Graph theory analysis shows that the lamin meshwork is not a random arrangement of filaments but exhibits small-world properties. Our results suggest that lamin filaments arrange to form an emergent meshwork whose topology dictates the mechanical properties of individual filaments. The quantitative insights imply a role of meshwork topology in laminopathies.The nuclear lamina—a meshwork of intermediate filaments termed lamins—is primarily responsible for the mechanical stability of the nucleus in multicellular organisms. However, structural-mechanical characterization of lamin filaments assembled in situ remains elusive. Here, we apply an integrative approach combining atomic force microscopy, cryo-electron tomography, network analysis, and molecular dynamics simulations to directly measure the mechanical response of single lamin filaments in three-dimensional meshwork. Endogenous lamin filaments portray non-Hookean behavior – they deform reversibly at a few hundred picoNewtons and stiffen at nanoNewton forces. The filaments are extensible, strong and tough similar to natural silk and superior to the synthetic polymer Kevlar ®Mechanical strength of in situ assembled nuclear lamin filaments arranged in a 3D meshwork is unclear. Here, using mechanical, structural and simulation tools, the authors report the hierarchical organization of the lamin meshwork that imparts strength and toughness to lamin filaments at par with silk and Kevlar The nuclear envelope (NE), comprising the outer (ONM) and inner nuclear membranes (INM) and the lamina underlining the INM4, forms a rigid-elastic shell protecting the genetic material6. The nuclear lamina—a filamentous protein meshwork at the interface of chromatin and the nuclear membrane7—functions as a scaffold for binding transcription factors9 and provides mechanical stability to the nucleus13. The principal components of the lamina in mammalian cells are mainly four lamins: lamins A and C, and lamins B1 and B2 (refs. 15). Lamins are classified as type V intermediate filaments (IFs), and share a conserved tripartite domain structure with other IFs, viz., a central α-helical coiled-coil rod domain flanked by a non-helical N-terminal head and an unstructured C-terminal tail domain that hosts immunoglobulin (Ig) domains16.During the life cycle of a cell, the nucleus experiences large mechanical variations and sustains fluctuating deformations and stress17 that affect the load-bearing tissues such as striated muscles leading to mechanical failure19. It is therefore important to understand the underlying principles of lamin mechanics in health and disease21. Intact nuclei of Xenopus laevis (X. laevis) oocytes22 and human fibroblasts were previously probed mechanically using the atomic force microscope (AFM)24 to understand the role of lamina in nuclear mechanics. Micropipette aspiration has successfully provided deformation characteristics of nuclei and the NE26. Intact cells and whole organisms have also been employed to quantify the physical properties of the nuclear lamina using stretchable substrates27 and microfluidic devices28. In an attempt to measure lamin mechanics directly, a sharpened tip of an AFM cantilever was pierced inside the nucleus through the plasma and nuclear membranes24.Mutations in lamins cause an important group of diseases termed laminopathies31.Studies performed on intact nuclei and entire organisms are informative but are influenced by the nuclear membranes, chromatin, and surrounding cells. Direct mechanical interrogation of native lamin filaments remains a pertinent goal towards understanding the mechanical properties of the lamina and the nucleus in health and disease32 derived from cryo-electron tomography (cryo-ET) of the nuclear lamina. A key finding is that the meshwork topology influences lamin filament mechanics. The study provides a general understanding of lamin filaments and nuclear lamina mechanics relevant to laminopathies.In this work, using a combined mechanical, structural, and simulation approach, we characterize in situ assembled lamin filaments by applying point loads and measure their deformation and apparent failure in the native meshwork Fig. . Our AFM34 that interact laterally to form tetrameric filaments36. The filaments form a dense meshwork attached to the INM21 at the nuclear lamina7. However, most lamins assemble into paracrystalline fibers in the test tube36. Therefore, native lamin filaments can only be studied using meshworks formed in situ.Lamin dimers assemble into staggered head-to-tail polar structures4  and HeLa cells by AFM. Owing to the size of the mammalian nucleus (10–15 μm diameter), its complexity, and tight connections of the chromatin with the lamina, opening a “window” into a mammalian nucleus is a daunting task. For this purpose, we developed a two-step procedure: in the first step, the cell membrane was de-roofed and the nucleus exposed and in the second step, the nuclear membrane was de-roofed opening windows into the nucleus and the lamin meshwork imaged  filaments has been visualized by electron microscopy in spread NEs of the X. laevis oocytes38. In this study, we placed oocyte nuclei on a poly-l-lysine-coated glass surface to firmly attach them prior to manual opening with a sharp needle and washing away the chromatin. The procedure ensured that the cytoplasmic face of the nuclear membrane  was attached to the poly-l-lysine surface and the lamin meshwork was exposed to the AFM cantilever tip (see “Methods”). The simple isolating and spreading of NEs from the oocytes in a physiological buffer without any detergent or fixative39 enabled AFM and cryo-ET characterization of native lamin filaments  and a condensed chromatin structure that is not associated with the laminants Fig. . Removin38, lamin filaments were arranged in a meshwork exhibiting a rectangular pattern or a less organized architecture interspersed and interacting with the NPCs  with an active closed-loop feedback, recording FE curves at 128 × 128 pixels of the sample at a force of 0.5–0.75 nN. As observed previously by electron microscopy methodsPCs Fig. . Since t44 including IFs46, and rather reflects a general mechanism of material deformation and failure47. Strain hardening was also reported for reconstituted lamin B networks and is dramatically higher than observed in networks of keratin, vimentin, or F-actin48.The FE curves showed an initial slow rise in the force with a plateau up to a yield point (low-force regime) denoting a structural change where plastic or permanent deformation occured in the lamin filament. At larger strains, the filaments showed stiffening resulting in apparent failure at nanoNewton forces (high-force regime) Fig. . FE curvX. laevis oocytes and MEFs (see “Methods”). Mechanical behavior of single lamin filaments was simulated by applying an out-of-plane pushing force on single points in the meshwork model. Similarities between the AFM and the in silico FE curves suggest that we probed single lamin filaments in vitro  sliding requires much larger forces than unfolding coiled-coil α-helices making unfolding the dominant event49. The inter-connectedness of the filaments at junctions may also resist sliding and promote strain-induced stiffening. The stiffening at high force represents the transition of α-helical coiled coils to β-sheets followed by failure. The force tolerated by a filament before failure in silico was comparable to that measured on lamin filaments assembled in situ. Simulations showed that the FE curve profiles did not depend on the size of the probe used to push the lamin filaments, and the transition from α-helix to β-sheet occurred through similar structural intermediates  simulations on single filaments in a meshwork model derived from cryo-electron tomograms of the nuclear laminae of tro Fig. . The sim50, stretched along the long axis52, or laterally dragged on a surface53. Interestingly, these studies showed that laterally pushing on cytoplasmic IF proteins in vitro required forces of 3–5 nN to break and low- and high-force regimes resembling our observation that lamin filaments can withstand forces of up to a 3–5 nN. Previous MD simulations of stretching lamins in an orthogonal meshwork32  in the low-force regime  experie nm Fig. . An engi58  of step unit ≈1.9 nm was detected between 1.0 and 1.5 nN   , α-keratin (45%), elastin (150%)61, but comparable to desmin (240%)50, vimentin (205%)52, and hagfish slime (220%)46.With a capacity to withstand an average deformation nm) Fig. , lamin f56. Interestingly, a direct correlation between the failure force and the stiffness implies that as a filament stiffens, its capacity to bear load increases  before stiffening. The first step of the protocol—pushing up to 0.5 nN—showed a plateau; however, the relaxation curve did not show a plateau (described previously for perfect spring proteins like myosin62). The second pushing step (up to 8 nN) showed the plateau again resulting in a hysteresis between the pushing and the relaxation curves . Interestingly, the plateau force did not depend on the loading rate  up to the apparent failure . However, the measured force (Fhigh) of 2–5 nN is similar to the strength of a single C–C bond65. Increasing the pushing speed of the cantilever 20-fold, and hence the kinetic energy imparted by the cantilever 400-fold, did not change the energy dissipated by lamin filaments. We therefore suggest, that the energy provided by the cantilever is not utilized for breaking covalent bonds but is expended in disrupting non-covalent interactions  involved in the unfolding of protein structures, including the force-induced α-helix to β-sheet transformation and the stick-slipping process of the β-sheets under tension49.The energy attributed to the hysteresis in the low-force regime was determined to be ≈107 J Fig.  increasiure Fig. . The eneT, of a lamin filament (r ≈ 2 nm) is estimated to be ≈147 MJ m−3  or ≈105 J kg−1. Remarkably, the toughness of a lamin filament is superior to that of high-tensile steel (6 MJ m−3), carbon fiber (25 MJ m−3), and Kevlar 49 fiber (50 MJ m−3), much higher than the toughness of natural materials such as elastin (2 MJ m−3), resilin (4 MJ m−3), tendon collagen (7.5 MJ m−3), and at par with that of wool (60 MJ m−3), Bombyx mori cocoon silk (70 MJ m−3), nylon (80 MJ m−3), A. diadematus dragline (160 MJ m−3), and viscid (150 MJ m−3) silks66. The strong, tough and extensible nature of lamin filaments offer promising possibilities for engineering lamin-based materials.Based on the total energy, the tensile toughness, Fload) of 0.75–3.0 nN. As in constant velocity experiments, discrete steps of 1.3 nm (Fload ≤ 1 nN), 4 nm, or 8 nm (Fload ≥ 2 nN) were detected . Next, we applied graph theory67 to compare and quantitate the meshwork topologies. To this end, the meshworks were converted to undirected 3D graphs; lamin filaments formed the links between the vertices (nodes) representing physical connections between the filaments  distribution of both the meshworks follows a power-law [P(k) ~ kλ−] with a scaling factor λ > 5. In other words, the meshworks consist of many nodes with connectivity 3 or 4 and a minor population of hubs of high degrees (5–17) , the lamin meshworks of both species share common topological features. Furthermore, in both meshworks, the average path-length scales with the meshwork size, i.e., the distance between the nodes scales with the number of nodes , cross-linking mass spectroscopy studies suggest that electrostatic interactions between the unstructured head and tail domains of adjacent lamin dimers may drive meshwork assembly63.Visually, the lamin meshwork appears as a random arrangement of filaments. Because a cryo-ET tomogram provides a 3D view of the overlapping areas of a meshwork, it is difficult to discern if the lamin meshwork is near-orthogonal or random (observed in 2D AFM and SEM images). Hence, we performed network analysis to bring out the hidden rules in the meshwork. The network analysis also provided a framework to: (i) compare the design features of the lamin meshwork in different species, and (ii) decipher the influence of meshwork topology on lamin filament mechanics. In a first step, we employed cryo-ET for visualizing the nuclear laminae of tes Fig.  and MEFsnts Fig. . Interes17) Fig. . It is ndes Fig. . This “s69 are suggested to influence the mechanical properties of the nucleus71. We conjectured that the meshwork topology may influence the mechanical properties of the lamina. To test this hypothesis, we performed MD simulations for hundreds of times by pushing single filaments in meshwork models of different topologies . For all meshwork topologies, the simulated profiles of the FE curves resembled those obtained in vitro  for 100 times  indicate the emergent nature of the meshwork may be a key design feature.Biological phenomena exhibiting emergent characteristics have been reported at the cellular78 suggest that the lamin meshwork topology determines filament and nuclear mechanics. To test this, we measured the resistance or counter-force of isolated MEFs nuclei by confining them between two parallel surfaces—a flat-wedged AFM cantilever and a glass surface79 80 show similar lamin meshworks; however, opening the nucleus and washing away the chromatin and associated proteins may have an effect on the lamin meshwork organization and the mechanical properties. Even if the meshwork is altered in our system, our work focuses on the mechanical properties of individual lamin filaments and shows that the meshwork topology dictates the mechanical properties of the lamina. This is a purely physical phenomenon and suggests that different lamin meshworks exhibit varying mechanical properties in agreement with previous studies with overexpression of lamins in different tissues83 and also in disease states71. Our study bridges the gap between in vitro and in vivo by mechanically characterizing the filaments in a lamin meshwork assembled in situ but without the influence of chromatin, nucleoplasm components and the nuclear membrane40.Mechanical measurements of isolated nucleiCombining mechanical and structural tools for interrogating individual lamin filaments offers a glimpse into the molecular mechanisms responsible for the mechanical properties of the lamin meshwork. The direct mechanical measurements of lamin filaments and meshwork simulations provide a mechanistic glimpse into their role in protecting the nuclear contents in response to external forces Fig. . The nonMechanisms protecting the lamin meshwork against mechanical force are integrated at each level in the hierarchical construction of the meshwork—starting from the basic building block α-helix coiled coil up to the higher order meshwork Fig. . The revλ from 0.5 to 5.6) , toughness (energy dissipated or stored) and the resilience of the filaments increased as additional hubs were introduced in a meshwork (increasing .6) Fig. . Our res78. The application of network theory can be applied to quantitatively correlate the organization of the nuclear lamina to its mechanical role in diseases and malfunction by debilitating mutations71 in cells and tissues83. The integrative approach is a promising step towards combining structural mechanics and visual proteomics of entire cells and cellular organelles for obtaining insights into the function of macromolecular assemblies in health and disease85. The results also have implications for material applications of lamins similar to silk and Kevlar®, and rationale design of protein-based meshworks with advanced mechanical functions86. For example, protein engineering of lamins combined with 3D printing technologies could be an area to explore in the near future.Here, we used the interactome of a subset of the nuclear lamin meshwork to model and simulate the meshwork mechanics for a direct comparison with experiments. The combination of AFM and cryo-ET together with network analysis opens possibilities to understand the structural basis of nuclear mechanics in health and diseaseX. laevis oocytes at stage VI were allowed to swell in a low-salt buffer (LSB)  for 20–25 min. A prick with a sharp needle punctured the oocyte and enabled the nucleus to slowly squeeze out. The intact nuclei were immediately transferred to Modified Barth’s Buffer (MBB) 2, TRIS to pH 7.5) and washed gently by a stream of the surrounding buffer repeatedly. The nuclei were then transferred to another Petri dish (World Precision Instruments) coated with poly-l-lysine (1 mg mL−1) which enabled the nuclei to stick firmly onto the glass surface of the dish. With a glass microneedle the nucleus was slightly pushed onto the surface while rolling the needle to break open the nuclear membrane such that the nucleoplasmic side was facing upward, i.e., INM. The nuclear contents including chromatin were gently removed and the stuck nuclear membrane washed with an ample volume of MBB (10–15 mL). If the ONM, i.e., the cytoplasmic side, were facing upward, we would not expect to see lamin filaments but only NPCs when imaged by AFM87. For the experiment with Benzonase® nuclease (Merck), the open nuclear membrane was incubated with 2500 U mL−1 of the nuclease for 1–2 h at room temperature.2) until a confluency of 75–90 % was obtained. The cells were prepared by washing the coverslips first with Ringer’s solution (+2 mM CaCl2) followed by Ringer’s solution without CaCl2. The coverslips were then exposed to hypotonic Ringer’s solution  to swell the cells and facilitate easy opening88. To open the nuclei, a two-step procedure was developed. In the first step, cells were opened by placing an Alcian blue-coated coverslip on the cell-coated coverslip for ~1 min. After ~30 s, the excess buffer between the coverslips was wicked using a filter paper. After a further ~30 s, the coverslips were separated by a stream of phosphate-buffered saline  (500–1000 μL) using a pipette. This facilitated the transfer of half-open cells with intact nuclei onto the top coverslip (Alcian blue-coated). The coverslips were then transferred to deionized water to swell the nuclei. The nuclei were then treated with Benzonase® nuclease (Merck) in PBS (supplemented with 2 mM Mg2+) to digest the chromatin, washed with a high-salt buffer , and then re-equilibrated in PBS. In the second step, the nuclei were opened to expose the lamina. For this, again an Alcian blue-coated coverslip was placed on the coverslip with nuclei, the excess buffer removed using a filter paper, and the coverslips separated by a stream of paraformaldehyde (4%) or PBS for experiments with unfixed nuclei. Both the coverslips were screened for nucleus and the one with higher density used for imaging. The procedure was carried out with and without protease inhibitors (Merck) without any noticeable effect on the lamin meshwork.Nuclear lamina of HeLa Kyoto and MEF cells were prepared for imaging with AFM by de-roofing the nuclei. Cells were seeded on autoclaved coverslips  and allowed to grow at 37 °C , the sample was not measured in those regions. Since we always imaged and did our measurements in the center of the membrane, we are certain that all our measurements were conducted on the INM. In the AFM images of the lamin meshwork we took before pushing on the filament, no ruffles were observed. Moreover, folds and ruffles in the nuclear membrane can break the lamin meshwork.As explained above, oocyte nuclei were attached onto poly-ver Fig. 87. If th−1. X. laevis oocyte nuclear lamina was imaged using AFM quantitative force imaging at 128 × 128 or 256 × 256 pixels. Random positions on lamin filaments were chosen in the closed-loop mode (feedback on); this is an entirely software-based “select and click” process that puts a cross-hair on the filament. Upon switching to the “force-spectroscopy” mode in the software, the tip of the AFM cantilever was pushed on the selected positions with a force of 8–10 nN at different velocities (0.05–5 μm s−1). AFM experiments were performed in an acoustically isolated, temperature-controlled enclosure (26 ± 1 °C). Importantly, the data were collected over a span of >2 years providing reproducible results ensuring that the quality and the selection procedure of the oocytes were maintained and did not affect the final results.Uncoated cantilevers of nominal tip radius ≈10 nm  were used for imaging and force spectroscopy. Before imaging and force spectroscopy, the cantilever sensitivity was determined by pressing the cantilever on a clean part (uncoated) of the glass surface. The spring constants measured using the in-built calibration module of the AFM  agreed with the typical range of the nominal spring constant of 0.03–0.13 N m89. Specifically, the stifness before failure and after α-helix transition to β-sheet was determined by fitting a linear function to the relevant region of the curve—at the region prior to the peak occurrence (failure). The goodness of fit was estimated by the R2 values (0.93–0.99) .The FE signals were exported in ASCII format from the JPK analysis software (v 4.2). The parameters: step unit (distance between the peak and the linear drop in the peak), failure force , stiffness , and deformation  were all determined manually using Punias 3D 4. Cryo-ET images from unswollen X. laevis oocyte nuclei also show that the native lamin meshwork is not always orthogonal  of the lamin meshwork was estimated to be similar in swollen and unswollen nuclei of X. laevis oocyte showing that the lamin meshwork is capable of large changes while maintaining its material properties5. We are therefore confident that in our open nucleus system, even if the meshwork was perturbed, the filament mechanics did not change drastically.In the MEF nucleus, the lamin meshwork is not in an orthogonal pattern Fig. 4. Cryo-E−1) and then retracted 1 μm from the filament at a specific velocity (0.1–2 μm s−1). With a maximum retract velocity 2 μm s−1 of the cantilever over 1 μm, the refolding time of the α-helical coiled coil is estimated to be ~500 ms. The cantilever was pushed again on the same spot until the failure peak was detected.Sample preparation and positioning of the AFM cantilever tip on lamin filaments were performed as mentioned above. For the repetitive protocol, the cantilever was first pushed on chosen positions on lamin filaments with a force of 0.5 nN at a specific velocity (0.1–2 μm s2 v/v), washed with 10 mL PBS and treated with trypsin for 3 min at 37 °C. Reaction was stopped by resuspending the cells in medium containing FCS; the suspension centrifuged at 1000 × g at 4 °C for 5 min. The cell pellet was washed with 5 mL cold (4 °C) hypotonic buffer 90 and centrifuged at 1000 × g for 5 min at 4 °C. The pellet was incubated in 5 mL hypotonic buffer containing 0.1% w/v digitonin on ice for 20 min, dounced 26 times and centrifuged at 1000 × g for 5 min at 4 °C. The pellet containing nuclei was resuspended in 5 mL cold hypotonic buffer, centrifuged , pellet resuspended in PBS (+2 mM MgCl2), and centrifuged again . The purified nuclei were resuspended in 0.5–1 mL PBS (+2 mM MgCl2) containing 1% w/v BSA.MEF cells were grown to 70–80% confluency in a T75 flask at 37 °C (5% CO® reagent (ThermoFischer Scientific). The bottom surface of the nucleus (attached to the glass) was focused and the cantilever approached to touch the glass surface close to the nucleus; this was set as 0 μm. The cantilever was retracted 20–25 μm away from the surface, positioned above the nucleus, and approached to touch the nucleus. Both, the piezo height and the force experienced by the cantilever, were monitored simultaneously in two different channels . Nuclei were stained with NucBlueX. laevis nuclear membranes were prepared in a similar manner as that for AFM experiments. Isolated nuclei were transferred onto a glow-discharged, perforated carbon copper grid (Quantifoil R2/1 200 mesh). The nuclei were opened manually and the NEs spread over the grid. The grid was washed three times with LSB to remove residual chromatin and oocyte debris. A 3 μL drop of BSA-conjugated 10 nm colloidal gold was applied, and the grid was vitrified by rapid plunge-freezing in liquid ethane. Tilt-series (+60o to −60o) of the nuclear membrane was collected using Serial EM (v 3.8) in a Titan Krios microscope (ThermoFischer Scientific) equipped with an energy filter and a K2 Summit direct electron detector using the dose fractionation mode at 5 fps. The projection images were acquired at a defocus of 6 μm at ×42,000 magnification, corresponding to a pixel size of 0.34 nm. The sample was exposed to a total dose of ≈52e− Å−2. The tomograms were reconstructed using TOM Toolbox and rendered in Amira software . Tomograms of MEF nuclear lamina were acquired and rendered following published procedures4.4 cells/10 μL sample buffer). Ten microlitres of each sample was loaded on a precast gradient gel (4–12%) and proteins were separated by SDS-PAGE according to their molecular weights. Next, the separated proteins were blotted on a PVDF membrane (1.1 mA cm−2 for 90 min). Afterwards, the membrane was blocked with 5% dry milk in PBS-T  for 30 min at RT and then incubated with primary antibody , anti-lamin B1 antibody : sc-374015, 1:500), diluted in 5% dry milk in PBS-T for 1 h to overnight at RT or 4 °C, respectively. The membrane was washed 3 × 10 min with 5% dry milk in PBS-T and incubated with secondary antibody  diluted in 5% dry milk in PBS-T for 1 h at RT. Then, the membrane was washed 3 × 10 min with 5% dry milk in PBS-T and once with PBS. To visualize the secondary antibody, Immobilon Western Chemiluminescent HRP substrate (Millipore) was added and the signal was detected using a Fujifilm LAS-3000 Imaging system.Cells  were harvested, counted with a hemocytometer and lysed in an appropriate amount of SDS sample buffer  and the parameters analyzed using the network analyzer module.Lamin filament density in individual slices of the tomograms were rendered manually using the Amira software. Thirteen tomograms from Scale-free model of the lamin meshwork: The mesoscopic model of the lamin meshwork used here is based on a combination of experimental and full atomistic data. The geometry of the meshwork was obtained from our experimental observations that give its topological feature to be scale free. The meshwork was initially generated by deciding the connectivity of the end nodes of all the lamin filaments with the node list and their connectivities built according to a Barabasi–Albert model. This connected node list was further modified by repeating a simple Monte-Carlo process for 1,000,000 times to randomly select a node and change its connectivity. This change will be more likely to retain if it makes the degree probability distribution closer to the desired Power-law distribution (P(k) ~ k−λ) with the desired scaling factor λ. We randomly assigned the coordinates of all the nodes in a two-dimensional plane that mimic the effect of nuclear membrane and treat each connection between the nodes as an existing lamin filament. Thereafter, we ran another simple Monte-Carlo process for 600,000 times and for each one we took the coordinates of a randomly selected node to have a random move. By doing so we ensured that the filaments in the meshwork have an average length of 12 ± 3 nm. The entire process was repeated to obtain meshwork models with different λ values. To account for the random effects introduced in the model generation, we performed individual loading tests on 100 meshworks and statistically studied the mechanical response from simulations.Multiscale-modeling of lamin filament: To address the failure mechanism of the filament meshwork, the interpretation of the dynamic fracture property of the entire meshwork of complex topology requires a large-scale meshwork model as well as accurate representation of the dynamical property of each filament and their interactions with atomic interaction detail (in the order of Å). Thereby, the multiscale modeling can be a strategy combining accuracy and efficiency, which includes the full atomic modeling for single filaments and the mesoscopic model for IF meshwork. The full atomic model provides an accurate description of the physical mechanisms governing the yielding  and stiffening  process of each filament under loading, and records the accurate description of the FE relation of the single filament during the loading history52. The atomic simulations were carried out using the CHARMM19 all-atom potential energy function with an effective Gaussian model for the water solvent91. We apply a constant temperature (300 K) controlled by a Berendsen thermostat. The alpha-carbon atoms at the two ends of a tetramer are pulled on by using steered MD with a pulling velocity of 0.1 Å ps−1 and a stiffness of 10 kcal mol−1 Å−2 using the CHARMM simulation package.r0 = 1 nm as the initial distance between neighboring beads as well as the equilibrium length of the springs. This length is much smaller than the persistence length of the lamin filament (≈1 μm). Therefore, each filament is modeled by a series of beads interacting according to nonlinear inter-particle multibody potentials (as the force field for the mesoscopic model) that is obtained from full atomistic simulations52. This force field of our mesoscopic system is given by3252 and the tensile force between two bonded neighboring beads is given bySimulation results in the current paper are obtained from a mesoscopic model, which inherits the physical properties of the atomic ones and provides a faithful estimation of dynamic fracture property in the large scale. We used mesoscopic beads to model each filament within the meshwork and fix r is the bond length and ki and 52, their value are given as −1 Å−2, −1 Å−2, −1 Å−2, −1 Å−3, −1 Å−4, −1, −1 Å−1, and −1 rad−2 relating to the bending stiffness of the IF EI. The non-bonded interaction −1 that prevents two filaments to overlap and penetrate during the simulation.In Eq. , r is thComputational experiments on the meshwork: Calculations were carried out in two steps: (1) relaxation followed by (2) loading. We modeled the effect of the nuclear membrane as a fixed plane substrate that has van der Waals interactions with the lamin filaments with a surface energy of 20 mJ m−2 at an equilibrium distance of 1.5 nm and cut-off distance of 4.0 nm. This energy is in the same order as vimentin protein adhesion on a silica surface and cell adhesion on a mineral surface92. Relaxation was achieved by heating up the system, then annealing the structure at a temperature of 300 K, followed by energy minimization. After relaxation, the system was maintained at 300 K in an NVT ensemble  and loading applied by displacing a point within a single filament . While the single point was under a pushing force, the rest of the beads within the cut-off distance interact with the substrate, giving the reaction force to deform the filament. This set-up resembles the AFM pushing at a single point on a lamin filament, continuously displacing particles in the boundary at a speed of 0.1 mm s−1. It is confirmed that the loading rate chosen here is slow enough that leads to quasi-static deformation conditions. Each of the computational simulations reported in this paper was carried out using the LAMMPS simulation package. Other steps, including network generation and post-analysis for statistical calculations, were performed by using customized scripts in MATLAB. More details on the modeling of each filament and numerical values of the physical terms can be found in previous papers52.Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Reporting Summary


Despite the advancement of the healthcare system, low birth weight (LBW) remains as one of the leading causes of under-five mortality. This cross-sectional study aimed to determine the prevalence of LBW and its associated factors among 483 third trimester pregnant women recruited from six selected public health clinics in the Federal Territory of Kuala Lumpur and the state of Selangor, Malaysia. Pregnant women were interviewed for information on socio-demographic characteristics, smoking behaviour, and second-hand smoke (SHS) exposure at home and in the workplace. Information on the obstetrical history and prenatal care visits history were retrieved from the maternal medical records, while infant’s birth outcomes were retrieved from infant medical records. The prevalence of LBW (<2.5 kg) in infants was 10.4%, with a mean birth weight of 3.0 [standard deviation (SD) 0.4] kg. Results from the multivariable logistic regression model showed that inadequate weight gained during pregnancy  and exposure to SHS at home  were significantly associated with LBW. In conclusion, pregnant women should monitor their rate of weight gain throughout pregnancy and avoid SHS exposure at home to reduce the risk of delivering LBW infants. Birth weight is a vital parameter that reflects maternal nutritional status and well-being before and during pregnancy, as well as foetal growth and development. LBW is defined as the weight taken at birth of less than 2.5 kg by the World Health Organization (WHO) . LBW is LBW is associated with a range of short- and long-term consequences, and these consequences may persist into adulthood . The “BaFindings from previous studies demonstrated that maternal factors including ethnicity, maternal age, educational level, employment status, monthly household income, household size, gravidity, and parity are important factors that are associated with LBW in infants ,23,24,25Therefore, the present study aims to determine the prevalence of LBW and its associated maternal and environmental risk factors among full-term infants. Identifying the risk factors of LBW could help to guide the development of prevention strategies in order to reduce the incidence of LBW and indirectly reduce childhood mortality and morbidity resulting from LBW.n = 11), incomplete maternal data (n = 3), and lack of data on birth outcomes (n = 38). The final sample size was 483 mother–infant pairs, with a response rate of 90.2%.This cross-sectional study is part of the Mother and Infant Cohort Study (MICOS) that focused on the contribution of early nutrition on the development of malnutrition and allergic diseases in infants during the first year of life, whereby this paper is focused on the risk factors associated with LBW among full-term infants. The protocol of MICOS has been described elsewhere . The pre2) was calculated as pre-pregnancy weight (kg) divided by the square of pre-pregnancy height (m2) and was then classified into underweight (<18.5 kg/m2), normal weight (18.5–24.9 kg/m2), overweight (25.0–29.9 kg/m2), and obesity (≥30.0 kg/m2) [Information on socio-demographic characteristics including maternal age, ethnicity, educational level, employment status, household size, and monthly household income were obtained from the pregnant women through a face-to-face interview. Meanwhile, information on obstetrical history including pre-pregnancy weight, height, last measured weight at the third trimester of pregnancy, gravidity, and parity were obtained from the maternal medical records. Pre-pregnancy BMI (kg/m0 kg/m2) . Total G0 kg/m2)  and cateInformation on prenatal care visit history including first prenatal care visit and the number of prenatal care visits were retrieved from the maternal medical records. First prenatal care visit was recorded as trimester of pregnancy in which the prenatal care was started, while the number of prenatal care visits was calculated as the total number of prenatal visits attended by the pregnant women (from the first attendance until the last attendance) at any primary healthcare facilities throughout their pregnancy.Information on maternal smoking and SHS exposure during pregnancy were assessed using the Global Adult Tobacco Survey (GATS) Core Questionnaires developed by the Centre for Disease Control and Prevention (CDC) . MaternaInformation on birth outcomes, namely, gestational age at birth, infant’s sex, and birth weight, were extracted from the infant medical records. Low birth weight was defined as weight at birth of less than 2.5 kg . Prior tp-value of less than 0.25 in the SLR model were included in the multivariable logistic regression model [p-value for the Omnibus test was less than 0.05, the percentage obtained in the overall classification table was more than 80.0%, the p-value for the Hosmer and Lemeshow test was greater than 0.05, and the p-value for multicollinearity and interaction was greater than 0.8 [p < 0.05.Data were analysed using IBM SPSS Statistics version 22.0 software . All continuous variables were tested for normality and the value of skewness within the range of ±2.0 is considered as normally distributed . Descripon model . The forthan 0.8 . ResultsCharacteristics of the pregnant women are shown in Birth outcomes of the full-term infants are presented in Results from the SLR analysis showed that low monthly household income , inadequate gestational weight gain , and being exposed to SHS at home  were significantly associated with an increased risk of LBW .p-value less than 0.25 were included in a forward multivariate logistic regression model. These variables were educational level, household size, monthly household income, total GWG, gravidity, exposure to SHS at home, and total number of prenatal care visits. Results of the multivariate logistic regression model  and explained 5.8% of the variances in LBW.Variables significantly associated with LBW in the SLR analysis with a on model  showed tThe present study revealed that about one in ten of the full-term infants were born with LBW. Inadequate GWG and exposure to SHS at home during pregnancy were the two main risk factors for LBW.The prevalence of LBW reported in the present study was slightly higher than those reported in the NHMS III in Kuala Lumpur (7.2%) and Selangor 8.3%), Malaysia . In cont%, MalaysThe present study showed that pregnant women with inadequate weight gained during pregnancy were more likely to deliver an LBW infant. The association between inadequate GWG and a higher risk of LBW was also been observed among pregnant women with a normal pre-pregnancy BMI recruited from a government hospital in Turkey . SimilarExcessive GWG was prevalent in our study population. A significant positive association between GWG during pregnancy and birth weight was reported in previous studies ,48,49, wThe present study shows that exposure to SHS at home was significantly associated with LBW. Consistent with the findings from several local studies in Malaysia, pregnant women who were exposed to SHS during pregnancy had a higher risk of delivering an LBW infant ,51. BailInconsistent with previous studies, we found no associations between any of the socio-demographic characteristics and obstetrical factors with LBW. It contradicted the findings from the previous studies in which pregnant women with older age, more household members, unemployed, and financially poor were associated with a higher risk of delivering an LBW infant ,51. SeveWe found no significant association between prenatal care visit history and LBW in the present study, which contradicted the findings from previous studies that reported a lower risk of LBW among pregnant women who started their prenatal care visit earlier and had at least 4 prenatal care visits ,51. ThisSeveral limitations need to be considered in the present study. Firstly, this was a cross-sectional study and the cause–effect relationships between the independent variables and LBW were unable to be determined. Secondly, this study was conducted among mother–infant pairs in Selangor and Kuala Lumpur, Malaysia, with an unbalanced proportion of ethnicity among respondents . Thus, findings from the present study may not be generalised to other populations. Thirdly, self-reported exposure to SHS may lead to underestimation or overestimation due to recall bias. Lastly, there is a discrepancy in the number of infants with LBW compared to those with normal birth weight. This discrepancy might reduce the power to detect some risk factors, for instance: socio-demographic characteristics , obstetrical history , and prenatal care visit history .To conclude, one in ten of the full-term infants were born with LBW. Inadequate GWG and exposure to SHS at home during pregnancy were associated with a higher risk of LBW in infants. The present study suggests that women should monitor their rate of weight gain throughout pregnancy and avoid SHS exposure at home in order to reduce the risk of delivering an LBW infant. In addition, pregnant women should be informed about the importance of gaining sufficient weight throughout pregnancy and the adverse effects of SHS exposure at home during their routine prenatal care visit.


Children used AKL-T01 for 4 weeks, followed by a 4-week pause and another 4-week treatment. The primary outcome was change in ADHD-related impairment ) after 4 weeks. Secondary outcomes included changes in IRS, ADHD Rating Scale (ADHD-RS). and Clinical Global Impressions Scale—Improvement (CGI-I) on days 28, 56, and 84. IRS significantly improved in both cohorts  after 4 weeks. IRS, ADHD-RS, and CGI-I remained stable during the pause and improved with a second treatment period. The treatment was well-tolerated with no serious adverse events. STARS-Adjunct extends AKL-T01’s body of evidence to a medication-treated pediatric ADHD population, and suggests additional treatment benefit.STARS-Adjunct was a multicenter, open-label effectiveness study of AKL-T01, an app and video-game-based treatment for inattention, as an adjunct to pharmacotherapy in 8–14-year-old children with attention-deficit/hyperactivity disorder (ADHD) on stimulant medication ( Attentional difficulties specifically are associated with impairment in several areas, such as academic functioning, shy and passive social behavior, and impaired adaptive functioning in children/adolescents3.Attention-deficit hyperactivity disorder (ADHD) is a neurodevelopmental disorder of persistent impaired attention, hyperactivity, and impulsivity that negatively affects daily functioning and quality of life. ADHD is one of the most commonly diagnosed pediatric mental health disorders, with a prevalence estimated to be 5% worldwide6. However, there are limitations to both modalities of treatment13. Based in part on these limitations, there has been considerable interest in additional approaches to augment multimodal ADHD management. Digital therapeutics may offer improved access, minimal side effects, and low potential for abuse while providing targeted treatment options for improving cognitive functions, such as attention. A previous randomized controlled trial (STARS-ADHD), for example, demonstrated AKL-T01, a video-game based digital therapeutic, administered for 4 weeks at home, significantly improved attentional functioning as measured by the Test of Variables of Attention (TOVA®) in children diagnosed with ADHD, and a greater proportion of children on AKL-T01 showed evidence of improved ADHD-related impairment (IRS)14 compared to a digital control intervention after 4 weeks of treatment. Although the IRS change scores did not achieve statistical significance, a trend was observed (p = 0.09) that favored the active treatment.Front-line treatment for ADHD includes pharmacological and non-pharmacological interventions, which have demonstrated short-term efficacyHowever, this trial noted several important limitations, including eligibility criteria that only included children not taking ADHD medication and scoring below a specific objective attention functioning threshold on the TOVA at study entry. In addition, this study only evaluated treatment effects after a single 4-week treatment regimen.The objective of the present trial, therefore, was to evaluate the AKL-T01 intervention as an adjunctive therapy in children currently taking stimulant medication for their ADHD, with no criteria pertaining to TOVA functioning at study entry. In addition, the present study evaluated a longer duration of intervention—specifically participants used AKL-T01 for 4 weeks (days 0–28), followed by a treatment break of 4 weeks (days 29–56) and a second treatment period of 4 weeks (days 57–84). By including a broader range of participants, another goal of the study was to evaluate the intervention under more “real-world” conditions and with a focus on clinical assessments related to ADHD-related impairment and symptoms. Specific to this point, we used the Impairment Rating Scale (IRS) as the primary outcome measure given findings from the previous study and its clinical relevance. It is important to note that this study was not designed to evaluate comparative efficacy and did not include a blinded control condition.A total of 236 participants were screened for inclusion in this study, with 206 meeting eligibility criteria Fig. . One hunOverall, 195/206 (95%) participants completed the study through the primary endpoint at the end of the initial 4-week AKL-T01 treatment period (Day 28); 186/206 (90%) completed through 8 weeks ; and 179/206 (87%) completed all 12 weeks of the study . The On Stimulants cohort remained on stimulants during the AKL-T01 treatment pause.In general, participants remained consistent with respect to their assigned treatment group throughout the trial. All children in the No Stimulants cohort remained off stimulants during the first AKL-T01 treatment period and the treatment pause period. Several participants in the On Stimulants cohort stopped their stimulant medication . Information on crossover is not available in the 2nd treatment period.14. In the present study, therefore, the 50% compliance criterion was applied during the first treatment month as an additional compliance indicator. For comparison in the present study, 79.1% of children met this criterion versus 85.6% in the previous RCT.All participants were instructed to play AKL-T01 for ~25 min/day, 5 days/week, for each 4-week treatment period. Within each day, the 25-min sessions were divided into five 5-min “missions.” As such, participants were recommended to play up to 100 missions over the course of a 4-week treatment (5 missions/day × 5 days/week × 4 weeks). In the first treatment month, the mean (SD) number of missions played in the intention-to-treat (ITT) population was 81.1 (28.37) missions for the On Stimulants cohort and 73.0 (33.83) missions for the No Stimulants cohort. Across the full study and 2 treatment months, the overall mean (SD) number of missions played in the ITT population was 135.2 (56.20) missions for the On Stimulants cohort and 116.4 (65.69) sessions for the No Stimulants cohort (of 200 recommended missions). In the previously published RCT, children were required to play 50% or more of the recommended missions as part of the per protocol populationSupplementary Figure p < 0.001) in the On Stimulants cohort and −0.5  in the No Stimulants cohort  (Table AKL-T01 significantly improved (lowered) ADHD-related impairment as measured with the IRS (clinician-rated) after the first 4-week treatment in both cohorts (primary endpoint): with mean changes from Baseline to Day 28 in IRS overall severity score of −0.7 : ; DOF: 127; Cohen’s d: .65; p < 0.001). These treatment effects increased to Day 84 from baseline in both cohorts  and further slightly improved at Day 84 in both cohorts.To evaluate the stability of effects after a 4-week treatment pause and the effects of an additional treatment period, we conducted exploratory analyses of our endpoints for Day 56 (after the treatment pause) and Day 84 (after the second 4-week AKL-T01 treatment). Mean changes from baseline to Day 56 in IRS overall severity score, ADHD-RS total score, and Inattention and Hyperactivity-Impulsivity subscale scores remained significantly improved for participants in both cohorts  in the On Stimulants cohort were classified as responders on the primary outcome of the IRS . In the No Stimulants cohort, 40.5% of participants were IRS responders. ADHD-RS symptom response rates were similar for children on and off ADHD medication with over 40% of responders across both cohorts after the second 4-week treatment. Table p = 0.041) but this difference was not found at Day 84 (p = 0.94). None of the other responder comparisons between groups were significant.In post hoc analyses, the response rate for children On Stimulants was higher in the IRS at Day 28 (after 4 weeks)  participants experienced a device-related adverse event (AE) during the 12-week trial. AEs were generally evenly distributed between the On Stimulants and No Stimulants cohorts. The most common device-related AEs were decreased frustration tolerance, headache, and irritability Table . All devThe present study adds to and extends the clinical evidence base for AKL-T01, a video-game based treatment for improving attentional functioning in children with ADHD. Results address limitations from previous studies in several ways. First, children who were concurrently treated with stimulant medication showed comparable benefits on impairment and ADHD symptoms compared to children not taking stimulant medication. Second, results show that effects persist during a 4-week treatment pause and further improve with a second 4-week treatment period. Finally, this study demonstrates improvement in clinically relevant (symptoms and impairment) outcomes for children without pre-existing attentional impairment, as measured by the TOVA in previous studies. Collectively, these results increase the generalizability of previous studies and provide further evidence that AKL-T01 is a viable option for improving attention functioning in children with ADHD, including children receiving concurrent pharmacotherapy.14. This previous study required children who were taking medication to discontinue use prior to randomization or to be naive to medication. Given that the majority of children diagnosed with ADHD take medication to help manage their condition12, it is important to evaluate the effects of new non-pharmacological interventions in the context of routine care. Moreover, based on existing literature15, practice guidelines typically recommend a multimodal approach for treating ADHD that includes both medication and behavior therapy16. Few studies have examined the effects of combining pharmacotherapy with other kinds of non-pharmacological interventions in children with ADHD. The few trials that studied cognitive training approaches as adjunctive to ADHD medication in children with ADHD show inconclusive results regarding additional benefits of such interventions in children with ADHD medication18. Results from the present study showed that the effects of AKL-T01 on ADHD-related impairment and ADHD symptoms were generally similar in children taking stimulant medication versus those not taking any medication.A previously published randomized controlled trial of AKL-01 (STARS-ADHD) reported significant improvements in attentional functioning (TOVA API) and evidence for improvement in ADHD-related impairment (IRS) after 28 days of treatment compared to a control group who received a tablet-based digital control word gameThe previous trial also excluded children whose objectively measured attentional functioning on the TOVA was not significantly impaired . This resulted in a number of children who otherwise met criteria for ADHD not being eligible. In the present study, there were no eligibility criteria for enrollment based on attentional functioning. By including both children who were taking medication and whose objectively measured attentional functioning was free to vary, the sample was more representative of the general population of pediatric patients with ADHD.14 was brief and the effects of continued, additional treatment periods with AKL-T01 were not explored. The current study addressed this by examining two separate 4-week bouts of AKL-T01 treatment with a treatment pause of 4 weeks in between the treatment periods. Results showed that outcome measures remained stable during the treatment pause and continued to improve following the second treatment period, and these patterns were seen in children on and off stimulant medication. These effects are similar to those observed in other open-label studies of both non-pharmacological  and pharmacological treatments22. In general, the magnitude of changes in ADHD symptoms after treatment were comparable to other non-pharmacological interventions. For example, a randomized, blinded, sham-controlled trial of trigeminal nerve stimulation—a treatment that has received FDA clearance for ADHD—reported effect sizes for ADHD-RS scores of approximately d = 0.5, compared to an effect size of d = 0.79 in the current, unblinded study23. Additional research is needed to better contextualize the comparative efficacy of AKL-T01 compared to other non-pharmacological treatment approaches for managing ADHD symptoms.The duration of treatment (4 weeks) in the original studyResults of the present study should be interpreted in light of several important limitations. First, the study was conducted without randomization or a blinded control group, thus making it impossible to rule out the possibility of placebo effects accounting for the findings or regression to the mean effects. But we observed similar outcomes in the IRS and ADHD-RS, with a similar magnitude of treatment effects for both the On Stimulants and No Stimulants cohorts, which was also comparable to those observed in the previously published STARS-ADHD RCT. This comparability suggests consistency in the degree to which AKL-T01 is associated with changes in parent and clinician-reported impairment and ADHD symptoms. In addition, the generally larger improvements in all measures during treatment, compared to the treatment pause suggest that effects were not merely regression to the mean. Moreover, given that one of the study aims was to examine longer-term treatment outcomes, maintaining participants on a control intervention for 3 months would have been challenging. Nevertheless, it is important to not interpret the present findings as evidence of efficacy in the absence of a control group, but rather as a demonstration that previously reported results are consistent in a sample more closely resembling that which would be seen in clinical practice, allowing a more clinically meaningful view on extended treatment regimens with AKL-T01.Second, the present study still excluded children with significant comorbidity. Although children with some conditions such as ODD and mild anxiety were allowed to participate  was a multisite, open-label study in children/adolescent 8–14 years old with a confirmed diagnosis of ADHD, primarily inattentive or combined subtype (per DSM-V and MINI-KID), experiencing suboptimal treatment response , at Screening. Participants had to be either stable on stimulant medication (On Stimulants cohort) or off any ADHD medication (No Stimulants cohort), and not present other significant comorbid psychiatric diagnoses. All participants had an IQ score ≥80 (per KBIT-II). The study was conducted in accordance with the International Conference on Harmonisation Regulations, and was approved by each site’s institutional review board . All participants and their caregivers provided written or verbal assent and written consent, respectively, prior to any study activities being conducted.The study planned to enroll 203 participants with a confirmed diagnosis of ADHD at 15 sites. Participants were divided between two cohorts: 130 participants were planned to enroll in the On Stimulants cohort, and 73 participants were planned to enroll in the No Stimulants cohort.During the first 4-week AKL-T01 treatment phase (Days 1 through 28) participants in the On Stimulants cohort received stimulants plus AKL-T01, and participants in the No Stimulants cohort received AKL-T01 only. During the 4 weeks AKL-T01 treatment pause (Days 29 through 56) between treatment phases, participants in the On Stimulants cohort remained on stimulant, and participants in No Stimulants cohort remained off ADHD medication. During the second 4-week AKL-T01 treatment phase (Days 57 through 84), participants in the On Stimulants cohort received stimulants plus AKL‑T01, and participants in the No Stimulants cohort received AKL‑T01 Fig. .Fig. 3STEligible patients were male/female children/adolescents aged 8–14 years old with a confirmed diagnosis of ADHD ), primarily inattentive or combined subtype (per DSM-V and MINI-KID), experiencing suboptimal treatment response, as reflected by ADHD-related impairment  at screening, and no significant comorbid psychiatric diagnoses that would make participation in the study difficult. All participants had an IQ score ≥80 (per KBIT-II). For the On Stimulants cohort, participants had to be on a stable dose of stimulant medication, at an approved dose, for at least 30 days prior to enrollment and show a moderate response on a stimulant, with room for improvement. For the No Stimulants cohort, participants had to be stable without a stimulant or any other ADHD medication for at least 30 days before the baseline visit. Complete inclusion and exclusion criteria are in Supplementary Note The AKL-T01 intervention was preloaded and administered on iPad mini 2 tablets . At the baseline visit, eligible patients were instructed to use AKL-T01 for about 10 min while a study coordinator monitored the session to ensure that patients could follow the rules of AKL-T01. Patients were further assessed on ADHD-related impairment (IRS) and symptoms, and attentional functioning at the baseline visit.Children then played the AKL-T01 digital treatment at home, for 4 weeks (Days 1–28), followed by a 4-week treatment pause (Days 29–56) and then a second 4-week AKL-T01 treatment (Days 57–84). Study visits were scheduled on Days 28, 56, and 84. During the AKL-T01 intervention periods, patients were instructed to use AKL-T01 at home for 5 days per week, for 4 weeks. A daily session consisted of five 4–5-min multitasking “missions” . Compliance was monitored electronically. AKL-T01 generated automatic reminders.33. In brief: users multitask by responding to a perceptual discrimination targeting task and a simultaneous sensory motor navigation task. Users advance by reducing interference costs (closes the performance gap between multitasking and single-tasking), and real-time and periodic recalibration occurs to maintain an optimal difficulty level.AKL-T01 is a digital therapeutic that uses a proprietary algorithm designed to improve attentional control, by training interference management (multitasking). AKL-T01 mechanisms have been described previouslyThe primary endpoint was the change in ADHD-related impairment as measured by the  from Baseline to Day 28. ADHD-related impairment is assessed across domains such as social functioning, academic progress, and self-esteem, including an overall impairment severity score.Secondary endpoints included the overall change in ADHD-RS Total Score from Baseline to Day 28 (parent-reported clinician-administered assessment of the child’s frequency of ADHD symptoms), the Clinical Global Impressions (CGI) is a clinician-rated assessment of a patient’s global functioning, scored from 1—very much improved to 7—very much worse, with 4 being no improvement. Secondary endpoints related to attention functioning (TOVA) were assessed but will be reported separately.Exploratory outcomes included clinical measures related to ADHD symptoms and impairment after the 4-week AKL-T01 treatment pause (Day 56) and after the second 4-week AKL-treatment phase (Day 84).All analyses were performed according to a prespecified statistical analysis plan, unless otherwise specified as post hoc. Unless otherwise indicated, statistical comparisons used a two-sided significance test evaluated at the 95% level of confidence.Unless otherwise specified, all outcomes were analyzed using an ITT methodology (defined as any participant enrolled in the study who received a device).t-test. Incorporating a 10% dropout rate required that at least 130 + 73 = 203 participants were planned to be enrolled in the study.Sample size estimation was performed and determined that an effective total of at least 66 non-medicated participants (No Stimulants cohort) would be needed to demonstrate an effect size of 0.40 with 90% power and 95% confidence. This target effect size was determined from our previous RCT study (STARS-ADHD), where the within-group effect size on the primary (IRS) was 0.38. Since there was no prior data to estimate an effect size for the medicated participants (On Stimulants cohort), we hypothesized that we might observe an attenuated effect (~25% decrease in effect size) and so this cohort was powered for a 0.30 effect size. This analysis revealed that at least 117 medicated participants (cohort 1) would be needed to demonstrate an effect size of 0.30 with 90% power at the 95% confidence level using a within-group All analyses were conducted using a complete case analysis. In no situation were missing data to be imputed.Statistical methodology for the primary, secondary, and exploratory endpoints are summarized below. For a complete list, please see Supplementary Note t-test evaluated at the 95% level of confidence.Within each cohort, the primary efficacy endpoint for each participant was the change in the clinician-reported IRS “overall severity of child’s problem in functioning and overall need for treatment” score from baseline to Day 28, defined as the score on Day 28 minus the score at baseline. Missing data were not imputed. If a participant was missing either the baseline or Day 28 IRS assessment, that participant was excluded from the analysis. The results of the primary efficacy analysis were summarized by cohort as mean change from baseline (95% CI). Significance was assessed with a two-sided paired The following secondary efficacy endpoints were tested using the same technique outlined for the primary efficacy analysis: (1) change in ADHD-RS from baseline to Day 28, (2) CGI-I score at Day 28, and (3) change in TOVA constituents from baseline to Day 28. TOVA results will be reported elsewhere.Family-wise error rate was controlled using a hierarchical testing strategy. Within each cohort, the maximum allowable Type I error rate was set to 0.05. For additional details, please see Supplementary Note Exploratory analyses included the second treatment month (Day 84) and interim pause time point (Day 56) for the primary and secondary endpoints , 30% improvement on ADHD-RS total score at Day 28 and score of 2 or better on the CGI-I at Day 28. Post hoc response rates were calculated for Day 0 to Day 84 (after the second 4-week AKL-T01 treatment).p = 0.152)).This was a multicenter study. The primary endpoint was assessed for poolability across sites within each cohort using a linear mixed model where site was a random effect (there was no significant effect of site and cohort (Further information on research design is available in the Supplementary InformationReporting Summary


Autism Spectrum Disorder (ASD) and Attention-Deficit/Hyperactivity Disorder (ADHD) represent two common neurodevelopmental disorders with considerable co-occurrence. Their comorbidity (ASD + ADHD) has been included in the latest diagnostic guidelines . The present study focuses on social visual attention that i) is a main aspect of social attention reflecting social cognition and ii) its atypicalities have been suggested as a potential biomarker for ASD. Considering the possible shared background of both disorders and their comorbidity, it is important to compare such traits directly. Here, 73 children and adolescents paired for age and IQ diagnosed with ASD (N = 12), ADHD (N = 21), comorbid ASD + ADHD (N = 15), and “typically developing” (TD) controls (N = 25), were shown static real-life social scenes while their gaze movements were recorded with eye-tracking. Scenes with two levels of social complexity were presented: low complexity (one person depicted) and high . Gaze fixation variables were investigated. Fixation duration on faces was significantly reduced only in ASD + ADHD which also required longer time to fixate all faces at least once. Fixation duration on faces in ASD was reduced, compared to TD, only when looking at scenes with high versus low social complexity. ADHD individuals did not differ from TD. Concluding, the observed alterations of social visual attention support the existence of possible dysfunctional particularities differentiating ASD, ADHD, and ASD + ADHD, which can be revealed with the new method of eye-tracking technique. The objective gaze measurements provided contribute to the development of biomarkers enabling early diagnosis, amelioration of care and further interventions specified for each group. Autism Spectrum Disorder (ASD) and Attention-Deficit/Hyperactivity Disorder (ADHD) are two of the most studied Neurodevelopmental Disorders . The maiThis co-occurrence has been under discussion both for clinicians and researchers even before DSM-V implemented the changes, in relation to symptom manifestation, both of autistic traits in ADHD individuals  and viceSocial cognition traits represent a main domain of shared and unique characteristics in ASD and ADHD . The twoIn ASD research, various eye-tracking studies including participants from infancy until adulthood exist –30. PartCompared to the ASD literature, a limited number of studies have emerged regarding ADHD and social visual attention . The fewEven less is known about participants with comorbid ASD and ADHD and to the best of our knowledge, there have been no eye-tracking studies about the visual exploration of social scenes in this group. However, it has been reported that ASD + ADHD participants showed deficits in attentional orienting as measured with Event-Related Potentials of face and gaze processing . MoreoveEye tracking measurements represent accurate, reproduceable measures that might serve as potential objective indications of medical states in order to predict the incidence and early diagnosis of a disorder as well as monitor outcomes of interventions . RegardiThe present study sets out to fill the described gap by directly comparing the ASD, ADHD, and comorbid ASD + ADHD groups in social visual attention to real-life scenes with different levels of social complexity. The study design enables us to differentiate the following scenarios: whether altered social visual attention measures are (a) specific to ASD and thus absent in those with ADHD symptoms; (b) present in ASD and more pronounced in comorbid cases; (c) a shared feature of these neurodevelopmental disorders. Additionally, interactions with the level of social complexity, early gazing behavior, and latencies until first facial fixations are explored.Results), paired for age and IQ  and theFor the purpose of this study, assessments of general and specific symptomatology in native language were applied additionally, characterizing the semiology of each group. Assessment of general emotional and behavioral problems was realized for all participants with the parent-rated Child Behavior Checklist (CBCL/6-18) , which hThe local institutional ethics committee approved the study protocol, which was conducted in accordance with the Declaration of Helsinki . All parThe experimental procedure was composed of two sessions, for the majority of the participants in following order: in the first session  an IQ test was performed  and pre® software . For data recording the iView RED250 remote eye tracker  was used with a sampling frequency of 120 Hz and a visual angle of 20° and binocular tracking mode. Preceding each task, a standard five-point calibration and four-point validation was conducted, as suggested by the software’s manufacturer, which was considered as successful if the deviance was lower than 0.5°  with the Presentationhan 0.5° . Event c applied .In addition to the above-mentioned selection criteria, data were examined separately in real time speed and only data fulling following thresholds were included: tracking ratio >75% (percentage of non-zero gaze positions divided by sampling frequency multiplied by run duration) and/or a total fixation duration ratio >50% , as previously described , 69.Areas of Interest (AOIs) were delineated for every picture annotating the whole image and differentiating the following categories: face, body and non-social, the latter including all non-social elements of the stimulus . This categorization on naturalistic scenes, including images of people during an interaction, has been used in previous studies . Eye traTwo basic types of fixation duration variables were calculated  for each participant: i) total fixation duration representing the sum of fixation durations over one stimulus, irrespective of AOI and ii) fixation durations for each AOI category, defined as the sum of fixation durations for each stimulus and representing the total fixation duration spent respectively in the three AOI categories. For the face AOIs, the analysis was additionally conducted for the first 5 and 10 seconds, representing the early gazing behavior of the visual exploration .Additionally, two latency variables were investigated for the face AOIs (in seconds) . FirstlyStatistical analysis was conducted with the statistical software R . Linear mixed effects models were implemented for the analysis of the interaction between fixation duration and clinical groups with the “lme4” library (v1.1-19) , while pFirstly, total fixation duration was examined and following fixed effects were entered into the model: i) group of participants , ii) level of social complexity , iii) interaction term between group of participants and level of social complexity, and iv) status of previous social training . A random intercept was used for each subject. Secondly, the fixation duration of each of the three AOI-categories  was examined with separate linear mixed effects models, structured accordingly. Here, the same explanatory variables as above were introduced as fixed effects, while individual total fixation duration for the respective stimulus was added as a covariate, in order to limit the effect of inter-individual differences in total fixation duration. Finally, similarly to the analysis of the whole run duration, the fixations to the faces were analysed separately for the first 5 and 10 s with total duration of fixations in the respective timespan as a covariate.2) with Yates’ continuity correction for categorical variables.For the latency variables two-way analysis of variance (ANOVA) was implemented, with the group of participants as the between-subject factor and stimuli as the within-subject factor. As only one value is available per stimulus, mixed effects models cannot be applied. For the statistical analysis of demographics, an ANOVA was used for continuous variables and a chi-square , three did not fulfil the IQ criterion  and one ADHD patient had not discontinued medication, leading to their exclusion from the analysis. Datasets of further nine participants with subthreshold values of tracking ratio and/or total fixation duration ratio  were excluded. Finally, datasets from n = 73 participants were included in this present study. Groups did not differ in age and IQ . There was a male predominance in the clinical groups . Previously accomplished social training in the ASD groups was equally balanced . In the ADHD group both main types were represented (N = 13 with F90.0 and 8 with F90.1). In the ASD group childhood autism and Asperger’s syndrome were the main diagnoses , while 1 subject was diagnosed with atypical autism (F84.1). In the ASD + ADHD group the distribution was similar  and regarding comorbidity, either it was diagnosed as such, or the criteria for both ASD and ADHD were fulfilled (for those diagnosed before the introduction of DSM-V). Confirmed diagnoses, as well as ADOS and ADI-R, are listed at the bottom of Descriptive statistics of diagnostic tests, questionnaire scoring results and results of statistical inference, as well as sociodemographic variables are shown in For the stimuli with low social complexity, the control group showed a total fixation duration of 109 ± 7.1 s , while the clinical groups showed lower total fixation durations: the ADHD group 104 ± 11.6 s, the ASD group 103 ± 12 s and the ASD + ADHD group 103 ± 10.6 s. For the two stimuli with high social complexity, the control group showed a mean total fixation duration of 104 ± 9.8 s, while the ADHD group spent 103 ± 7.6 s fixating, the ASD 100 ± 12.3 and the ASD + ADHD 98 ± 13.1 s. Boxplots did not show any extreme outliers.post-hoc test < 0.001). For the ASD + ADHD group and both levels of social complexity, total fixation duration was significantly lower by 6.2 s , while the two other groups did not differ compared to TD . Consistent with the results for the total run duration, the other clinical groups did not show any significant differences. The interaction observed between social-complexity and the ASD group for the total run duration, was at trend level at early gazing . Tabular results of fixed effects parameter estimates for 5 and 10 s can be found in For the first 5 and 10 s, analogous mixed effects analysis of fixations on faces was implemented. For the first 5 s, no group difference was evident. At 10 s, the total fixation duration on face AOIs across all groups was on average 2.6 ± 1.3 s for low versus 0.9 ± 0.7 s for high social-complexity stimuli. In the ASD + ADHD, the fixation duration on faces was significantly lower for both social-complexities by 0.3 s compared to the TD group . The mixed effects analysis with the non-social elements AOI category did not show any group differences or any interactions of group with social complexity. There was a significant effect of social complexity, with participants fixating more on non-social elements in low social complexity stimuli . Detailed fixed effects parameter estimates can be found in No significant group effects and no interactive effects were observed for the AOIs of bodies. Similar to the face AOI the fixation duration on high social complexity stimuli was distributed among the bodies of the four depicted persons  = 1.0, p = 0.392; all contrasts versus TD: p-values non-significant) and no interactive effects with social complexity. By contrast, the latency to completion of face fixations was 5.7 s higher in the ASD + ADHD compared to the TD group  = 3.49, p = 0.017, p-value of contrast 0.003). The other two clinical groups did not show prolonged latencies compared to the TD group.All four groups showed similar latencies to first face fixation  and the State Funded Doctoral Scholarship of Baden-Wuerttemberg. The article processing charge was funded by the University of Freiburg in the funding programme Open Access Publishing.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Executive functions have been previously shown to correlate with empathic attitudes and prosocial behaviors. People with higher levels of executive functions, as a whole, may better regulate their emotions and reduce perceived distress during the empathetic processes. Our goal was to explore the relationship between empathy and executive functioning in a sample of children and adolescents diagnosed with Attention Deficit and Hyperactivity Disorder alone or associated with comorbid Disruptive Behavior Disorders and/or Autism Spectrum Disorder. We also aimed to examine the role of empathic dimensions and executive skills in regulating externalizing behaviors. The 151 participants with ADHD were assigned to four groups according to their psychiatric comorbidity (either “pure” or with ASD and/or ODD/CD) and assessed by means of either parent- or self-reported questionnaires, namely the BRIEF−2, the BES, and the IRI. No questionnaire was found to discriminate between the four groups. Affective Empathy was found to positively correlate with Emotional and Behavioral Regulation competences. Furthermore, Aggressiveness and Oppositional Defiant Problems were positively associated with Executive Emotional and Behavioral Regulation competences. On the other hand, Rule-Breaking Behaviors and Conduct Problems were negatively associated with Affective Empathy and with Behavioral skills. Our study provides an additional contribution for a better understanding of the complex relationship between empathic competence and executive functions, showing that executive functioning and empathic attitudes interact with each other to regulate aggressive behaviors. This study further corroborates developmental models of empathy and their clinical implications, for which externalizing behaviors could be attenuated by enhancing executive functioning skills. Feeling empathy for someone means understanding his/her emotions and/or personally experiencing the same; it means creating a customized space in one’s own inner world to host the world of the other. In other words, it refers to the ability to share and comprehend another person’s thoughts and moods . FeelingIn light of the close association of empathy with contextual factors, early childhood experiences and social behaviors, it is direct to assume that psychopathological conditions are often convoyed by empathy deficits ,6. EmpatThe reduction or absence of empathy represents the cornerstone of Conduct Disorders (CD) characterized by disruptive and antisocial behaviors . Adults Some evidence has also shown that empathy is compromised in a proportion of children with Attention Deficit and Hyperactivity Disorder (ADHD); in particular, lower levels of social perspective taking are observed ,21,22. IDespite the identification of selective or global impairments of empathy in clinical settings, functional studies, aimed at assessing brain correlates of the experience of feeling the emotions of others, found that empathic attitudes are activated through an emotional processing which is regulated both by bottom-up and top-down circuitry within the prefrontal and limbic cortex . An earlAccording to the Russian Doll Model, several components of the empathic response, which have been added layer by layer during evolution, remain functionally integrated . At its A stepwise transition from immature forms of emotional contagion to more sophisticated expressions of prosocial attitudes, as brilliantly theorized by Hoffman’s developmental theory of empathy , may be It should be noted, however, that in these studies, EF were variably measured through different neuropsychological tasks, such as the Go/No-Go test and the Wisconsin Card Sorting Test, which assess specific EF such as Cognitive Flexibility and Working Memory in laboratory settings. No study, however, used caregiver-reported measures of behavioral patterns for children and adolescents to evaluate EF in everyday life environments. Given the centrality of EFs in controlling behaviors in everyday life, relying only on laboratory measures of EF performances, detected with clinical tests, may limit the confidence and completeness of the clinical evaluation. The measures based on performances, in fact, depict only limited aspects of the EF system in a narrow time frame and do not fully capture the integrated multidimensional decision-making process based on an analysis of the priority which is often that of real life situations .A recent meta-analysis  summarizThe present study aims to explore possible relationships between the different facets of empathy and the specific subcomponents of EF in a clinical sample of children and adolescents primarily diagnosed with ADHD, compared to children with comorbid ASD or ODD/CD or both. EF profiles have been evaluated through the Behavior Rating Inventory of Executive Function (BRIEF)  questionMoreover, the current study examined the role of empathic dimensions and executive skills in regulating the externalizing behaviors typical of some clinical manifestations of ADHD and ODD/CD, such as for instance aggression, oppositional behaviors and rule transgression. It is believed, in fact, that emotional regulation plays an important role in inhibiting aggressive behaviors by implementing perspective-taking abilities and empathic concern towards the others .Our study included 151 drug-naïve participants  recruited in our third-level Department of Child and Adolescent Psychiatry from March 2019 to December 2019. Subjects underwent a multi-dimensional assessment, through individual clinical evaluations and observations of social interactions within a group of peers, in order to thoroughly investigate ASD symptomatology. The diagnoses were made according to the Diagnostic and Statistical Manual of Mental Disorders–Fifth edition (DSM−5) , based oThe Child Behavior Checklist for ages 6 to 18 years (CBCL–6/18) ,58 is a Patients were included if they received a diagnosis of ADHD, with or without comorbid psychiatric conditions, including ODD/CD and ASD. The latter was suspected based on either medical history and clinical observations or a total SCQ score above the cut-off value and later confirmed through the administration of the Autism Diagnostic Observation Schedule–Second Edition (ADOS-2) . The excWe identified four clinical groups in our samples: “pure” ADHD group , including 64 subjects ; comorbid ADHD + ODD/CD group (here-in-after referred as the ADHD+ODD/CD group), including 43 subjects ; comorbid ADHD + ASD group (here-in-after referred as the ADHD+ASD group), including 19 subjects ; comorbid ADHD + ASD + ODD/CD group (here-in-after referred as the ADHD+ODD/CD+ASD group), including 25 subjects . All participants and parents were informed about assessment instruments and participated voluntarily in the study after written informed consent was obtained for assessment procedures from parents of all children. The study conformed to the Declaration of Helsinki and the Regional Ethics Committee for Clinical Trials of Tuscany, Pediatric Ethics Committee section, approved the study .Patients’ clinical profiles were also assessed by means of several questionnaires, either through self or parental reports. Particularly, the Italian versions of the following measures were used: the Behavior Rating Inventory of Executive Functions–Second version (BRIEF-2)  administThe Behavior Rating Inventory of Executive Function–Second version (BRIEF-2)  is the uThe Antisocial Process Screening Device (APSD)  is a 20-The Basic Empathy Scale (BES)  is a sel®  and RStudio®  software. For each clinical variable with continuous distribution, outliers were defined as observations lying outside the range between [first quartile − 1.5 * interquartile range] and [third quartile + 1.5 * interquartile range] and removed. For each BRIEF-2 subscale-related variable, observations were removed whether the corresponding values at either the Infrequency or the Inconsistency scale was higher than 99° percentile of normalized data. As for the BES and the IRI questionnaires, z-scores were computed for each subscales, i.e., the Cognitive and the Affective Empathy scales; thus, z-scores of the two questionnaires were merged together so that a single pair of variables assessing cognitive and affective empathy was available for all subjects irrespectively of the age.Statistical analyses were performed by means of MatLabp-value < 0.05) between clinical variables with continuous distribution. A Tukey post-hoc test was used whenever the ANOVA led to a statistically significant result in order to identify significant comparisons between variables. Spearman’s ranks correlation coefficients were estimated to detect significant relationships between rank values of questionnaire variables. Benjamini and Hochberg’s False Discovery Rate (FDR) correction method [Analyses of Variance (ANOVA) were used to assess significant differences  were included as dependent variables of the models, whilst the three main indexes of the BRIEF , the z-scores of the two subscales of the merged Empathy Questionnaire (Affective and Cognitive Empathy) and the SCQ total scores were used as independent variables of the models.Scores obtained by the four clinical groups in the aforementioned questionnaires are reported in Significant correlations between BRIEF-2-related subscales and APSD, SCQ and Empathy questionnaires are illustrated in Pearson’s linear correlation coefficients were here represented as colored boxes to show only significant relationships between continuous variables of selected questionnaires. The traditional significance level of 5% was corrected by means of Bonferroni’s correction method for multiple comparisons. A color legend bar is displayed on the right.Finally, a linear regression model was applied to identify statistical relationships between four aggressive/disruptive behavior-related CBCL subscales , as dependent variables, and several subscales from the other questionnaires, as independent variables, namely: the three main indexes of the BRIEF-2 , the two Empathy subscales  and the SCQ total score.As displayed in The first aim of the present study was to explore possible relationships between the different aspects of empathy and specific subcomponents of EF in children and adolescents with ADHD alone, ADHD and ODD/CD and/or ASD. To this aim, our effort was to achieve at least three major objectives: (1) to assess empathic attitudes, CU traits, antisocial behaviors and socio-relational skills in our four groups of patients; (2) to identify potential relationships between these variables and the EF profiles; (3) to explore the interrelated role of the cognitive and affective dimensions of empathy and the EF in regulating antisocial behaviors and aggressiveness. To the best of our knowledge, this is the first study of the kind performed in such a multi-structured sample of children and adolescents.First, none of the questionnaires used in our study was individually able to discriminate between the four clinical groups. The SCQ is a clinical checklist, based on parents or caregivers report, aimed at identifying the presence of abnormal social and communicative behaviors. Although the SCQ is used as a screening tool for ASD symptoms, the lack of significant differences in our sample between ADHD patients with or without ASD suggests that the social functioning deficits reported in ADHD patients , at leasSimilarly, no significant differences emerged between groups in the two questionnaires assessing CU traits. Our finding may suggest that these traits may be trans-nosographic and thus be present not exclusively in ODD/CD, but also in ADHD  and in AThe principal goal of the present study was to assess the reciprocal relationship between empathic attitudes and EF in ADHD patients. A recent meta-analysis , mainly In our sample, affective empathic competences, assessed through the BES and the IRI questionnaires, are negatively correlated with Emotional and Behavioral Regulation impairments, identified through the BRIEF-2 questionnaire. The greater the difficulties in “hot” EF, the lower the empathic attitudes, or, in other words, individuals with severe deficits in the EF profile exhibited lower scores on Empathy questionnaires. Nonetheless, no significant correlations were found neither for EF metacognitive domains nor with the cognitive empathy subscale.Our results suggest that inhibitory and emotional control play an important role in regulating externalizing behavior, even controlling for empathic competences. As stated above, empathic attitudes are activated through an emotional processing which is regulated both by bottom-up and top-down circuitry within the prefrontal and limbic cortex ,77. ThisOur results indicate that EF are more strongly related to the affective empathy than to the cognitive one, which is in disagreement with the results of the aforementioned meta-analysis . NonetheFurthermore, significant positive relationships were found between several variables of the APSD questionnaire and the BRIEF-2 subscales related to the Emotional and Behavioral Regulation domains. Namely, the higher the scores in CU traits-related questionnaires, the higher the impairment in the Emotional-Behavioral Regulation competences. This finding is in line with a recent study assessing the relationship between CU traits and parent ratings of EF . In partFinally, in our research, we tried to investigate, in a clinical sample of ADHD patients with psychiatric comorbidities, the relationships between two of the fundamental psychological grounds in the neuropsychologic developmental milestones of children and adolescents, namely EF and empathic attitudes, and how they reciprocally interact to regulate behavioral self-regulation and aggressiveness. Our results confirm a strong and finely structured relationship between these variables, being aggressive behaviors and related disturbances significantly influenced by these underlying processes. Our work highlights two different interactions between EF and empathy to regulate social behaviors in ADHD, where a dysfunction of these elements is essential for aggressive and antisocial behaviors to be carried out towards the others.A first model identifies aggressiveness and oppositional problems, as indexed through the CBCL questionnaire, mainly associated with difficulties in executive emotional-behavioral regulation processes (such as impulse control and the ability to appropriately regulate one’s behavior according to the context), but not with dimensions of empathy. Interestingly, reactive aggressiveness usually emerges as an impulsive response to hostile-perceived environmental events, often precipitated by irritability and tantrums . This tyOn the other hand, rule-breaking behaviors and conduct disorders, as indexed through the CBCL questionnaire, likely relate to a proactive type of aggressiveness, which is associated to the activation of self-oriented behaviors to take advantage for personal purposes to the achievement of benefits at the emotional expense of the others’ perspective . Our stuOur results are thus in line with previous studies and further elucidate the complex and intriguing relationships between empathic attitudes and EF. In other words, it seems that both impaired behavioral self-regulation and difficulties in the emotional sharing of others’ internal state may lead to a down-regulation of proactive aggressiveness inhibition systems, while emotional and behavioral regulation functioning systems are essential in preventing more reactive forms of aggressiveness towards the others. Thus, the multifaceted interactions of both “hot” EF and empathic attitudes have a central role in regulating prosocial behaviors.Our study displays, however, a number of limitations that might undermine the robustness of our conclusions; notably, a marked discrepancy in group size, particularly between the “pure” ADHD and ADHD+ODD/CD+ASD groups, and the absence of a control group of healthy children. The purpose of this preliminary study was, indeed, to explore the issues addressed above; therefore, further studies on larger samples, possibly including heathy controls, a greater number of girls and ASD patients, and children with limited prosocial emotions, will be performed to confirm the results.In conclusion, our study provides a further contribution for a better understanding of the complex and intriguing relationship between empathic competence and executive skills. These evidences could be beneficial for the definition of treatment strategies aimed at attenuating externalizing behaviors. Aggressive behaviors would, indeed, be modified by an empathic attitude-oriented approach, which should focus on the underlying executive dysfunction. To sum up, we showed that executive functioning and empathic attitudes interact with each other to regulate aggressive behaviors, being the former more related to reactive aggressiveness and the latter to proactive aggressiveness.