity .2-Fe3O4, and silver, as well as carbon and titanium dioxide nanotubes.Large molecule intercalation. Due to the high surface area of GO nanosheets, the interlayer electrostatic repulsion may destabilize the GO membranes. To this end, the insertion of large molecules that may intercalate between layers can manage to address the issue and stabilize the structure. This process can also be used to tune the membrane effectiveness by adjusting spacing and enhancing molecular separation . The mosChen et al. created a hierarchical  graphene oxide/covalent organic framework (GO/COF) membrane through the intercalation of COF particles, which possess a hollow structure and nanopores at the almost 2 nm range, as well as multiple functional groups that render it extremely hydrophilic, thus offering additional fast transport nanochannels. The fabricated membrane has outstanding flux at 1 bar and excellent rejection efficiency of dyes. Furthermore, the intercalation of COF is able to considerably reduce the swelling, thus improving membrane stability when exposed to either water or strong acidic/base solutions .Impact of Interlayer Spacing. The spacing between the layers of graphene is approximately 0.34 nm, whereas that of GO is about 0.8 nm . If the Impact of Crosslinking. Crosslinking GO layers, adapting the temperature levels and functionalization, are some of the few factors that can impact the preferred layer spacing. Hung et al. created a GO through treatment with diamine based catalysts . The finStability. The large-scale applications of graphene membranes is a daunting task, requiring the careful preparation of large area porous films, taking into account parameters such as flexibility, ability to handle easily and pore uniformity while designing the process . CleaninIn this work, we explicitly discuss the progress of membranes based on graphene. A large number of research works over the past few years verified that they exhibit outstanding permeability, allowing for very fast and highly-selective transfer of water and thus have very successful wastewater treatment applications through various synthesizing techniques such as the coating, layer by layer, vacuum/pressure-assisted and annealing methods. Nevertheless, there are still certain challenges to address regarding the spacing between layers and the maintenance of the structure in order to be able to exclude small-sized ions. Furthermore, avoiding membrane swelling when dissolved in wet environments is an additional issue that could hinder its usage. Important advancements have been achieved on the separation mechanisms, and various successful liquid separation applications have been highlighted. (a) Their long-term stability is still a significant issue. For large-scale projects, the testing period for their stability ranges between a few hours and a few days, which does not correspond to industrial settings. Thus, the subject needs to be further explored. (b) Secondly, the fabricated membranes must withstand chemical cleaning, since they are required to address fouling. There are still only a few membranes based on graphene that can tolerate such processes. (c) Lastly, it remains difficult to maintain the efficient separation of the membranes in liquids. The spacing of graphene sheets is affected in wet environments.The inability of graphene-based membranes to extract a broad variety of pollutants from wastewater in just one step is an important hindrance that translates to requiring additional exploration to fix the water treatment issue. The diminishing membrane performance owing to fouling is a serious concern that needs to be appropriately addressed. In spite of the technological growth in terms of production methods, the full elimination of defects is crucial before implementations at a larger scale.


To improve the graphene/copper interfacial bonding and the strength of the copper matrix, Cu–Cr–Mg alloy powder and graphene nanosheets (GNPs) have been used as raw materials in the preparation of a layered graphene/Cu–Cr–Mg composite through high-energy ball-milling and fast hot-pressing sintering. The microstructure of the composite after sintering, as well as the effect of graphene on the mechanical properties and conductivity of the composite, are also studied. The results show that the tensile strength of the composite material reached a value of 349 MPa, which is 46% higher than that of the copper matrix, and the reinforcement efficiency of graphene is as large as 136. Furthermore, the electrical conductivity of the composite material was 81.6% IACS, which is only 0.90% IACS lower than that of the copper matrix. The Cr and Mg elements are found to diffuse to the interface of the graphene/copper composite during sintering, and finely dispersed chromium carbide particles are found to significantly improve the interfacial bonding strength of the composite. Thus, graphene could effectively improve the mechanical properties of the composite while maintaining a high electrical conductivity.  Copper alloys are widely used in electrodes, rail transit, electronic packaging, and electrical contact. The most optimal properties of the copper alloys include high strength while providing excellent electrical and thermal conductivities. The fast development of the mechanical, electronic, and transportation industries has given rise to high expectations of the properties of copper alloys . HoweverHowever, the experimental mechanical properties of graphene/copper composites are usually much lower than the theoretical expectations. This is mainly due to the uneven dispersion of graphene and the weak interfacial bonding between graphene and copper. At present, considerable results have been achieved for the dispersion of graphene in a Cu matrix. In contrast, there are few reports on the optimization of the graphene/copper interface in the composite. The decoration of a graphene surface with metal particles is, at present, a method that can be used to improve the binding within the graphene-copper interface. Since the modified graphene and copper matrix show good chemical affinities, the problem with poor interfacial wettability can be well improved. According to the current reports, Cu, Ni, and Ag can significantly enhance the interfacial binding and mechanical properties of the graphene/Cu composite ,8,9. AnoIn the present study, Cu–Cr–Mg alloy powder and graphene nanosheets were used as raw materials in the preparation of a GNPs/Cu–Cr–Mg composite. The method of high-energy ball-milling was used in this preparation. The problems presented above were then solved by including Cr and Mg elements in the alloy powder. The interfacial bond strength in the composite was improved by the addition of Cr, and the added Mg element did not only play a major role in the solid solution strengthening but also caused segregation in the areas where the Cr element was enriched (to control the size of the carbides).2/g), stearic acid , absolute ethanol , and argon (99.99%). Alloy powder was prepared by gas atomization method using high purity electrolytic copper (99.99%), cast Cu-10Cr (wt.%) intermediate alloy, and cast Cu-10Mg (wt.%) intermediate alloy as raw materials. The resulting powders were sieved, and the powders with average particle size less than 50 μm were collected. The chemical composition of the Cu–Cr–Mg alloy powder is listed in The raw materials that were used in the experiments were a gas-atomized Cu–Cr–Mg alloy powder, graphene nanosheets . Moreover, the density of the composite was measured by using the Archimedes drainage method . The phase composition of the composites was characterized by X-ray diffraction . The electrical conductivity of the composite was tested by using a digital eddy current metal conductometer . Furthermore, field emission scanning electron microscopy  was used to analyze the microscopic morphology of the composite powder, as well as the microstructure and tensile fracture of the composite material. The surface elemental analysis of the prepared composite blocks was carried out by using an electron probe microanalyzer . Moreover, the composite was subjected to room temperature tensile tests by using a universal testing machine  with a strain rate of 1 mm/min.The composite was sintered in fast hot-pressing sintering . Raman spectroscopy  was used to analyze the structure of the GNPs and the composite at different stages by using a light source with a wavelength of 532 nm and a scanning rate in the range of 1000–3000 cmTo understand the microstructure of the sintered GNPs/Cu–Cr–Mg composites, the samples were wire-electrode cut in parallel with the sintering pressing direction. The XRD results of the composite are shown in An EPMA analysis was performed to further study the distribution of graphene and alloying elements in the composite. The EPMA surface scan results can be seen in −1), the G peak (~1580 cm−1), and the 2D peak (~2700 cm−1). The D peak is known as the defect band, and the 2D peak is known as the G0 peak. The D peak has been caused by the vibration of the defects in the graphene plane, or on the edge, which reveals the structural defects in graphene. Moreover, the G peak corresponds to the in-plane vibration of the sp2 hybridized carbon atoms. The intensity ratio of the D-to-G peak can be used to evaluate the defects and quality of graphene [D/IG) to describe the change in graphene defect density, which has also been reported in other studies [D/IG of the pristine GNPs was found to be as small as 0.17, which indicates that the quality of graphene is high. However, the ID/IG of graphene was found to increase to 0.45 after ball-milling, which indicates that the sp2 structure of graphene has been damaged to some extent. The reason is that in the process of high-energy ball-milling, the graphene and ZrO2 balls will collide with each other, causing some damage to the surface and edges of graphene [D/IG value of graphene did slightly increase to 0.55 as a result of sintering. It can, in part, be explained by the plastic deformation that takes place in the powder during the sintering process. On the other hand, Cr in the alloy matrix can react with amorphous carbon at the edge of the graphene during the sintering process, which increases the number of defects in the graphene [The structures of pristine GNPs, as well as the structures of graphene before and after rapid hot-pressing sintering, were analyzed by using Raman spectroscopy. Three characteristic peaks of graphene can be seen in the Raman spectrum in graphene ,20. In a studies ,21. To agraphene . Moreovegraphene ,23,24.The relative density and electrical conductivity of the composites are listed in The conductivity of the Cu–Cr–Mg matrix material was found to have a value of 82.4% IACS. Moreover, the conductivity of the GNPs/Cu–Cr–Mg composite had a value of 81.5% IACS, which is only 0.9% IACS lower than that of the matrix. This decrease in conductivity is small compared with other studies ,8,25. ThThe tensile properties of the GNPs/Cu–Cr–Mg composite are shown in c and σm are the yield strengths of the composite and the matrix, respectively, and Vf is the volume fraction of graphene. The strengthening efficiency (R) of a reinforced phase is an important parameter in the evaluation of the strengthening effect of a reinforced phase in the composite. It is defined as presented in Equation (1) as follows:Furthermore, (1)Flake Cu–Cr–Mg powder was successfully prepared by controlling the parameters of ball-milling. The microstructure of the composite material was also a layered structure, and the directional distribution of graphene was realized. The Cr atoms were found to react with the amorphous carbon at the edge of graphene during the sintering process. Moreover, the Mg atoms were found to limit the size of the carbides by segregating to the surface of the carbide, which effectively improved the binding strength of the graphene/copper interface;(2)D/IG was 0.55, which indicates that the structure of graphene remains intact during the preparation process, and there are few defects;Graphene maintained sharp G peaks and smaller D peaks after ball-milling and sintering. The value of I(3)The tensile strength of the composite reached a value of 349 MPa, which was 46% higher than that of the matrix. Moreover, the enhancement efficiency of graphene was 136. Furthermore, the conductivity of the composite became 81.5% IACS, which was only 1% IACS lower than that of the matrix. This can be attributed to the improvement of the graphene/copper interface by the Cr and Mg elements, as well as the high strength and high conductivity of graphene.By using Cu–Cr–Mg alloy powder and graphene nanosheets as raw materials, a GNPs/Cu–Cr–Mg composite has been prepared by using high-energy ball-milling and fast hot-pressing sintering. The microstructure of the composites, as well as the effect of the alloying elements and graphene on the mechanical properties and electrical conductivity of the composite, have been especially studied. The following results could be made:


Particle shape, size, and concentration were found to be major factors that affected overall biocompatibility of graphene.Graphene-based materials have been shown to have advantageous properties in biomedical and dental applications due to their high mechanical, physiochemical, antibacterial, and stem cell differentiating properties. Although graphene-based materials have displayed appropriate biocompatible properties when used in implant materials for orthopedic applications, little research has been performed to specifically test the biocompatibility of graphene for dental applications. The oral environment, compared to the body, varies greatly and must be considered when evaluating biocompatibility requirements for dental applications. This review will discuss  One such structure that carbon can form is graphene, which is a two-dimensional structure of carbon atoms and one of the strongest materials in existence . Fr. Fr91]. 0.1 w/v% .S. mutans bacteria in vitro and demonstrated good mechanical performance without decreasing adhesive strength. Reduced nano-graphene oxide and graphene nanoplates doped with silver nanoparticles were prepared by another research group, which found that the graphene nanoplates doped with silver nanoparticles displayed good adhesive properties that improves the resin-bonded dentin interface. In addition, cell viability of the adhesive was found to be above 85% -1,3-benzene sulfonate) colorimetric cell proliferation assay using human gingival fibroblasts (HGF-1) to test local biocompatibility up to 6 months periment .Another study was conducted to improve polyetheretherketone (PEEK) nanofillers for bone restoration in orthopedic and orthodontic/dental applications. The researchers created novel surface-porous nanofillers made of PEEK combined with hydroxyapatite (HA) and graphene oxide via a heated injection mold process. The surface morphology, mechanical properties, and cellular responses of the nanofillers were investigated. It was found that the inclusion of the hydroxyapatite and graphene oxide significantly improved the overall cell adhesion and proliferation on the PEEK surface showing notable promise in improving tissue integration of PEEK nanofillers for orthopedic and dental/orthodontic applications . The autFrom these studies, graphene oxide and graphene-based materials are promising as additional filler materials for uses in dental composites and resins in restoratives dentistry and orthodontics. The addition of graphene-based materials as fillers has been shown to provide good mechanical performance in dental composites. Graphene-based materials have also shown enhanced bonding strength in dental adhesives, offer good protection from dentin demineralization in infected teeth, and possess good biocompatibility in such dental applications.in vivo applications, biocompatibility of materials must be assessed. Therefore, the following studies included in vitro biocompatibility tests to further assess graphene's biocompatibility for potential dental applications.Osseointegration and differentiation of stem cells are important factors to consider when creating biomaterials for restorative dental applications and bone tissue engineering applications. Graphene has previously been used in various bone tissue engineering applications to promote cell attachment, proliferation, differentiation, and osteogenesis for dental applications. Graphene-based materials also possess optimal tissue engineering properties by having high surface area, mechanical strength, and ease of functionalization , 103. VaUnderstanding and controlling mesenchymal stem cell (MSC) differentiation mechanisms is an important area of research for tissue engineering. One study investigated the effects of graphene and graphene oxide substrates on osteogenic differentiation of MSCs isolated from adult bone marrow. The chemical interaction between the substrates was investigated to determine molecular insights into proliferation enhancement mechanisms. From the results, higher osteogenic differentiation of MSCs was observed on graphene films in comparison to graphene oxide films. The authors found that the graphene films could preconcentrate osteogenic inducers, dexamethasone and Β-glycerolphosphate, which accelerate differentiation of MSC toward osteogenic differentiation . Graphenμg/ml concentrations and rGO sheets with dimensions of 3.8 ± 0.4 μm at high concentrations of 100 μg/ml displayed high cytotoxic effects after 1 hr of exposure. The likely causes were due to the sharp edges from the graphene and oxidative stress created when interacting with cells. In addition, rGONPs could penetrate into the nucleus of human MSCs and cause genotoxicity at low concentrations around 0.1 to 1 μg/ml after 1 hr.Human MSCs isolated from an infant USB were used to evaluate the concentration and size-dependent cytotoxic and genotoxic effects of graphene sheets and nanoplatelets . Reducedin vivo study was later performed by the same group [in vivo and further investigate the integrin/FAK mechano-transduction pathway that potentially causes osteogenic differentiation  has shown promise in creating graphene films and scaffolds for tissue engineering. Dental pulp stem cells (DPSCs) are commonly used to test the biocompatibility, cell differentiation, and proliferation of graphene incorporated materials using a CVD method for dental applications. DPSCs possess a multi-lineage differentiation potential which allows them to differentiate into odontoblasts that assist in tooth dentin regeneration or osteoblasts which form bone matrices in the tooth . In addime group  to assesntiation . A mesenGraphene has been used in various materials to promote and support the differentiation of human periodontal ligament stem cells (hPDLSCs) as well. Graphene oxide combined with silk fibroin-based scaffolding material was found to promote hPDLSC differentiation into osteoblast-like cells . GO coatin vitro tests using bone mesenchymal stem cells (BMSCs), they found that the cells had good attachment and proliferation on the GO-modified groups compared to control groups. In addition, in vivo studies using rat models of the material found no toxic systemic effects, and the GO-modified scaffold allowed for higher calcium deposition of the BMSCs compared to control groups [Porphyromonas gingivalis bacteria and promoted cell proliferation in embryonic osteoblast MC3T3-E1 cells. They also found that the addition of GO enhanced osteogenic differentiation, in vitro, compared to unmodified composite materials [Polymer substrates and scaffolds are commonly used to promote bone tissue regeneration, and graphene has been incorporated into polymer scaffolds to assist in cell proliferation, differentiation, and osteogenesis. However, most polymer substrates, on their own, possess poor mechanical properties and are hydrophobic, which decreases cell attachment and proliferation. Therefore, graphene is often added into polymer scaffolds to increase hydrophilicity and mechanical, thermal, and electrical properties . A studyl groups . Anotheraterials .in vitro and in vivo. From in vitro tests, they found that the graphene CS/PCL scaffold displayed almost double ALP activity compared to other scaffolds, which indicates early osteoblast differentiation. From in vivo tests, they found that the graphene CS/PCL scaffolds promoted proliferation and osteogenesis of Wharton's jelly MSCs [μm was necessary to promote sufficient bone regeneration and osteoconductivity [Porous polymer scaffolds are used in bone tissue engineering for orthopedic and regenerative tissue engineering in dentistry. Huang et al.  fabricatlly MSCs . A hybrilly MSCs . The poructivity , 120.Collagen is among the main components in soft and hard tissues and is widely distributed throughout bones, tendons, skin, and teeth , 122. Itμg/ml [μg/ml seemed to significantly decrease cell viability, but concentrations below 62.5 μg/ml displayed negligible differences. However, the rGO-modified BCP coating displayed significantly higher new bone formation during in vivo rat studies [Calcium hydroxyapatite (HAP) is another material used in bone tissue engineering due to its excellent biocompatibility and osteoconductivity . Howeverμg/ml . In addiμg/ml . Higher  studies .in vitro cytological examination, researchers found that the composite had good wettability and bond strength that increased cell viability and differentiation potential for bone MSCs. In vivo studies also showed that osseointegration enhancement of the material was observed when implanted into rats [A chitosan HAP composite modified with GO was studied to increase cell adhesion, viability, differentiation, and mineralization in osteogenic properties . From innto rats . In addinto rats . From thGraphene-based materials have been used as coatings for titanium implants. Various layers of graphene oxide have been added to titanium implants and have been shown to increase antibacterial properties, osteogenic cell behavior, and cell proliferation . GO and In general, graphene oxide and reduced graphene oxide materials have displayed relatively good biocompatibility for various bone tissue engineering applications. The addition of graphene oxide has been shown to improve cell attachment, proliferation, and differentiation of DPSCs and hPDLSC in various porous polymer scaffolds and hydroxyapatite composites. It assists in better osseointegration of implants. However, there is still debate over the optimal concentration of GO and rGO in various scaffolds and composites to achieve optimal cell viability and cell attachment. Overall, substrates with graphene-based materials included allow for proper cell adhesion, viability, and proliferation directly related to biocompatible aspects of the overall material .Graphene-based nanoparticles are considered a topic of great interest in the field of dentistry. They are being studied for applications such as dental adhesives, fillings, and tissue engineering. Graphene-based materials have been shown to have promising results as antibacterial agents. Antibacterial and antibiofilm properties for dental implants are crucial since bacterial infections are often the causes for implant failure and integration with the bone. The addition of graphene-based materials in dental composite fillings has offered antibacterial properties to decrease bacterial adsorption onto composite material to prevent further tooth decay. Additionally, graphene nanoparticles have been found to have adequate initial biocompatibility in the mouth when it comes to bone and tissue engineering. They have been shown to possess good osteoblastic differentiation tendencies to increase osseointegration and improve bone tissue development. Increased proliferation and differentiation of stem cells is a major factor in showing graphene's biocompatibility for dental applications.in vitro tests, there is still a lack of long-term cytotoxicity studies of graphene nanoparticles in the body, as well as a lack of understanding as to how easily they can be excreted from the body. Many factors such as particle size, shape, and concentration appear to play a large role in how toxic these nanoparticles can be, and it would seem that the cytotoxicity varies on a case-by-case basis. Standardization of biocompatibility requirements for dental applications is also needed with long-term in vitro and in vivo studies to fully understand the biocompatibility of these nanoparticles and their long-term health effects.Although graphene has been shown to display relatively good biocompatible qualities in various dental applications from


The cell adhesion molecule L1 is essential not only for neural development, but also for synaptic functions and regeneration after trauma in adulthood. Abnormalities in L1 functions cause developmental and degenerative disorders. L1’s functions critically depend on proteolysis which underlies dynamic cell interactions and signal transduction. We showed that a 70 kDa fragment (L1-70) supports mitochondrial functions and gene transcription. To gain further insights into L1-70’s functions, we investigated several binding partners. Here we show that L1-70 interacts with topoisomerase 1 (TOP1), peroxisome proliferator-activated receptor γ (PPARγ) and NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2). TOP1, PPARγ and NDUFV2 siRNAs reduced L1-dependent neurite outgrowth, and the topoisomerase inhibitors topotecan and irinotecan inhibited L1-dependent neurite outgrowth, neuronal survival and migration. In cultured neurons, L1 siRNA reduces the expression levels of the long autism genes neurexin-1 (Nrxn1) and neuroligin-1 (Nlgn1) and of the mitochondrially encoded gene NADH:ubiquinone oxidoreductase core subunit 2 (ND2). In mutant mice lacking L1-70, Nrxn1 and Nlgn1, but not ND2, mRNA levels are reduced. Since L1-70’s interactions with TOP1, PPARγ and NDUFV2 contribute to the expression of two essential long autism genes and regulate important neuronal functions, we propose that L1 may not only ameliorate neurological problems, but also psychiatric dysfunctions. The cell adhesion molecule L1 regulates important functions in the developing and adult nervous system under physiological and pathological conditions ,3. In huStudies in mice have shown that L1 acts through homophilic and heterophilic interactions and that the proteolytic cleavage of L1 is crucial for its neural functions ,29,30,31Stimulation of L1 signal transduction by the function-triggering L1 antibody 557 generates a transmembrane C-terminal fragment of 70 kDa (L1-70)  via a prAnother transmembrane C-terminal L1 fragment of 55 kDa (L1-55) is generated by metalloproteases, the β-site of amyloid precursor protein cleaving enzyme and γ-secretase ,38. InteBy mass spectrometric analysis upon affinity chromatography using the intracellular domain of L1 (L1-ICD) and a nuclear brain extract we have identified TOP1 as a nuclear molecule that could be associated with L1-ICD . To follWe also utilized affinity chromatography to find other nuclear and cytoplasmic molecules that could interact directly or indirectly with L1-ICD, including splicing factor proline/glutamine-rich (SFPQ), non-POU domain-containing octamer-binding protein (NonO), paraspeckle component 1 (PSPC1), WD-repeat protein 5 (WDR5), histone H1.4 (HistH1e), nucleoporin 93 kDa (Nup93), heat shock cognate protein 71 kDa (Hsc70), synaptotagmin 1 (SYT1), and importin β1 (impβ1) . ImmunopFor the present study, we investigated whether the aforementioned molecules interact with L1-70. Using immunoprecipitation and proximity ligation assay, we found that TOP1, PPARγ and NDUFV2, but not the other tested molecules, interact with L1-70. Thus, these interactions were chosen for further investigation of their functional consequences.ELISA showed that the interactions of TOP1 and PPARγ with L1-ICD are mediated via distinct L1-ICD sequences. Reduction of TOP1, PPARγ and NDUFV2 expression by siRNAs or inhibition of topoisomerase activity with topotecan and irinotecan reduced the L1-dependent neurite outgrowth as well as neuronal survival and migration. Reduction of L1 levels by siRNA downregulated the expression levels of the long autism genes Nrxn1 and Nlgn1 and of the mitochondrial gene ND2. Nrxn1 and Nlgn1 mRNA levels were also reduced in mutant mice deficient in L1-70. Our results indicate that the interactions of L1-70 with TOP1, PPARγ and NDUFV2 regulate L1-dependent neuronal functions.Because of the functional importance of the L1-70 fragment ,34,35,36Because we were able to show, through ELISA, that NDUFV2 directly binds to L1-ICD , we thenWe were able to show that L1-70 was not generated in gene-edited mice expressing either L1 with a mutated dibasic sequence in the third FNIII domain at position 858–863  or L1 with a mutation of arginine 687  . Thus, wWhen performing proximity ligation with L1 antibody and antibodies against SFPQ, ERα and RXR, which forms heterodimers with PPARγ , we obseTo gain more evidence for L1-70’s association with TOP1, PPARγ and NDUFV2, immunoprecipitation was performed with immobilized antibodies and a non-nuclear brain fraction of wild-type and L1-70-lacking mutant mice. Western blot analysis indicates one L1-immunopositive band of 70 kDa in the TOP1, PPARγ and NDUFV2 immunoprecipitates of the non-nuclear wild-type brain fraction, whereas no 70 kDa band was seen in the immunoprecipitates of the non-nuclear brain fractions from the L1 mutants c. No banTo investigate whether the interaction of L1 with TOP1, PPARγ and NDUFV2 plays a role in L1-mediated neurite outgrowth, cortical neurons were mock-transfected or transfected with TOP1, PPARγ and NDUFV2 siRNAs or control siRNA and then treated without and with L1 antibody 557 which induces L1-dependent neurite outgrowth ,28,29. Tn = 3) in neurons after transfection with the corresponding siRNA relative to the levels in neurons transfected with control siRNA (n = 3), respectively  the interactions of TOP1, PPARγ and NDUFV2 are reduced when the MBP-mediated generation of L1-70 is inhibited by the serine protease inhibitor aprotinin; (2) TOP1, PPARγ and NDUFV2 co-immunoprecipitate L1-70 from brain fractions of wild-type, but not L1-70-lacking mutant mice; and (3) L1 antibody 557-induced generation of L1-70 ,28 leadsWe have previously reported that NDUFV2 binds to the KDET motif in L1-ICD . We showSince TOP1 inhibitors and knockdown of TOP1 have been shown to reduce the expression levels of the Nrxn1 and Nlgn1 genes ,43,44, aTOP1 is a key transcriptional regulator in postmitotic neurons ,44 and dTOP1 has also been found in mitochondria, and TOP1-deficient murine embryonic fibroblasts showed a higher mitochondrial membrane potential, increased expression of mitochondrial genes, including ND2, decreased ATP levels and enhanced levels of reactive oxygen species (ROS) . BecauseIn utero electroporation of NDUFV2 shRNA in the cerebral cortex of mouse embryos resulted in an impaired neuronal migration and accumulation of ectopic neurons in lower cortical layers: most NDUFV2 shRNA-expressing neurons were found in the intermediate and ventricular zones, whereas control cortical neurons migrated into the upper cortical plate . MoreoveIn mice lacking the cytoplasmic polyadenylation element binding protein 1, the expression of NDUFV2 in total brain extracts and the amount of NDUFV2 in mitochondrial complex 1 was reduced . This rePPARγ agonists have been considered for the treatment of Alzheimer’s and Parkinson’s disease ,79,80,81There is increasing evidence that mitochondrial dysfunction and oxidative stress are involved in the pathogenesis of many neurodegenerative diseases and neurodevelopmental disorders, such as autism, attention deficit hyperactivity disorder, schizophrenia, Down’s syndrome and Fragile X syndrome. In particular, abnormalities in the mitochondrial respiratory electron-transport chain during development in utero, infancy, childhood and adulthood may be involved in the etiology of neurodevelopmental disorders and neurodegenerative diseases ,109. TheL1-deficient mice  and geneThe following antibodies were from Santa Cruz Biotechnology : mouse L1 antibody C-2  against the intracellular L1 domain, mouse PPARγ antibody E-8 , mouse Topo I antibody C-21 , mouse NDUFV2 antibody F-5 , mouse importin β1 (karyopherin β1) antibody H-7 , goat TOP1 (Topo I) antibody C-15 , goat SYT (synaptotagmin 1) antibody N-19 , rabbit SFPQ (PSF) antibody H80 , rabbit WDR5 antibody H-36 , rabbit Nup93 antibody H-300 , rabbit AR (androgen receptor) antibody N-20 , rabbit VDR (vitamin D receptor) antibody C-20 , rabbit RXR (retinoid X receptors) antibody C-20 , and rabbit ER (estrogen receptor) α antibody HC-20 . Rabbit antibodies against PSPC1 , NonO , NDUFV2 , Hsc70 , and Histone H1  were from ChromoTek and Proteintech Germany . Rabbit antibodies against PPARγ , MeCP2 , HP1ɣ , and GAPDH  were acquired from Cell Signaling Technology Europe . Anti-L1CAM antibody ab12399 against the intracellular L1 domain was acquired from Abcam . Rat monoclonal function-triggering L1 antibody 557 has been described . SecondaProduction and purification of recombinant His-tagged L1-ICD  has been described .Recombinant full-length human NDUFV2  was from OriGene Technologies . Recombinant human DNA Topoisomerase-I  and recombinant full-length human PPARγ  were from Biozol .® HD Transfection Reagent (Cat #E2311) was from Promega .Synthetic L1 peptides P1 (CFIKRSKGGKYSVKDKEDTQVDSEARPMKDETFGE), P2 (RPMKDETFGEYRSLESDNEEKAFGSSQPSLNGDIK), P3 (GDIKPLGSDDSLADYGGSVD), P4 (CSVDVQFNEDGSFIGQYSGK), P5 (CSGKKEKEAAGGNDSSGATSPINPAVALE) and tat-P4 (YGRKKRRQRRRSVDVQFNEDGSFIGQYSGK) were from Schafer-N . Mouse L1 siRNA , mouse TOP1 siRNA , mouse PPARγ siRNA , mouse NDUFV2 siRNA (sc-149892), and control siRNA  were from Santa Cruz Biotechnology. FuGENEAprotinin (Cay14716-10), topotecan hydrochloride (Cay14129-10), and irinotecan hydrochloride (Cay14180-25) were from Biomol  and 4′,6-diamidino-2-phenylindole (DAPI) was from Thermo Fisher Scientific .2 and CaCl2  (PBS), treated with blocking solution  for 2  h, washed again with PBS containing 0.005% Tween 20 (PBST), and incubated with increasing concentrations of recombinant His-tagged L1-ICD as ligand for 1 h under gentle agitation. For competition ELISA, 2.5 µM L1-ICD were preincubated for 1 h without or with a five-fold molar excess of L1 peptides P1, P2, P3, P4 or P5. The mixtures were then incubated with substrate-coated recombinant proteins. After washing two times with PBS and three times with PBST, L1 antibody C-2 (1:500) in blocking solution was applied for 1 h followed by two washes with PBS and three washes with PBST and incubation with horseradish peroxidase-coupled anti-mouse antibody (diluted 1:2000 in blocking solution) for 1 h. Wells were washed again with PBST and 1 mg/mL ortho-phenylenediamine dihydrochloride (Thermo Fisher Scientific) was used for detection of bound L1-ICD. The reaction was terminated by addition of 25 µL 2.5 M sulphuric acid. Absorbance was measured at 492  nm with an ELISA reader .For ELISA, 25 µL of 10 or 20 µg/mL recombinant proteins were incubated overnight at 4 °C in 384-well microtiter plates with high binding surface . The following steps were performed at room temperature. Wells were washed with Dulbecco’s phosphate-buffered saline with MgClg for 15 min. Cells were then diluted in Neurobasal A medium  supplemented with 2 mM L-glutamine (Invitrogen), 4 nM L-thyroxine (Sigma-Aldrich), 0.1 mg/mL BSA (Sigma-Aldrich), 12.5 μg/mL insulin (Sigma-Aldrich), 30 nM sodium selenite (Sigma-Aldrich), 100 μg/mL transferrin, 0.1 mg/mL streptomycin and 100 U/mL penicillin (Invitrogen). For the proximity ligation assay, cells were seeded onto poly-L-lysine-coated 12 mm glass coverslips in a 24-well plate at a density of 2.5 × 105 cells per well. For determination of neurite outgrowth, cells were seeded at a density of 5 × 104 cells per well of a 48-well plate coated with poly-L-lysine (Sigma-Aldrich), and for neuronal survival analysis cells were seeded at a density 1.25 × 105 cells per well of a 48-well plate coated with poly-L-lysine. For analysis of RNA levels cells were seeded at a density of 3 × 106 cells per well of a 6-well plate coated with poly-L-lysine.Cerebellar neurons were prepared from cerebella of six- to eight-day-old mice. Cerebella were incubated with 10 mg/mL trypsin and 0.5 mg/mL DNase I (Sigma-Aldrich) in Hanks’ balanced salt solution (HBSS) for 15 min at 37 °C, washed with HBSS, mechanically dissociated and centrifuged at 100× Cerebellar explants were prepared from six- to seven-day-old mice. Cerebella were forced through Nitrex nets with pore sizes of 300, 200 and 100 μm and tissue pieces were plated on plastic coverslips coated with poly-L-lysine and maintained for 16 h in culture medium containing 20% horse serum. The culture medium was then replaced by serum-free medium.5 cells per well. For determination of neurite outgrowth, cells were seeded at a density of 5 × 104 cells per well of a 48-well plate coated with poly-L-lysine (Sigma-Aldrich). For Western blot analysis, cells were seeded onto poly-L-lysine-coated 12-well plates at a density of 1.5 × 106 cells per well and for analysis of RNA levels cells were seeded at a density of 3 × 106 cells per well of a 6-well plate coated with poly-L-lysine.For culturing of cortical neurons, cerebral cortices were dissected from 15.5- to 16.5-day-old embryos and incubated in 0.025% trypsin (Sigma-Aldrich) in HBSS at 37 °C for 30 min. The cortices were then incubated in HBSS containing 1% BSA (Sigma-Aldrich) and 1% trypsin inhibitor  at 37 °C for 5 min. After washing in HBSS, the tissue was mechanically dissociated and the dissociated cells were cultured in Neurobasal medium (Invitrogen) supplemented with 1% B-27 (Invitrogen), 2 mM L-glutamine (Invitrogen), 100 U/mL penicillin (Invitrogen) and 100 μg/mL streptomycin (Invitrogen). For the proximity ligation assay and immunostaining, cells were seeded onto poly-L-lysine-coated 12 mm glass coverslips in a 24-well plate at a density of 1.25 × 10For stimulating L1 functions, neurons were treated with 50 µg/mL L1 antibody 557. For aprotinin treatment, neurons were treated 2 h after seeding with 1 µM aprotinin. Treatment of neurons with 50 µg/mL tat-P4 peptide was performed 30 min after seeding. Treatment with 300 nM topotecan or irinotecan was performed 30 min after seeding (neurite outgrowth) or 30 min before addition of hydrogen peroxide . Explants were treated with 300 nM topotecan and irinotecan directly after medium change and then 30 min later with 50 µg/mL L1 antibody 557. Following this, explants were maintained for a further 32 h.For transfection of cortical neurons, cortical neurons were seeded onto poly-L-lysine-coated coverslips in 24-well plates (proximity ligation and immunostaining), or onto 6-well pates , 12-well plates  and 48-well plates (neurite outgrowth) and maintained for 2 h before transfection with 1 µL (48-well), 2 µL (24-well), 4 µL (12-well) or 8 µL (6-well) 10 µM siRNA and 1–4 µL FuGENE transfection reagent per well. L1 antibody 557 (50 µg/mL) was added to the cultures 24 h after transfection  or 2 h after transfection (neurite outgrowth), and 48 h after transfection the cells were analyzed for neurite outgrowth and cell survival, or used for immunostaining, Western blot analysis, RNA isolation or proximity ligation assay.https://imagej.nih.gov/ij/index.html; RRID:SCR_003070, accessed on 8 March 2022). Numbers of red dots and numbers of DAPI-stained nuclei were determined using ImageJ and the number of red dots per image was divided by the number of nuclei per image. The average values of red dots per cell (=nucleus) were determined in ten images per condition.Cultures were fixed for 15 min at room temperature in 4% formaldehyde, washed with PBS and used for immunostaining or subjected to proximity ligation assay using Duolink PLA products according to the manufacturer’s protocol  with minor modifications. For the proximity ligation assay, cells were incubated with 1% Triton X-100 in PBS for 30 min, washed once with PBS and blocked with Duolink Blocking solution for 30 min before being incubated for 24 h at 4 °C with mouse L1 antibody C-2 and goat or rabbit antibodies against TOP1, SYT, SFPQ, WDR5, Nup93, PSPC1, NonO, NDUFV2, HistH1e, ERα, RXR, PPARγ, AR or VDR or with rabbit L1 antibody ab12399 and mouse antibodies against impβ1, TOP1, PPARγ and NDUFV2, all diluted 1:10 in Duolink Antibody Diluent. Cells were washed twice using Duolink Wash Buffer A and incubated with a mixture of secondary antibodies conjugated with oligonucleotides (Duolink PLA Anti-Rabbit or Anti-Goat Probe MINUS and Duolink Anti-Mouse PLA Probe PLUS). The proximity ligation reaction was then performed according to the manufacturer’s protocol using the Duolink In Situ Detection Reagent RED. Thereafter, the coverslips were incubated in 5 µg DAPI/mL PBS for 15 min, washed twice with PBS and mounted in Immuno-Mount (Thermo Fisher Scientific). Ten images per condition were taken using an Olympus F1000 confocal microscope  and analyzed using ImageJ software .Cells and explants were washed gently with pre-warmed culture medium, fixed in 2.5% glutaraldehyde for 30 min at room temperature, and stained with 1% toluidine blue and 1% methylene blue in 1% sodium tetraborate for 30 min at room temperature. Neurite outgrowth and neuronal migration were analyzed by measuring the total length of neurites in an Axiovert microscope with the AxioVision 4.6 imaging system  or with Image J counting the number of cells that had migrated out of the explant core. For neurite outgrowth analysis for each condition at least 100 neurons were counted and migrating cells were counted from 12–15 explants per condition.2O2 for 24 h. Live and dead cells were then stained with calcein-AM (Thermo Fisher Scientific) and propidium iodide (Sigma-Aldrich) and imaged with a Zeiss AxioObserver.A1 microscope (Carl Zeiss) with a 20× objective (aperture 0.4) and the AxioVision 4.6 software (Carl Zeiss). Live and dead cells were counted in five images (containing 350–400 cells each) from each of three wells per condition and experiment using ImageJ.To determine cell death, neurons were maintained overnight in serum-free medium and then treated with 50 µg/mL of L1 antibody 557 and exposed to oxidative stress by the addition of 10 µM HFor the preparation of nuclear and non-nuclear fractions, the Subcellular Protein Fractionation Kit for Tissue (Thermo Fisher Scientific) was used. Fractions in cytoplasmic extraction buffer (CEB) and membrane extraction buffer (MEB) were pooled and taken as non-nuclear fraction. Fractions in nuclear extraction buffer (NEB) were taken as nuclear fraction. Nuclear and non-nuclear fractions were used for immunoprecipitation.For immunoprecipitation, Protein G magnetic beads (25 μL per sample) were washed twice in phosphate-buffered saline (PBS), pH 7.4, and incubated in dilution buffer  for 10 min at 4 °C under rotation. The mouse NDUFV2, PPARγ and TOP1 antibodies (10 μg per sample) were diluted in dilution buffer. The diluted antibody solutions were incubated with the beads for 1 h at 4 °C under rotation. The beads were washed in dilution buffer for 5 min at 4 °C under rotation. A freshly prepared 13 mg/mL stock solution of dimethyl pimelimidate (DMP) was 1:1 diluted with wash buffer (0.2 M triethanolamine in PBS). After washing the beads in PBS, the diluted DMP solution (pH 8–9) was added to the beads which were then incubated for 30 min at room temperature (20–24 °C) under rotation. The beads were then washed in wash buffer for 5 min at room temperature under rotation and incubated in DMP solution and wash buffer twice more. Finally, the beads were incubated twice in quenching buffer (50 mM ethanolamine in PBS) for 5 min at room temperature under rotation. After washing, the beads were incubated with brain fractions overnight at 4 °C under rotation. The beads were then washed twice with lysis buffer and once with PBS before being boiled for 5 min in sample buffer .® TGX™ Precast Protein Gels . PageRuler™ Plus Prestained Protein Ladder (Thermo Fisher Scientific) was used as molecular weight marker. Proteins were then transferred to 0.45 μm Protran™ nitrocellulose membranes  and stained with Ponceau S to control protein loading. The membranes were then incubated for 1 h in blocking solution   with 0.05% Tween 20 (TBST) and then incubated overnight with primary antibodies (1:1000) in blocking solution at 4 °C with shaking. After washing five times for 5 min in TBST, the membranes were incubated for 1 h with horseradish peroxidase-conjugated secondary antibody . Bands were detected using enhanced chemiluminescent solution  and a CCD camera .For Western blot analysis, samples were run on 4%–20% Mini-PROTEAN® II reverse transcriptase (Thermo Fisher Scientific) was used. qPCR was performed in triplicate using reverse transcribed mRNA, the 7900HT Fast Real-Time PCR System (Thermo Fisher Scientific), the qPCR kit QuantiNova SYBR Green , primers for determination of the mRNA levels of Nrxn1 , Nlgn1 , Cntnap2 , Dlg2 , COX1 , COX2 , COX3 , CytB , ATP6 , ND1 , ND2 , ND3 , ND4 , ND4L , ND5 , ND6 , and the reference gene actin . All primers were from Metabion . The SDS 2.4 software  was used for analysis of the qPCR data. The mRNA levels of Nrxn1, Nlgn1, Cntnap2, Dlg2, COX1, COX2, COX3, CytB, ATP6, ND1, ND2, ND3, ND4, ND4L, ND5, and ND6 relative to the mRNA levels of the reference gene actin were calculated. Data of relative gene expression  were used for statistical analysis.For reverse transcription, oligoT18 primer and SuperScript6) were scraped off in 300 µL buffer A  and incubated for 15 min on ice. After centrifugation for 8 min at 1000× g and 4 °C, the pellet was resuspended in 300 µL buffer B , and 100 µL 4 M NaCl was added. An aliquot was saved for determination of the protein concentrations. After incubation for 30 min on ice, the samples were centrifuged for 10 min at 9000× g and 4 °C. The supernatant was collected and 400 µL glycerol was added, and the samples were thoroughly mixed and stored as nuclear extracts at −80 °C until use.Cultured neurons  for 30 min at 37 °C. Stopping buffer was added to a final concentration of 1% sodium dodecyl sulfate, 15% glycerol, 50 mM EDTA, pH 8, and 0.5% bromophenol blue, and the samples were subjected to electrophoresis on a 1% agarose gel  and to ethidium bromide staining.In a total volume of 25 µL, nuclear extracts (50–100 ng protein) were incubated with 100 ng supercoiled pUC19 plasmid DNA  in reaction buffer . The types of tests are indicated in the legends.