The results obtained in the course of development and the results of testing of the graphene-based sensor for detection of protein molecules are also presented. The biosensor was fabricated by the technology previously developed for the gas sensor. The working capacity of the biosensor was tested with an immunochemical system constituted by fluorescein and monoclonal antibodies (mAbs) binding this dye.This work is devoted to the development and optimization of the parameters of graphene-based sensors. The graphene films used in the present study were grown on semi-insulating 6H-SiC substrates by thermal decomposition of SiC at the temperature of ~1700 °C. The results of measurements by Auger and Raman spectroscopies confirmed the presence of single-layer graphene on the silicon carbide surface. Model approach to the theory of adsorption on epitaxial graphene is presented. It is demonstrated that the Green-function method in conjunction with the simple substrate models permit one to obtain analytical results for the charge transfer between adsorbed molecules and substrate. The sensor structure was formed on the graphene film by laser. Initially, a simpler gas sensor was made. The sensors developed in this study demonstrated sensitivity to the NO The discovery of graphene and the study of its properties is one of the brightest pages in the development of solid-state physics over the past 15 years. This discovery sparked the emergence of a whole class of two-dimensional structures, of which graphene is still the most-studied material. The exfoliation method, used by K. Novoselov and A. Geim in their first work on the preparation and study of graphene, is reduced to the separation of a one-atom-thick flake from a graphite crystal. Until now, graphene samples obtained by this technology have had the best structural perfection. It was on these samples that the most important results were obtained, which confirmed the two-dimensional nature of this material. However, their small size and irregular and unpredictable geometrical shape in advance do not allow the exfoliation method in industry.Graphene films obtained by thermal destruction of the surface of silicon carbide are the second in terms of structural perfection. Thus, it is possible to obtain structures up to industrially important dimensions; in this case, the dimensions are limited only by the initial SiC substrate, i.e., up to 6 inches (150 mm) in diameter.High mobility of charge carriers in combination with their low concentration;The maximum possible ratio of surface area to volume;Low noise level.Theoretical and experimental studies show that graphene has a unique set of electrophysical properties:The combination of these properties leads to the fact that the addition of a minimal amount of impurity to the graphene surface can noticeably change the conductivity of the graphene film. Thus, graphene is a very promising material for the manufacture of various types of sensors.In this work, we discuss the technology of forming a biosensor based on a graphene film obtained by thermal destruction of the surface of silicon carbide. A brief review of the model approach to the theory of adsorption on epitaxial graphene is presented. New results obtained by detecting various types of biological molecules with a graphene sensor are analyzed.After appearing in a previous paper , where iWe begin with the adsorption on single-layer free-standing graphene. As far as we know, the first corresponding model was put forward in ,16. In tgiven in ,16).At zero temperature the occupation number or ω<−3t . If initNow turn to the epitaxial graphene (epigraphene) model description. The simplest approach is described in . The fir3a) . Such a (ω) from ,16.General expression for the epigraphene DOS is given byHere given in .There are two limiting regimes for the graphene–substrate interaction: Strong coupling, when given in . It is cIt is easy to understand now that for the DOS of particle adsorbed on epigraphene, formula (1) has to be rewritten in the formshown in  that thee is the elementary charge, l is the adparticle bond length, and Up to now, we considered adsorption of a single particle at zero temperature. For finite coverages nd A¯~10 . It is wussed in .n-type substrate or, conversely, decrease (increase) the conductivity of a p-type substrate. Second, the adsorbed particles (adparticles) serve as additional scattering centers, which generally must influence the surface mobility The effect of adsorption on the substrate appears in mainly two effects. One is the change l oxides ,24. A thloped in ,26, wherAnalysis ,26 of exIn , we haveInterest in graphene flared up after the publication of K.S. Novoselova, A.K. Geim et al., In which they demonstrated the possibility of obtaining graphene sheets using micromechanical cleavage of bulk crystalline graphite . This wa8 Ω cm. Schottky diodes based on the graphene/SiC structure, obtained by thermal destruction of SiC substrates, are characterized by high uniformity of characteristics.Until now, graphene samples obtained by this technology have the best structural perfection. It was on these samples that the most important results were obtained, which confirmed the two-dimensional nature of this material. However, their small size, irregular, and unpredictable geometrical shape in advance do not allow their use in industry. Graphene films obtained by thermal destruction of the surface of silicon carbide are the second in terms of structural perfection. Thus, it is possible to obtain structures up to industrially important dimensions; in this case, the dimensions are limited only by the initial SiC substrate, i.e., up to 6 inches (150 mm). The orientation of the obtained graphene films is specified by a SiC substrate, which makes it possible to obtain graphene films with a large area with a preferential azimuthal orientation of domains. This method is based on the dissociative evaporation (sublimation) of SiC components from the surface of a single crystal and the formation of a graphene film from residual carbon atoms  The phenGraphene films grown by CVD on a metal foil are in third place in terms of their structural perfection . This meThe main disadvantage of the method of thermal destruction of the SiC surface is the high cost of SiC substrates. However, the high structural perfection of graphene obtained by the thermal destruction method, as well as the possibility of growing graphene on high-resistance substrates, neutralize this disadvantage.Dissociative evaporation or sublimation of SiC is one of the most important processes that determine the growth of crystals and epitaxial films of this material from its own vapors. The work of S.K. Lilov  presentsAn important step in obtaining a graphene film is the selection of a SiC substrate that will be used for the thermal destruction process.The first criterion for choosing substrates is their crystal perfection. To obtain homogeneous graphene, substrates with a homogeneous crystal structure are required; therefore, it is preferable to use single-crystal SiC substrates. Various structural defects, such as dislocations, micropores, stacking faults, polytypic inclusions, low-angle boundaries, etc., can also negatively affect the uniformity of graphene growth on the substrate surface; therefore, it is necessary to choose substrates with a minimum density of structural defects.The second criterion for choosing substrates is the surface roughness on which graphene growth is planned. Today there are several stages of surface treatment of SiC wafers: Grinding, mechanical polishing, and chemical-mechanical polishing.H and 4H-SiC substrates with a minimum misorientation angle (α ~ 0), the growth was carried out on the (0001) face (Si face). We used semi-insulating (high-resistivity substrates), since they did not require the transfer of a graphene film onto a non-conducting substrate in the further manufacture of sensors.The third criterion for choosing substrates is the crystallographic orientation of the substrate surface, as well as the angle of deflection of the substrate surface from the basal plane . In our experiments, we used 62 and 11 × 11 mm2 in size. Modern commercial SiC wafers are in standard sizes of 2, 3, 4, or 6 inches. The use of such large samples for laboratory research is not profitable, because, firstly, the cost of the material is quite high, and secondly, the characterization of large samples takes a lot of time. Therefore, in order to carry out experiments on developing the technology of graphene growth, the wafers were cut using special equipment for cutting semiconductor substrates into typical specimens 5 × 5 mmxCy components from the substrate surface. Various surface contaminants or surface irregularities of the substrate can contribute to the sublimation process, leading to nonuniform sublimation of molecules from the substrate surface. For the successful development of graphene growth technology, a necessary condition is a high-quality preparation of the SiC surface, which reduces the effect of contamination and surface inhomogeneities on the sublimation process. The growth of graphene on the SiC surface is accompanied by the sublimation of the SiPre-growth etching in a hydrogen atmosphere was used for preliminary cleaning of the SiC substrate surface. The method has been known for a long time ,9 and isTo enable the etching of SiC substrates “in situ” before graphene growth, this technology was adapted to a technological unit for the growth of graphene on SiC. The adaptation consisted in changing the composition of the gas mixture in which the etching is performed, as well as in the selection of a certain technological mode of etching.3H8 [Typically, for etching, the chamber is purged with either pure hydrogen  or with 3H8 . In our The grown graphene samples were characterized by using a combination of three methods: Raman spectroscopy, atomic-force microscopy (AFM), and Kelvin-probe force microscopy (KPFM).2 with spatial resolution of 1 μm. The measurements were carried out at room temperature using a T64000 Raman spectrometer  equipped with a confocal microscope. The spectra were excited using the 532 nm Nd:YAG laser. . To avoid heating and damage to the sample, the laser power was limited to 4 mW. Two main features in the presented spectra, namely the sharp and intense G and 2D lines, are originating from the graphene film [−1 range corresponds to the buffer layer, which is an interface carbon layer located between the graphene film and the 4H-SiC substrate [Raman spectroscopy is widely used as a graphene characterization tool. Analysis of the data obtained by these methods allows one to determine such properties of graphene as number of layers, degree of defectiveness, strain and doping levels, and many more. ene film , while tubstrate .D line, the appearance of which is indicative of presence of structural defects [The  defects , in our D line allows one to easily distinguish monolayer graphene from bi- and multilayer by analyzing the shape of this line. D line FWHM distribution. In the regions with FWHM (2D) between 35 and 40 cm−1, this line had a symmetric Lorentzian form, which pointed to the monolayer nature of graphene in corresponding areas [D line FWHM was increased, the line had an asymmetric shape similar to that reported for bilayer graphene [Double-resonance nature of the 2ng areas . In the graphene  and coul2 area of the sample is shown on An AFM topography map of the 12 × 12 μm2 area of the studied sample is shown in Distribution of surface potential is also capable of providing information on graphene thickness. It is known that the surface potential of bilayer graphene is ~100 mV higher than that of monolayer . The KPFIt was shown in  that graTo clean the graphene detector, a current of about 10 mA is passed through it this is enough to heat the structure so that the gas particles are desorbed. This cleaning mechanism does not affect the degree of efficiency of gas detection: The process of sorption-desorption of gases is completely reversible, that is, it is a reusable detector. It should be noted that, in ,42 Figu. Excess o is the initial resistance in the absence of a detectable gas in the incoming air flow.Purified air was used as a carrier gas. Sensor sensitivity (r) was expressed as a percentage, %, and is defined as the relative change in the sample resistance in the presence of a recorded gas in the gas mixture:2 in the gas mixture (periods of gas supply are indicated as light gray stripes) at 20 °C. Since the NO2 desorption rate at room temperature is very low, annealing at 110 °C was used to return the sensor to its original state after each exposure period [e period ,44.2 concentration of 10 ppb, the amplitude of the sensor response was about 3% when exposed to the gas mixture for 1 h Such sensitivity of the sensors is quite sufficient for environmental monitoring.For NOIt should be noted that one of the serious disadvantages of the graphene gas sensor is the lack of selectivity. Indeed, it is impossible to tell from the change in conductivity which molecule was adsorbed on the graphene surface. Moreover, some molecules give contributions of the opposite sign, so the total change in resistance can be close to zero.A distinctive feature of the graphene film is that the molecules or a group of atoms adsorbed on its surface act as a donor or an acceptor, leading to a change in its electronic state (resistance), which can be detected . This spHowever, the graphene film itself is not a selectively sensitive sensor and can attach various substances and biomolecules to its surface. To use graphene as a biosensor, a special treatment is used, which increases the selectivity of chemical reactions on the graphene surface and creates additional covalent bonds for chemical reactions with other molecules that need to be detected.The antibody–antigen (Ab-Ag) interaction is fundamental to the functioning of the human immune system . ReactioThe concept of the graphene/SiC biosensor developed in his work is based on the creation of conditions for a controlled Ab-Ag interaction on the graphene surface in graphene/SiC chips. It is this interaction that leads to a change in the electronic state of graphene (its resistance), which can be registered. The developed concept of a biosensor is universal for the detection of protein compounds and viruses Only complementary (related) antibodies and antigens take part in the interaction reaction, which achieves the selectivity of the biosensor. To provide biosensing ability, graphene/SiC films are undergoing multistep processing and treatments.H-SiC a size of 11 × 11 mm2. The growth process was carried out in a graphite crucible with induction heating in an argon atmosphere (720–750 Torr) at a temperature of 1700–1800 °С [Graphene films were grown by thermal decomposition of semi-insulating substrates (0001) ± 0.25° 4–1800 °С . The metD) (AFM) images of graphene/SiC samples. A set of elongated terraces having different width are clearly seen in the morphology image of the surface. These steps can facilitate attachment to graphene of large  antibodies of viruses.The presence of the graphene monolayer on the SiC substrate was confirmed by Raman spectra. A specific feature of the morphology of SiC surface after graphene growth is the presence of terraces on the surface, the width and height of which depends on the conditions of graphene growth. As example, 2 chips, which after that were mounted on a holder and welded with gold wires. After that, all current-carrying parts of the holder and contact pads of the chip were covered by a protective varnish. The protective varnish is a compound based on thermo reactive resins with various fillers and additives (VT-25-200). Sensing area of the chip was 1 × 1 mm2 or so.After the graphene film growth and characterization, graphene/SiC chips (dies) were processed for manufacturing biosensors. The processing includes standard photolithography using dry etching, metallization, cutting the substrate into individual chips, and mounting them on a holder. A rectangular chip topology was formed on the surface of a graphene/SiC sample by laser photolithography in combination with ion-reactive etching in argon and etching in oxygen plasma. Ti/Au contacts (5 nm/50 nm) were created by vacuum deposition and explosive photolithography. Then, the graphene/SiC sample was cut into individual 1.5 × 2 mmI-V) characteristics of the manufactured chips were linear, which indicated ohmic contacts and the absence of potential barriers on the contacts that could affect the measurement results.The current-voltage  and their subsequent reduction to phenylamine groups (aminobenzene C6H4NH2) using a two-steps cyclic voltammetry (CV) process [In this project, the process of functionalization of the graphene surface in the chip will be carried out in two stages by creating covalent bonds during the deposition of nitrophenyl groups  (0.025 mg in 50 mL of acetonitrile) and tetrafluoroborate (tetrabutylammonium) tetrafluoroborate (TBA 1.65 g in 50 mL of acetonitrile). The electrochemical reaction between the graphene surface in the chip and the anhydrous electrolyte was carried out in a cell, of which the design allows purging the electrolyte and the space above it with a clean and dry inert gas (argon) to remove traces of moisture from the ambient air. The process was carried out according to a three-electrode scheme. The graphene surface in the chip was a working electrode, a platinum plate served as a counter electrode, and a silver wire (Ag/Ag+) served as a reference electrode.The reaction of attaching of nitrobenzene occurred during cycling of the potential at the working electrode (the graphene surface) in the range from 0 mV to −600 mV and vice versa. In this case, the process current was recorded. At the second step of CV process, the reduction of nitrobenzene attached to graphene to aminobenzene was carried out already in an aqueous solution of 0.1 M KCl and ethyl alcohol (9:1) in an open flask also according to a three-electrode scheme. The electrodes were: A standard silver chloride reference electrode (Ag/AgCl) Esr-10101 , the graphene surface in the chip was a working electrode (anode), and a platinum plate counter electrode. The reduction reaction was also carried out in the mode of cycling the potential on the working electrode from 0mV to −1000 mV and vice versa. After the process, the samples were washed in deionized water and dried in argon. Typical CVs of reduction processes are shown in After functionalization, the deposition (or immobilization) of antibody molecules took place on the created covalent bonds. Then, the graphene surface was passivated (blocked) in a solution of 0.1% BSA in PBS  for 1 hour, followed by washing in pure PBS for 5 minutes. After that, the graphene chip is ready to apply as a biosensor.First, the response of the chip (current through the chip) was investigated during the reaction of complementary antibodies and antigens of the influenza A virus (antibody A-antigen A) on the prepared graphene surface. We used strains of influenza viruses obtained from the Smorodintsev Research Institute of Influenza, St. Petersburg, Russia: influenza virus A/California/07/09 (H1N1pdm09) and influenza virus B/Brisbane/46/15.At the beginning, antibodies to influenza A or B virus were immobilized on chips with functionalized graphene surface for 3 h at 37 °C. The monoclonal antibodies to influenza A or B viruses used in the experiment are directed to nucleoprotein (NP), a highly conserved major protein of the influenza virus associated with RNA (ribonucleic acid). Then, the graphene surface was passivated (blocked) in a solution of 0.1% BSA in PBS  for 1 h, followed by washing in pure PBS for 5 min The graphene chips (sensors) prepared in this way were used in the detection experiments.−15 g/mL to 1 × 10−10 g/mL , 6 dilutions and 12 tubes in total. Since it was initially assumed that chips based on graphene monolayers could be very sensitive sensors, the range of selected concentrations of antigens to be detected was chosen below the detection limit of the PCR (polymerase chain reaction) test widely used in enzyme-linked immunosorbent assay for detecting viruses. This should confirm the advantage of graphene sensors in terms of sensing ability. The experiments described below showed that this choice of the ultra-low concentration range of antigens was justified, and the chips showed their sensitivity down to a concentration of 1 × 10−15 g/mL (1 fg/mL). Note that the work did not set the task of determining the sensitivity limit of the chips being developed for the detection of influenza viruses.Before the sensing experiments, two groups of dilutions were prepared in PBS for influenza A virus and influenza B virus at different concentrations from 1 × 10First, the direct reaction of the complementary antibody and antigen on the surface of graphene in the chip was investigated. The prepared graphene sensor was first incubated with pure PBS , then its response (current passing through the chip) was recorded at DC conditions when, depending on the sensor resistance, a sequence of DC voltage of 20 mV, 40 mV, 60 mV, and 80 mV, was applied between chip contact pads for 20–30 s. Then, a graphene sensor was incubated into a tube with the PBS solution of an antigen A (or B) virus for 20–30 s and its response was recorded. The chip was sequentially incubated in the different antigen virus solutions one by one from low to high concentration of the antigen virus. First, the direct reaction of complementary antibodies and antigen on the surface of graphene in a chip was investigated. An example of the timing diagrams of the chip response during its sequential incubation in PBS solutions of influenza A virus (AV) antigens is given in The data in 2 = 0.96. The dependency in 2 becomes smaller for a DC voltage of 80 mV due to the scatter of the data. We think this scatter reflects the specificity of reactions on graphene surfaces with terraces, which, according to AFM scanning, have the width of up to 1100 nm for chips of this series (EG313). The logarithmic dependence was observed earlier for graphene-based biosensor when studying its response on egg albumin solutions in PBS [The data in s in PBS . Note, iIn continuation of these studies, additional passivation of graphene in an albumin solution (0.1% BSA in PBS) was performed after immobilization of the antibody during the preparation of the chips for the next experiments. In addition, the chips had different widths of terraces on the graphene surface. The timing diagrams of the chip’s response (current through the chip) were similar to those shown in The designations of the chips in this work at different stages of experiments are presented in the −12 g/mL and above. In contrast, EG331-61 chip with wider terraces (up to 4 microns) showed a decrease in chip response with an increase in the concentration of virus A virus antigen with a clear saturation of the response at concentration of 1 × 10−12 g/mL (1 pg/mL) and above.EG313-41 chip, which has a terrace on the surface of graphene about 1 µm wide, shows the dependence of the increased response of the chip (current through the chip) with the concentration of antigen A in the solution that is well approximated by logarithmic function with the parameter R2-0.94. In contrast, the chip with wider terraces (up to 4 microns) showed a decrease in chip response with an increase in the concentration of virus A virus antigen with a clear saturation of the response at concentrations of 1 × 10During the functionalization of graphene, unsaturated covalent bonds are created on its surface that promote to the attachment (immobilization) of the necessary antibodies. At the detection process, only complementary (related) antigens can react with immobilized antibodies noticeably changing the electronic state of graphene. This is the main principle of operation the sensors that are being developed. The interaction of the remaining non-complementary molecules with the graphene surface does not have the character of a chemical reaction, but rather occurs due to weak van der Waals forces, possibly due to defects and imperfections of the graphene/SiC substrate composition. This interaction can be further screened to reduce its effect on the electronic properties of graphene by passivating the graphene surface with neutral molecules like albumin. However, the attachment of molecules to the graphene surface that are non-complementary to the immobilized antibody can have a significant effect on the resulting chip response (current through the chip).We believe that a decrease and saturation of the chip response (current through the chip) in Too small of a number of sites with an immobilized virus A antibody creates conditions when the virus A antigens, the concentration of which is higher than the A virus antigens, are forced to directly attach to the graphene surface due to van der Waals forces, thus significantly affecting the nature of the change in the chip response. Apparently, the graphene surface treatment and graphene functionalization conditions should take into account the specifics of graphene morphology, in particular, the width and height of terraces on its surface.Special experiments were performed to demonstrate specificity of the antibody–antigen interaction on the graphene surface. The experiments were carried out in two stages.The procedure for the first stage of the experiment was the same as described above with one exception. The only difference was that the prepared chip with the immobilized antibody was immersed (incubated) in solutions of non-complementary antigens of viruses. In other words, the chip with the immobilized antibodies of the virus A was immersed in solutions with the antigens of the virus B and its response (current through the chip) was recorded. Similarly, the chip with the immobilized antibodies of virus B was incubated in solutions with the antigen of the virus A.At the second stage of the experiment, the same chip was immersed for 20 s in other solutions but with virus B antigens (second incubation) that were complementary (related) to the antibody virus B immobilized on the chip. The chip response (current through the chip) was also recorded. After the experiments, the responses of graphene chip (current through the chip) were compared and studied.As before, the timing diagrams of the chip response (current through the chip) obtained at both stages of the experiment were similar to those shown in In this case, the spread between the maximum and minimum response values for the EG260-9 chip with narrow terraces (400–600 nm) reaches less than 200 nA, and up to 530–550 nm for other chips with large terraces width.2 0.9–0.97.The results of the second stage of the experiments are shown in −10 g/mL (100 pg/mL), which is possibly due to the saturation of antibodies on the graphene surface by the reaction with antigens in a PBS solution. When approximating the experimental results, this point was not taken into account.For the EG260-9 chip results in For the EG313-15 chip with moderately wide steps on the surface (1 μm), a similar logarithmic dependence was observed in 2 to 0.9 when approximating the experimental results. It should be noted that in this study the EG313 series chips most often showed the dependence of the response on the concentration of antibodies A or B in the form of a logarithmic function (parameter R2 more than 0.9) over the entire range of investigated concentrations.The scatter of the data reduced the parameter R−15 g/mL (1 fg/mL) in PBS solution. A similar maximum response value was observed for the EG331-61 chip in The EG332-13 chip in We attribute this feature to the fact that in highly dilute solutions the antigen interacts not only with immobilized complementary antibody molecules on the surface, but also directly deposited on the graphene surface if the sites with antibodies are located at a great distance from each other or there are simply few of them. The presented results for chips with wide steps in Note also that the passivation of the graphene surface in chips with albumin is necessary rather to saturate the covalent bonds that have not attached the antigen than to passivate the graphene surface . Real pa−15 g/mL to 1 × 10−10 g/mL is shown.In conclusion, a series of experiments on the detection of virus antigens A and B with chips (sensors) based on graphene monolayer films on SiC was carried out. The sensitivity of the chips for the detection of diluted solutions of virus antigens in PBS solutions in the concentration range from 1 × 10The specificity of the reaсtion antibody A—antigen B and antibody B—antigen A on the graphene surface was demonstrated. The results confirm the principle of operation of the sensor based on the reaction of interaction of a complementary antibody and an antigen on the graphene surface. The surface morphology of a graphene monolayer, in particular the width of characteristic terraces, affects the response of the chip (current through the chip) when detecting influenza viruses.The advent of graphene opened a new page in solid state physics—the production and study of two-dimensional materials. The development of research rather quickly raised the question of the possibility of practical application of these materials. It was obvious that the method of mechanical exfoliation, which produced the first samples of graphene, was not suitable for industrial production.In this work, we considered the most promising technology for producing graphene films, namely, thermal destruction of the silicon carbide surface. This technology makes it possible to obtain films with a sufficiently high structural perfection, and when using semi-insulating SiC, there is no need to transfer the grown graphene to a dielectric substrate. To create devices, one can use post-growth technologies common for semiconductor production.The presence of electrophysical parameters of graphene such as the maximum ratio of area to volume, a low concentration of carriers in combination with their high mobility, and a low noise intensity makes it possible to obtain supersensitive resistive sensors on its basis. The practical application of graphene gas sensors is hindered by the lack of selectivity in the registration of various gases. The use of an antigen—antibody pair allows one to solve the problem of biosensor selectivity and opens up very wide possibilities for the use of graphene-based sensors in medicine and biology. This approach may lead to the creation of portable biosensors capable of detecting diagnostically significant markers of diseases in biological fluids in the express analysis mode.


In this study, the three-phase structure consisting of epoxy resin, carbon nanotubes (CNTs), and graphene, which is assumed to be the surface of carbon fiber, was simulated using molecular dynamics. Models in which the CNT number and initial position of CNT are varied were prepared in this study. Relaxation calculation for each three-phase model was implemented, and the movement of molecules was investigated. When CNTs are located between the graphene and epoxy at initial, how the epoxy approaches to graphene was discussed. Besides, interaction energies between CNT/graphene, CNT/epoxy, and graphene/epoxy were evaluated after relaxations. The value of the interaction energy between two individual molecules  among three-phase structure were obtained, respectively, and those mechanisms were discussed in this study. Carbon fiber reinforced plastic (CFRP) uses resin as a base material and carbon fiber as a reinforcing agent. Lightweight, high strength, and high stiffness are the essential properties of CFRP. Therefore, it is used in aircraft, wind turbines, satellites . The excRecently, many studies have been conducted using molecular dynamics (MD) simulations, including several studies on the molecular interface that is related to a component of materials ,37,38,39Yu B et al.  emphasizThere are some studies on the interface of the graphene/CNTs/epoxy resin model ,43. ZhanIn this study, we focused on the interaction between two individual molecules in a three-phase structure and calculated the interaction energy between two individual molecules, considering the influence of another molecule on the three-phase structure. We emphasized the interaction between epoxy resin and CNTs, which are arranged in parallel with graphene, and also emphasized the π–π interaction between benzenes. We observed the behavior of CNTs and epoxy resin and compared the interaction energy between CNTs-epoxy resin and CNTs-graphene. Our overarching aim was to clarify the structure of graphene/CNTs/epoxy resin.x, y, z) =  (Å); CNTs  and , with diameters of 0.5420 and 1.084 nm, respectively. J-OCTA 5.0 software  was used for analysis and VSOP  as the MD engine. J-OCTA5.0 enabled us to create CNTs and to input force field parameters easier. The DREIDING force field parameters were used [We created a full atomic model. To model the three-phase structure, we prepared each molecule: epoxy resin (number of particles: 8960); three graphene sheets of cell size:  =  (Å). We created the graphene with 1944 carbon atoms. Subsequently, three graphene sheets were stacked in the z-direction with cell size:  =  (Å). The molecular structure is shown in We made epoxy resin using bisphenol A diglycidyl ether of bisphenol A (DGEBA), with a molecular weight of 344.4, as an epoxy monomer, and ethylenediamine (EDA), with a molecular weight of 60.10, as the curing agent. The structural formula of each reagent is presented in z-direction and created a total of six types of three-phase structures: (a) two CNTs ; (b) three CNTs ; (c) two CNTs ; (d) two CNTs , considering temperature changes; (e) four CNTs  parallel arrangements; (f) four CNTs  vertical arrangements. In the actual structure, CNTs adhered to the surface of the carbon fiber in a dispersed state. Models with CNTs  parallel and vertical arrangements were prepared, respectively, to simulate a similar structure. The molecular structures are shown in x, y, z) =  (Å). For the model with CNT  in z-direction was 190.0 Å. CNT  had a larger diameter than CNT . Thus, we made room for the entire structure by extending the z-direction of the cell. The graphene placed at the bottom was fixed. We considered periodic boundary conditions in the x, y, and z directions. We arranged the epoxy resin, CNTs, and graphene from top to bottom in the The relaxation calculation procedure for each three-phase structure is presented below:(1) The canonical ensemble which is performed with constant volume, temperature, and number of particles (NVT ensemble) was created at a temperature of 300 K. Step time was 0.5 fs. The total number of steps was 1000. Relaxation time was 2 ns.z-direction. The step time was 0.5 fs. The total number of steps was 1000. Relaxation time was 2 ns. There was an exception; the initial temperature was set to 600 K for one model with two CNTs . Compression was performed, while the temperature was lowered from 600 K to 300 K to investigate the behavior and interaction energy difference when a temperature change from high to low occurred at constant pressure.(2) The isothermal-isobaric ensemble (NPT ensemble) was set at a temperature of 300 K and an atmospheric pressure of 0.1 MPa in the (3) The NVT ensemble was created at a temperature of 300 K. Step time was 0.5 fs. The total number of steps was 1000. Relaxation time was 2 ns. The model with four CNTs  arranged vertically was also examined with the relaxation time increased to 10 ns.The procedure used the Nose-Hoover method for temperature control and the Parrinello-Rahman method for pressure control.After relaxation, the behavior of each molecule was observed, and the interaction energy was derived. The interaction energy between one molecule and other molecules was calculated using Equations (1)–(3), respectively.We also calculated the interaction energy between two individual molecules. The interaction energy between epoxy and graphene in the system was After calculating the interaction energy values, we used absolute values for each of the interaction energy values to make it easier to compare them.We observed the molecular behavior during relaxation in the NVT ensemble. In all six structure types, immediately after starting relaxation, the epoxy resin above the CNTs was drawn toward the fixed graphene. Next, we confirmed the cell compression in the NPT ensemble. The structure after relaxation in the NVT ensemble is shown in xy plane than that of a small-diameter CNTs, and thus we inferred that this was caused by more stable π–π interactions with graphene. Considering temperature, the model subjected to temperature change showed a larger value than the model subjected to a constant temperature. However, it was a numerical value that could be treated as an error. Finally, the amount of interaction energy changed depending on the arrangement of the CNTs. The interaction energy of the model with four CNTs  vertical arrangement was lower than that of the model with parallel arrangements. This is because the epoxy resin did not reach the CNTs at the bottom of the structure, and consequently, the amount of interaction with graphene was lower. The values of We obtained the values of the interaction energy between one molecule and other molecules. The values of xy plane was arranged parallel to the benzene in the graphene. The benzenes arranged in parallel show strong π–π interaction, whereas the interaction between the non-parallel benzenes shows weak π–π interaction [The interaction energy values for eraction . Consequeraction . ConsequWe analyzed a three-phase structure composed of epoxy resin, CNTs, and graphene using MD simulation. First, we confirmed the structure of the molecules after relaxation. The epoxy resin penetrated in between the small-diameter CNTs but did not penetrate in between the large-diameter CNTs. In the model with the CNTs arranged vertically, it was observed that the epoxy resin didn’t reach the graphene because the CNTs become an obstacle. Furthermore, we obtained the interaction energy between two individual molecules. The interaction energy value increased as the CNT diameter increased and also as the number of CNTs increased. Based on


Background: In light of the COVID-19 pandemic and resulting demand for innovative hospital management we organized an interactive online discussion for medical students and healthcare professionals about hospital management during the crisis.Objective: The event offered an opportunity to learn from a hospital crisis management. We looked at how this new online format compares to a traditional discussion event. Methods: We used an online platform with four guests, a moderator and about 100 attendees. During the event we gathered demographic facts through an interactive questionnaire tool and an extensive evaluation afterwards.Results: The event was rated with an overall grade of 1.4  and participants agreed that this format should be organized again . 70% of audience members preferred the online format of the event. Due to the high volume, only about 30%  of the questions posed by the audience were addressed.Conclusion: Firstly, most participants preferred the event to be online, contrary to our expectation. Secondly, the handling of the amount of individual questions posed significant challenges.Finally, the number of attendees and questions suggested a continuing demand among students and physicians for further education regarding hospital management, especially regarding COVID-19. These findings also require a critical look at future formats and topics of podium discussions in medical education. The online format might be a good alternative to face-to-face lectures.  MeCuM-SiGma  is a course designed to teach the basics of healthcare management and policy to medical students , 2], , , 5]..5].The COVID-19 pandemic made increasingly clear that hospital management is vital in preventing shortages, allocating staff efficiently and providing the best care possible in an overextended healthcare system , 7]. It. It7]. IThe entire event was held via an online platform (Zoom). The audience was muted for the duration of the event and only submitted questions via chat. The total length of the event was about 2 hours. During the event, we gathered demographic facts about the audience using an interactive questionnaire (60% response rate). Participants also completed a post-event evaluation form (30% response rate).142 people registered for the event. The maximum number of simultaneously logged in guests was 100. At 1,5 hours some participants left the chatroom, 70 participants remained until the end. 80% of the respondents of our live survey were medical students and 16% were physicians. Most participants did not have any hospital management experience (69%). In the follow-up evaluation (n=30) the mean age was 30 years (SD 10) and 47% of participants were female. The event was rated with an overall grade of 1.4  and participants agreed that this format should be organized again next year . The grading was comparable to four similar in-person events . 69% of participants stated that they were doing something else on their computer and they generally rated the possibility of parallel work as positive . Surprisingly, 70% of audience members preferred the online format. Participants indicated that they were unhappy with the handling of the posed questions. 35 questions were asked, 40% about hospital management during the crisis, 20% about future planning, 20% about policy and 20% other categories. Few questions were asked about personal or student topics. Due to the high volume of questions the moderator could only address about 30% of the questions posed.This case report is about a one-time event; hence we are cautious to draw general conclusions. However, our results show some interesting trends. The event was evaluated positively. Participants favored being able to complete other tasks simultaneously. It has been shown before that online multitasking is more common than at in-person events , also thThe handling of questions posed significant challenges. Contrary to classroom events, where questions are asked individually, new online formats, especially chat boxes, allow a simultaneous posing of questions. Although many audience members asked questions (ca. 35%), indicating high engagement, the participants were unhappy that many questions went unaddressed. Based on our experience organizing similar in-person events, less questions are asked in person even when the overall ratings of the event are comparable.The number of attendees and questions asked suggests a robust demand among medical students and physicians for further education regarding hospital management, especially on the topic of COVID-19. These findings require a critical look at the format of future podium discussions in medical education. Many participants preferred the online format and topic. Online discussions can be a good alternative to, or additional feature of, in-person lectures even post-COVID-19. It is advisable to have a concept to deal with a high volume of questions during online discussions. In the future we will collect unanswered questions and ask guests to answer them in written form or as a podcast after the event. In regard to learners’ attention and amount of questions, it remains to be examined whether the anonymity of an online event is advantageous.The authors declare that they have no competing interests.


The investigated genes displayed a unique impact on TDP-43 aggregation, generating distinct types of cytoplasmic inclusions, similar to those already described as resembling prion strains, which could represent the basis for neurodegenerative disease heterogeneity.Amyotrophic lateral sclerosis is a progressive neurodegenerative disorder, characterized by cytoplasmic inclusions of RNA-binding protein TDP-43. Despite decades of research and identification of more than 50 genes associated with amyotrophic lateral sclerosis (ALS), the cause of TDP-43 translocation from the nucleus and its aggregation in the cytoplasm still remains unknown. Our study addressed the impact of selected ALS-associated genes on TDP-43 aggregation behavior in wild-type and aggregation prone TDP-43 in vitro cell models. These were developed by deleting TDP-43 nuclear localization signal and stepwise shortening its low-complexity region. The SH-SY5Y cells were co-transfected with the constructs of aggregation-prone TDP-43 and wild-type or mutant ALS-associated genes  N-terminal oligomerization domain, a classical bipartite nuclear localization signal (NLS), two RNA recognition motifs (RRM1 and RRM2) and a low-complexity domain (LCD) [Amyotrophic lateral sclerosis (ALS) is a fatal progressive neurodegenerative disease pathologically characterized by cytoplasmic deposits of misfolded proteins in the affected neurons. The main component of these inclusions represents ubiquitinated, phosphorylated and cleaved TAR DNA-binding protein 43 (TDP-43) . Under pin (LCD) ,6. Whilein (LCD) ,8,9. So in (LCD) ,19,20,21in (LCD) . Still, in (LCD) .N-terminal domain in TDP-43 aggregation is somewhat controversial: while some argue that the oligomerization of TDP-43 via its N-terminal domain allows interactions with other partner proteins and RNA, thereby antagonizing its pathological aggregation, others suggest that it enhances the propensity of the intrinsically disordered C-terminal region towards aggregation [N-terminal domain reduces propensity of TDP-43 to undergo LLPS and to form aggregates [Recently, a liquid-liquid phase separation (LLPS) of RNA-binding proteins, such as TDP-43, has been implicated as a possible inception mechanism for protein aggregation and subsequent self-templating, ultimately resulting in various types of irreversible inclusions, i.e., amyloid-like fibrils in neurodegenerative disease . The mairegation ,32. It hgregates ,34. Moregregates ,37,38,39Mounting evidence suggests that cytoplasmic accumulations of TDP-43 exhibit prion-like characteristics . In addiN-terminal region of TDP-43 intact, as it is necessary for its oligomerization and phase-separation, although some studies have achieved TDP-43 aggregation with only the C-terminal region present [First, we developed a novel in vitro TDP-43 aggregation model in the neuroblastoma SH-SY5Y cell line. To achieve TDP-43 cytoplasmic aggregation, we eliminated NLS from the full-length TDP-43 sequence. Then, we stepwise shortened its LCD (containing IDR and CR domains), to disrupt the physiological conformation of TDP-43 dimers. We left the  present ,48. FullTDP-43 constructs: TDP-43 was subcloned from previously published plasmid  into mKOALS-linked genes: First, a gBlock DNA fragment (IDT) containing HRV 3C site, Gly-Ser-Gly linker and 3xHA was amplified by Phusion™ High-Fidelity DNA Polymerase (primer sequences are listed in ® CRL-2266™) were cultured as a monolayer in DMEM/F-12 (Thermo Fisher Scientific), supplemented with 10% (v/v) FBS (Thermo Fisher Scientific) and penicillin-streptomycin solution (Thermo Fisher Scientific) at 37 °C, in a humidified atmosphere containing 5% CO2. For immunocytochemistry, 1.1 × 105 cells/well were plated on coverslips into 24-well plates, 24 h before transfection. For western blot analysis, 2.2 × 105 cells/well were plated into 12-well plates and 6.5 × 105 cells/well were plated on 6-well plates, 24 h before transfection. Cells were transiently transfected using Xfect transfection reagent according to manufacturer instructions . Co-transfection experiments were carried out with plasmids DNA in ratios 1:1 amounting to 1 µg total DNA/well for 24-well plates, 2.5 µg total DNA/well for 12-well plates and 7.5 µg total DNA/well for 6-well plates. All of the co-transfections were carried out side-by-side at the same time, to allow comparisons between different experimental conditions.Neuroblastoma cells SH-SY5Y  was added to the pellets. The pellets were resuspended by pipetting and centrifuged again at 21,000× g for 30 min at 22 °C. The supernatants were collected as an UREA-soluble fraction. To both RIPA- and UREA-soluble fractions, the 6x SDS loading buffer with 0.3 M DTT was added, and RIPA-soluble fractions were boiled at 100 °C for 10 min. All samples were loaded to Novex WedgeWell 8–16% Tris-Glycine gels (Invitrogen Thermo Fisher Scientific) and run in SDS running buffer at 120 V for 90 min. Proteins were transferred onto Trans-Blot Turbo mini-size nitrocellulose membrane  by Trans-Blot Turbo Transfer System (Bio-Rad) in Trans-Blot Turbo Transfer buffer (Bio-Rad). Membranes were washed once in TBS, blocked in 5% skim milk in 0.1% TBST for 30 min, and then incubated overnight with primary antibodies in 5% skim milk in 0.1% TBST . The next day, the membranes were washed 3 times in 0.1% TBST and incubated with secondary antibodies in 5% skim milk in 0.1% TBST for 1.5 h . Upon three washes in 0.1% TBST, the fluorescence signal was detected using Chemidoc (Bio-Rad).For total cell lysates, 24 h after transfection, cells in 12-well plates were washed 3x with ice-cold PBS, and lysed in 2x SDS loading buffer with 0.1 M DTT. Samples were boiled at 100 °C for 10 min. For RIPA-soluble fraction, cells in 6-well plates were washed 3x with ice-cold PBS and lysed in 200 µL RIPA buffer , containing protease inhibitors  and phosphatase inhibitors . Samples were sonicated using an ultrasonic probe  and centrifuged at 21,000× Twenty-four hours after transfection, cells were washed 3x with PBS, fixed in 4% PFA for 30 min and washed with PBS again. Then, they were permeabilized with 0.1% TX-100 in PBS for 10 min, blocked in 3% BSA in PBS for 30 min and incubated with primary antibodies (anti-HA-tag (C29F4) rabbit polyclonal Cell Signaling #3724, 1:500), overnight at 4 °C. The next day, the coverslips were washed, incubated with secondary antibodies  for 2 h, incubated in DAPI solution (0.1 μg/mL) (Thermo Fisher Scientific) for 15 min, and mounted on microscope slides with ProLong™ Gold Antifade Mountant (Thermo Fisher Scientific).SEM. The difference between averages was tested by ordinary one-way ANOVA followed by Tukey multiple comparisons test, using GraphPad Prism 6. The p-value < 0.05 was considered statistically significant. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.Slides were analyzed by confocal microscopy , using ZEN software. Three experiments were performed in duplicates for each experimental condition, and a minimum of 15 visual fields, each containing between 50–70 cells from each duplicate were taken. Overall, between 550 and 3400 transfected cells were analyzed per each experimental condition. The percentage of transfected cells harboring TDP-43 aggregates was quantified by manual counting. A further analysis of the number of aggregates per cell and their size were performed with ImageJ Shape Descriptors plugin . All data were recorded as averages ± C-terminal domain on TDP-43 aggregation, SH-SY5Y cells were transfected with various mKO2 tagged TDP-43 constructs, lacking NLS (dNLS) and different parts of LCD —see C-terminal deletions of CR (dNLSd299) or IDR1 (dNLSd267) of TDP-43, decreased the percentage of cells with dNLSd299 (6.9 ± 0.57%) and dNLSd267 (4.1 ± 1.37%) aggregates. Further aggregate analyses within each individual cell revealed that the expression of dNLS resulted in the highest number of cytoplasmic aggregates per individual cell (14.1 ± 0.80), compared to the expression of any other TDP-43 truncated form (2) and dNLSd299 (4.8 ± 0.61 µm2) constructs  was used as a baseline control of the aggregate formation  and dNLSd343 (1.2 ± 0.14 µm2) aggregates in comparison with the cells co-transfected with control HAonly (dNLS (2.1 ± 0.34 µm2) and dNLSd343 (3.9 ± 0.39 µm2)). The decreased aggregate size appeared more pronounced in case of dNLSd343 than dNLS cells co-transfected with either wild-type or mutant hnRNPA1  and MATR3mut (3.7 ± 0.30 µm2) evidently increased the size of aggregates in cells co-transfected with dNLS  and VCPmut (4.2 ± 0.24 µm2) co-transfected cells, compared to HAonly (2.1 ± 0.34 µm2) (2) and VCPmut (2.5 ± 0.35 µm2) with dNLSd343, suggesting the involvement of IDR2 domain of TDP-43 in VCP interactions, and its necessity for TDP-43 aggregate maturation.The co-transfection of dNLS or dNLSd343 with VCPmut C resulte.34 µm2) C,D. Addi.34 µm2) C. No sucSimilar to VCPmut, the co-transfection of TDP-43 dNLS construct with UBQLN2mut  39.0 ± A. On theDespite the overwhelming majority of ALS cases being characterized by cytoplasmic deposits of TDP-43 protein in the affected cells, ALS patients display large phenotypic variability . As was N-terminal domain spatially separates aggregation-prone LCD [C-terminal sequence. The TDP-43 fragments were designed based on the borders of TDP-43 LCD segments determined by Schmidt and colleagues [Therefore, our study aimed to explore the influence of ALS-related genes and their mutations on aggregation pattern of TDP-43 in vitro. First, we developed a TDP-43 aggregation model in neuroblastoma SH-SY5Y cell line. To ensure cytoplasmic localization of TDP-43, we deleted NLS from the full-length sequence. As physiological TDP-43 oligomerization via its rone LCD , we trielleagues . Like otlleagues ,55, we slleagues , our expExpression of TDP-43 dNLS and dNLSd343 exhibited two dissimilar yet distinct aggregation patterns: dNLS displayed numerous dispersed and small aggregates, whereas dNLSd343 displayed fewer large bulk aggregates. Therefore, we further investigated both of them in the context of the influence ALS-associated genes may have on TDP-43 aggregation. We selected four different genes involved in ALS pathogenesis, each of them with a distinct role within the cell. Four chosen ALS-associated genes in their wild-type and mutant form were co-expressed with TDP-43wt, dNLS, dNLSd343 and KO2only in SH-SY5Y cells. The expression of all transfected constructs was confirmed by Western blotting. Even though the co-transfections of ALS-associated genes’ constructs with dNLSd343 displayed expression at really low levels, as compared to other TDP-43 constructs, they produced higher percentage of transfected cells with aggregates.C-terminal part of TDP-43 directly interacts with C-terminal domain of hnRNPA1 [C-terminal domain form smaller aggregates compared to a full-length protein [C-terminal domains spatially separate aggregation-prone LCDs and reduce the number of large aggregates, as observed in our study. Interestingly, even though hnRNPA1 undergoes LLPS and can form irreversible fibrils, which are enhanced by ALS-linked mutation D262V [Notably, hnRNPA1 is a member of the hnRNP protein family, involved in splicing, mRNA stabilization, miRNA biogenesis and transcriptional regulation ,57,58,59 hnRNPA1  and that hnRNPA1 , we can  protein . This ison D262V ,27,63, wN-terminal sequence of MATR3 exhibits low complexity and is able to undergo phase separation to form liquid-like droplets in the nucleus [MATR3 is also an RNA-binding protein, localized in the nuclear matrix and involved in various tasks concerning nucleic acids, such as chromatin organization, DNA replication, transcription and repair, RNA processing, transport, stability and alternative splicing ,65,66,67 nucleus ,71. The  nucleus , possiblIn contrast to previously discussed proteins mainly associated with RNA processes, one of VCP’s main duties is facilitating the proteasomal degradation of damaged or misfolded proteins by binding to polyubiquinated proteins and delivering them to the proteasome complex ,74,75. SLike VCP, UBQLN2 participates in proteolytic processes, such as delivering ubiquitinated proteins to the proteasome. Besides, it is also involved in autophagy, cell signaling, cell cycle progression and cytoskeletal association . SimilarOver the recent years, the extent of ALS heterogeneity, as well as its clinical, pathological and genetic overlap with several other degenerative disorders has become increasingly evident . NeverthHowever, determining the influence of ALS-associated genes on the aggregation pattern of TDP-43 remains the first step in elucidating the mechanisms of TDP-43 aggregation. It would be important to confirm these differences in TDP-43 aggregation patterns directly in the aggregates extracted from post mortem tissue or from motor neurons derived from ALS patients, with distinct mutations in ALS-associated genes. Additional studies, revealing the effects of TDP-43 interactors with the potential to reduce or accelerate TDP-43 aggregation in combination with different mutations of ALS-associated genes, are definitely needed.In summary, our results underline the significance of comparative research in the ALS field and provide an understanding of how different mechanisms interact at the molecular level and lead to neurodegeneration that may prove crucial in future for the successful development of therapeutics.


We discover that m6A ‘writer’ METTL3 facilitates L1 retrotransposition, whereas m6A ‘eraser’ ALKBH5 suppresses it. The essential m6A cluster that is located on L1 5′ UTR serves as a docking site for eukaryotic initiation factor 3 (eIF3), enhances translational efficiency and promotes the formation of L1 ribonucleoprotein. Furthermore, through the comparative analysis of human- and primate-specific L1 lineages, we find that the most functional m6A motif-containing L1s have been positively selected and became a distinctive feature of evolutionarily young L1s. Thus, our findings demonstrate that L1 retrotransposons hijack the RNA m6A modification system for their successful replication.L1 retrotransposons can pose a threat to genome integrity. The host has evolved to restrict L1 replication. However, mechanisms underlying L1 propagation out of the host surveillance remains unclear. Here, we propose an evolutionary survival strategy of L1, which exploits RNA m 6A modifications on L1 RNA increase translation efficiency and retrotransposition in human cells. M6A motifs are more enriched in evolutionary young L1s.L1 is a group of active retrotransposons in humans. Here the authors show that m The average human genome contains 80–100 copies of retrotransposition-competent L1s3. A retrotransposition-competent L1 is 6 kb in length and consists of a 5′ untranslated region (UTR) containing an internal promoter4, two open reading frames (ORF1 and ORF2), and a short 3′ UTR. ORF1 encodes a nucleic acid chaperon protein (ORF1p)5, while ORF2 encodes a protein with endonuclease and reverse transcriptase activity (ORF2p)7. ORF1p and ORF2p associate preferentially with their parental mRNA to form an L1 ribonucleoprotein (RNP) particle8. The L1 RNP enters the nucleus and then generate the progeny through de novo insertion of its cDNA10. The mobility of L1s contributed to a source of genetic variation, but also pose a threat to genome integrity13. Although several host factors have evolved to suppress L1 retrotransposition, the youngest L1 subfamilies are still active and replicated continuously16. To date, however, the mechanism of how L1s have propagated under host surveillance remains unknown.Long interspersed element-1 (L1) is currently an active autonomous retrotransposon, and constitutes ~17% of the human genome6A) is the most prevalent internal modification in eukaryotic mRNAs, which determines RNA function and fate17. Several enzymes dynamically process the m6A modification of mRNA. The methyltransferase-like enzyme METTL3, which is the catalytic subunit of the RNA methyltransferase complex, adds m6A at the consensus motif DRAmCH 19. Conversely, m6A is removed by the demethylases α-ketoglutarate-dependent dioxygenase AlkB homolog 5 (ALKBH5) or fat mass and obesity-associated protein (FTO)21. Emerging studies have revealed that m6A modifications in viral transcripts affect the gene expression and replication of viruses such as HIV-122. Despite the critical role of m6A in pathogenic viral transcripts, it remains unclear whether m6A participates in the regulation of the endogenous parasites, L1 retrotransposons.N6-methyladenosine  for efficient translation and promotes the formation of L1 RNP, which are essential for L1 mobility. Lastly, we traced a recent episode of human- and primate-specific L1 evolution and revealed that the most functional m6A site (A332 residue in L1 5′ UTR) first appeared ~12 million years ago. During the primate evolution, A332 m6A-positive L1s have been selected and became a distinctive feature of evolutionarily young L1s, which suggests that the acquisition of m6A motif has acted as an evolutionary driving force for L1 retrotransposons.Here, we show that L1 retrotransposon exploits m6A modification affects L1 retrotransposition, we evaluated the effects of the RNA m6A machinery on L1 retrotransposition using a cell-based engineered L1-reporter assay23. For the assay, we used the pJJ101-L1-dn6 2.2 construct (hereafter referred to as pL1Hs) that contains a blasticidin S deaminase gene (mblastI) within the 3′ UTR antisense to the SV40 promoter25  in HeLa cells and transfected pL1Hs vector. In METTL3-depleted cells, the number of blasticidin S-resistant colonies, which represent successful L1 retrotransposition, was reduced by >2-fold compared to that of control siRNA . As anticipated, ALKBH5 could successfully restrained L1 mobility to levels that were comparable to that suppressed by a reverse transcription inhibitor , whereas ALKBH5H204A overexpression did not result in the restriction of L1 mobility  using human embryonic stem cells (H9 hESCs) that express endogenous L1 at sufficient levels30. Through qRT-PCR analysis of the MeRIP eluates, we detected the enrichment of L1 RNA at a level comparable to that for known m6A-modified SON and CREBBP mRNA, but much more than negative control HPRT1 mRNA 33. SRAMP analysis revealed that the 9 peaks found in the ORF1, ORF2, and 3′ UTR regions do not contain m6A motifs, and that only the 6 peaks located at 5′ UTR have potential m6A motifs  into HeLa cells treated with m6A machinery-targeting siRNA and monitored ORF1p expression. Intriguingly, the knockdown of m6A enzymes did not affect ORF1p expression in the absence of 5′ UTR  through MeRIP-seq analysis in either hESCs or L1-reporter-expressing HeLa cells  markedly inhibited L1 mobility  induced approximately 50% decline in L1 mobility compared to that induced by the wild-type L1  and MALAT1 (abundant in the nucleus), over 80% of the L1 mRNA was present in the cytoplasmic fraction and the m6A-deficient mutation did not affect the cellular localization of L1 RNA  of eIF3 subunits a, b, d, and g38. By mapping the reads from PAR-CLIP of the eIF3 subunits along the endogenous L1Hs, we revealed that eIF3 exhibits preferential binding to the L1 5′ UTR  to gauge the reverse transcriptase activity of ORF2p41 . In L1PA2 and younger lineages, the number of A332 m6A positive L1s increased drastically (92.9%)  first appeared in L1PA3 or older lineages, which indicates that the productive potential of m6A has allowed positive selection of A332 m6A-positive L1s during the evolution from the common ancestor.Over the last 40 million years of human evolution, L1 subfamilies have frequently acquired novel 5′ UTRs9%) Fig. , and the9%) Fig. . On the 9%) Fig. . We inve600 Fig. . As in ttif Fig. . In summ6A acquisition in ancestral L1 5′ UTR, we generated a chimeric pL1 construct that contained L1PA2 5′ UTR and L1Hs ORF1/2 with the mblastI reporter  did not exert the same effect  that was obtained in the past 12 million years. This suggests the potential role of m6A as a driving force in L1 evolution , which have evolved with L1s, suppress the old L1 transcription in a sequence-specific manner16. However, L1Hs, which is the youngest L1 lineage in the human genome, escapes KRAB-ZFP restriction and is not recognized by any KRAB-ZFPs16. Instead, the host defense utilizes post-transcriptional suppression mechanisms, such as small RNA interference  or APOBECs, to restrict the replication of L1s56; however, the youngest L1s are still active. Our findings provide clues on how the youngest L1s continuously replicate under host surveillance. The emergence and the propagation of the A332 m6A-positive L1s suggest that 5′ UTR m6A modification was a countermeasure against the host post-transcriptional restriction.L1s have been continuously active since the origin of mammals2.HeLa cells were cultured in DMEM supplemented with 10% (v/v) fetal bovine serum  and 1% (v/v) GlutaMAXI (Gibco). Human embryonic carcinoma PA-1 cells were cultured in RPMI 1640 supplemented with 10% (v/v) FBS (HyClone) and 1% (v/v) GlutaMAXI (Gibco). hESCs  were cultured in defined hESC culture medium (Stem Cell Technology) on hESC-qualified extracellular matrix (Corning)-coated culture dishes (Corning) or on tissue culture wall plate . The cultures were incubated at 37 °C in 5% COXbaI and PmeI (NEB). The site-directed mutagenesis of FH-ALKBH5 to catalytically inactive mutant (H204A) construct was performed using the Phusion High-Fidelity polymerase kit (Thermo Fisher Scientific).The FLAG-HA-pcDNA3.1-derived plasmids used in this study were named using FH as a prefix with the respective protein names specified. AcGFP, ALKBH5, and FTO cDNA were cloned into FLAG-HA-pcDNA3.1  for overexpressing N-terminally FLAG-HA-tagged protein. FH-based plasmids were generated by restriction enzyme cloning using 25. For the mutagenesis of L1 5′ UTR m6A sites, the L1-dn6 5′ UTR and ORF1 region containing NotI and AgeI restriction sites was recloned into plasmid pCMV14. Using site-directed mutagenesis PCR, the following mutants of pCMV14 L1 5′ UTR ORF1 plasmids were prepared: Δ5′ UTR, A332T, A495T, A600T, A332/600T, and A332/495/600T (named as m6A mut). Next, NotI-AgeI fragments of pCMV14 L1 5′ UTR ORF1 mutant constructs were amplified and subcloned into pL1Hs. To generate pL1PA25′ UTR, we synthesized the L1PA2 5′ UTR region based on reported consensus sequences using gene synthesis (Cosmogenetech). We then replaced the L1Hs 5′ UTR of pL1Hs with L1PA2 5′ UTR, as described above.pJJ101-L1-dn6 2.2, which is referred to as pL1Hs in this study, is a pCEP4-based plasmid that contains an active human L1(L1-dn6) and was generously provided by J. L. Garica-Perez45. pAD3TE1 was gift from Aurélien J. Doucet. The generation of L1 5′ UTR m6A mutant constructs of pAD3TE1 was performed according to the method for pL1Hs mutant construct generation.pAD3TE1 is an L1.3 plasmid containing the T7 gene 10 epitope tag on the carboxyl-terminus of ORF1p, TAP tag on the carboxyl-terminus of ORF2p, and 24 copies of the MS2 loop repeat in the 3′ UTR26. pYX014 encodes L1 constructs under the L1 native 5′ UTR promoter. pYX015 carries a retrotransposition-defective mutation in L1 ORF1. pYX014 and pYX015 plasmids contain a Renilla luciferase cassette to normalize transfection efficiency levels. To generate pYX014 L1Hs and m6A mut constructs, NotI-PmlI fragments that were 2166 bp in length, including those spanning from the L1 5′ UTR to the forepart of ORF2 in pL1Hs and pL1 m6A mut, were subcloned into pYX014 via restriction enzyme cloning.L1-firefly luciferase-tagged plasmids pYX014 and pYX015 were gifts from W. AnL1-neo-TET, a codon-optimized synthetic L1 construct, was generously provided by Astrid Roy-Engel . The L1-neo-TET lacks a 5′ UTR. To generate a 5′ UTR-containing L1-neo-TET construct, the 5′ UTR of pL1Hs was amplified using PCR and the amplicon was inserted downstream of the CMV promoter of L1-neo-TET.Renilla luciferase gene encoded by pYX014. Site-directed mutagenesis was used to generate the following m6A motif-abrogating mutants of pFR-based plasmids: A332T, A495T, A569T, A600T, A679T, A758T, A839T, A332/600T, and A332/495/600T.pFR-L1Hs 5′ UTR plasmids were generated by restriction enzyme cloning. The L1 5′ UTR of pL1Hs and firefly luciferase of pGL3-Basic (Promega) were cloned into pCMV14 downstream of the CMV promoter. Thereafter, the neomycin-resistant gene located downstream of the SV40 promoter was substituted with pDEST HA-derived plasmids were named using HA as a prefix with the respective proteins, YTHDF1 and YTHDF2. YTHDF1 and YTHDF2 cDNA were cloned into pDEST HA vector using pENTR/D-TOPO vector (Invitrogen) and Gateway® LR Clonase® II Enzyme mix (Invitrogen). HA tag sequence is located in 5′ end of insert for overexpressing N-terminally HA-tagged protein.siRNAs directed against METTL3 (L-005170-02), ALKBH5 (L-004281-01), FTO (L-004159-01), or nontargeting siRNAs (D-001210-01-50) were purchased from Dharmacon. All siRNA transfections were performed using the DharmaFECCT 1 transfection reagent (Dharmacon) according to the manufacturer’s instructions.57. Uncropped blots were provided in the source data file.The cells were lysed in RIPA buffer , 0.5% sodium deoxycholate, 0.05% SDS, 1 mM EDTA, 1 mM DTT) supplemented with cOmplete protease inhibitor cocktail (Roche) for 15 min on ice. The lysates were cleared by centrifugation and mixed with Laemmli sample buffer. The mixture was then boiled at 98 °C for 10 min, separated by SDS-PAGE in 10% gels, and transferred onto nitrocellulose blotting membranes (Amersham). The membranes were blocked by incubating with 5% skim milk in Tris-Buffered Saline Tween-20 (TBST) for 30 min and incubated overnight at 4 °C with the respective primary antibodies at 1:1,000 dilution, except for anti-EIF3b antibody at 1:2000. Subsequently, the membranes were washed thrice with TBST and incubated with HRP-conjugated secondary antibodies  at 1:5000 dilution in 5% skim milk/TBST. After washing thrice with TBST, the immunocomplexes were imaged using SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific). Band intensity quantification in Supplementary Fig. TM qPCR 2X PreMIX (Enzynomics) was used for subsequent qPCR reactions. The qPCR primers used in this study are listed in Supplementary Table Total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. For the removal of genomic or plasmid DNA, total RNA was treated with recombinant DNase I (Takara) for 1 h at 37 °C, followed by purification using the NucleoSpin RNA Clean-up kit (Macherey–Nagel). cDNA synthesis was performed using the ReverTra Ace qPCR RT Kit (Toyobo) according to the recommended protocol. TOPreal2, 100 μg/ml of cycloheximide) were prepared a day before polysome fractionation. For polysome fixation, 7–10 × 106 cells were incubated for 10 min in a media containing 100 μg/ml of cycloheximide at 37 °C and were collected by scrapping with PBS containing 100 μg/ml of cycloheximide. After centrifugation at 1200 × g, 4 °C for 5 min, the cell pellets were lysed in 100 μl of polysome extraction buffer  supplemented with RNase inhibitor (Enzynomics), protease/phosphatase inhibitor cocktail , and 1 mM DTT. The cells were incubated in the buffer for 10 min at 4 °C and centrifuged at 12,000 × g, 4 °C for 10 min to remove debris and nuclei. The protein concentration was measured using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific). Five hundred to 600 μg of the lysate was introduced at the top of the linear sucrose gradient and centrifuged at 222,000 × g, 4 °C for 2 h using the SW41Ti rotor of the Beckman ultracentrifuge. Fifty microgram of the lysate was saved as input RNA. After centrifugation, 1 ml fractions were collected from the top to the bottom of the gradient using the BioLogic LP system and fraction collector (BioRad) with UV absorbance at 260 nm. Next, 250 μl of each fraction was mixed with 750 μl of TRIzol LS reagent (Invitrogen) and 20 ng of spike-in RNA (synthesized firefly luciferase mRNA). RNA extraction and qPCR were performed as described above. The levels of RNA in each fraction were normalized to those of spike-in RNA and input RNA.Ten milliliter of 10–50% linear sucrose gradients in base solution  at 800 ng per well using Lipofectamine 3000 (Invitrogen) following manufacturer’s instructions. Two days later, 200 μg/ml hygromycin B (Invitrogen) was added to the media to select the transfected cells. Cell selection continued for 4 days, and the hygromycin B-resistant cells were reseeded at 2.5 × 104 per well in a 6-well plate. The next day, blasticidin S (Invitrogen) was added to a final concentration of 8 μg/ml and the cells were cultured for 7–9 days in its presence. The colonies were stained with crystal violet and counted using Colony, version 1.1 (Fujifilm). Retrotransposition assays were performed using RNA interference targeted toward METTL3, ALKBH5, and FTO with slight modifications in the process described above. For this, 6 × 104 HeLa cells were seeded into 12-well plates with 40 nM siRNA-Dharmafect1 (Dharmacon) mixture. After 24 h, the cultures were divided equally and plated into two wells in 12-well plates. The next day, the cells were transfected with pL1Hs at 500 ng per well. Four days after transfection, the cells were plated at 6 × 104 cells per well in 6-well plates and selected using 8 μg/ml of blasticidin S. Retrotransposition assays with the overexpression of AcGFP, ALKBH5, and FTO were performed as described above. Briefly, HeLa cells were transfected 500 ng FH-plasmid and 700 ng of L1 plasmid, and 4 days after transfection, 6 × 104 cells were reseeded into a well in a 6-well plate and selected after treatment with blasticidin S for 7–9 days.HeLa cells were plated at 8 × 104 cells per well in 12-well plates. The next day, the cells were transfected with 800 ng per well of the pFR vector (pCMV-L1 5′ UTR-firefly luciferase) using Lipofectamine 3000 (Invitrogen). Two days later, the transfected cells were harvested and luminescence was measured using the Dual-Luciferase Reporter Assay System (Promega) according to manufacturer’s instruction. Briefly, 250 μl of passive lysis buffer was used to lyse cells in each well in 12-well plates. Next, 20 μl of the lysate was mixed with 100 μl of the Luciferase Assay Reagent II, and the luminescence of firefly luciferase was measured using a microplate luminometer (BERTHOLD). Renilla luciferase activity was subsequently measured after administering 100 μl Stop & Glo Reagent.HeLa cells were plated at 8 × 1034 with some modifications. For each experiment, 1.2 × 106 HeLa cells were plated on two 100 mm dishes each. The next day, the cells were transfected with 6 μg of L1 plasmid per dish using Lipofectamine 3000 (Invitrogen). Two days later, the cells were washed twice with cold PBS, and allowed to form UV crosslinks on ice under 150 kJ/cm2 of UV 254 nm light . The cells were scraped and transferred to PBS and pelleted by centrifugation at 1000 × g, 4 °C for 3 min. The pellets were resuspended in 1 ml of lysis buffer . The lysate was passed through a 21 G needle ten times and shock-frozen using liquid nitrogen. The lysate was thawed on ice and centrifuged at 15,000 × g for 15 min. The supernatant was further cleared by filtering through a 0.22-μm membrane. From each lysate, 5% was retained as input. For immunoprecipitation, 10 μl of Dynabeads Protein G (Invitrogen) was washed twice with lysis buffer and incubated with 3 μg of eIF3b antibody  on a rotating wheel at room temperature for 1 h. The cell lysates were mixed with the antibody-bead complex and rotated overnight at 4 °C. The beads were washed five times in high-salt buffer . The antibody-lysate mixture and the conserved input lysates were resuspended in 100 μl of 1× Proteinase K buffer . Next, 1 mg Proteinase K (Macherey–Nagel) was added into the suspensions. Protein digestion was conducted at 50 °C for 2 h in a shaking incubator. After incubation, 100 μl of 7 M Urea (w/v)-1X Proteinase K buffer was added into the immunoprecipitation samples, and the samples were re-incubated at 50 °C for 2 h in a shaking incubator. RNA was extracted using TRIzol LS supplemented with 20 ng of spike-in RNA.eIF3-RNA CLIP-qPCR was performed as described previously58 with some modifications. HeLa cells were plated on two 100 mm dishes at 1.2 × 106 cells per dish. After 18 h, the cells were transfected with 8 μg of pL1Hs per dish. After 48 h, poly (A) + RNA was extracted using the Poly (A) purist Mag kit (Invitrogen). The poly (A) + RNA was mixed with RNA fragmentation reagents (Invitrogen) and fragmented into oligonucleotide that was 50–150 nt in length by heating to 75 °C for 5 min. Fragmented RNA was purified by ethanol precipitation. Next, 6 μg of fragmented RNA was incubated with 4 μg of anti-m6A antibody  in MeRIP buffer  on a rotating wheel for 2 h at 4 °C. After that, the immunoprecipitation mixtures were mixed with Dynabead protein A (Invitrogen) and incubated overnight on a rotating wheel at 4 °C. After washing five times with MeRIP buffer, RNA was eluted twice by incubating in elution buffer on a rotating wheel for 1 h at 4 °C . The eluted RNA was purified by ethanol precipitation. cDNA libraries were prepared as previously described59. Briefly, RNA was dephosphorylated using calf intestinal alkaline phosphatase (NEB) and labeled with γ-32P-ATP using T4 polynucleotide kinase (Takara). RNA was separated by 10% urea-PAGE and purified from the excised gel corresponding to 50–150 nt RNA fragments. The extracted RNA was ligated to a 3′ adapter using T4 RNA ligase 2, truncated KQ (NEB). The RNA was then purified from free 3′ adapters by repeated gel excision. The 3′ adapter-ligated RNA was ligated to a 5′ adapter using T4 RNA ligase 1 (NEB) and subsequently reverse transcribed using SuperScript III reverse transcriptase (Invitrogen). The cDNA library was amplified by PCR using Phusion HF polymerase (Thermo Fisher Scientific), separated by 6% acrylamide gel electrophoresis, and purified by gel excision. The libraries were sequenced to 2 × 100 base-pair reads on the Illumina HiSeq 2500. The sequence of the 3′ and 5′ adapters, reverse transcription primer, and 5′ and 3′ PCR primers are listed in Supplementary Table MeRIP was performed as described earlier60 (cutadapt -g TACAGTCCGACGATC -A TGGAATTCTCGGGTGCCAAGG). The 3′ and 5′ adapter sequences in the first and second read in a pair (owing to the short insert size) were further trimmed and the read pairs with either reads < 18 bp were discarded. The remaining reads were then aligned to the combined human genome (hg19), and reporter L1 (pL1Hs) sequence using Spliced Transcripts Alignment to a Reference (STAR)61 and peak calling was performed using MACS262. For analyzing the m6A modifications in endogenous L1, the sequence reads from human embryonic stem cells were retrieved31 (accession code: GSE52600) and were aligned against L1Hs consensus sequence using STAR. The codes are available from https://github.com/hastj7373/merip-seq.For MeRIP-seq analysis, the adapters were trimmed using Cutadapt6A mut were transfected into HeLa cells. The following day, the cells were reseeded on sterile coverslips where 200 μg/ml hygromycin B was added for selection. After 3 days, the cells were fixed with 3.7% formaldehyde in PBS for 10 min, and the fixation was quenched by adding 0.1 M glycine in PBS for 10 min. The fixed cells were permeabilized in 70% ethanol for at least 3 h to 1 week at 4 °C. The cells were then rehydrated with PBS for 30 min and incubated in a prehybridization solution  for 30 min at 37 °C. Hybridization was performed overnight at 37 °C in 50 μL of hybridization solution containing 10% formamide, 2× SSC, 10% dextran sulfate, 50 μg yeast tRNA, 0.2% BSA, 0.1 M DTT, 50 units RNase inhibitor (Enzynomics), and 10 ng of MS2-Q670 probe  at 37 °C. Next, the cells were washed twice with a prehybridization solution for 30 min. For the immunofluorescence experiment, the hybridized cells were incubated in the blocking solution  for 1 h, followed by incubation with anti-T7 primary antibody  diluted in the blocking solution (1:200) for 2 h. The cells were washed twice with the prehybridization solution for 15 min and incubated with FITC-conjugated anti-goat secondary antibody  diluted in blocking solution (1:200) for 1 h. The cells were washed twice as described above, and the coverslips were mounted on slide glasses using the Vectashield antifade medium with DAPI (Vector Laboratories). The samples were imaged using an inverted microscope Nikon Eclipse Ti2 equipped with a 1.45 numerical aperture Plan apo λ ×100 oil objective and a sCMOS camera (Photometrics prime 95B 25 mm). For each field of view, stacks of images of 6 μm were captured at every 0.3 μm in the DAPI395, GFP488, and Alexa647 channels using the NIS-Elements software.The L1-MS2-stem-loop constructs pAD3TE1 L1Hs and pAD3TE1 L1 mThe sequence of the RNA FISH probes are: MS2LK20 (5′ TTTCTAGAGTCG ACCTGCAG 3′), MS2 LK51-1 (5′ CTAGGCAATTAGGTACCTTAG 3′), and MS2 LK51-2 (5′ CTAATGAACCCGGGAATACTG 3′). Each probe was labeled with two Quasar 670 dyes at both ends. The mixture of the three probes were used for RNA FISH of L1 RNA tagged with the MS2 loops.57. Protein and mRNA particles from z-stack images were detected using the TrackNTrace software64. After the detection of particles, the protein-mRNA pairs with an intermolecular distance of 330 nm (three pixels) were considered as colocalizing pairs. The intensities of proteins colocalizing with mRNA were determined based on the amplitude of the fitted 2D Gaussian function from the TrackNTrace software.Binary masks of cells were generated using the ROI manager in ImageJ41. Briefly, HeLa cells were plated (4 × 106 cells in 60 mm dishes); the following day, the cells were transfected with 3 μg of L1 plasmid (pL1Hs) using Lipofectamine 3000 (Invitrogen). After 48 h, 200 μg/ml hygromycin B was added to the media to select the cells carrying the L1 plasmid. After 2 days of selection, the cells were lysed with CHAPS lysis buffer , 1 mM MgCl2, 1 mM EGTA, and 10% glycerol) supplemented with 1 mM DTT and the cOmplete protease inhibitor cocktail (Roche) and cleared by centrifugation . The cleared lysates were loaded on a sucrose cushion  and 6 ml of 17% sucrose (from the bottom) solutions) in 13.2 ml Ultra-Clear tubes (Beckman Coulter) and centrifuged at 178,000 × g, 4 °C for 2 h in a SW41Ti rotor of Beckman ultracentrifuge. The colorless pellets were suspended by pipetting in 100 μL of RNase-free water supplemented with 1× protease inhibitors. Pierce BCA Protein Assay (Thermo fisher Scientific) was conducted to determine the protein concentration. Three microgram of the RNP samples were retained and used later in RNA isolation and immunoblotting experiments. Seven hundred and fifty nanogram of each RNP sample was mixed with the LEAP assay reaction buffer , 40 units of RNase inhibitor (Enzynomics), total reaction volume: 50 μl) and incubated at 37 °C for 1 h. One microliter of LEAP cDNA products were subsequently amplified using 0.4 μM of L1 LEAP primer with the Phusion High-Fidelity polymerase kit (Thermo Fisher Scientific). PCR amplicons were separated and visualized in EtBr-stained 2% agarose gel.The LEAP assay was performed as described previously38. Briefly, the authors immunoprecipitated eIF3b from 4-thiouridine-and-UV-treated 293 T cells to capture the eIF3-RNA complex. After high-salt washing and RNase digestion, they separated individual eIF3-RNA complexes through denaturing gel electrophoresis. eIF3a, b, d, and g were identified from four separate bands using mass-spectrometry and the interacting RNAs were purified and sequenced. Although three replicates were generated for each protein, only the first replicate was used for each. After retrieving the raw sequence files from NCBI (accession code: GSE65004), reads with low basecall qualities were excluded using the fastq_quality_filter from FASTX Toolkit . PCR duplicates were also excluded using fastx_collapser. Moreover, we excluded the reads that were shorter than 10-nt after trimming primer IDs and 3′ adapters from further analysis . The remaining reads were mapped to the L1Hs consensus sequence, wherein upto three mismatches were allowed using bowtie265 . For mean coverage analysis of 5′ UTR, ORF1, ORF2, and 3′ UTR, the number of reads that begin and end within each region were counted and the number was divided by the length of the corresponding region. The codes used for analyzing PAR-CLIP and mapping data are available from https://github.com/schanbaek/eif3_par-clip.We utilized previously published PAR-CLIP data for eIF3a, b, d, and g47 with a computational approach. Only L1s of which insertion sites and two flanking regions are supported to be unique to the human, chimpanzee or gorilla genome by BLAT and liftOver were included in the further analyses. Then, we eliminated certain ambiguous elements containing gap sequence in the reference genome data and those that were less than 5.5 kb. The flanking sequences  of each species-specific L1 candidate were manually compared to the orthologous loci in human , chimpanzee , gorilla , and orangutan  genomes. The flanking sequences were used to identify the orthologous positions in the other genomes using BLAST-Like Alignment Tool (BLAT). We collected and retrieved the species-specific full-length L1s. We then classified the L1 subfamilies  using RepeatMasker utility66. Multiple sequence alignment of species-specific full-length L1s in each genome was performed using MUSCLE (MUltiple Sequence Comparison by Log- Expectation) under the default option67. The conserved sequence motifs at the three sites  were visualized using the program Weblogo68. Species-specific L1 loci are listed in Supplementary data To identify the species-specific full-length L1s in human, chimpanzee, and gorilla genome, we used BLAT-based and liftOver-based methodst-test was used for unpaired data. Two-sided Kolmogorov–Smirnov test was used to assess the quantification of number and intensity of colocalizing puncta in Fig. P-values <0.05 were considered significant.GraphPad Prism 7.00 was used for statistical analysis. Two-sided student’s Further information on research design is available in the Supplementary InformationPeer Review FileSupplementary Data 1Supplementary Data 2Supplementary Data 3Reporting Summary


Staying up to date and answering clinical questions with current best evidence from health research is challenging. Evidence-based clinical texts, databases, and tools can help, but clinicians first need to translate their clinical questions into searchable queries. MacPLUS FS (McMaster Premium LiteratUre Service Federated Search) is an online search engine that allows clinicians to explore multiple resources simultaneously and retrieves one single output that includes the following: (1) evidence from summaries , (2) preappraised research , and (3) non-preappraised research , with and without validated bibliographic search filters. MacPLUS FS can also be used as a laboratory to explore clinical questions and evidence retrieval.Our primary objective was to examine how clinicians formulate their queries on a federated search engine, according to the population, intervention, comparison, and outcome (PICO) framework. Our secondary objective was to assess which resources were accessed by clinicians to answer their questions.We performed an analytical survey among 908 clinicians who used MacPLUS FS in the context of a randomized controlled trial on search retrieval. Recording account log-ins and usage, we extracted all 1085 queries performed during a 6-month period and classified each search term according to the PICO framework. We further categorized queries into background  and foreground questions . We then analyzed the type of resources that clinicians accessed.P=.51). Queries included a median of 3 search terms (IQR 2-4), most often related to the population and intervention or test, rarely related to the outcome, and never related to the comparator. About half of the resources accessed  were summaries, 24.4% (149/610) were preappraised research, and 24.1% were (147/610) non-preappraised research.There were 695 structured queries, after exclusion of meaningless queries and iterations of similar searches. We classified 56.5% (393/695) of these queries as background questions and 43.5% (302/695) as foreground questions, the majority of which were related to questions about therapy , followed by diagnosis , etiology , and prognosis . This distribution did not significantly differ between postgraduate residents and medical faculty physicians (Our results, from a large sample of real-life queries, could guide the development of educational interventions to improve clinicians’ retrieval skills, as well as inform the design of more useful evidence-based resources for clinical practice.ClinicalTrials.gov NCT02038439; https://www.clinicaltrials.gov/ct2/show/NCT02038439 Web-based searches have become the norm when looking for information and answers to most of our questions in daily life. This has also become true in the practice of medicine; online medical resources to access evidence are increasingly considered “as essential as the stethoscope” . While fPhysicians are typically familiar with only a few of these resources, likely those to which they have been exposed in training or by peers, and often ignore most of the ecosystem and architecture of published evidence. Yet, their daily practice triggers, on average, five to eight questions every 10 patients -10. ClinHow physicians translate their clinical questions into searchable queries remains poorly known. How many search terms do they use? How often do their queries fit the PICO framework ,13? Do eThe type of search engine or evidence resource may also influence the way we conduct queries. Google and Wikipedia tend to retrieve relevant answers, albeit selective, with intuitive, less-structured search strategies -16. SomeWe, therefore, explored how clinicians formulate their queries in a federated online search engine, namely MacPLUS FS (McMaster Premium LiteratUre Service Federated Search). MacPLUS FS allows clinicians to explore multiple resources simultaneously, retrieving one single output that includes the following: (1) evidence from evidence-based summaries , (2) preappraised research , and (3) non-preappraised research , with and without validated search filters see . In thisWe conducted an analytical survey of clinical search queries among 431 postgraduate medical trainees and 477 medical faculty members registered to a federated search engine, MacPLUS FS. The service was freely available to registered users from any computer with an internet browser throughout the clinical setting or elsewhere.badges and reputation scores. Participating clinicians were randomized to each of the three interventions in a factorial design (A × B × C).Participating clinicians consented to be enrolled in 6-month, MacPLUS FS, randomized controlled trials , which tFor each clinician, utilization of MacPLUS FS was recorded through accounts tracking log-ins and usage, including their detailed search queries. Registration to the service was free, and access to each evidence resource was through clinicians’ academic institutions, mostly McMaster University, Hamilton, Canada. Clinicians were categorized according to their baseline search levels and specialty types .We extracted all 1085 search queries performed by clinicians during the conduct of the MacPLUS FS trials. Two authors (AS and TA) assessed each query individually, counting the number of search terms—counting all words —and documenting all abbreviations and Boolean terms . Search queries were then classified into (1) structured searches, (2) searches for specific articles , (3) iteration of structured searches, namely a group of related structured queries with a similar PICO question within the same log-in session, and (4) undetermined searches .summaries, preappraised research, and non-preappraised research (see The same two authors (AS and TA) classified structured queries into background or foreground questions see , accordiarch see .We examined types of questions  according to the level of training as well as clinicians’ specialties and baseline frequencies of search . We then examined the number and type of search terms across each type of question. We compared distributions using chi-square parametric tests when relevant and Kruskall-Wallis tests for nonnormal distributions. Data abstraction was done using Microsoft Excel 2016, version 15.29, and data analysis was performed using SPSS Statistics for Windows, version 23.0 (IBM Corp).regular searchers (≥1 search per month), occasional searchers (<1 search per month), or alert-only users (no searches).Participants were postgraduate residents and medical faculty members who had registered in MacPLUS FS prior to the trial. Of the 678 postgraduate residents and 753 medical faculty members, 431 (63.6%) and 477 (63.3%), respectively, were deemed eligible after the exclusion of 247 postgraduate residents and 266 medical faculty members, who either never logged in to MacPLUS FS during the year prior to the study or quit the institutions served by MacPLUS FS [The 908 clinicians made 1085 search queries, of which 235 (21.66%) were subsequent iterations of the same search, 124 (11.43%) were a search for a specific article, and 31 (2.86%) could not be classified and remained undetermined. A total of 695 out of 1085 queries (64.06%) were structured queries following the PICO format, with 480 out of 695 (69.1%) single queries, whereas 215 (30.9%) included a group of related queries. This corresponds to an average of 2.1 attempts per group query.P=.51) (see .51) see .P=.009). There were no differences between specialty types (P=.67).P<.001). Indeed, there were 70.2% (276/393) of background questions with 2 or fewer terms versus 18.2% (55/302) of the foreground questions, and 81.8% (247/302) of foreground questions with 3 or more terms versus 29.8% (117/393) of the background questions.Overall, 72.5% (504/695) of structured queries see  containeP<.001). Although 35.7% (248/695) of structured queries did not result in any resource access, 39.9% (277/695) led to one resource accessed, 11.8% (82/695) led to two, and 12.7% (88/695) led to three or more resources. Across all 1085 queries, the average number of resources accessed was 0.88 (SD 1.42). When users attempted a second search on the same clinical question , 7.2% (17/235) resulted in one or more resources accessed, while 92.8% (218/235) led to an end of their search query with no additional resource accessed. When searching for a specific article, 37.9% (47/124) led to one resource accessed and 12.9% (16/124) led to two or more resources accessed.The number of evidence-based resources that clinicians accessed for each type of query  are displayed in P<.001), and family physicians looked at significantly more resources than did internists and specialized physicians (P<.001).Among 1085 queries made by 908 clinicians, 695 were structured queries. A small majority were related to background questions, and most foreground questions were questions about therapy, rather than diagnostic or prognostic questions. Structured queries included a median of 3 terms, most often related to the population and intervention or test, rarely related to the outcome, and never related to the comparator. Explicit Boolean terms were rarely used; of note, the search engine assumed by default a Boolean “AND” between search terms. About half of the resources accessed were summaries, while the rest were equally divided between preappraised and non-preappraised resources.We found no difference between searches made by postgraduate resident trainees and medical faculty members. As they are in training, one could have expected postgraduate residents to have more background questions, whereas faculty members were expected to have more foreground questions, for example, in comparing the effectiveness or risks of management strategies. Our results did not confirm this assumption, as faculty members had more than half of their searches on background questions as well. This may be due to the complexity of patient care. A given faculty member may be an expert in a given field but adopt a learning strategy to rapidly get the big picture, to understand uncommon situations. Their high level of access for summary resources, such as UpToDate or DynaMed, likely supports this explanation. Similarly, family doctors also accessed more summary resources, not only because of their need for quick clear answers to questions arising within short appointments with patients, but also, perhaps, because they provide care for patients across an entire age spectrum.Another issue relates to the frequency of searches clinicians are able to perform in daily life. In our study, 908 clinicians performed only 1085 queries in 6 months. Other studies have shown that clinicians tend not to search in order to answer the questions that arise in their daily clinical practice -20. In oAnother observation from our study is that clinicians’ queries tend to remain relatively simple: few search terms, often covering few PICO concepts, mostly population and intervention. While simple strategies work well for high-level summaries, they are much less efficient with large databases like PubMed. Our daily habits for searching on the Web may explain clinicians’ tendencies for simple queries. Strictly from a user’s perspective, we have all become very efficient in searching for information mostly through Google and Wikipedia, just by typing a few intuitive keywords in the free-text bar at the top of a webpage. Medical search engines may misguide the user in having them assume the engine will work similarly to Google .One area for improvement of search engines could be to invite users to structure their queries according to the PICO framework. Schardt et al  have fouThe main limitation of our study was that clinicians likely used other means than MacPLUS FS to answer some of their daily questions. Our design also did not assess the clinical impact of the answers retrieved. This would have required mixed methods approaches to estimate the number of patients needed to benefit from information , as described by Pluye et al .Finally, our sample of searches was recorded in the context MacPLUS FS randomized controlled trials , and it Strengths include the direct record of queries in one of the largest samples of physicians from different specialties and levels of practice. It is also the first study on a federated search engine, which allowed us to show that clinicians access all resources and not only summary-level evidence.A constant flow of new articles overwhelms clinicians who are continuously exposed to them. To keep up and to answer our clinical questions, it is essential to clarify and translate clinical questions into searchable queries. Our results could lead to the development of educational and clinical interventions on how to increase searching skills . These cOur findings also highlight the potential role of federated search engines over the use of single resources to meet clinicians’ needs . A federOther avenues of research include the improvement of search functionalities and clinical interventions to meet users’ expectations in navigating through the evidence, in order to rapidly find the most relevant and least-biased answers for better clinical practice and patient care.