Here, we describe a synthetic molecular mechanism that transforms a rotary motion of an asymmetric camshaft into reciprocating large-scale transitions in a surrounding stator orchestrated by mechanical deformation. We design the mechanism using DNA origami, characterize its structure via cryo-electron microscopy, and examine its dynamic behavior using single-particle fluorescence microscopy and molecular dynamics simulations. While the camshaft can rotate inside the stator by diffusion, the stator’s mechanics makes the camshaft pause at preferred orientations. By changing the stator’s mechanical stiffness, we accelerate or suppress the Brownian rotation, demonstrating an allosteric coupling between the camshaft and the stator. Our mechanism provides a framework for manufacturing artificial nanomachines that function because of coordinated movements of their components.Biological molecular motors transform chemical energy into mechanical work by coupling cyclic catalytic reactions to large-scale structural transitions. Mechanical deformation can be surprisingly efficient in realizing such coupling, as demonstrated by the F Biological molecular motors convert chemical energy into mechanical motion by coupling catalytic reactions to large-scale structural transitions. Here, the authors report the design of a rotating DNA nanomechanism that comprises a camshaft whose rotary motion can be transformed into reciprocating large-scale transitions in the structure of the surrounding stator. Intriguingly, the F1 motor of F1FO ATP synthase is reversible5: it can either function as a rotary motor powered by the chemical energy of ATP hydrolysis or it can use the mechanical energy of the central shaft rotation to catalyze synthesis of ATP. The fact that the F1 ATP synthase can be both a motor and a chemical generator reflects the microscopic reversibility of elementary chemical processes and is a unique feature of molecular scale machines. Realizing a similar degree of mechanochemical coupling in a synthetic nanoscale system remains a landmark technological goal.Macroscopic machines commonly rely on a coordinated motion of multiple rigid components to perform their function. For example, an internal combustion engine uses a rotating camshaft to cyclically open or close the peripheral valves for fuel injection and exhaust gas removal; the coordination of the valves’ operation is paramount to the engine’s function. Nanoscale biological machines also often consist of multiple components that move in a coordinated fashion. For example, the rotation of the central shaft in F12. DNA nanotechnology has also already provided a range of mechanical systems including pivots, hinges, crank sliders, and rotary mechanisms17 that can be reconfigured using strand displacement reactions (SDR)18 or by changing environmental parameters such as pH, ionic strength, temperature, and external fields24.The construction of artificial molecular machines by chemical synthesis has previously provided important insights regarding how to create molecular mechanisms with internal degrees of freedom, such as catenanes and rotaxanes, and how to power molecular motions using chemical fuels, light, and other stimuli27. As such, DNA origami nanomachines may offer additional opportunities for the assembly of mechanisms of coordinated mechanical power transmission. In this work, we describe the construction, computational characterization and experimental validation of a rotary mechanism with user-defined power and motion transmission. We conceive this object by combining macroscale machine design concepts with functional and structural aspects of the ATP synthase, and consider it to be a stepping stone toward creating artificial machines that achieve and generalize functionalities observed in biological motors.Whereas the molecular mechanisms generated by chemical synthesis tend to include on the order of 100 atoms, DNA nanostructures, in particular DNA origami objects, are much larger and can encompass hundreds of thousands of atoms29.We designed our rotary mechanism as a tetramer composed of a camshaft-like rotor in a surrounding stator. Rotations of the camshaft will induce reciprocal deformations of the structural elements in the stator Fig. . We apprThe stator comprises three similarly shaped components, consisting of 46 helices packed in parallel on a honeycomb-like lattice. One stator unit possesses an asymmetric feature  to study the dynamical behavior of our rotary complex. We anchored the stator to a glass slide covered by polyethylene glycol (PEG) and biotin by extending the stator unit 1 with a helix bundle (6hb) domain at the top of one of the pawls Fig.  and 20. Imaging of rotary mechanisms with released camshafts revealed particles performing rotary random walks in addition to stationary particles Fig. . We desi32 . In these simulations, we enforced the relative orientation of the camshaft and the stator via a harmonic potential that acted on the dihedral angle formed by the centers of geometry of the four regions Fig. . By chanWe generalized the simulation analyses to variants 3 and 6, revealing that all variants exhibit qualitatively similar deformations of the pawls with respect to the cam despite the different inter-pawl connections  thermal cycling devices. The reactions were left at 65 °C for 15 min and were subsequently subjected to a thermal ramp from 60 to 44 °C (1 °C/h). The folded structures were stored at room temperature (RT). Sequences are reported in Supplementary Data 1-7.The folding reaction mixtures contained 40 nM scaffold and 160 nM (for structures with p9072 scaffold) or 200 nM (for the other structures) staples . The folding buffer included 5 mM TRIS, 1 mM EDTA, 5 mM NaCl and 10–20 mM MgCl30. For concentrating the monomers, ultrafiltration was used, while ultracentrifugation  was used for concentrating the higher-order structures.After folding, all samples were purified via PEG purification, ultrafiltration or physical extraction from agarose gels2. After mixing, the solution was centrifuged at 16k × g for 20 min. The supernatant was removed and the remaining pellet was diluted in a buffer containing 5 mM TRIS, 1 mM EDTA, 5 mM NaCl and 5 mM MgCl2.For PEG purification, the DNA origami sample was diluted in a 1:1 ratio with a solution containing 15% PEG 8000. The salt concentration was adjusted to reach a minimum of 10 mM MgCl2. An equilibration step with only buffer was performed at 2k × g for 5 min at RT. Then 0.5–2 mL sample (depending on the filter size) was added and centrifuged at 10k × g for 5 min (for the 0.5 mL filters) or 7k × g for 10 min (for the 2 mL filters). Another two rounds of centrifugation were performed adding buffer up to the maximum volume of the filter. To retrieve the sample, the filter was turned upside down in a new tube and centrifuged for 5 min.For ultrafiltration, Amicon Ultra 0.5 mL or 2 mL Ultracel filters, 50 K and 100 K were used with buffers containing 5 mM TRIS, 1 mM EDTA, 5 mM NaCl and 5 mM MgCl2 for 1–5 h at 80–90 V. The gels for the monomers were cooled by a water bath, while the gels for the higher-order structures were cooled using a cooling system (Hailea). The desired bands were cut from the gel using an X-tracta Generation 2 hand punch (Biozym). The gel slices were manually squeezed and then centrifuged at 2k × g for 5 min in a Freeze ‘N Squeeze DNA Gel Extraction Spin Column with pore size 0.45 µm (BioRad).For physical extraction from agarose gels, the samples were electrophoresed on 2% gels containing 0.5× TBE and 5.5 mM (for the monomers) or 22 mM (for the higher-order structures) MgCl2 and left at 40 °C for 16–24 h. The other stator units and the lever arm were added in stoichiometric conditions at 40 mM MgCl2 and left at 40 °C for 1 day/monomer addition. To set the central shaft free to rotate, invader strands were added in excess  and the reaction mixture was left in a shaker for 16–24 h at room temperature.For the dimerization between stator unit 1 and shaft, a 1:1 solution of the monomers was mixed with 20 mM MgCl2 at different concentrations: typically, for monomers and dimers 5.5 mM MgCl2 was used, while 22–33 mM MgCl2 was necessary for higher order structures. For the latter samples, the temperature was kept at around 10 °C with a cooling system (Hailea). The gels were scanned using a Typhoon FLA 9500 laser scanner  at a resolution of 50 µm/pixel. For clarity, gel intensities in this paper were auto leveled.The DNA nanostructures were electrophoresed on 2% agarose gels containing 0.5× TBE and MgClPurified structures were adsorbed onto glow-discharge Cu grids with carbon support  and stained with a 2% aqueous uranyl formate solution containing 25 mM NaOH. Samples were incubated for different time lengths depending on the concentration. In general, structures with concentrations in the order of tens of nM were incubated for 30 s, while lower concentrated samples (5 nM or below) were incubated for 5 to 10 min.40 without CTF correction.Images were acquired using a Philips CM100 operating at 100 kV or a Tecnai 120 (Thermo Fisher Scientific) at 120 kV. Negative stain 2D class averages were computed using RELION30 as described in the section “Purification and concentration of the DNA origami nanostructures” until reaching concentrations around 1 µM.For cryo-EM analysis of the monomers, the samples were folded and ultrafiltratedFor higher order structures, monomers were folded and gel extracted in order to ensure to have only the right monomeric species. The samples were ultrafiltrated for increasing the concentration. The monomers were polymerized as described in the previous section. The polymers were ultracentrifuged at 110k × g for 30 min at RT. The supernatant was pipetted away leaving only a small volume of sample with the desired high concentration. If needed, the invader strands were added at a 2× excess to binding site for 16-24 h at room temperature.The samples were incubated onto glow-discharged C-flat 1.2/1.3, C-flat 2/1 or lacey carbon with ultrathin carbon support grids and plunged-frozen using a Vitrobot Mark IV (Thermo Fisher Scientific). The parameters used are given in the table below .The camshaft was manually imaged using a Tecnai 120 (Thermo Fisher Scientific). All the other samples were imaged automatically with a Titan Krios (Thermo Fisher Scientific). Imaging parameters are listed in the table below.41. All the micrographs were CTF corrected with CTFFIND442. The camshaft particles were picked manually, while for the other samples particles were picked with RELION40 or with crYOLO43. Other processing steps were performed with RELION . Multiple runs of 2D and 3D classifications were typically performed to exclude incompletely folded particles or particles lying on the carbon film. The 3D classes presenting most features were further refined and post-processed.The movies acquired with the Titan were subjected to motion correction using MotionCor2For the complex set free to rotate, a 3D classification without alignment was performed after refinement, using multiple maps as references, i.e., the three reconstructions of the structure with the camshaft fixed to the stator units. With this method, three different positions of the central camshaft could be found.31, where each of the pawls and the bearing were treated as a separate body. Similarly, the higher order structures were subjected to multibody refinements where each of the monomers composing the complex was considered a separate rigid body.The stator units were further subjected to a multibody refinement44. One helix per pawl in the first extreme map was identified and the distance to the same helix in the second extreme map was calculated with UCSF Chimera.For the analysis of the pawls’ motion for the sample with the released camshaft, all the particles in the final three 3D classes showing different conformations were considered. Ten rigid bodies were defined: the camshaft, the 6 pawls (2 per each stator unit) and the 3 bearings (1 per each stator unit). Multibody and principle component analysis were used to identify the main motions. Two main pawls’ motions could be determined. The extreme maps were then overlapped in UCSF ChimeraMonomers were folded and purified using physical gel extraction. The samples were ultrafiltrated for increasing the concentration. The monomers were polymerized as described before. If needed, the invader was added at a 4× excess to binding site for 16-24 h at room temperature. Biotinylated oligos were incubated with a 32× excess neutravidin and then added to the polymers in an 8× excess to binding site for 1–2 h at RT. The resulting reaction mixture was gel purified for extracting only the correct species. Sample concentrations varied between 100 and 700 pM. Samples were left at RT for at most 2 days and then imaged at the microscope.2O and then sonicated for 5 min in a 2% Hellmanex solution. The slides were rinsed in ddH2O, sonicated in ddH2O for 5 min, rinsed again and then sonicated in ethanol (99%). The slides were dried at 70 °C for 1 h. A solution of 0.5% bioPEG-silane solved in ethanol with 1% acetic acid was incubated on the slides at 70 °C for 30 min. The slides were then rinsed with ddH2O, dried with N2, and stored protected from light45.Microscope cover slides (Sigma Aldrich) were cleaned in 2 M NaOH for 30 min, rinsed with ddH2 for 3-4 times. 50 µL sample was incubated for 5 to 20 min depending on the sample concentration. The chamber was washed again 3–5 times with a buffer containing 5 mM TRIS, 1 mM EDTA and 500 to 1000 mM NaCl. Afterwards, the chamber was washed 3 times with imaging buffer containing an oxygen scavenging system  prior to data acquisition. Enzymes, Trolox and glucose were purchased from Sigma Aldrich. Movies were acquired at room temperature with a custom-built objective-type TIRFM45. Movies were acquired for 2–20 min at a frame rate of 20 frames/s and a laser on time of 5 ms.An acrylic glass template containing 4 chambers was sealed to the cover slide with vacuum grease. The chambers were washed with a buffer containing 10 mM TRIS, 2 mM EDTA, 10 mM NaCl, 40 mM MgCl46). All successive steps were performed using a custom MATLAB (R2019b) script. Moving particles were manually picked and their frame-by-frame standard deviation was computed. Defective particles or particles showing no motion were sorted out by inspecting the standard deviation images. The remaining particles were further processed by tracking the position of the lever arm in each frame using a virtual window center of mass approach (VWCM). The obtained spots were clustered in 3 groups indicating the three preferred positions of the central camshaft. From the tracked spots, angular positions, RMSD and angular velocities (Ω) were calculated according to:N is the number of frames, i indicates the i-th frame, θ0 is the angle at t = 0, and Δt indicates the time difference between 2 consecutive frames. A histogram of the angular velocities was calculated.Movies were drift-corrected using a FIJI plugin  was placed on a dihedral angle formed by the centers of geometry of the following four groups of particles: (i) beads constituting the central 60 bp of each of the six-helices that formed the “cam” of the rotor; (ii) beads in the same 60-bp-thick plane of the caDNAno design that formed the 24-helix shaft of the rotor; (iii) a 60-bp-thick slab of beads in the shaft of the rotor near the bearing region separated by 85 bp (center-to-center) from the other group of beads on the shaft; and (iv) a 40-bp thick slab of beads in the center of the second subunit of the stator (between 50 and 90 bp from the bottom edge of the bearing). For each variant, two simulations were performed with the rest angle of the harmonic potential increasing in one and decreasing in the other. The simulations were performed for a sufficiently long period of time to observe three complete rotations in each direction.Selected variants  were additionally simulated with an applied bias to drive the rotation. The simulations were performed as described above, except a harmonic potential (Supplementary InformationDescription of Additional Supplementary FilesPeer Review FileSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Supplementary Movie 6Supplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Supplementary Data 8Supplementary Software


Advanced architecture and rational design of electrode materials for electrochemical sodium‐ion storage are well developed by researchers worldwide. MXene‐based materials are considered as one of the most potential electrode materials for sodium‐ion‐based devices, such as sodium‐ion batteries (SIBs), sodium–sulfur batteries (SSBs), and sodium‐ion capacitors (SICs), because of the excellent physicochemical characteristics of MXenes. Here, in this review, the recent research work and progress, both theoretical and experimental, on MXene‐based materials including pure MXenes and MXene‐based composites in application of SIBs, SSBs, and SICs are comprehensively summarized. The sodium storage mechanisms and the effective methods to enhance the electrochemical performance are also discussed. Finally, the current critical challenges and future research directions on the development of these MXene‐based materials for electrochemical sodium‐ion storage are presented. This review provides an in‐depth summary of the application of MXene‐based materials in the sodium‐ion storage, including the detailed sodium‐ion storage performances and mechanisms. Moreover, the relation between the structures and electrochemical performances and the corresponding effective strategies for further optimization are also discussed. In addition, the perspectives on dealing with current challenges are proposed. Thus, new chemical structure and architecture of sodium accommodable materials should be developed to improve the efficiency of sodium storage.Sodium‐ion storage is the strong alternative to lithium‐ion storage for large‐scale renewable energy storage systems due to the similar physical/chemical properties, higher elemental abundance, and lower supply cost of sodium to lithium. Unfortunately, compared with lithium, sodium has larger ion radius (0.102 nm), higher standard reduction potential (−2.71 V vs standard hydrogen electrode (SHE)), and lower electronegativity (0.93), leading to the sluggish sorption or/and insertion kinetics and large volume expansion.1 Ther The MXenes family shares a general composition of Mn+1XnTx, where M represents a transition metal like Ti, V, Mn, Mo, Nb, Cr, Sc, etc., X is C and/or N, and Tx stands for terminal surface groups ‐O, ‐F, and/or ‐OH, which is typically prepared by selectively etching of A layers, such as Al, Si, Ga, from the corresponding Mn+1AXn phases. Owing to their outstanding metallic conductivity, tunable surface chemistry, and 2D layered structure, MXenes have been considered as the promising candidates for supercapacitors, rechargeable batteries, catalysts, oxygen reduction and evolution, water purification, electromagnetic interference shielding, pressure sensor, and field‐effect transistors applications.Recently, a new large group of 2D transition metal carbides, carbonitrides, and nitrides labeled as MXenes has attracted tremendous attention.3 The  Based on the density functional theory (DFT) calculations, the surface termination groups have a great effect on the properties and performance of MXenes. For example, sodium ions can be well adsorbed on monolayer bare MXenes or O‐terminated MXenes due to the good negative adsorption energies but cannot well absorbed on the monolayer F‐terminated and OH‐terminated MXenes. Simultaneously, the monolayer bare MXenes or O‐terminated MXenes also exhibit low diffusion barrier and open‐circuit voltage (OCV) for sodium ions, suggesting that they are expected to be the promising anode materials with high capacities and good rate capabilities. However, experimentally, MXenes have the tendency to aggregate or stack, which impedes the charge transport through the electrodes, resulting in limited capacity values. To address these issues, many strategies including single‐/few‐layer MXenes, expanded interlayer spacing, 3D porous structures have been proposed to accelerate the electrochemical kinetics and enhance the capability.In fact, many research have been conducted on MXenes as electrode materials for sodium‐ion‐based devices.18 Bas Therefore, many researchers have focused on designing the MXene‐based composite materials. For one thing, MXenes can offer the intertwined conductive network and then significantly increase the electronic conductivity. For another, secondary materials are expected to prevent the aggregation of individual nanosheets. What is more, the unique structures and synergistic effects are beneficial for the electrochemical performance.Additionally, compared with the existing anode materials for SIBs, pure MXene electrodes do not perform the satisfying reversible capacity, limiting their further application in energy storage fields.21 TheFigure In this review, the recent research work and progress carried out on the MXene‐based materials for sodium‐ion storage are systematically and comprehensively summarized. We put the emphasis on their synthesis conditions, structures, ion intercalation chemistries, and detailed sodium‐ion storage performances based on the experimental and theoretical investigations. Particularly, the application of pure MXenes and MXene‐based composite materials in the electrodes for SIBs, SSBs, and SICs are both introduced, and the corresponding effective methods to optimize their performance are highlighted and discussed in depth Figure 1. In ad22.1 Since their first discovery in 2011, over 20 species of MXenes have been created such as Ti3C2, Ti2C, Nb2C, Nb4C3, V2C, Mo2C, Zr3C2, Hf3C2, Ta4C3, Mo2TiC2, Ti4N3, V2N. They are currently mainly synthesized by selectively etching of A layers in the corresponding MAX phases by various etchants, including aqueous hydrofluoric acid (HF), ammonium bifluoride (NH4HF2), LiF‐HCl (in situ forming HF), etc., resulting in the mixture of O‐, OH‐, and F‐terminated functional groups. For instance, the exfoliated 2D Ti3C2Tx with accordion‐like morphology could be obtained by treating the Ti3AlC2 powders in HF for 2 h. The large‐volume synthesis of Ti3C2Tx using this method  could be obtained by selectively etching the ScAl3C3 precursor using the organic base, tetramethylammonium hydroxide (TMAOH), as etchant. The Cl‐terminated MXenes including Ti3C2Cl2 and Ti2CCl2 can be achieved by the reaction of the strong Lewis acidic molten salts ZnCl2 with Ti3ZnC2 and Ti2ZnC, respectively . Nb2CTx has been delaminated by inserting isopropylamine (i‐PrA) between layers followed by mild sonication in water  strategy combining with sonication .42 Nb2r Figure .[26]n Figure .[44]33X2 system contains quintuple layers stacked in a sequence of M‐X‐M‐X‐M, which can be seen as three M atomic layers intercalated with two X atomic layers 2. As shown in Figure 2X, where no X atom is available for hollow sites A and an X atom is available for hollow sites B. Therefore, four possible configurations for surface groups of M2X system have been proposed  calculated by DFT, most bare MXenes are intrinsic metallic and have high density of carries, suggesting their good electronic conductivity. However, when terminated with ‐F, ‐O, or ‐OH surface groups, the resultant monolayers can be changed to semiconductors. For example, the metallic Lu2C could transform into the semiconductor after the functionalization of ‐F and ‐OH groups. Since ‐F and ‐OH surface groups may decrease the ionic conductivity and impede the ion transport, it is believed that decreasing the concentration of them could improve the electrochemical properties of MXene‐based materials, while both the bare and O‐terminated MXenes are highly desirable for the electrode materials.More importantly, the electronic properties of MXenes are strongly related with the terminal surface groups generated during the synthetic processes.52 Acc3C2F2 and III‐Ti3C2F2 show semiconducting states with the bandgap of 0.04 and 0.03 eV, respectively, while II‐Ti3C2F2 is a metal. In addition, I‐Ti3C2(OH)2 and III‐Ti3C2(OH)2 have semiconductor characters possessing the bandgap of 0.05 and 0.07 eV, respectively, while II‐Ti3C2(OH)2 is metallic , the sodium‐ions intercalation chemistry in T3C2Tx has been clarified. The XRD patterns  during the severe charging–discharging process. When more than two sodium layers are intercalated, the inner sodium will be bonded to their neighbors by weaker metallic bonding, combining with the outer sodium layer shielding the O2− anions. Moreover, there is a continuous expansion in the spacing of MXene sheets with the increasing sodium content.Recently, the studies about the mechanism of sodium intercalation in Vn Figure , indicat  of TiO groups.66 By 3C2 MXenes with enlarged interlayer distance of 7 nm. The calculated diffusion energy barriers on bare, OH‐, F‐, and O‐terminated Ti3C2 MXenes are 0.02, 0.013, 0.19, and 0.20 eV, respectively, indicating that sodium ions can easily migrate on the interlayer‐expanded Ti3C2 MXenes. Moreover, according to the calculated theoretical storage capacities, the interlayer‐expansion approach could improve the sodium storage capacities of MXene‐based SIBs.The interlayer spacing also plays an important role in determining the capacities of rechargeable batteries. The stable multilayer sodium adsorption can be obtained on the bare and O‐terminated Ti of 7 nm.69 The3C2 with ‐O groups, whereas the effects become less obvious in bare, F‐, and OH‐terminated systems. This phenomenon stems from the localization of electronic states and is strongly associated with the surface chemistry. The Fermi level (Ef) for the bare, F‐, and O‐terminated Ti3C2 MXenes shift upward while the OH‐functionalized nanosheets shift downward, suggesting the existence of strong electronic interaction between the surfaces of MXenes and the adsorbed sodium atoms. The sodium adsorption also made Ef upward shift in the Zr2CO2 MXene, making the semiconductor Zr2CO2 MXene switch to a metal and then ensuring the fast electron transport in the electrode for SIBs.To get more insight into the interaction between the surfaces of MXenes and sodium ions, many works have been carried out on the electronic structures of system before and after sodium adsorption. The sodium‐ion adsorption has significant impact on the electronic transport of Ti systems.70 Thi2CO2/graphene and V2CO2/graphene heterostructures are encouraging in the application of SIBs due to the low diffusion energy barriers for sodium and estimated high capacities. The heterostructures composed of Ti2CT2  and MoS2 exhibit more negative adsorption energies and larger electrical conductivities than the pure components monolayer. The diffusion barrier for sodium and theoretical capacity for SIBs are calculated as 0.37 eV and over 430 mAh g−1, respectively. Furthermore, the excellent mechanical flexibility and large ultimate tensile strains are favorable to the application of flexible batteries. Tang et al. constructed 16 different heterostructures of bare or O‐terminated Ti, V, Nb or Mo‐based MXenes with MoS2 or VS2. By first principles calculations, they found that sodium ions could be intercalated into the interlayer of structures containing O‐terminated MXenes, whereas the intercalation of sodium ions in the structures with bare MXenes is energy unfavorable. Meanwhile, only the structures of O‐terminated MXenes with VS2 allow five layers of sodium ions, while the others have the distortion. Furthermore, the diffusion barriers for sodium ions of the first layer and interlayer and OCVs of these heterostructures are 0.086–0.221 eV, 0.0002–0.033 eV, and 0.16–0.36 V, respectively, making them promising candidates for SIBs. Besides, the ultralow overpotential for ηORR/ηOER of Ti2CO2/VS2 heterostructure proved that it also has a great potential in Na‐O2 batteries.Additionally, Tipacities.76 The5.2Table Like most of 2D materials, MXenes suffer from the tendency to stack during the experiment, which limits the penetration of electrolyte and impedes the ionic transport. Therefore, in this section, three main strategies to make full use of their electrochemical performance are summarized including 1) synthesizing single‐/few‐layer MXenes, 2) increasing the interlayer spacing of MXenes by introducing intercalation agents, and 3) creating 3D porous structures. 5.2.1Figure  The Ti3C2Tx MXenes and Ti3AlC2 used for SIBs showed the reversible capacity of 100 and 16.8 mAh g−1, respectively. The enhancement can be credited to the expanded interlayer spacing due to the removal of Al layer and the increased active storage sites. Meanwhile, at a current density of 0.8 or 0.2 A g−1 after 1000 cycles, a reversible capacity of 53.7 or 68.3 mAh g−1 could be obtained , which showed the continuous intercalation of sodium ions between layers and various charge‐transfer resistance at different potentials. The results demonstrated that both pseudocapacitive and diffusion capacitive occur. The rectangular shape of cyclic voltammetry (CV) curves  was achieved due to the high pressure and temperature, which can be observed in high‐resolution transmission electron microscopy  with a uniform height of 4 or 7 nm  electrodes with the first layer of delaminated nanosheets with a compact morphology and second layer of multilayer particles with an open structure  results suggested that a relatively high ratio of ‐O terminations in Hf3C2Tx are favorable to the capacities. These findings indicate that the delaminated MXenes with 2D structures are strong candidate electrode materials for energy storage, especially for applications where size is important.As a typical instance in SICs application, the bistacked Tie Figure  were fab @Ti3C2 @Ti3C2 was used as the matrix for sodium metal anode, which accommodated the deposited sodium by pillar effect and effectively guided the nucleation and growth of sodium within the interlayer space, preventing formation of sodium dendrite and benefiting for the uniform sodium deposition. As a consequence, the CT‐Sn(II)@Ti3C2 electrode endowed a high areal capacity of 5 mAh cm−2 after 500 cycles at 10 mA cm−2. Subsequently, they introduced S atoms into the interlayer of Ti3C2 after preintercalation of CTAB with thermal diffusion of elemental S and annealing process . The CT‐Ti3C2 matrix, redox reaction between Na+ and S bonds as well as the pillar effect contributes to the enhanced interface‐dominated pseudocapacitance. DFT calculations revealed that the absorption of Na atoms on the Ti3C2S2 and Ti3C2O2 were lower than that of Ti3C2 and Ti3C2F2 and S‐doped MXenes could accommodate two layers of sodium ions, directing improving the sodium‐ion storage capability. Moreover, the fabricated SICs with AC cathode exhibited excellent long‐term cycling performance with the capacity retention of 73.3% after 10 000 cycles at 2 A g−1 with around 100% Coulombic efficiency  followed by Sn2 Figure .88]882+ ps Figure , forming5.2.4Assembling the 2D materials into 3D porous structures could prove a promising solution to the problems of poor charge transport in electrode materials, hence leading to the devices with high performance.3C2 by continuous shaking treatment of HF‐etched Ti3C2 in alkaline KOH solution (a‐Ti3C2 MNRs), which not only increasing the interlayer spacing to 1.25 nm but also resulting in the porous frameworks of nanoribbons , creating many irregular large pores with diameters of 100–400 nm . As shown in Figure 3C2Tx colloidal solution, followed by the addition of HCl to precipitate sulfur on MXene nanosheets and finally heat‐treated at 400 °C to remove sulfur. The resulted open morphology provided the interconnected ion storage reservoirs and improved the electrolyte/electrode interfacial interaction, which simultaneously ensuring the fast electron transfer and the efficient ionic transport. Thus, good capability of 124 mAh g−1 at 10 A g−1 and cycling stability for 1000 cycles of SIBs were achieved. According to the CV analysis, the predominate nondiffusion‐limited charge storage mechanism together with the high conductivity of MXenes afforded the ultrafast sodium‐ion storage, overcoming the trade‐off between the energy and power densities. Interestingly, the wrinkled structure with enlarged layers of Ti3C2Tx can be achieved by employing a mixture of S and Al during preparing the MAX phase rather than pure Al  via sacrificial melamine formaldehyde (MF) nanospheres templates. The surface modification of nitrogen atoms was favorable to the electrical conductivity of MXenes and redox reactivity. The interconnected nanosheets with porous framework could form the continuous ion pathway and guarantee the sufficient electrolyte penetration, shortening the ion diffusion length and accelerating the kinetics of electrochemical reactions. Therefore, the sodium‐ion storage performance including the rate capability and cycling stability can be significantly promoted. As a result, the fabricated 3D‐printed SICs based on N‐Ti3C2Tx anode and AC cathode 3 nanoparticles have been developed, where the ultrafine nanoparticles are homogenously embedded on MXene flakes. When applied in SIBs, the designed Ti3C2Tx/CoS nanocomposites delivered a remarkable capacity of 267 mAh g−1 after 1700 cycles at 2 A g−1  uniformly distributed in the MXene networks. MXenes provide the highly ionic and electronic conductive network for Sb2O3 nanoparticles and Sb2O3 nanoparticles prevent the MXenes flakes from restacking and serve as sodium‐ions reservoir. Meanwhile, the voids generated during preparation process could accommodate the volume expansion. As expected, the hybrid anodes delivered a capacity of 472 mAh g−1 after 100 cycles at 0.1 A g−1 and 295 mAh g−1 at 2 A g−1. Chen et al. decorated Ti3C2Tx MXenes with Sb nanoparticles (5–10 nm) through a solution‐phase method. The hybrid materials delivered a capacity of 200 mAh g−1 after 500 cycles at 0.1 A g−1. Yang et al. achieved Ti3C2Tx/Bi2S2 composites with a sandwich‐like stereochemical structure  nanocomposites with BPQDs (sub‐10 nm) homogenously anchored on Ti3C2 nanosheets  with fast, complete, and stable alloying reaction as a typical battery‐type anode and increased pseudocapacitive capacity because of the pseudocapacitive component of MXenes. Moreover, the reinforced battery‐capacitive DMES guaranteed the outstanding comprehensive performance of batteries. Li et al. also prepared Ti3C2/BP composites with Ti3C2 nanoflakes and black phosphorus (BP) nanoparticles. As expected, when used as anodes for SIBs, the composites delivered a capacity of 100 mAh g−1 after 200 cycles at 0.1 A g−1. Du et al. reported the composites with FeS2 nanodots (10 nm) uniformly covered on the surface of MXenes. For sodium‐ion storage, the hybrid presented a reversible capacity of 563 mAh g−1 after 100 cycles at 0.1 A g−1. The electrochemical kinetics characterizations including EIS and CV were performed to analyze the charge‐storage mechanism, indicating that the hybrid have the smaller charge transfer resistance and enhanced capacitive character. These electrochemical behaviors were attributed to the large surface area and extraordinary electrical conductivity of the unique Ti3C2Tx/FeS2 heterostructure.Usually, due to the unique desorption behavior of MXenes, researchers have successfully obtained the composites by mixing the two aqueous solutions coupled with longtime stirring and/or sonication. After the self‐assemble process, the composites were collected by centrifugation or vacuum‐assisted filtration. For instance, Meng et al. fabricated the Tis Figure 11a.[113C2Tx/phosphorene hybrid anode for fast and stable sodium storage . Wu et al. constructed a heterolayered structure of Ti3C2Tx flakes composited with SnS2 nanoplates  (PDDA)‐modified layered black phosphorene (BP). The modification of PDDA not only made BP positively charged but also improved their dispersity and stability. As expected, the heterostructure electrodes for SIBs exhibited an ultralong cycling stability of 658 mAh g−1 within 2000 cycles at 1 A g−1 with 0.05% capacity decay of per cycle  films for sodium storage by self‐assembly of negatively charged Ti3C2Tx and positively charged CNTs with CTAB modification, which achieved a capacity of 89 mAh cm−3 at high current density of 5 A g−1. Particularly, Sun et al. reported the freestanding and flexible hard carbon (HC) films  (GpTiC). As depicted in Figure −1, the resulting electrodes delivered a high capacity retention of 84.8% and 75.8% after 1000 and 2000 cycles, respectively.Furthermore, considering their 2D layer structure and easy construction of film electrode, MXene‐based materials have attracted extensive research interest as freestanding, flexible electrodes for next‐generation sodium storage. Xie et al. first reported the freestanding, porous Ti 5 A g−1.120 Pas Figure  for SIBss Figure , effectis Figure . Zhang e5.3.31.5O8.3, Na2Ti3O7@C, or TiO2/C through oxidation or alkalization processes, with careful design and reaction, great advances in the transformation strategies have been made to in situ obtain the MXene‐based composites. For example, Yang et al. constructed accordion‐like Ti3C2/TiO2 nanohybrid with expanded interspacing by facile hydration strategy, where TiO2 nanoparticles are in situ formed and homogenously decorated on the surface of resultant MXenes 3 cubes are uniformly covered on Ti3C2 nanosheets and coated with a carbon layer through in situ solvothermal transformation followed by calcination. Due to the nanoscale integration of MXene and NaTi2(PO4)3, the cells showed a superior rate capacity of 102–208 mAh g−1 at 10–0.5 A g−1 3 and pseudocapacitance‐type MXene. Huang et al. proposed the sandwich‐like Ti3C2/Na0.23TiO2 architecture consisting of short nanobelts on nanosheets  surface. The well‐preserved 2D architecture could guarantee the high conductivity and large electrode/electrolyte contacting area as well as accelerate the sodium‐ion migration. Besides, the robust whole structure can accommodate the volume expansion during repeating charge/discharge processes and ease the peeling off of NTO. Consequently, a capacity of 82 mAh g−1 after 1900 cycles at 2 A g−1 was obtained for SIBs.Other than the partial oxidization of MXenes to achieve the corresponding MXene/metal oxide composites, MXenes can be acted as the titanium source for the fabrication of a new material and simultaneously in situ assembled with MXenes. Yang et al. reported an anode material denoted as MXene@NTP‐C for SIBs, where NaTicination.129 Du1 Figure . Electros Figure  by one‐s2/CNT/TiOxNy where a composite of CoS2, TiOxNy nanoparticles, and CNT are uniformly decorated on the surface of MXene Generally, the dominated acid etching methods obtaining MXenes have the low yield and high risk. In this regard, it is important to develop a controllable, efficient, green, and safe synthetic strategy to synthesize MXenes with controlled number of layers, tunable surface groups, enlarged interlayer spacing, and outstanding quality. F‐free and bottom‐up strategies like chemical vapor deposition, atomic layer deposition should be given more attention in the future.2)As mentioned earlier, the surface chemistries of MXenes could dramatically influence the properties of MXene‐based materials. Different etchant could result in MXenes with different surface groups. So far, MXenes without any surface groups have not been obtained. Besides, the precise surface chemistries of MXenes and the interaction between the surface groups and materials are still not very clear. It has been suggested that the bare/‐O terminated MXenes have better physicochemical properties. Thus, more researches are necessary to be carried out to regulate the surface chemistries and explore their application in the electrochemical storage.3)3C2Tx. Looking for new MXenes with different functions to develop sodium‐ion storage has great potential. For instance, according to the calculations, TiC3 exhibits better anode material performance than Ti3C2 due to the large absorption area and strong adsorption ability for sodium ions. Ti2C MXenes give a capacity of 536.84 mAh g−1. The heterostructures of VS2 with Ti2CO2 and V2CO2 have huge capacities and small OCVs simultaneously. We also hope that these materials can be applied in the promising directions through further optimization.Little works have been published on the MXenes beyond Tiium ions.47 Ti24)The long‐term development of MXene‐based materials is strongly hindered by the restacking problem and instability of MXenes in the oxygen environments. In order to alleviate this situation, efforts can be focused on the rational structural design and accurate morphological control of the electrodes such as the 3D porous architectures, aerogels, and coating techniques.5)Theoretical studies must take into consideration of the nonuniform, incomplete, and mixed coverage of surface groups and stacked multilayer of MXenes to precisely predict their properties and in turn guide experiments. Considering these superior complexities, it is necessary to combine the simulations with the advanced computations including machine learning, classical molecular dynamics, and high‐throughput computation to sufficiently understand the surface chemistries and interfaces. Moreover, comprehensive theories and predictive models as well as the computing software and hardware should be further developed to narrow the gap between the predictions and experimental results at low cost.6)The sodium‐ion storage mechanisms underlying the electrochemical reaction and the component/structural evolution during charge/discharge processes should be in‐depth investigated through in situ works, such as in situ XRD, in situ Raman, in situ scanning electron microscopy (SEM), in situ TEM, and so on. In addition, further optimizing current sodium‐ion storage systems including the electrode configuration, electrolyte type, electrode/electrolyte interactions, and interfaces is also important for desirable performance.In this review, we have highlighted the recent progress on the synthesis, structures, electronic properties, and intercalation chemistries of MXene‐based materials and their applications in SIBs, SSBs, and SICs based on the theories and experiments. On the one hand, the synthetic strategies for pure MXenes such as F‐containing and F‐free etching method could result in different surface groups and interlayer spacing, strongly influencing their structures and properties. The association between them is systematically introduced. Besides, the application of pure MXenes including the multilayer MXenes, single‐/few‐layer MXenes, MXenes with expanded interlayer spacing, and 3D porous structures in the sodium‐ion storage is comprehensively summarized. On the other hand, to make the most of MXenes, a variety of nanomaterials from 0D to 2D have been successfully combined with MXenes, forming the MXene‐based composites with unique architectures. Based on the synthetic strategies, MXene‐based composites are classified into three types: growth on MXenes, self‐assembly, and in situ transformation reaction. The enhanced sodium‐ion storage performance can be ascribed to the synergistic effects between MXenes and the other materials. First, MXenes form the robust and conductive transfer pathway for electrons/ions, dramatically facilitating the transportation kinetics during the electrochemical reactions. Second, the high specific surface area of MXenes matrix ensures the uniform distribution of other active materials, effectively accommodating the volume expansion. Third, the hybrid materials can provide more active sites for sodium‐ion absorption and prevent aggregation of individual nanomaterials. Fourth, the established 3D porous architecture effectively shortens the diffusion path of sodium ions and accelerates the penetration of electrolytes. Therefore, the fabricated sodium‐ion storage systems could deliver the higher rate capability and better cycling stability than the reference devices. Although MXene‐based materials for electrochemical sodium‐ion storage have been extensively studied and show expected prospects, there are still several critical obstacles that need to be further optimized for their practical applications as shown below:The engineering of nanostructured electrode materials is promising in the applications, which could lead to the enhanced electrochemical performance due to their unique nanostructures, large surface areas, and tunable compositions. Ether‐based electrolytes could provide a higher initial CE and longer cycling life in SIBs, while the aqueous electrolyte is a good choice for green, safe, and scale sodium‐ion storage systems. Besides, a more stable and thinner electrode/electrolyte interface is highly expected to buffer the volume change and improve the sodium storage performance. To satisfy the commercial requirements of SIBs in large‐scale energy storage system, comparable performance to state‐of‐the‐art LIBs such as long cycle life and high energy density as well as the lower production cost should be intensively progressed. We believe that success in addressing above‐mentioned problems will contribute to the future evolution of MXene‐based materials in electrochemical sodium‐ion storage applications.The authors declare no conflict of interest.


Human anamorsin is an iron–sulfur (Fe–S)‐cluster‐binding protein acting as an electron donor in the early steps of cytosolic iron–sulfur protein biogenesis. Human anamorsin belongs to the eukaryotic CIAPIN1 protein family and contains two highly conserved cysteine‐rich motifs, each binding an Fe–S cluster. In vitro works by various groups have provided rather controversial results for the type of Fe–S clusters bound to the CIAPIN1 proteins. In order to unravel the knot on this topic, we used an in cellulo approach combining Mössbauer and EPR spectroscopies to characterize the iron–sulfur‐cluster‐bound form of human anamorsin. We found that the protein binds two [2Fe–2S] clusters at both its cysteine‐rich motifs. A combined in cellulo Mössbauer‐ and EPR‐based approach is applied to address controversial aspects of Fe–S cluster‐binding properties of human anamorsin, a protein involved in the biogenesis of cytosolic [4Fe–4S] cluster‐binding proteins. We show that human anamorsin binds two [2Fe–2S] clusters in‐cell in two highly conserved cysteine‐rich motifs typical of the eukaryotic CIAPIN1 protein family, with different electron‐spin‐relaxation properties. The spectra recorded on the cells expressing M2‐ and WT‐anamorsin , and of low‐spin ferrous heme and diamagnetic [4Fe–4S]2+ clusters, the latter two species presenting similar nuclear parameters, and usually denoted as the Central Doublet (CD). Accordingly, four doublets were considered to reproduce the spectra of control cells. A fifth doublet was introduced to fit the spectra of induced cells. This additional doublet corresponds to the difference spectrum obtained by subtracting the control cells spectrum from that of the induced cells . The simulations (see Supporting Information for details) and the experimental spectra are shown in Figure Mössbauer spectra of 2+ clusters.2+ clusters detected upon anamorsin expression. Consequently, the incorporation of a [4Fe–4S]2+ cluster in the M1 or M2 motif of WT‐anamorsin is essentially negligible, and it can be safely concluded that both WT‐ and M2‐anamorsin accommodate [2Fe–2S]2+ clusters.The nuclear parameters of the additional Fe site detected upon induction were typical of oxidized [2Fe–2S]clusters.18, 24in cellulo Mössbauer spectra, we acquired continuous wave (CW) in cellulo EPR spectra of E. coli cells expressing WT‐ or M2‐anamorsin and of the related control cells, all treated with 10 mM sodium dithionite under anaerobic conditions . The in cellulo EPR difference spectra (obtained as reported in the Supporting Information) of reduced E. coli cells expressing M2‐anamorsin exhibited a rhombic spectrum over a wide range of temperatures . When the microwave power was increased from 0.5 mW to 5 mW at 10 K, or the temperature was further lowered to 5 K the signal was easily saturated  and high‐spin FeIII (SFeIII=5/2) ions, leading to a S=9/2 total spin ground state.S=1/2 spin suggests that the partial electron delocalization observed in the reduced [2Fe–2S]+ cluster bound to M2 motif is not strong enough to make the maximal total spin S=9/2 value as the ground state. However, it could allow the lowering in energy of the excited S>1/2 spin states, thus leading to a faster electron spin relaxation rate for the ground S=1/2 state compared with those typically observed in ferredoxin‐type [2Fe–2S] clusters, thus more closely mimicking an electron spin relaxation rate value typical of fast relaxing [4Fe–4S] clusters.This behavior is consistent with the presence of a K Figure . This pein FeII SFeII=2 aE. coli cells expressing WT‐anamorsin showed more complex in cellulo EPR difference spectra, arising from the presence of two rhombic EPR signals , that were previously assigned to the two [2Fe–2S]+ clusters bound to the M1 and M2 motifs of WT‐anamorsin, respectively.in cellulo EPR difference spectrum was dominated by the signal arising from the S=1/2 spin of the [2Fe–2S]+ cluster bound to the M1 motif of WT‐anamorsin.+ cluster increased, as showed by the increase in the intensity of the signal at g=2.016 and g=1.890 , which is responsible, in eukaryotic cells, for the reduction of the [2Fe–2S] cluster bound to the M1‐site of anamorsin[E. coli cells.Here, we have shown by anamorsin11, 12The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


Drosophila Robo2 is a member of the evolutionarily conserved Roundabout (Robo) family of axon guidance receptors. Robo receptors signal midline repulsion in response to Slit ligands, which bind to the N‐terminal Ig1 domain in most family members. In the Drosophila embryonic ventral nerve cord, Robo1 and Robo2 signal Slit‐dependent midline repulsion, while Robo2 also regulates the medial‐lateral position of longitudinal axon pathways and acts non‐autonomously to promote midline crossing of commissural axons. While Robo2 signals midline repulsion in response to Slit, it is less clear whether Robo2's other activities are also Slit‐dependent. To determine which of Robo2's axon guidance roles depend on its Slit‐binding Ig1 domain, we used a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9‐based strategy to replace the endogenous robo2 gene with a robo2 variant lacking the Ig1 domain (robo2∆Ig1). We compare the expression and localization of Robo2∆Ig1 protein with full‐length Robo2 in embryonic neurons in vivo and examine its ability to substitute for Robo2 to mediate midline repulsion and lateral axon pathway formation. We find that the removal of the Ig1 domain from Robo2∆Ig1 disrupts both of these axon guidance activities. In addition, we find that the Ig1 domain of Robo2 is required for its proper subcellular localization in embryonic neurons, a role that is not shared by the Ig1 domain of Robo1. Finally, we report that although FasII‐positive lateral axons are misguided in embryos expressing Robo2∆Ig1, the axons that normally express Robo2 are correctly guided to the lateral zone, suggesting that Robo2 may guide lateral longitudinal axons through a cell non‐autonomous mechanism. Robo1 and Robo2 cooperate to signal midline repulsion in ipsilateral and post‐crossing commissural axons  can influence development in ways other than by acting as canonical midline repulsive Slit receptors  family are widely conserved among bilaterian animals, and their canonical role is to regulate midline crossing of axons by signaling midline repulsion in response to Slit ligands. In groups such as insects and vertebrates, where multiple family members are present, some Robo receptors have acquired additional or alternative activities. In 1.1Drosophila Robos, share a characteristic arrangement of eight extracellular structural domains: five immunoglobulin‐like domains (Ig1–Ig5) plus three fibronectin type‐III domains (Fn1–Fn3). The cytoplasmic regions of Robo receptors are more divergent, but share some or all of four conserved cytoplasmic (CC) amino acid motifs (CC0–CC3). Specific biochemical roles have been identified for some individual ectodomain elements: the N‐terminal Ig1 domain is the primary Slit‐binding domain in most Robo receptors  is individually dispensable for midline repulsion  it prevents midline crossing of ipsilateral and post‐crossing commissural axons in response to the repellant ligand Slit, (b) it promotes midline crossing of commissural axons non‐autonomously by antagonizing Slit‐Robo1 repulsion, and (c) it regulates the medial‐lateral position of longitudinal axon pathways. Robo2 acts alongside Robo1 to signal midline repulsion during the early stages of axon guidance in the embryonic VNC. Genetic data show that this activity of Robo2 is Slit‐dependent, as robo1,robo2 double mutants display more severe midline crossing defects than robo1 or robo2 single mutants, and the robo1,robo2 double mutants phenocopy Slit null mutants  to specify the medial‐lateral distance of longitudinal axon pathways from the midline in response to a midline‐secreted Slit gradient , we have begun a systematic structure/function analysis of ectodomain elements within Robo2. Here, we describe our initial set of experiments using a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9‐based gene replacement approach to examine the requirement for the Robo2 Ig1 domain for the receptor's endogenous roles in midline repulsion and lateral pathway formation. We show that each of these activities is disrupted by deletion of the Robo2 Ig1 domain, and we also show that, in contrast to Robo1, the Ig1 domain of Robo2 is also important for proper axonal localization of the Robo2 protein in embryonic neurons in vivo.To determine the requirements for individual ectodomain elements for the various axon guidance roles of 22.1robo2 locus to express structural variants of Robo2 ‐tagged full‐length robo2 cDNA. In our robo2 modified alleles, the endogenous robo2 promoter, transcriptional start site, first exon , and first intron remain unmodified. Spitzweck et al.  and downstream (3′H) flanking sequences to serve as a template for homology‐directed repair (HDR). The robo2 coding sequence in this donor construct is flanked by restriction sites, allowing us to swap out the full‐length robo2 sequence for any alternative coding sequence. Using this approach, we should be able to generate many different robo2 gene replacement variants using the same set of gRNAs and the same homologous donor backbone. For the CRISPR‐modified alleles described here (robo2robo2 and robo2robo2∆Ig1), each donor construct was co‐injected along with the pCFD4 gRNA construct into Drosophila embryos expressing Cas9 under the control of the germline‐specific nanos promoter (nos‐Cas9.P)  expression plasmid using the pCFD4 gRNA backbone , deletions within the donor coding sequence , and partial replacements that retained all or part of the last exon . Sequencing of genomic DNA fragments from flies in which the 3′ end of the robo2 gene was not replaced revealed sequence polymorphisms relative to the reference genome sequence for robo2 .In addition to the correctly modified 2 Figure , which a2.3Drosophila Robo1 does not affect its expression pattern, axonal localization, clearance from commissural axon segments, or regulation by the endosomal sorting protein Commissureless (Comm)  homozygous for our modified CRISPR alleles  to label all of the axons in the VNC and reveal the overall architecture of the axon scaffold.We have previously shown that deleting the Ig1 domain from s Figure . We alsorobo2robo2 allele reproduces Robo2's normal expression pattern in the VNC of late‐stage embryos: the protein is primarily localized to neuronal axons and restricted to the lateral‐most longitudinal axon pathways in the neuropile , and the original polyclonal anti‐Robo2 antibodies , similar to those present in robo2/leak mutant embryos  of robo2 are essential for viability, these observations suggest that multiple functions of robo2 may be disrupted by deletion of its Ig1 domain. In this initial report, we will focus on the midline repulsion and lateral pathway formation activities of robo2.In addition to these nerve cord defects, we observed embryonic head development defects in 2.4robo2robo2 and robo2robo2∆Ig1 embryos, we used an anti‐FasII antibody to label a subset of longitudinal axon pathways in the VNC. Robo2 is required for guidance of FasII‐positive axons in the contexts of midline repulsion and longitudinal pathway formation: in robo2 mutants, medial FasII‐positive axons ectopically cross the midline (reflecting a lack of midline repulsion), and FasII‐positive lateral axon pathways fail to form correctly  embryos, FasII‐positive longitudinal pathways form correctly in three distinct zones within the neuropile of the embryonic VNC , and FasII‐positive axons do not cross the midline , we observed ectopic midline crossing of FasII‐positive medial axons in 20.6% of abdominal segments (Segments A1–A7) and breaks in the lateral pathway and/or fusions between the lateral and intermediate pathways in 44.4% of abdominal hemisegments , suggesting that Robo2∆Ig1 is not able to substitute for full‐length Robo2 in the contexts of midline repulsion or lateral pathway formation. The observation that lateral FasII‐positive axon pathways are defective in robo2robo2∆Ig1 embryos, while Robo2‐positive axons appear to be positioned correctly within the lateral zone, suggests that lateral positioning of FasII‐positive and Robo2‐positive lateral axons may occur independently.In contrast, we observed ectopic midline crossing (24.2% of segments) and lateral pathway defects (37.6% of hemisegments) in 3Drosophila Robo2 axon guidance receptor and used this approach to show that the Slit‐binding Ig1 domain of Robo2 is required for two distinct axon guidance roles of Robo2 during development of the Drosophila embryonic VNC . We have also shown that the Ig1 domain contributes to the proper localization of Robo2 in embryonic neurons, suggesting a possible role for Robo2 Ig1 in protein trafficking to and/or retention in neuronal axons that is not conserved in Drosophila Robo1. The tools and approach we describe here will facilitate additional structure/function and gene replacement studies of Drosophila robo2.Here, we have described a CRISPR/Cas9‐based gene replacement approach to characterize the functional importance of structural elements in the 3.1Drosophila Robo1 protein  rescue approach employing a large (83.9 kb) robo2‐containing BAC to examine the role of Robo2's Ig2 domain in promoting midline crossing , orthologs from other species, or chimeric/variant receptors, which would facilitate further structure/function or comparative/evo‐devo studies. For example, we have used an equivalent approach to replace Drosophila Robo3 with its Tribolium ortholog Robo2/3 to compare their axon guidance activities  identifying/isolating regulatory sequences is not required, as endogenous regulatory sequences are used instead; (b) the genetics of introducing markers and/or other mutations into the modified background is simplified, as there is no need to track an inactivating mutation plus a separate rescue transgene; and (c) the laborious recombineering and difficult transgenesis with very large BAC DNA fragments can be avoided. This CRISPR/Cas9 gene replacement approach could also be used to replace s Evans, .3.2Drosophila Robo1 is not required for proper expression or axonal localization of the Robo1 protein in the embryonic VNC , suggesting that the Ig1 domains in Robo2 and Robo3 play a role in protein localization that is not shared by the Ig1 domain in Robo1. Importantly, deleting the Ig1 domain from Robo2 does not appear to affect its translation or protein stability, as Robo2 and Robo2∆Ig1 proteins are expressed at equivalent levels and detectable at the cell surface in cultured Drosophila S2R+ cells  are still tightly restricted to the lateral region of the neuropile and are not apparently misguided into the intermediate or medial zones. We have observed that HA‐positive axons similarly remain restricted to the lateral zone in robo2robo1 embryos, in which the robo2 coding sequence has been replaced by robo1, even though these embryos also display lateral FasII pathway defects similar to robo2 null mutants  may not be the same as the lateral axons that normally express Robo2. In other words, Robo2 may also act non‐autonomously to regulate lateral position of FasII‐positive axon pathways. Distinguishing between these possibilities will require examining both FasII and HA expression in the same embryos , and/or generating additional markers to label Robo2‐expressing lateral axons independently of Robo2 expression to examine their lateral positions in wild‐type, robo2 mutant, and robo2 gene replacement embryos. These results also demonstrate that the Ig1 domain of Robo2 is not required for guidance of Robo2‐expressing longitudinal axons to lateral pathways, which indicates that the lateral axons that normally express Robo2 may not require its activity to form and/or join lateral pathways, or that Robo2 directs the medial‐lateral positioning of these axons through an Ig1‐independent mechanism.When 44.14.1.1robo2robo2 donor construct was assembled from four PCR fragments via Gibson assembly (New England Biolabs #E2611). The four fragments were derived from pBluescript (plasmid backbone), the wild‐type robo2 genomic locus (5′ and 3′ homology regions), and an HA‐tagged robo2 cDNA plasmid (4 × HA epitope tag and robo2 coding region). The robo2 coding sequence in the robo2robo2 donor construct is flanked by NheI and NotI restriction sites. To make the robo2robo2∆Ig1 donor, the full‐length robo2 coding sequence was excised with NheI–NotI and replaced with the robo2∆Ig1 coding sequence. Donor plasmids contain engineered mutations in PAM and/or protospacer sequences to prevent cleavage by Cas9. Modified robo2 HDR alleles include the following amino acid residues after the N‐terminal 4 × HA epitope tag, relative to Genbank reference sequence AAF51375: robo2robo2 (E89‐V1463), robo2robo2∆Ig1 (E89‐N90/L187‐V1463). The entire donor regions including coding sequences and robo2 flanking regions were sequenced prior to injection.The 4.1.2robo2 gRNA sequences were cloned into the tandem expression vector pCFD4 , robo2123 and robo2135 , and y1M{w[+mC] = nos‐Cas9.P}ZH‐2A w* (nos‐Cas9.P)   were identified by testing two pools of three F1 females per G0 cross by genomic PCR with primers 458 and 325 (for robo2robo2) or 458 and 327 (for robo2robo2∆Ig1), which produce 1.5‐kb products only when the respective HDR alleles are present. From each identified founder, 5–10 F1 males were then crossed individually to Sco/CyOwg virgin females. After 3 days, the F1 males were removed from the crosses and tested by PCR with the same set of primers to determine if they carried the modified allele. F2 flies from positive F1 crosses were used to generate balanced stocks, and the modified alleles were fully sequenced by amplifying the entire modified locus (approximately 6 kb) from genomic DNA using primers 458 and 545 or 458 and 594, then sequencing the PCR product after cloning via CloneJET PCR cloning kit (Thermo Scientific).The 4.3Drosophila embryo collection, fixation, and antibody staining were carried out as previously described  is reported as “relative cortex HA levels” in Figure robo2robo2 embryos) is reported as “relative axon scaffold width” in Figure d Patel, . The fold Patel,  and Adobd Patel, , anti‐HAFIGURE S1 Expression of HA‐tagged Robo2 proteins in CRISPR‐modified embryos. Ventral nerve cords (VNCs) from embryos homozygous for robo2robo2 (top) or robo2robo2∆Ig1 (bottom) CRISPR‐modified alleles were stained with anti‐HRP  and anti‐HA . Anti‐HA staining in robo2robo2 embryos reproduces the normal endogenous Robo2 expression pattern, with Robo2 protein primarily localized to longitudinal axons in the lateral region of the neuropile (arrows) and a low level of signal detectable in neuronal cell bodies in the VNC cortex (area with asterisk). In robo2robo2∆Ig1 embryos, HA‐tagged Robo2∆Ig1 protein is detectable on lateral longitudinal axons (arrows) and also present at elevated levels on or in neuronal cell bodies within the cortex compared to robo2robo2 embryos (asterisk).Click here for additional data file.TABLE S1 Supplementary TableClick here for additional data file.TABLE S2 Supplementary TableClick here for additional data file.TABLE S3 Supplementary TableClick here for additional data file.


Proliferative vitreoretinopathy (PVR) is a refractory vitreoretinal fibrosis disease, and epithelial‐mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is the key pathological mechanism of PVR. However, few studies focused on the role of METTL3, the dominating methyltransferase for m6A RNA modification in PVR pathogenesis. Immunofluorescence staining and qRT‐PCR were used to determine the expression of METTL3 in human tissues. Lentiviral transfection was used to stably overexpress and knockdown METTL3 in ARPE‐19 cells. MTT assay was employed to study the effects of METTL3 on cell proliferation. The impact of METTL3 on the EMT of ARPE‐19 cells was assessed by migratory assay, morphological observation and expression of EMT markers. Intravitreal injection of cells overexpressing METTL3 was used to assess the impact of METTL3 on the establishment of the PVR model. We found that METTL3 expression was less in human PVR membranes than in the normal RPE layers. In ARPE‐19 cells, total m6A abundance and the METTL3 expression were down‐regulated after EMT. Additionally, METTL3 overexpression inhibited cell proliferation through inducing cell cycle arrest at G0/G1 phase. Furthermore, METTL3 overexpression weakened the capacity of TGFβ1 to trigger EMT by regulating wnt/β ‐catenin pathway. Oppositely, knockdown of METTL3 facilitated proliferation and EMT of ARPE‐19 cells. In vivo, intravitreal injection of METTL3‐overexpressing cells delayed the development of PVR compared with injection of control cells. In summary, this study suggested that METTL3 is involved in the PVR process, and METTL3 overexpression inhibits the EMT of ARPE‐19 cells in vitro and suppresses the PVR process in vivo. PVR is characterized by the formation of proliferative membranes.The EMT of RPE cells is considered as the key pathologic mechanism of PVR.It was reported that epigenetic modifications including DNA methylation and histone acetylation, could regulate the EMT of RPE cells.In this study, we found differences in METTL3 expression between human PVR membranes and normal RPE layers, and then we investigated the effect of METTL3 overexpression on EMT of ARPE‐19 cells and wnt/β‐catenin pathway. Additionally, to determine the role of METTL3 in the EMT of ARPE‐19 cells in vivo, we injected cells overexpressing METTL3 into rat vitreous cavities and assessed their capabilities to form PVR membranes. Our data uncovered the critical function of METTL3 in regulation of proliferative vitreoretinopathy, and the elucidation of this regulatory mechanism will provide new ideas for the treatment of PVR.22.1The study protocol involving human patients was approved by the Ethics Committee of Zhongshan Ophthalmic Center , in agreement with the Declaration of Helsinki, and written informed consent was obtained from each subject. ERMs and SRMs were obtained from PVR patients who underwent vitreoretinal surgery. Surgery for all patients strictly followed the indications of operation. RPE layers were obtained from normal donor eyes from Guangdong Eye Bank . Characteristics of the patients/subjects were listed in Table ® Plus 555 and donkey anti‐rabbit Alexa Fluor® Plus 488 , at 37°C for 90 min. The nuclei were labelled with DAPI , and images were taken under a confocal laser scanning microscope . Sections incubated in the buffer containing no primary antibodies were used as a negative control to examine the specific staining of target proteins.Upon dissection, the specimen was fixed by immediately immersing in 4% paraformaldehyde  for 24 h. The fixed specimen was embedded in optimum cutting temperature compound (OCT), frozen and sliced into 8‐ μm thick sections, and those sections were stored at −80°C. To perform immunofluorescence staining, the sections were thawed at room temperature for 2 h, washed with phosphate‐buffered saline , then punched with 0.3% Triton  for 10 min. Then, the sections were blocked with 10% normal goat serum at room temperature for 90 min and incubated with a mixture of primary antibodies, including pan‐cytokeratin antibody , αSMA antibody , and METTL3 antibody  at 4°C overnight. Subsequently, the sections were incubated with a mixture of secondary antibodies, including goat anti‐mouse Alexa Fluor2.2A human retinal pigment epithelial cell line (ARPE‐19) was purchased from American Type Culture Collection  and cultured in Dulbecco's Modified Eagle's Medium/Nutrient Mixture F‐12  supplemented with 10% fetal bovine serum  at 37°C in a humid atmosphere containing 5% CO2. Recombinant human TGFβ1 was purchased from Novoprotein. SKL2001, the agonist of wnt/β‐catenin pathway, was purchased from MedChemExpress.The pCDH‐Vec control vectors and pCDH‐METTL3 overexpression vectors as well as pLKO.1‐TRC‐Vec control vectors and pLKO.1‐TRC‐shMETTL3 knockdown vectors used for stable transfection were kindly provided by Prof. Shuibin Lin.2.3S is the amount of input sample RNA in ng.The level of m6A in ARPE‐19 cells was detected using the EpiQuik m6A RNA Methylation Quantification Kit (Fluorometric)  according to the manufacturer's instructions. Briefly, total RNA was bound to the bottom of the 96‐well plate with the Binding Solution (BS) and incubated at 37°C for 90 min. Then, samples were captured with the Capture Antibody , by incubation at room temperature for 60 min. Subsequently, the Detection Antibody  was added to the plate. After incubation at room temperature for 30 min, the Enhancer Solution (ES) was added to the plate, and the samples were incubated at room temperature for 30 min, Relative Fluorescence Units (RFU) in Fluoro‐Development Solution were measured using the SpectraMax® fluorescence microplate reader at 530 and 590 nm excitation and emission wavelengths, respectively. The total m6A level was calculated based on the standard curve using the following equation:2.4® Plus 488 .Immunofluorescence staining for cells was performed as described previously.2.5Apoptosis, cell cycle detection and MTT assay were performed as described previously 2.64 cells. The lower chambers were filled with 500μl of ARPE‐19 culture medium containing 20% FBS. After incubation for 12 h and 24 h, cells that passed through the membrane were fixed to the membrane with methanol  and then stained with crystal violet  at room temperature for 15 min. Images were captured using a light microscope . Cell migration ability was assessed based on the cell numbers that passed through the polycarbonate membrane, which were analysed and calculated using the Image J software.The transwell assay was conducted using a 24‐well transwell plate with a polycarbonate membrane . ARPE‐19 cells were digested into a cell suspension of 200 μl without FBS and seeded in the upper chambers at a density of 8 × 102.7ARPE‐19 cells were seeded in 6‐well plates. When the cell density reached 80%, scratches were made by a p200 pipette tip. Cells were observed and photographed under an optical microscope at 0, 24 and 48 h. The cell migration ability was reflected by the wound healing area, which was analysed using the Image J software and calculated as follows:0A is the area of the wound at 0 h, and 24/48A is the area of the wound at 24/48 h.2.8Western blot analysis was performed as described previously,2.9Quantitative real‐time PCR was performed as described previously 2.105 per μl in PBS [pH 7.4]) overexpressing METTL3 or control vector cells or with an equal volume of PBS (pH 7.4). The experimental animals were anaesthetized with chloral hydrate, then an antibiotic and a topical anaesthetic were applied to the right eye after pupillary dilation. Subsequently, a 33‐gauge insulin needle was used to pierce through the sclera, and 3 μl of cell suspension or PBS was injected into the vitreous cavity with a 33G Hamilton syringe fitted with a 33‐gauge microneedle. After operation, the right eye was coated with an antibiotic eye ointment. Vitreoretinal conditions of all injected eyes were evaluated and graded by two masked observers at 1, 3, 7, 10 and 14 days after the operation, and fundus photographs were acquired using the retinal imaging system (Phoenix Micron IV). Because one rat injected with the control vector cells developed cataract after the injection, this rat was not involved in fundus observation. The identification of rat PVR stages (0‐3) follows the rat PVR grading scale : 0, no proliferative response; 1, intravitreal proliferation; 2, epiretinal membrane formation with retinal folds; 3, white dense membrane covering the retina, with retinal folds and localized retinal detachments with or without a localized posterior capsular cataract.All animal experiments in this study have been approved by the Animal Care and Use Committee of Sun Yat‐sen University and strictly adhered to the National Institutes of Health (NIH) guide for the care and use of laboratory animals. The study was carried out as described previously,2.11All rats were sacrificed on day 14 by cervical dislocation. Eyeballs of the dead rats were removed, fixed in 10% neutral buffered formalin  for 24 h and dehydrated using reagent alcohol by gradient. The dehydrated samples were embedded in paraffin and sliced to a thickness of 5 μm. Haemotoxylin and eosin (H&E) staining was performed for the histological analysis of the retina and proliferative membranes, and immunofluorescence of αSMA was carried out to further identify proliferative membranes at the molecular level.2.12‐test was used for comparison between two groups, and one‐way ANOVA followed by Bonferroni's post hoc test was applied for multiple comparisons. Mann‐Whitney U test was used for ordinal data suitable for non‐parametric tests. *P <.05, **P <.01, ***P <.001, ****P <.0001, #P >.05.Statistical analysis was carried out using IBM SPSS 21.0. All data are presented as mean ± standard deviation (SD) of at least three independent experiments unless otherwise specified. Unpaired two‐tailed Student's t33.1Immunofluorescence staining showed that METTL3 was positively expressed on PVR membranes including ERMs and SRMs, and coexisted with cytokeratin, which is a marker of RPE cells Figure . However3.2We used TGFβ1 to induce EMT in ARPE‐19 cells and then detected total abundance of m6A, which was found to be down‐regulated after 48h of EMT induction Figure . To iden3.3The expression of METTL3 was significantly elevated at the protein level after lentiviral transfection with METTL3 overexpression plasmid Figure . ImmunofWe assessed toxic effects of METTL3 overexpression on ARPE‐19 cells through apoptosis assay and observed no significant difference between the METTL3 overexpression group and the vector group Figure . However3.4PVR is essentially an excessive wound‐repair response, and enhanced migration capabilities of RPE cells are vital for EMT process. We used TGFβ of 10ng/ml to induce EMT in ARPE‐19 cells and detected in transwell assay that migration speed of the METTL3 overexpression group was visibly lower than that of the vector group at 12h and 24h after cell seeding, whether in the existence of TGFβ1 or not, as shown by the number of cells migrating through the filter Figure . Consist3.5It has been well established that wnt/β‐catenin signalling pathway is a highly conserved pathway through evolution, who plays a crucial part in cell migration. We have demonstrated that METTL3 inhibited migration of ARPE‐19 cells together with down‐regulating the expression of MMP9, the downstream gene of wnt/β‐catenin pathway. Furthermore, we explored the role of METTL3 in the wnt/β‐catenin pathway and it was indicated by western blot that the protein level of pGSK3β, β‐catenin and cyclinD1 was strikingly down‐regulated in METTL3 overexpression cells, even in the absence of TGFβ1 Figure . In addi3.6P <.01) (Table P <.01)  and pre‐retinal proliferation accompanied with punctate infiltration detected in 11 rats Stage 2), and tractional detachment of the retina in 1 rat (Stage 3). By contrast, among the rats injected with METTL3‐overexpressing cells, 13 had not yet started the PVR process (Stage 0). 4 showed intravitreal proliferation (Stage 1), and 3 showed pre‐retinal proliferation (Stage 2). The difference in PVR development between the two groups was statistically significant  Table .To identify the pathological features of rat retinas in three groups, all rats were sacrificed on day 14 and their eyeballs were carefully collected. The results of H&E staining showed that in the vector group, the retina was subjected to a centripetal pulling force, leading to the shrinkage and detachment of the retina accompanied with swelling and thickening. Figure . Additio3.7As METTL3 overexpression suppressed cell proliferation and TGFβ1‐induced EMT, we next investigated whether knockdown of METTL3 could facilitate proliferation and EMT in ARPE‐19 cells. We down‐regulated the expression of METTL3 effectively with 2 separate, specific shRNAs Figure . Apoptos4In this study, we first observed in human tissues that METTL3 expression decreased in PVR membranes compared with normal RPE layer, and we further verified in ARPE‐19 cells that total amount of m6A as well as the expression of METTL3 was down‐regulated after the induction of EMT. In the further exploration of the regulation mechanism of METTL3 on EMT in vitro, we uncovered that METTL3 overexpression inhibited cell proliferation and migration as well as the expression of EMT markers via wnt/β‐catenin pathway. Finally, it was elucidated in vivo that METTL3 delayed the initiation and development of PVR, which reinforced our findings in vitro.RPE cells are the dominant cellular component in PVR membranes,Recent studies suggest that METTL3 influences cell apoptosis in several cancer cells.METTL3 could play different roles in the EMT of different tumour cells, and we observed an inhibitory effect of METTL3 on the EMT of ARPE‐19 cells. One reason for this diversity may be that m6A modification has diverse decisions on RNA fate, which depends on the m6A reader family YTHDFs.Overall, our data demonstrated for the first time that the epigenetic modification of mRNA is involved in the pathological process of PVR, and overexpression of the m6A methyltransferase METTL3 could inhibit the EMT of ARPE‐19 cells in vitro and restrain the PVR process in vivo. The study highlights a new perspective in the clinical treatment of PVR, and provides a theoretical basis for development of new drugs.The authors confirm that there are no conflicts of interest.Xinqi Ma: Data curation (lead); Methodology (lead); Writing‐original draft (lead). Chongde Long: Data curation ; Methodology . Fangyu Wang: Data curation ; Methodology (supporting). Bingsheng Lou: Formal analysis ; Resources (supporting). Miner Yuan: Data curation (supporting); Resources . Fang Duan: Validation (supporting); Writing‐review & editing . Yao Yang: Funding acquisition (supporting); Writing‐review & editing (supporting). Jiaqing Li: Resources . Xiaobing Qian: Methodology (supporting); Resources (supporting). Jieting Zeng: Data curation (supporting); Methodology (supporting). Shuibin Lin: Methodology (supporting); Writing‐review & editing (supporting). Huangxuan Shen: Conceptualization ; Investigation ; Supervision ; Writing‐review & editing . Xiaofeng Lin: Conceptualization (lead); Funding acquisition (lead); Project administration (lead); Supervision ; Validation (lead).Figure S1Click here for additional data file.


To specify the concentration of the liquid antibiotics to be added to polymethylmethacrylate (PMMA) and its impact on the quality of the spacer is the purpose of this study with liquid clindamycin added to different cements.Staphylococcus aureus, Staphylococcus epidermidis, Cutibacterium acnes and methicillin‐resistant Staphylococcus aureus (MRSA) with a 0.5 McFarland turbidity standard.In the present study, eight different cement mixtures were prepared and investigated. In the following, number 1 indicates the references, 2 all cements after liquid clindamycin was added to the liquid cement compound, 3 all cements after liquid clindamycin was added to the cement powder, and 4 all cements after liquid clindamycin was added to the cement dough. After curing, cements were filled into metal moulds and a pressure of 3 bar was maintained for 30 min. Mechanical investigations were carried out according to ISO 5833 (2002) and DIN 53435 (2007). For microbiological tests, standardized cylindrical mouldings  were produced and incubated in 10 ml buffer solution at room temperature for 24 h. All eluates were generated by spreading previously established suspensions of Apparently, we found that in all investigated cases, the admixture of liquid antibiotic negatively affected the mechanical characteristics of the cement mould. Among the various test groups, the influence on the ISO compression strength and ISO flexural modulus of the investigated test groups was only minimal when liquid clindamycin was added to cement liquid. Compared to admixing of liquid clindamycin into cement powder or dough ISO compression strength and ISO flexural modulus and flexural strength showed the maximum reduction. The efficacy against chosen germs was reduced as well when liquid antibiotic was admixed instead of powder. This admixture of liquid anti‐infective agents resulted in a 234% enhanced elution after 10 days 29 a negative effect on the inhibition zones were detected during the previous period.The admixture of powdery antibiotic is preferable to liquid antibiotics. If no powdery antibiotic is available, we can recommend the admixture of liquid antibiotic to liquid cement prior to dough production in case powdery antibiotics cannot be used. However, we discourage the admixture of liquid antibiotic to cement powder or cement dough during early low viscose phase. Experimental arrangement with Palacos R leading to the results. Endoprosthetic revision surgeries follow specified algorithmsThe manual addition of AB is generally necessary if no customary PMMA cements with the required antibiotics for a particular surgery are commercially availableAnti‐infective agents are often applied perioperatively during the cement preparationet al. (2011)Adding of liquid AB is commonly discouraged because it has a considerable impact on the mechanical stabilityet al. (2014)Candida albicans. Currently, there are no specifications concerning concentration and dose of the liquid AB to be added, let alone its impact on the quality of the spacer in vivo.If a suitable sterile AB powder is not available, use of a liquid AB is necessary. Furthermore, if anti‐infective agents (e.g. antimycotics) are characterized by a poor elution for physical (hydrophobic) or chemical (cross‐links with PMMA) reasons, a liquid agent may be a possible alternative. Antifungal drugs have not received much attention when it comes to admixing.In this study, liquid clindamycin was added in low concentrations (1 g powder in 1 mL sterile water) to the cement liquid, the cement powder, and the cement dough during the early low viscose phase. The cements used were PALACOS R and PALACOS R + G. As reference, powdery clindamycin (1 g) was admixed. All manually manufactured cements were investigated mechanically according to International Standard Operations (ISO) 5833 (2002)In the present study, eight different cement mixtures were prepared and investigated. In what follows, number 1 indicates the references, 2 all cements after liquid clindamycin was added to the liquid cement compound, 3 all cements after liquid clindamycin was added to the cement powder, and 4 all cements after liquid clindamycin was added to the cement dough. : 1a R = reference, 2a R, 3a R, and 4a R. For product designation Basis PALACOS R + G (b): 1b R + G = reference, 2b R + G, 3b R + G and 4b R + G.Each reference cement contained 1 g clindamycin powder; all test cements contained 1 mL clindamycin solution. All cements were produced by the PALAMIX System.In the cases of reference groups 1a R and 1b R + G, 1 g clindamycin powder was mixed with 40 g PALACOS R or 40.8 g PALACOS R + G each and subsequently transferred to the PALAMIX System. Cements were produced according to the manufacturer's specifications. After curing, cements were poured into metal molds and a pressure of 3 bar was maintained for 30 min.For all test groups, 1 g clindamycin powder was dissolved in 1 mL distilled water.In test group 2, 1 mL clindamycin solution was mixed with 20 mL monomer liquid with continuous stirring and then produced by the PALAMIX System with the appropriate cement powder.In test group 3, the PALACOS R or PALACOS R + G polymer powder was mixed with 1 mL clindamycin solution by careful stirring in the PALAMIX System. Afterwards, the dough was produced with the monomer liquid.In test group 4, the respective cements were produced by the PALAMIX System according to the manufacturer's specifications. After 15 sec the PALAMIX System was opened and 1 mL clindamycin solution was added and mixed with the mixing paddles for another 15 s.Mechanical investigations were carried out according to ISO 5833: Implants for Surgery‐Acrylic Resin Cements (2002) and German Industry Standard/DIN 53435 (2007): Testing of Plastics. ISO 5833 specifies three different mechanical tests , whereby special standardized molded bodies are used. The tests are carried out using special equipment with a number of repetitions (statistics) specified by the standard. The mean values are used.Industry Standard/Deutsche Industrie Norm/DIN 53435 (2007) describes two different mechanical tests , whereby special standardized molded bodies are used. The tests are carried out using special equipment with a number of repetitions (statistics) specified by the standard. The mean values are used.Mechanical investigations were carried out at the Heraeus Medical laboratories in Wehrheim, Germany.For microbiological inhibitory tests, standardized cylindrical moldings  were produced and incubated in 10 mL buffer solution at room temperature for 24 h. All eluates were stored at 8°C in the fridge.Staphylococcus aureus, Staphylococcus epidermidis, Cutibacterium acnes, and methicillin‐resistant Staphylococcus aureus (MRSA) with a 0.5 McFarland turbidity standard. A 6‐mm hole was punched centrally into the agar and 50 μL of one eluate was applied per plate. After inoculation, the plates were incubated for 24 h at 37°C.Agar plates were generated by spreading previously established suspensions of Microbiological tests were carried out at the Medical University of Graz, Austria.www.graphpad.com).Data presentation, statistics, and graphs were carried out with GraphPad Prism 5.01 for Windows  compared to cases in which powdery AB were added (reference groups).Among the various test groups, the ISO compression strength of groups 2a R and 2b R + G was like references 1a R and 1b R + G, respectively. Group 3a R differed only slightly from the reference, with a compression strength reduction of only 1.8%, while group 4a R expressed the greatest reduction (8.8%). Within group b, 3b R + G expressed the largest difference compared to reference 1b R + G, with a reduction of 6.3%. Test group 4b R + G showed a reduction of 1.7%.The influence on the ISO compression strength of the investigated test groups was only minimal when liquid clindamycin was added to cement liquid. However, if liquid clindamycin was added to cement powder or dough, the strength values were comparatively lower Fig. .Regarding the ISO flexural modulus, all analyzed test groups differed from the reference groups. Group 2a R showed a reduction of 4.9%, group 3a R 12.9%, and group 4a R 11.5%. Within group b, group 2b R + G expressed the minimal difference compared to the reference, with a reduction of 1.4%, while test groups 3b R + G and 4b R + G showed a reduction of 12.5% each.With respect to the ISO flexural modulus, the strength values were again comparatively lower when liquid clindamycin was added to cement powder or dough instead of cement liquid Fig. .The determined strength values according to ISO flexural strength of test group 2a R and 2b R + G were the closest to the respective reference, with a reduction of 7.44% in the case of 2a R and 6.55% in the case of 2b R + G. Within group a, test group 4a R showed a strength reduction of 7.9% and test group 3a R of 10.9%. In group b, test group 3b R + G expressed the maximum reduction with 14.3%. Test groups 2b R + G and 4b R + G showed a reduction of 6.6% and 10.3%, respectively..After addition of liquid clindamycin, the maximum reduction in flexural strength was detected when mixed directly with cement powder or cement dough Fig...An analysis of the DIN impact strength revealed that the references showed the maximum impact strength compared to all test groups. The minimal difference to the corresponding reference was detected in test group 3a R, with a reduction of 15.5%. Test group 2a R showed a reduction of 16.4% and 4a R of 22.4%. Within group b, test group 3b R + G was the closest to its reference, with a reduction of only 2.2%, while test group 2b R + G showed a reduction of 11.4% and 4b R + G of 5.3% Fig. .Regarding the DIN flexural strength, test groups 2a R (−6.9%) and 2b R + G (0.7%) were the closest to their corresponding references. Compared to reference 1a R, test groups 3a R and 4a R showed a reduction in flexural strength of 17.2% and 12.6%, respectively. Furthermore, test groups 3a R + G and 4a R + G showed a reduction in flexural strength of 17.2% and 12.6%, respectively, when compared to reference 1a R + G.The minimal DIN flexural strength was detected when liquid clindamycin was added directly to cement powder or cement dough Fig. .In group a, the inhibition zone diameter was reduced by approximately 17.1% in test group 2a R compared to reference 1a R. Further on, the inhibition zone diameter continued to decrease by 27.3% in test group 3a R and by even 30.7% in 4a R.While a reduction of efficacy was detected in all test groups of group a (cement without gentamicin), the inhibition zone diameters of test groups b (cement with gentamicin) were similar to their reference 1b R + G. They were slightly reduced, by 5.2%, in test group 3b R + G and 6.9% in 2b R + G. The only exception was test group 4b R + G (admixture to cement dough), with a reduction of 14.2%  and group b (PALACOS R + G) clearly demonstrated a reduced inhibition zone diameter when clindamycin was used without gentamicin.Within group a, test group 2a R showed a reduction of 29.8% in the inhibition zone diameter and was, thus, the closest to the reference. In test group 3a R, a reduction of 45.5% was detected. 4a R even expressed a further reduction of 52.4%. However, in group b, test group 3b R + G achieved the maximum inhibition zone diameter of all test groups, with a reduction of only 9.1% compared to the reference. The inhibition zone diameter was reduced by 11.8% in test group 2b R + G and by 31.7% in 4b R + G  were always reduced compared to those detected in group b (PALACOS R + G).In group a, test group 2a R was the closest to reference 1a R, with a reduction of 14.9%. Test groups 3a R and 4a R showed a reduction of 17.5% and 24.8%, respectively. Meanwhile, in group b, the closest test group to reference 1b R + G was 3b R + G, with a reduction of 10.1% in inhibition zone diameter, followed by a reduction of 11.6% in 2b R + G and 17.7% in 4b R + G Fig. .In the present study, 1 mL liquid clindamycin was admixed manually into cement liquid, cement powder, and cement dough during the early phase of low viscosity.Subsequently, the influence of the different ways to mix AB with cement on the mechanical stability of the implant was tested according to ISO 5833 or DIN 53435, and on microbiological efficacy by applying the inhibition zone testThe results clearly demonstrated that the addition of liquid anti‐infective agents to PMMA cement negatively affected mechanical and microbiological properties compared to the addition of powdery anti‐infective agents. This negative effect could already be detected at a low dose (1 mL of clindamycin) and is expected to increase significantly if higher doses are used. Furthermore, we expect that different types of anti‐infective agents will elicit a similar reduction of mechanical stability and microbiological efficacy if added as a liquid to PMMA cement.Not all admixed (powdery) agents reduce the mechanical characteristics of PMMATo assess the microbiological efficacy of admixed AB, reproducible standards are essential. However, if AB are admixed as liquids, a homogenous distribution within the cement remains questionable. In addition, the exact location of the admixed liquid AB within cured PMMA cement is unclear; if AB intercalate between polymer chains of the matrix, diffusion towards and release from the surface of the cement might be rather limited. Furthermore, the heat development during cement curing can lead to different evaporation rates of liquid AB. The temperature peaks are a serious issue, especially for the production of PMMA spacers outside the body, where the heat development cannot be buffered by prostheses, blood stream, and cement thickness. Liquid AB exposed to extreme heat during the spacer manufacturing process and temperature‐sensitive agents can easily be destroyed.Low doses of liquid clindamycin were manually admixed to PALACOS cement in three different ways to examine at which doses the addition of liquid AB is feasible and how successful the procedure is. This exploration intends to establish a recommendation for surgeons, who may case‐dependently need to mix liquid AB, analogous to the preparation with powdery AB.The admixture of the polar liquid clindamycin to the nonpolar methyl methacrylate (MMA) of the cement liquid will result in two practically immiscible phases (test group 2). How immiscible liquid plus cement powder distributes is strongly influenced by radically polymerizing, transforming liquid MMA into solid PMMADuring the wetting process of cement powder (polymer) with liquid clindamycin, the AB solution will be sucked up immediately by the dry polymer (test group 3). Thus, homogeneous wetting is not possible. The inhomogeneous distribution of AB is certainly one of the major reasons for the weakening of the cement matrix detected by the mechanical tests and weaker performance in the microbiological tests. A reproducible, homogeneous admixture of liquid anti‐infective agents during the early dough phase  is impeded by the polymerization process, which has already started. A lot of polymer chains have already formed. Belated penetration of liquid AB is only selectively possible by using a paddle. Consequently pores will form in the cement matrix after curing, regarded as weak pointsWe could demonstrate that admixing liquid anti‐infective agents to the cement liquid had the smallest negative effect on mechanics and efficacy.The efficacy of AB containing PMMA cements is especially dependent on the diffusion ability of the anti‐infective agents used from the cement matrixC. acnes and MRSA, gentamicin alone is not or is rarely effective. The synergistic effect of the gentamicin/clindamycin combination was remarkable. The synergistic effect was best when clindamycin was added as a powder. Manual admixture of AB to PMMA cement is recommended for revision surgery upon infection and for therapy support. A concentration of 6 g powdery AB per 40 g PMMA cement has been shown to be sufficientCombining gentamicin and clindamycin meets the needs of local endoprosthetics because their germ spectra is complementary with a synergistic effectAdmixing of up to 12 mL of liquid anti‐infective agents to the PMMA cement is possible. This admixture of liquid anti‐infective agents resulted in a 234% enhanced elution after 10 daysThe present work confirms that an admixture of powdery AB is preferable to an admixture of liquid AB, which concurs with previous studiesIf sterile powdery antibiotics are not suitable or available for the admixture to PMMA, liquid antibiotics may be used as an effective alternative. The best study results were achieved in test group 2 (liquid AB to liquid monomer), both mechanically and microbiologically.If admixture of liquid AB is inevitable, the admixture of the liquid AB to the liquid monomer should be considered, followed by blending with the polymer powder. We discourage admixing liquid AB to PMMA powder or cement dough with low viscosity.This study is limited because only one liquid antibiotic (clindamycin) was tested. A low concentration of clindamycin was admixed and investigated. Other antibiotics with various concentrations may have a different impact on the PMMA cement qualities.Prof. Dr Kühn reports personal fees from Heraeus Medical, outside the submitted work. This enabled mechanical investigations to be carried out at the Heraeus Medical in Wehrheim (Germany). None of the other authors has or may receive payments or benefits from Heraeus Medical related to this work.