The analysis of potassium PMFs in terms of forces revealed a conflicting influence on permeation of van der Waals and electrostatic forces that both undergo an inversion of their direction as pore charge is increased. Even if the most important transition involves the interplay between the electrostatic forces exerted by graphene and water, the simulations also revealed an important role of the changing distribution of potassium and chloride ions. The influence of pore charge on the orientation of water molecules was also found to affect the van der Waals forces they exert on potassium.Selective permeation through graphene nanopores is attracting increasing interest as an efficient and cost-effective technique for water desalination and purification. In this work, using umbrella sampling and molecular dynamics simulations with constant electric field, we analyze the influence of pore charge on potassium and chloride ion permeation. As pore charge is increased, the barrier of the potential of mean force (PMF) gradually decreases until it turns into a well split in two subminima. While in the case of K Increase of graphene pore charge determines decrease of PMF barrier that turns into well: current increases, reaches plateau and declines. This technology paves the way to a wide range of potential applications in the fields of desalination of seawater,8 wastewater purification9 and DNA sequencing.10 These applications take advantage of the ultra-thinness of graphene which allows fast water transport while excluding ions or selecting specific ion types. The performance of these technologies critically relies on precise control of pore size. In fact, atomically precise pore sizes would allow the development of molecular sieves capable of separating compounds with atomic scale differences in size and shape. Significant advances in this field have been achieved by Gilbert et al.11 who demonstrated the fabrication of individual nanopores in hexagonal boron nitride with atomically precise control of pore shape and size, and by Thirumaran et al.12 who, using controlled Ga+ ion irradiation, introduced a population of sub-nanometer pores in a MoS2 membrane, realizing atomic transport measurements. Despite these recent achievements, experiments on transport across nanopores in 2D materials such as graphene, boron nitride, molybdenum disulfide and tungsten disulfide, are still lacking and most studies infer the conductance and sub-nanometer pore diameters indirectly from computational modelling.Graphene is a thin membrane consisting of spet al.13 showed that for small nanopores the sieving effect can be ascribed to steric hindrance of the pore edge. More specifically, it was shown that in order to effectively exclude salt ions, the diameter of a graphene nanopore cannot exceed 5.5 Å. Since the creation of small nanopores is technologically more challenging than drilling larger ones,14,15 alternative selectivity strategies suitable for larger pores have been actively sought. In particular, it was shown that placement of charges on the pore edge by chemical functionalization makes the pore selective to counter-ions. Using this strategy Sint et al.8 showed that replacement of carbon atoms of the graphene pore with nitrogen and fluorine makes the pore cation selective while addition of hydrogen make it anion selective. Moreover, Zhao et al.16 showed computationally that even when the pore radius is much larger than the hydrated radius of the ion, negatively charged nanopores still exhibit remarkable selectivity. This finding was confirmed by Konatham et al.17 that however observed that the selectivity induced by charged groups becomes less effective as the pore diameter is increased.In a pioneering Molecular Dynamics (MD) study Cohen-Tanugi 18 For instance, K+ channels like KcsA pass potassium and sodium with a ratio 1000 : 1  of KcsA. A similar approach was employed by He et al.22 who designed bio-inspired graphene nanopores containing four carbonyl groups (4CO) mimicking KcsA SF, or four carboxylate groups (4COO) arranged as in the SF of the NavAb Na+ channel.The main research question addressed by the many research groups working on graphene nanopores is the following: what is the ideal functionalization providing high water flows while discriminating against specific solutes? Biological ion channels, shaped through millions of years of evolution, are characterized by flow rates comparable to the free diffusion limit while retaining high selectivity.1000 : 1  while Na22 that the 4CO construct by He et al., as expected, was potassium selective. Surprisingly however, the 4COO construct that was expected to mimick the properties of NavAb, was not sodium but potassium selective and the selectivity of another construct with three carboxylate groups turned out to be voltage-tunable. Moreover, when the pore diameter is less than 5 nm, the confined liquid structure in the nanopore affects various transport properties of the ions.23 Since such properties may deviate from the bulk medium behaviour, ion dynamics in a nanopore confined region or an atomically thin membrane is still not well understood. Another open problem is related to the fact that functionalization can lead to a high density of charge along the pore rim. This in turn, leads to the accumulation of a concentration polarization layer in the neighborhood of the graphene membrane.24 The influence of this Debye layer on ion current is still not clear.The design principles of biological ion channels, however, are far from being completely understood. For instance, MD simulations showed+ and Cl− ion permeation through a pore drilled in a graphene monolayer. This enabled us to identify general trends useful for the design of functionalized nanopores. The current–voltage curves derived from MD simulations in the presence of a constant electric field were analyzed in terms of the potential of mean force (PMF) computed through umbrella sampling (US) simulations.25 We discovered that, as pore charge is increased, the barrier of K+ PMF gradually decreases until it turns into a well split in two sub-minima. An analysis of electrostatic energy and desolvation of the ion biased in US simulations, showed that the approximately constant desolvation cost is better and better compensated by the energy of electrostatic interaction with the charged pore. Thus this scenario, appears to be consistent with theoretical models by Eisenman26,27 and Zwolak.28,29 However, the analysis of the forces acting on the biased ion revealed a richer picture where a relevant role is also played by the potassium and chloride ions whose tendency to accumulate in concentration polarization layers significantly affects the electrostatic forces. Also, we observed that pore charge influences the orientation of water molecules affecting the van der Waals force they exert on the solvated K+ ion. Finally, the study of Cl− permeation revealed that, even if the PMF profiles are extremely powerful tools for the analysis of ion permeation, they must be used with extreme care. In fact, asymmetric ion distributions arising from the application of high voltages, cannot be accounted for by a PMF computed in the V = 0 regime, but they have a profound influence on ionic currents.In this paper we study the dependence on pore charge of KI–V curves and PMF profiles is reconciled in terms of the effect of voltage-induced asymmetric ion distributions. Finally, we draw conclusions.The paper is organized as follows. After the description of our computational methodology, we study potassium permeation devoting particular attention to the analysis of the forces and orientational effects that are discussed in specific subsections. The Results section concludes with the analysis of chloride permeation where a seeming discrepancy between rp 4.5 Å. The pore is thus larger than those of typical biological ion channels. However, the radius is below the 5.5 Å cutoff for unselective ion transport16 so that permeation and selectivity are mainly determined by the rim charge. The pore was generated choosing a reference carbon atom with coordinates  and removing all the graphene atoms whose distance from the reference atom  was below the chosen pore radius. The pore was either left uncharged or assigned an overall charge −1e, −2e, −3e, −4e, −5e, −6e or −7e. The pore charge was evenly spread over all the atoms lining the pore rim  run for 0.5, 1.0 and 2.0 ns respectively. Each equilibration stage was preceded by 1000 steps of conjugate gradient minimization. The short-range van der Waals and electrostatic interactions were cut off at 12.0 Å with a switching distance of 10.0 Å. The long-range electrostatic interactions were computed with the particle mesh Ewald method. Periodic boundary conditions were imposed in all directions and coordinates were stored every 2 ps.The system studied comprises a single graphene layer with a nanopore of radius 33 corresponding to potential differences ranging from −0.5 to −4 volts at 0.5 V intervals. The constant field was computed as Ez = V/Lz where V is the desired potential difference, while Lz is the length of the simulation box along the z-axis. Following where the sum runs over all the ions of a given type, Lz is the length of the simulation box in the direction of the channel axis, α is a conversion factor to express the current in amperes, qi and zi are the charge and position of ion i and the displacement is computed over two sampling intervals δt. In order to perform a statistical analysis through block-averages, the 40 ns long trajectories, run in the NVT ensemble at the temperature of 300 K, were split in 4 10 ns-blocks. Currents are computed as averages, and errors as standard deviations over the four blocks.After equilibration the system was simulated in the presence of a uniform electric field25 was employed. In these simulations the axial coordinate z of a single K+ was harmonically restrained  while the radial position was subject to a semi-harmonic wall at 4.5 Å from the pore axis. We considered 81 axial windows from z = −20.0 Å to 20.0 Å of width 0.5 Å. In the calculation of the K+ PMF each window was run for 6.0 ns with the first 1.0 ns considered as equilibration and excluded from the analysis. In the calculation of the Cl− PMF, due to slower convergence, each window was run for 16 ns with the first nanosecond discarded as equilibration. Using the implementation by Grossfield35 the Weighted Histogram Analysis Method (WHAM)36 was used to calculate the PMF with a tolerance of 10−7.In order to obtain the potential of mean force (PMF) of the permeating potassium ion the umbrella sampling method+ permeation, we ran MD simulations with an external applied potential in the range from −0.5 V to −4.0 V at 0.5 V intervals  the force exerted by the other K+ ions; (ii) the force exerted by Cl− ions; (iii) the force exerted by water molecules; (iv) the force exerted by graphene. + pushing it away from the bulk and towards graphene. By contrast, the negatively charged chlorides attract the reference K+ ion pulling it into the bulk. Due to the 1 : 1 stoichiometry of KCl it is not surprizing that the forces exerted by potassium and chloride ions are almost symmetrical and tend to balance each other. However, at high values of the pore charge  there is the appearance of a high local density of potassium ions on either side of the graphene layer. This local layer of potassium ions repels the reference K+ ion, pushing it away from graphene and reinforcing the effect of chloride ions. The force exerted by K+ and Cl− ions is however, comparatively small with respect to that exerted by graphene and water. When the pore is neutral, graphene exerts a zero electrostatic force. However, as the pore charge is increased, graphene exerts a stronger and stronger attracting force on the reference K+ ion. Finally, the force exerted by water pulls the K+ ion strongly into the bulk. In summary, the reversal in the sign of the total electrostatic force occurs when the graphene force becomes dominant compared to the water force and the potassium force starts pushing the reference K+ ion into the bulk.Both the van der Waals and electrostatic forces acting on the K+ ion is pulled closer and closer to the charged pore entering into the repulsive branch of the Lennard-Jones potential of the carbon atoms. Another notable feature of the van der Waals forces is that the force exerted on the reference K+ by the other potassium ions is always negligible. This is due to the fact that the repulsive K+–K+ electrostatic force keeps the potassium ions far away from the reference K+ so that the short-range van der Waals force is vanishing. Other trends can easily be identified, but they are more difficult to interpret. For instance, the van der Waals force exerted by Cl− ions always pushes the reference K+ ion towards the graphene pore. Moreover, the van der Waals force exerted by water molecules, at intermediate distance from graphene pulls the reference K+ ion into the bulk, but it pushes it towards the pore at closer distances from the graphene layer. The pattern exhibited by the chloride and water van der Waals force is discussed in more detail in the next section. As a final note, we can now explain the behaviour of the total van der Waals force. In particular, the reversal in the direction of this force occurs when the repulsive force exerted by graphene becomes dominant with respect to the force exerted by water that tends to push the reference K+ ion towards the pore.The van der Waals force also shows some interesting trends. First of all, note that graphene exerts a repulsive force, increasing with the pore charge. This is due to the fact that, as the pore charge is increased, the reference K+ has negligible Lennard-Jones interactions with the hydrogen atoms belonging to water molecules of both the first and second shell. This is because the peak of the first two shells (determined from the RDF profiles) corresponds to the flat tail of the Lennard-Jones curve. For the same reason K+ experiences vanishing Lennard-Jones forces from the oxygen atoms belonging to the water molecules of the second shell. Conversely, potassium is subject to repulsive forces from the oxygen atoms of the first water shell because the peak of this shell is located in the repulsive branch of the LJ potential.The pattern exhibited by the van der Waals force of water can be explained in terms of the several factors. First of all, the analysis of the Lennard-Jones (LJ) potential of the potassium/water-oxygen and potassium/water-hydrogen interactions  and close to the pore but not so close as to exit from the bulk phase and lose their hydration shells. On the other hand, when the pore charge becomes very high, the coulombic attraction is so strong that a close contact with the charged pore becomes more energetically convenient than remaining in the bulk solution to maintain the hydration energy.In order to explain this behaviour we plot the number density of potassium and chloride ions in axial bins with height 0.5 Å and radius 4.5 Å  choosing as an example the simulation at −4.0 V. The potassium profile in ESI Fig. SF6(a)+ ions. When Qp = 0 there is a single chloride density peak at z = −4.0 Å. This is due to the fact that the electric field is oriented towards the negative z-axis. This means that the negatively charged chlorides are pushed towards the positive z-axis. However, since graphene is not very permeable to chloride, they tend to accumulate just below the pore. As the rim charge is increased up to Qp = −4, the chloride peak decreases and shifts further away from the pore at z = −5.0 Å due to the increasing electrostatic repulsion exerted by the pore. However, when the charge is increased to Qp = −6 and −7 there will be the appearance of three high density peaks at z = −7.0 Å, z = −5.0 Å and z = −2.0 Å. This behaviour is clearly a consequence of the build-up of two high potassium density peaks close to the pore. The high local potassium concentration screens the negative charge of the pore and the K+/Cl− attraction allows the appearance of a high Cl− concentration. Moreover, the shielding of the pore charge allows an increase of the Cl− current as shown in the current plot ent plot  at Qp = ESI Fig. SF7+ PMF except that each window was run for 16 ns instead of 6 ns to reach convergence. The first nanosecond of the simulations was discarded as equilibration and the analysis was performed on the remaining 15 ns. The PMF profiles appearing in Qp = 0 to Qp = −3 and then drops from Qp = −4 to Qp = −7. This behaviour is seemingly at odds with the current/charge plots shown in Qp = 0 to Qp = −1 and Qp = −2 that does not seem to be justified by the very similar peaks of the PMF profiles. A clue to help in disentangling this contradiction is provided by the current plot in Qp = 0 to Qp > 0 becomes more and more pronounced as the applied voltage increases. This suggests that the high current observed at Qp = 0 could be due to some peculiar ion distribution induced by the external electric field. To test this hypothesis in ESI Fig. SF8+ and Cl− ions in the simulation with an applied potential of −4.0 V. It can be noticed that at Qp = 0 the ion distribution around the pore is highly asymmetrical. Specifically, the Cl− concentration below the pore is much higher than the concentration of the same ion above the graphene layer, while the potassium concentration above the pore is only slightly higher than the K+ concentration below the pore. This distribution determines that, in the neighborhood of the pore, there is a large availability of chloride ions that could be involved in permeation. This results in a high chloride current even if the free energy barrier of the PMF is high. It must be stressed explicitly that, since the PMF was computed in absence of external field, this concentration polarization effect could not be accounted for by the PMF profiles. As a comparison, we also computed the ion density profiles for the system with pore charge Qp = −2 in the presence of an external potential of −4.0 V. In this case it can be observed that the chloride density below the pore is only moderately higher than that above the pore. Thus, the gradient of concentration of Cl− is smaller than in the Qp = 0 case explaining why the current at Qp = −2 is much smaller than that at Qp = 0 even though the peaks of the PMFs are almost identical. As a final note, it can be observed that the shallow free energy wells appearing on either side of the barrier at Qp = −6 and Qp = −7 are also due to the high local density of K+ ions that attract the incoming chloride.More insight into the permeation mechanism of chloride can be attained from the analysis of the PMF profiles. The PMF was again computed through umbrella sampling simulations using a protocol identical to the one employed for the calculation of the Ket al.16 showed that even large graphene nanopores, if negatively charged, can enhance K+ transport while completely rejecting Cl−. Even if this work explores a wide range of values of pore charge and radius, no attempt was made to study the influence of the charge on potassium and chloride PMFs. Similarly, Li et al.37 recently showed that a charged graphene surface can induce selectivity of Li+ and Na+ over K+. However, these authors, instead of analyzing classical pores generated by removal of carbon atoms, considered ion flow through the stretched aromatic rings of a graphene membrane under mechanical strain.One of the many potential applications of graphene concerns water desalination and purification. While the sieving capability of small graphene pores depends on steric effects, the selectivity of larger pores critically depends on the charge. Yet, only a couple of studies systematically addressed the issue of selectivity/charge relationship. Zhao I/V curves and the PMF profiles fills a gap in the scientific literature. As a cautionary note, it must be observed that we chose to use a simplified pore charge distribution . In realistic functionalized graphene nanopores the charge distribution can be different. Remarkable examples include the bio-inspired nanopores studied by Kang et al.21 and the graphene-based crown ethers analysed by Guo et al.38 and by Smolyanitsky et al.39 where the electrostatic potential maps critically depend on the placement and orientation of the ether dipoles. Despite its simplicity however, our approach can qualitatively reproduce the properties of realistic systems. This is due to the fact that many patterns are significantly robust with respect to the fine details of the charge distribution. As an example, Zhao et al.16 who used a charge model similar to ours, showed that, as long as the total charge is kept constant, charging all pore atoms or only alternating ones led to comparable ion fluxes. Similarly to what is reported in +, we found that, as the pore charge is increased, the barrier of K+ PMF decreases until it turns into a well that partially traps potassium, reducing the current. This trend can be explained in terms of an increasing electrostatic compensation of a roughly constant desolvation cost. This is in agreement with the seminal theory developed by Eisenman26,27 that identifies two driving forces for ion permeation: (i) the ion–pore electrostatic interactions and (ii) the desolvation cost the ion incurs when crossing the pore. More formally, an ion with charge q and radius r has a hydration free energy represented by the Born approximation: Ghydr = q2/8πεwr where εw is the dielectric constant of water and its electrostatic interaction with a site within the channel of charge qs and radius rs is Gint = qqs/4πεw(r + rs). Ion permeation can only occur if Gint ≥ Ghydr. It is noteworthy that use of the Born formula to express the dehydration cost, amounts to assuming that the ion is completely desolvated upon entering the pore. Eisenman's ideas were further developed by Zwolak and coworkers28,29 who expressed the desolvation cost as a function of the pore radius. This approach accounts for partially desolvated states since dehydration occurs in a quantized way, a shell at a time. According to this model desolvation only depends on the geometric properties of the pore, namely its radius. Our results however, show that this picture is oversimplified. Even if the large number of water molecules lost by the ion upon entering the neutral pore confirms the importance of geometry, + but also for Na+ and Li+.Our systematic exploration of the influence of the pore charge on both the 40 on the grounds of theoretical modelling and Brownian dynamics simulations suggested that knock-on barrierless conduction can be considered as a general mechanism of transport in ion channels with multiple occupancy. Similar conclusions have been reached by the Ionic Coulomb Blockade model41 predicting that conduction bands occur when the charge of the selectivity filter balances the charge of the ions already inside the channel plus the image charge of a further potentially incoming ion. Our simulations show that barrierless conduction can be tuned using pore charge as an adjustable parameter. In fact, when the rim charge is high enough, the ion/pore interaction almost perfectly balances the desolvation cost flattening the permeation barrier. Recent computational studies have shown that barrierless conduction can be induced using other control parameters. For instance Fang et al.42 showed that a modest mechanical strain on graphene-embedded crown ether pores reduces the K+ release barrier, significantly increasing the current. Interestingly, in this case also, the barrier arises from both the desolvation cost and ion–pore electrostatics. The mechanical pore expansion flattens the barrier simultaneously weakening the ion/pore interactions and strengthening the ion/water ones.The decrease of the free energy barrier as a function of increasing pore charge leads to the issue of near-barrierless permeation. In the literature it is widely debated whether barrierless conduction must be considered a universal mechanism of ion channels or a peculiar property of KcsA-like channels where two- and three-ion occupation states are isoenergetic. Yesylevskyy and Kharkyanen+ ion towards the pore while electrostatic forces tend to keep it in the bulk. However, as the pore charge is increased, the directions of both forces are reversed. The reversal of the van der Waals force is due to the build-up of an increasing repulsive force exerted by graphene that becomes dominant over the LJ forces exerted by chloride and water. The reversal of the electrostatic force depends on two main factors. First of all, as pore charge increases, the attractive electrostatic force exerted by graphene overcomes the repulsive force exerted by water. The interplay between these two forces is the one expected based on simplified models like Eisenman's and Zwolak's. The simulations however, also reveal an important but unexpected role of potassium and chloride ions. For small values of the pore charge these ions are evenly spread in the bulk phase so that their forces (the reference K+ ion is attracted to the bulk by chloride and pushed to graphene by the other potassium ions) tend to balance each other. At high pore charges however, the formation of a concentration polarization layer of K+ ions switches the sign of the electrostatic force that potassium ions exert on the reference K+ ion. The formation of concentration polarization layers is even more significant in the case of Cl− permeation reconciling the seeming mismatch between the modest decrease of the barrier of the chloride PMF (when Qp varies from 0 to −3) and the significant drop of the current.The atomistic level insight provided by molecular dynamics not only sheds light on the details of the desolvation process, but it also reveals a rich picture not foreseen by simplified physical models. Our simulations show that van der Waals and electrostatic forces exert opposite effects on the permeation process . For smaet al.43 observed a surprisingly large K+/Cl− selectivity in pores even as large as 20 nm in diameter. A careful examination of the results attributed this counter-intuitive result to the elevated concentration of mobile cations near the electronegative graphene surface. More recently, Hu et al.24 showed through MD simulations that sodium and potassium form concentration polarization layers on either side of a graphene sheet. In their work with a neutral pore, Na+ forms symmetrical density peaks on either side of the graphene pore similarly to what happens with K+ in our simulation with Qp = 0. Chloride, on the other hand, forms a single very high density peak just above the membrane. This mismatch with our simulations where the Cl− density peak is below the pore, is simply due to the different orientation of the electric field in their work.Concentration polarization, the non-uniform ion distribution close to a sufficiently small pore is a widespread phenomenon also highlighted in other studies. For instance Rollings In summary, we have characterized the influence of pore charge on the permeation of potassium and chloride across a graphene nanopore. While the simulations basically confirm the validity of simplified physical models, they also reveal a much richer phenomenology where the asymmetry of ion distributions and the orientational effects induced by the applied potential and pore charge significantly affect conduction, thus calling for more detailed and comprehensive modelling.There are no conflicts to declare.RA-009-C9RA03025E-s001


Drosophila mdg4-ERV (gypsy) or the human ERV, HERV-K (HML-2) are each sufficient to stimulate cytoplasmic aggregation of human TDP-43. Viral ERV transmission also triggers TDP-43 pathology in recipient cells that express physiological levels of TDP-43, whether they are in contact or at a distance. This mechanism potentially underlies the TDP-43 proteinopathy-caused neurodegenerative propagation through neuronal tissue.Inter-cellular movement of “prion-like” proteins is thought to explain propagation of neurodegeneration between cells. For example, propagation of abnormally phosphorylated cytoplasmic inclusions of TAR-DNA-Binding protein (TDP-43) is proposed to underlie progression of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). But unlike transmissible prion diseases, ALS and FTD are not infectious and injection of aggregated TDP-43 is not sufficient to cause disease. This suggests a missing component of a positive feedback necessary to sustain disease progression. We demonstrate that endogenous retrovirus (ERV) expression and TDP-43 proteinopathy are mutually reinforcing. Expression of either  Drosophila or human endogenous retroviruses (ERVs) is sufficient to cause TDP-43 protein aggregation, and viral transmission of the ERVs triggers TDP-43 pathology in recipient cells. This mechanism may underly spread of neurodegenerative effects in a Drosophila model.Expression of  Mutations in TDP-43 are responsible for a fraction of the inherited forms of ALS, providing a direct causal link between this gene and neurodegenerative cascades. But the vast majority of cases involve the mis-localization and aggregation of TDP-43 of normal amino acid sequence1. Thus, initiation of TDP-43 protein pathology is triggered by upstream factors, likely ones that accumulate with age. On the other hand, disease progression after diagnosis is relatively rapid, and is thought to involve inter-cellular propagation of disease pathology from a point of stochastic focal origin. This inflection in rate of disease progression after decades of apparently normal physiology is consistent with involvement of a self-sustaining biological processes, a positive feedback loop6.Cytoplasmic aggregation of abnormally phosphorylated TDP-43 (pTDP-43) is seen in both neurons and glia in the affected brain regions of 97% of patients with ALS, ~40% of cases with FTD, in many cases of Alzheimer’s disease (AD), and in patients with an AD-like presentation 6. In this model, pathological forms of misfolded TDP-43 are able to template misfolding in trans of normal wild-type TDP-43, much as has been demonstrated for the prion protein (PrP) that underlies infectious prion diseases8. Such misfolded TDP-43 is proposed to be released from neurons and/or glial cells and then to be taken up by recipient cells, where the prion-like templating process is repeated. Indeed, numerous biochemical experiments support the idea that TDP-43 protein misfolding can template in trans, leading to propagation of pathological forms of TDP-43 in vitro, and there are elegant experiments that demonstrate inter-cellular spread, both in cell culture and by injection into cortex of mice that also over-express a sub pathological level of transgenic human TDP-43 variants11. These experiments reveal that pathological cytoplasmic hyperphosphorylated TDP-43 appears in cells far from the site of injection. But unlike the case of PrP, injections of pathological TDP-43 does not cause robust propagation of proteinopathy in recipient animals, and does not cause neurodegenerative cellular effects, unless those animals also over-express transgenic human TDP-43 in addition to the endogenous mouse gene11. We and others have documented that TDP-43 protein pathology or knockout of TDP-43 are each sufficient to cause dysfunctional expression of ERVs and retrotransposable elements (RTEs). This has been shown in Drosophila, human neuroblastoma cells, and cortical tissue from human subjects21. Here, we provide evidence that ERVs provide a hitherto unknown component of a positive feedback loop to sustain propagation of TDP-43 pathology and to spread neurodegenerative effects through neuronal tissue.One model that has been proposed to explain such a self-sustaining inter-cellular propagation is the prion hypothesis24. Indeed, enteroviral infection can cause pathological accumulation of TDP-4324. We therefore tested whether expression of ERVs could impact the localization and phosphorylation of TDP-43. We first tested the impact of expressing HERV-K, a human ERV whose expression has been observed in cortical tissue of patients that exhibit TDP-43 pathology16. We generated a HERV-K reporter construct that was tagged with a nuclear localized H2B-mCherry (HERV-K-H2B-mCherry)  Fig. . To accory) Fig. . In contry) Fig. .Fig. 1Hu26. We also observe cytoplasmic mislocalization of TDP43 using a pan-TDP-43 antibody  is sufficient to trigger cytoplasmic accumulation of pathologically phosphorylated human TDP-43 . Indeed, by 24 h after induced expression of a V5-tagged human TDP-43, 64.31% of cells that were transfected with an mCherry tagged mdg4-ERV exhibited redistribution of TDP-43-V5 to the cytoplasm compared with just 35% of cells transfected with a control mCherry  has been replaced by a human TDP-43 cDNA with a 3xFlag tag fused at the N-terminal (abbreviated as TDP-43WT-KI)28. In this strain, only the human TDP-43 gene is expressed, at physiological levels via the endogenous fly ortholog’s promoter28. Although knockout of the endogenous fly ortholog is lethal, this humanized TDP-43WT-KI fly strain exhibits normal longevity that is indistinguishable from that of the laboratory wild-type strain28  only in a spatially restricted glial subtype called the subperineurial glia (SPG)  Fig. . It is wset Fig. . This pets), so that we could induce spatially restricted TDP-43 expression after allowing normal development (induction relies on shifting the growth temperature from 21 °C to 29 °C). The use of an inducible expression system also provides the means for observing effects over a time-course after initiating expression. We found that Gal4 mediated induction of hTDP-43 in SPG (SPGts > hTDP-43) greatly reduces median survival of animals that contain the TDP-43WT-KI replacement of the fly ortholog. Median survival of this strain was just 18 days compared 31 days for animals containing just the TDP-43WT-KI and 34 days for animals that contain the SPGts and TDP-43WT-KI, but no UAS-hTDP-43 , we observed only nuclear Flag-labeled TDP-43 in neurons, with no evidence of pTDP-43 accumulation. This was true over a 15 day time course after shifting to induction temperature  Fig. . This efure Fig. . By conture Fig. . This efure Fig. . For exaure Fig. , and theure Fig. . We alsoure Fig. . We alsoure Fig. . This in36. DNA damage has been observed in post mortem brain tissue from patients with TDP-43 related disorders36, in iPSC derived neurons from C9orf72 familial patients that exhibit TDP-43 pathology37, and in animal models including Drosophila when TDP-43 is over-expressed34. We therefore wondered whether we would detect DNA damage foci in neurons that exhibited pTDP-43 pathology due to focal induction of hTDP-43 over-expression in SPG. Importantly, such neurons exhibit pTDP-43 due to induced hTDP-43 over-expression in a neighboring cell type while only expressing human TDP-43 at physiological levels themselves  rather than the humanized TDP-43WT-KI knock in 39 in SPG using the same Gal80ts approach. This allowed us to monitor both pTDP-43 signal and the RFP signal independently. We also co-expressed a membrane tethered mCD8-GFP in the SPG in order to mark the membrane, yielding an outline of SPG morphology. As with induction of the untagged TDP-43, induction in SPG of the TDP-43-RFP (SPGts > hTDP-43-RFP in TDP-43WT-KI) shortened the animal’s lifespan to a median survival of 15 days  resulted in a strong cell autonomous effect on TDP-43 pathology, resulting in robust pTDP-43 signal in all SPG at D7, and this persisted to D15    Fig. . Such ecWT-KI instead of the fly gene  and in a higher fraction of cells at (29.02% vs 12.12%) at D15 than at D7 , we significantly attenuated these non-cell autonomous effects on pTDP-43 in neurons , we greatly reduced the appearance of DNA damage in the nearby neurons both at D7 and D15  was sufficient to greatly ameliorate the loss of neurons  that are capable of transmitting between cells grown in culture. Indeed, both of these ERVs contain an enveloped glycoprotein that is required to mediate such intercellular transmission. We previously established Drosophila S2 cell culture assays to detect transmission of mdg4-ERV between cells grown in contact, or between cells that were separated by a 0.4 µm filter in a transwell system40. We used these assays to test whether viral transmission of mdg4-ERV was sufficient to mediate propagation of TDP-43 pathology to recipient cells.Both the 40. In contrast, the H2B-mCherry control was never seen to transfer to the hTDP-43-V5 expressing population of cells that are grown in contact  were co-transfected into S2 cells 59 and in Aicardi-Goutieres syndrome61. RTE and ERV expression has the potential to contribute to many of the cellular impacts that are thought to be at play across these disorders, including inflammatory signaling, DNA damage and defects in RNA metabolism and proteostasis65. But our findings raise the possibility that RTE and ERV expression provides a feedback amplification to sustain the proteinopathy in other disorders as well. This suggests the potential of clinical interventions to block RTE/ERV expression or function, as a means to interrupt a positive feedback mechanism driving disease progression.Normal aging is associated with a progressive rise in RTE and ERV expressionNo statistical methods were used to predetermine sample size. Animal age for each experiment was noted throughout the text. The experiments were not randomized, and the investigators were not blinded to allocation during experiments and outcome assessment.Drosophila Stock Center. The hTDP-43-KI28 and UAS-hTDP-43-RFP39 were generous gifts from Professor David Morton  and Professor Jane Y. Wu (Northwestern University School of Medicine). The following stocks, UAS-GFP-IR, UAS-mdg4-IR , UAS-hTDP-43, tub-Gal80ts and UAS-mCD8-GFP were used in our previous studies43. To prevent artifacts from genetic variation between groups, all strains used in this study were backcrossed to our laboratory wild-type strain, Canton-S derivative w1118 (isoCJ1), for at least five generations. Male flies were chosen as the experimental subjects throughout the study. All flies for TARGET66 temperature-shift experiments  were raised in a 21 °C incubator from the embryonic stage66. All flies for TARGET experiments were immediately shifted to the permissive temperature at 29 °C after eclosion, which was designated as day 0. These TARGET flies were then incubated at 29 °C for lifespan assays or dissected at desired time points for analyzing the patterns of molecular markers dependent on the experimental designs.We obtained SPG-Gal4  and UAS-myr-RFP (#7119) from Bloomington 40 and moxBFP  constructs and then fused by PCR to generate the H2B-BFP fragment. The P2A was separately added to the C-terminal of H2B-BFP and N-terminal of hTDP-43 by PCR. The final H2B-BFP-P2A-hTDP-43 was then created by PCR fusing H2B-BFP-P2A and P2A-hTDP-43 fragments and then cloned into the inducible pMT-V5-6xHis plasmid. The pAc-H2B-mCherry-HA, pAc-mdg4-H2B-mCherry-HA and pAc-mdg4-H2B-mCherry-HAΔEnv constructs were generated described previously40.To generate the inducible hTDP-43-V5 construct, the full length of hTDP-43 was PCR amplified but the stop codon was removed in order to generate an in frame fusion protein with the V5 tag provided by the pMT-V5-6xHis plasmid . To generate the pMT-H2B-BFP-P2A-hTDP-43-V5 construct, the H2B and BFP were separately PCR amplified from pAc-H2B-mCherry13 and used as the template to generate the constitutive pcDNA3.1-HERV-K-P2A-H2B-mCherry-HA plasmid. In short, the P2A-H2B-mCherry-HA fragment was PCR amplified and integrated downstream of Env of pcDNA3.1-HERV-K while removing stop codon of Env.To generate the constitutively expressing pcDNA3.1-H2B-mCherry-HA, the H2B-mCherry-HA was moved from pAc-H2B-mCherry-HA by restriction enzyme NotI and reinserted the H2B-mCherry-HA DNA fragment into the NotI site of pcDNA3.1 . The backbone of HERV-K (pcDNA3.1-HERV-K) was obtained from Professor Nath’s laboratory14. In short, ten adult flies of a given genotype were housed within one vial, and >100 flies in total for experimental group were used for each experiment. The surviving flies from each vial were flipped into fresh vials with fly food every other day, and dead flies at specific time points were recorded for the final survival curve analysis. The Log-Rank (Mantel-Cox) test and the Gehan-Breslow-Wilcoxon test were used to compare the significance of survival curves.Adult male flies with desired genotypes raised at 21 °C were collected immediately after eclosion and incubated in a 29 °C incubator for survival assay as addressed in previous studies12. Adult brains were dissected in ice-cold phosphate-buffered-saline (PBS) and then transferred into 4% paraformaldehyde (Electron Microscopy Sciences #15713) PBS solution ) and incubated for 30 min under vacuum twice. After fixation, dissected brains were washed three times for 10 min with 1XPBST wash solution (1XPBS with 1% Triton-X-100 and 3% NaCl). Brain samples were then incubated in blocking solution  overnight at 4 °C on a nutator. After blocking, dissected brains were transferred into primary antibodies and incubated overnight at 4 °C with the following dilutions: mouse anti-Repo , rat anti-Elav , mouse anti-Elav , mouse anti-Flag , rabbit anti-pTDP-43 , rabbit anti-pTDP-43  and rabbit anti-γH2Av  in 1XPBST washing solution with 10% normal horse serum. Samples were then washed with 1XPBST four times, 15 min each. Fly brains were incubated in secondary antibody solution  at 4 °C overnight. Secondary antibodies were combined from the following lists: donkey anti-mouse Alexa Fluor 488 , donkey anti-rabbit Alexa Fluor 488 , donkey anti-rat Alexa Fluor 594 , donkey anti-mouse Alexa Fluor 647 , donkey anti-rat DyLight 405 . After washing with 1XPBST for 4 × 15 min, fly brains were mounted in FocusClear , imaged using a Zeiss LSM 800 with Airyscan mode, and acquired images were processed by the Zeiss Zen software package.Adult brains at specific time points for each experiment were dissected, and immunofluorescent staining was performed as previously described2 (77.99 µm x 71.75 µm) area  were maintained in Schneider’s Drosophila medium  supplemented with 10% fetal bovine serum (FBS)  and 100U/ml Penicillin-Streptomycin  in a 25 °C incubator. Human neuroblastoma cells, SH-SY5Y , were grown in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12)  with 10% FBS and 100U/ml Penicillin-Streptomycin in a 37 °C incubator with 5% CO2 supplementation.42. 5 × 105 cells were seeded onto coverslips coated with 0.5 mg/ml concanavalin A (ConA)  and placed in the 6 well culture plate overnight prior to the transfection. Single or co-transfections with desired construct combinations  were performed with 1 µg of DNA each and Effectene transfection reagents . Post 24 h transfection, transfection complexes were washed away and supplied with the fresh complete medium containing 700 µM CuSO4 to induce hTDP-43-V5 expression.Conditions for S2 cells transfection were modified from our previous studies5 cells were seeded onto coverslips coated with 0.1 mg/ml Poly-D-Lysine and placed in 6 well culture plates overnight prior to transfection. Single transfection was performed under the manufacturer’s guideline with a mixture of 4 µg pcDNA3.1-H2B-mCherry or pcDNA3.1-HERV-K-P2A-H2B-mCherry with 12 µl TransIT-2020 reagent in a 1:3 ratio . After 24 h of incubation, transfection complexes were washed away, and fresh complete medium was supplied daily until the desired time points for analysis.Transfection conditions of SH-SY5Y were as follows: 8 × 105 of donor (pAc-H2B-mCherry or pAc-mdg4-P2A-H2B-mCherry) and recipient (pMT-hTDP-43-V5) cells were seeded at a 1:1 ratio onto the ConA-coated coverslip and maintained for 72 h to enhance the entrance of VLPs into recipient cells. At 20 or 24 h prior to the experimental time, the culture medium was replaced with a medium containing 700 µM CuSO4 to induce hTDP-43-V5 expression in recipient cells. For transwell assays, 5 × 105 of recipient (pMT-hTDP-43-V5) cells were plated onto ConA-coated coverslips and placed in the bottom well. At the same time, 1 × 106 of donor (pAc-H2B-mCherry or pAc-mdg4-P2A-H2B-mCherry) cells were placed into the upper well. 20 or 24 h prior to analysis, CuSO4 was added to induce hTDP-43-V5 expression in recipient cells.In order to expand the donor cell population and enhance the production of virus-like particles (VLPs), S2 cells were transfected with pAc-H2B-mCherry or pAc-mdg4-P2A-H2B-mCherry 48 h before co-culture or transwell assays were performed  PBS solution ) for 10 min on a nutator. After fixation, cells were washed three times with 1XPBST wash solution (1XPBS with 1% Triton-X-100 and 3% NaCl) and then incubated for 1 h in blocking solution  at room temperature. Cells were transferred into primary antibodies in 1XPBST washing solution with 10% normal horse serum after blocking and incubated for 1 h at room temperature with the following dilutions: mouse anti-V5 , rabbit anti-TDP-43  and rabbit anti-pTDP-43 , rabbit anti-pTDP-43 , mouse anti-HERV-K-Env , rat anti-mCherry  and rabbit anti-STMN2 . Samples were then washed with 1XPBST three times for 10 min each. Cells were incubated within secondary antibody solution  with DAPI at room temperature for 1 h. After washing three times with 1XPBST for 10 min each, cells were mounted in ProLong Diamond Antifade Mountant (Thermo Fisher Scientific), imaged using a Zeiss LSM 800 with Airyscan mode, and acquired images were processed by Zeiss Zen software package.25. To obtain the relative fold changes of POLDIP3α, values from each group were further normalized to the levels in pcDNA3.1-H2B-mCherry transfection at Day 2 in order to obtain the relative levels.Total RNA was extracted from pcDNA3.1-H2B-mCherry or pcDNA3.1-HERV-K-P2A-H2B-mCherry transfected SH-SY5Y cells using Trizol reagent  at different time points, and the total RNA extractions were treated with RQ1 DNase  before performing reverse transcription. 1 µg of total RNA from each sample was used to synthesize cDNA with SuperScript IV VILO Master Mix . qPCR was then performed using PowerUp SYBR Green master mix  on an Applied Biosystems QuantStudio 3 Real Time PCR System. The primer sets against POLDIP3α (Forward: 5'-CCAAAACCATCCAGGTTCCACAGCAG-3' and Reverse: 5'-GTGGTGGAGAAAGCCGCCTGAG-3') and GAPDH (Forward: 5'-GTTCGACAGTCAGCCGCATC-3' and Reverse: 5'-GGAATTTGCCATGGGTGGA-3') used in this study were as previously described27. Transfected S2 cells were washed, collected with 1XPBS, and then homogenized in ice-cold RIPA buffer  with protease inhibitors  and phosphatase inhibitors . Cell lysates were centrifuged at 16000 g for 30 min at 4 °C, and the supernatant was saved as the RIPA-soluble fraction. The RIPA-insoluble pellets were washed with RIPA buffer by centrifuging at 16000 g for 15 min at 4 °C. Supernatants were discarded and the pellets were extracted and homogenized with urea buffer . The lysates were centrifuged at 16000 g for 30 min at 4 °C. The supernatant was kept as the RIPA-insoluble and urea-soluble fraction.The soluble and insoluble TDP-43 fractions within the S2 cells were sequentially extracted dependent on the solubilities in the extraction buffers as previously descripted30 µg protein samples from each cellular fraction were separated by 10% polyacrylamide gel  and followed by a canonical Western Blot protocol. In short, membranes were blocked in 1XTris Buffered Saline-Tween-20 (TBST) (1XTBS + 0.2% Tween-20) with 5% SlimFast (chocolate flavor) at room temperature for 30 min. After blocking, membranes were incubated with primary antibodies mouse anti-GFP , and rabbit anti-pTDP-43  in 5% SlimFast -TBST for 1 h at room temperature or overnight at 4 °C. After washing three times with 1XTBST, membranes were incubated with secondary antibodies  or goat anti-rabbit-HRP  at 1:1000 or 1:2000 dilutions) for 1 h at room temperature. Signals were developed in chemiluminescent HRP substrate  and imaged with Sapphire Biomolecular Imager (Azure Biosystems).p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.Statistical analyses for different experimental setups were performed using GraphPad Prism 9. Specific statistical analyses used for comparing the significance between experimental groups were addressed in the corresponding figure legends and the experimental reproducibility was also listed in the figure legends. The significant levels were indicated as star numbers in the following format: *Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


The antagonism between strength and corrosion resistance in graphene‐reinforced aluminum matrix composites is an inherent challenge to designing reliable structural components. Heteroatom microstructural modification is highly appreciated to conquer the obstacle. Here, a bottom‐up strategy to exploit the heterogeneous phase interface to enable high corrosion durability is proposed. Deformation‐driven metallurgy derived from severe plastic deformation is developed to produce Mg‐alloyed fluorinated graphene structures with homogeneous dispersion. These structures allow for absorbing corrosion products, forming a dense protective layer against corrosion, and local micro‐tuning of the suppression of charge transfer. This results in superior corrosion resistance with an outstanding strength‐ductility balance of the composites via ultrafine‐grained and precipitation strengthening. The anti‐corrosion polarization resistance remains 89% of the initial state after 2‐month immersion in chloride‐containing environment, while the ultra‐tensile strength and elongation of 532 ± 39 MPa and 17.3 ± 1.2% are obtained. The economical strategy of heteroatom modification broadens the horizon for anti‐corrosion engineering in aluminum matrix composites, which is critical for the design of carbonaceous nanomaterial‐reinforced composites to realize desired performances for practical applications. A bottom‐up strategy is proposed to exploit the heterogeneous phase interface to enable high corrosion durability via deformation‐driven metallurgy. The anti‐corrosion polarization resistance remains 89% of the initial state after 2‐month immersion in chloride‐containing environment, which is critical for the design of carbonaceous nanomaterial‐reinforced composites to realize desired performances for practical applications. This barrier characteristic and chemical stability make it not surprising to shield the metals underneath it from unexpected reactions and corrosion. However, when these nanomaterials are applied to prepare aluminum matrix composites in practical applications, especially for high specific‐strength functions, fatal acceleration of the corrosion rate is often to be obtained instead of corrosion improvement. The undesirable anti‐corrosion weakening of graphene reinforced aluminum matrix composites can be attributed to three major inherent issues: (a) Graphene can trigger corrosion‐promotion activity (CPA) to enhance the localized galvanic corrosion due to the heterogeneous corrosion potential, such as graphene  and aluminum matrix (−1.1 V vs SCE). When coupling with a relatively active metal, the electrochemical stable and high electrically conductive graphene tends to behave like noble cathode, contributing to the formation of micro‐galvanic corrosion that initiates localized corrosion and boosts the anodic dissolution; (b) Small molecules such as O2 and H2O may infiltrate from the edge defects of graphene over the long‐term application, which consequently accelerated the corrosion rate; (c) Graphene is easy to agglomerate and exhibits poor compatibility due to its large specific surface area and strong van der Waals interaction, which severely reduces their dispersibility in the aluminum matrices and deteriorates the corrosion resistance performances.Sparked by its in‐plane impermeability to nearly all molecules,1 grap It can reduce the electrical conductivity by increasing the electron scattering in graphene lattice, as well as modifying the local electron density to inhibit the electrochemical activity of graphene. Fluorinated graphene is promising to enhance the anti‐corrosion performance for the insulating nature induced by F doping, as well as the molecule impermeability inherited from graphene. An alternative to suppressing the corrosion rate is to design microstructures that are intrinsically electrochemically homogeneous . However, the combination of these two approaches loses effectiveness since the composites reinforced by fluorinated graphene are susceptible to weak interfacial bonding and low load‐bearing capacity due to the chemical inertness of this modified graphene. The desirable interfacial bonding cannot be simply obtained by the high‐value plastic strain induced by severe plastic deformation. Developing cheap but effective microstructure solutions that enable both high corrosion resistance and mechanical performances thus remains a fundamental challenge.In light of these, several tailoring strategies were proposed to suppress the CPA behavior toward highly applicable aluminum matrix composites. Heteroatom tailoring of graphene shows great potential for changing the electronic density to tailor its electronic and electrochemical properties.9 It cHere we propose a bottom‐up strategy based on the principle of exploiting great interfacial bonding between fluorinated graphene nanoplatelets (F‐GNPs) and aluminum matrices via Mg alloying and severe plastic deformation. A severe plastic deformation technique, deformation‐driven metallurgy (DDM), was applied to obtain the homogeneous dispersion of F‐GNPs to realize extraordinary corrosion resistance. Corrosion‐suppression activity (CSA) induced by the synergistic strategy of active metal and heteroatomic modification was studied. This work aims to examine the effectiveness of synergistic modification and DDM techniques in terms of corrosion behavior in an aggressive chloride‐containing environment. Microstructural factors, interfacial characteristics, reinforcement dispersion, and electrochemical response were evaluated and discussed in detail.22.1Figure Based on the principle of heteroatom modification toward high corrosion durability, we designed a type of carbonaceous nanomaterial‐reinforced aluminum matrix composites with great interfacial bonding via Mg‐alloyed F‐GNPs and severe plastic deformation. Figure ID/IG A higher ID/IG value refers to the more broken graphene, indicating that there are more active open edges to provide necessary conditions for subsequent metallurgical bonding. Compared with the original (F)‐GNPs, an increase in this value can be seen in all the ball milling routes. Due to the more significant lubricating effect of the fluorinated graphene, the crushing effect caused by ball milling is weakened in the AM+F and MF+A routes. On the one hand, the integrity of F‐GNPs themselves is better preserved, resulting in a more efficient load transfer effect for mechanical performances and surficial absorbing integrity for corrosion resistance. On the other hand, it also means the reduction of the active reaction sites. It is also worth noting that the characteristic peak of MgF2 is observed in Figure 2 has high chemical stability and bond energy, the existence of this substance implies the strong chemical bonding between the carbonaceous phases and the matrices. This phenomenon compensates for the adverse effects of fewer active sites during the ball milling process. In addition, the ball milling routes of preferential mixing of (F)‐GNPs in the first step have a low ID/IG value, indicating that the physical isolation effect between Al and Mg brought by (F)‐GNPs can reduce the overreaction of the ball‐milled powders. Figure ID/IG values have decreased correspondingly. Obviously, graphene cannot be re‐spliced into a larger piece via severe plastic deformation. The decrease in this value indicates that the decrease in the thickness and layers of the (F)‐GNPs, and finally results in the increase of the specific surface area of graphene. This provides better prerequisites for the load transfer effect. In addition, the results of X‐ray diffraction (XRD) tests ‐GNPs and the matrices.To access the influence of different bottom‐up preparation routes on the microstructural factors of the (F)‐GNPs and the matrices, we conducted Raman spectroscopy, as shown in s Figure  show tha3 to sp2 ratio, which is in line with the bonding form of F‐GNPs, that is, one C atom forms a bond with two in‐plane C atoms and one out‐of‐plane F atom. Besides, there are basically no C‐Al bonds in the F‐GNPs DDM composites, which proves that the existence of F atoms isolates the direct reaction between Al matrix and C atoms, thereby avoiding the formation of the easily hydrolyzable compounds Al4C3. Since this substance is detrimental to the structural integrity of materials exposed to the aqueous environment, this phenomenon provides the possibility to improve corrosion resistance. Figure ). This is consistent with our vision properly, which is to achieve indirect bonding between aluminum and (F)‐GNPs through Mg alloys, thereby avoiding the CPA effect and improving the comprehensive corrosion resistance of the DDM composites.X‐ray photoelectron spectroscopy (XPS) was also applied to analyze the microstructural evolution and further illustrate the bonding behavior of the DDM composites. Figure e F atom.17 BesFigure  which significantly promotes the flow intensity of the composites during the severe plastic deformation process. More obvious dynamic recovery and recrystallization are triggered by the enhanced lattice distortion and defect energy; (2) by adding (F)‐GNPs in the first step of the ball milling process, the excessive reaction of Al and Mg can be avoided, thereby reducing the dynamic viscosity of the thermo‐plasticized composites during DDM process. This makes the material more prone to plastic deformation and further improves the grain refinement effect. From the above two explanations, it can be found that the DDM composites obtained via the MF+A route has achieved the finest microstructures with an average grain diameter of 1.14 ± 0.45 µm. We further observed the grain characteristics of the MF+A DDM composites, as shown in Figure  From the energy spectrum results, the distribution area of Mg and F‐GNPs overlaps, indicating that a self‐assembled nanoscale structure is formed between Mg and graphene. As shown in Figure 4C3, and enhances the bonding strength between the Al matrix and the reinforcements, since F‐GNPs are relatively chemically inert to aluminum. In addition, due to the poor electronic conductivity of the fluoride, it also provides a necessary condition for reducing the CPA effect on the composites.Figure 22 Fro2.2Figure  the precipitation/solid solution strengthening produced by the Mg element, the sound interfacial bonding between the matrix and the reinforcements, and the Hall‐Petch strengthening brought by the severe plastic deformation, the DDM composites we prepared has obtained the extraordinary mechanical performances. We further studied the comprehensive mechanical responses with different Mg addition. One can see that the tensile strength performance with all the compositions is several times that of pure aluminum, which confirms the good mechanical properties. According to our previous research, the comprehensive mechanical performance has reached the highest known level in related carbonaceous nanomaterial‐reinforced aluminum matrix composites. In the two preferred preparation routes, the mechanical properties of the composites basically increase gradually with the increase in the Mg addition. Regrettably, the elongation has shown a downward trend. Since ductility is also the primary component of the comprehensive mechanical properties, we need to strictly consider the balance of strength and ductility. As such, a 5.5% Mg addition can achieve the relatively ideal mechanical responses.Since our strategy is to conquer the antagonism between strength and corrosion resistance in graphene‐reinforced aluminum matrix composites, we tested the mechanical performances of the DDM composites with four different preparation routes. Figure 25 the2.33+ at the electrical double layer formed at the corrosion surface, while the second is the relaxation and exfoliation of the corrosion products at the exposed surface. The passive oxide film of pure aluminum is porous in the chlorine‐containing solution, resulting in low electrical resistance. The two response loops of pure aluminum are prone to be separated. In contrast, due to the dense protective film formed by the (F)‐GNPs and heteroatom‐doped oxide film according to our previous research, the electrochemical responses in this paper cannot be distinguished, indicating that the impedance value of the doped oxide film is relatively high and shows great protection against corrosion. Besides, the larger diameter of the capacitive loop means a bigger electrochemical impedance. The corrosion resistance of MF+A DDM composites gradually increases and decreases over time. It is worth noting that even when the exposure time reaches 1440 h, that is, 2 months later, the diameter of the capacitive loop still does not show a sharp weakening trend, which shows that our composites can show good anti‐corrosion performance under long‐term service conditions. We further conducted EIS tests on various preparation routes, as well as pure aluminum, and fitted them according to the equivalent circuit in Figure Figure  surface.30 The), the corrosion current densities are significantly lower than that of pure aluminum. The Tafel curves only show a slight change over time, indicating that the corrosion rate is not sensitive to time. Figure Figure 33), t2.4 undesirable anti‐corrosion weakening is easily triggered due to the CPA effect, high‐density graphene defects, and agglomeration. The problem of agglomeration can be well solved by the severe plastic deformation process. Our bottom‐up DDM process can realize homogeneous distribution of (F)‐GNPs in the matrix and even further reduce the thickness of graphene through interlayer shear to improve the service performance of graphene. Nevertheless, how the corrosion acceleration caused by the CPA effect and high‐density defects can be avoided or even achieved the CSA effect is still puzzling. We therefore performed elemental distribution analysis and characterization of the electrochemical corroded surface, as shown in Figure  At the junction between F‐GNPs, due to the existence of the F‐doped oxide film, these F‐GNPs form a tight bond with the oxide film, thereby avoiding the problem of the decrease in the diffusion distance of the corrosive medium caused by the microscopic defects. In a word, this layer composed of F‐GNPs and F‐doped oxide film and coated by Mg alloying finally realizes the CSA effect with a low corrosion rate better than pure aluminum over long‐term service.Electrochemical results and hydrogen evolution rate measurements seem to prove that the corrosion resistance of the DDM composites is higher than that of pure aluminum. However, according to Sun et al.,34 und2O3 (0001) slab, the F‐GNP@Al2O3 (0001) slab, and the F‐GNP+Mg@Al2O3 (0001) slab, respectively. The existence of F‐GNP significantly improves the work function of the composites, indicating that the difficulty of electrons removed from the matrix is increased. Moreover, although F‐GNP without Mg alloying seems to have a higher work function, we can see from the electron density difference image  calculation to clarify the electron transfer characteristics of the protective layer containing Mg‐alloyed F‐GNPs. The detailed calculation method can be found in Supplementary Information ncreased.37 More Figure  that the3We designed a novel bottom‐up strategy to achieve charming carbonaceous nanomaterial‐reinforced aluminum matrix composites with a better balance between corrosion resistance and mechanical performances. This strategy is to conquer the corrosion promotion activity caused by heterogeneous corrosion potential, graphene defects, and agglomeration. Mg‐alloyed fluorinated graphene nanoplatelets were introduced via severe plastic deformation and designed preparation routes. Homogeneously dispersed Mg‐alloyed fluorinated graphene nanoplatelets and ultrafine‐grained microstructures contributed to the formation of dense and strong protective film again chloride‐containing aqueous environment. Corrosion suppression activity was discovered in the designed composites. The anti‐corrosion polarization resistance remained 89% of the initial state after 2‐month immersion in chloride‐containing environment, while the ultra‐tensile strength and elongation of 532 ± 39 MPa and 17.3 ± 1.2% were achieved. The microstructural characterization and density function theory calculation confirmed the existence and rationality of the dense anti‐corrosion layer composed of fluorinated graphene nanoplatelets and F‐doped oxide film and coated by Mg alloying. Moreover, this work is significant for conquering the antagonism between mechanical performances and corrosion resistance, which is indeed critical for the design of carbonaceous nanomaterial‐reinforced aluminum matrix composites to realize some desired performance for practical applications.The authors declare no conflict of interest.Supporting InformationClick here for additional data file.


Understanding COVID-19 contagion among poor populations is hampered by a paucity of data, and especially so in remote rural communities with limited access to transportation, communication, and health services. We report on the first study on COVID-19 contagion across rural communities without road access. We conducted telephone surveys with over 400 riverine communities in the Peruvian Amazon in the early phase of the pandemic. During the first wave , COVID-19 spread from cities to most communities through public and private river transportation according to their remoteness. The initial spread was delayed by transportation restrictions but at the same time was driven in unintended ways by government social assistance. During the second wave , although people’s self-protective behaviors (promoted through communication access) helped to suppress the contagion, people responded to transportation restrictions and social assistance in distinct ways, leading to greater contagion among Indigenous communities than mestizo communities. As such, the spatial contagion during the early phase of the pandemic in tropical forests was shaped by river transportation and social behaviors. These novel findings have important implications for research and policies on pandemics in rural areas. In rural remote regions where people have limited access to transportation, communication, health services, and testing for COVID-19, available data are scant which hampers much-needed understanding10.Understanding COVID-19 contagion among poor populations in low- and middle-income countries and people’s reactions to the pandemic and corresponding government policies is critical to design effective policies to address post-pandemic problems and future pandemics2, or about 2.3 times the area of Costa Rica) 16.Our study examined the spread and evolution of COVID-19 during the early phase of the pandemic across rural communities without road access in the Peruvian Amazon. In this remote environment where rivers serve as roads, transportation restrictions could potentially prevent the spread of the virus by effectively isolating communities. Immediately after a national lockdown was lifted, we conducted telephone surveys with over 400 communities in four major river basins—the Amazon, Napo, Pastaza and Ucayali .Our study has three related objectives. First, we assess the spatial contagion of COVID-19 across rural communities relying solely on river transportation. Extant works on the spatial spread of disease highlight the role of human movement through air and ground transportation, especially across and within urban areas, suggesting policies for mitigation targeted towards highly connected areas29. We add to the literature by showing how these two factors shaped the spatial contagion of COVID-19 across rural communities through public and private river transportation in interrelated ways.Second, we examine both government policies restricting human movement  and local people’s self-protective behaviors  which are known to be critical in restricting person-to-person interactions to suppress contagionribereños) are descendants over many generations of Iberian and Indigenous peoples living in the region30. Concerns about the fate of Indigenous peoples, especially in Amazonia, are warranted and have been prominent in media reports and research on COVID-19, especially during early stages of the pandemic, but tend to overlook the larger mestizo population34. Our large-scale community surveys allow us to make comparisons between these two populations across communities, which is uncommon in the broad literature on Amazonia. We find that transportation restrictions and self-protective behaviors shaped the spatial contagion of COVID-19 in distinct ways for these two populations.Lastly, we compare Indigenous and mestizo communities (‘Study area’ in Methods). Mestizos  and a follow-up survey in August 2020 (during the second wave) (‘Surveys’ in Methods). Our analysis sample is a balanced panel of 435 communities  . Mortality was highest early during the first wave, especially in the cities of Iquitos and Pucallpa. A national lockdown was declared on March 16, 2020, lasting until early May, when restrictions were gradually relaxedWe focus on any incidence of COVID-19 case in the community, including suspected ones . With limited health facilities and testing for COVID-19 in the study area , information about confirmed cases is incomplete and data on the number of cases can be inaccurate. Although suspected case incidence may be also inaccurate, any case captures people’s perceived risk of infection in the community that would have underlain their behaviors. The first COVID-19 case had occurred in most communities (91%) by June, i.e., within three months, during the first wave Fig. A. We estWithout road access in the study area, local residents rely solely on river transportation for travelling. The river network distance from cities  varies markedly by community . The timing of initial spread across communities depended on their remoteness: the nonparametrically predicted probability of spread by April and May shows negative steep distance gradients , it took longer for the virus to reach Indigenous communities (until May), but by the time of the baseline survey in July the difference between these two types of communities had vanished . Public river transportation is effectively the only way for local people to travel from remote communities to cities and was available for 80% of all communities (Indigenous and mestizo) before the pandemic ; it was persistent and not prevalent in 18% and 43%, respectively, over time : (1) preventive measures mostly based on individual behaviors  before the baseline survey (mid-March-July), which captures initial adoption, and at the time of the follow-up survey, and (2) social restrictions consisting of community-level restrictions  at the time of each survey . Over time, preventive measures became weaker and social restrictions became somewhat stronger . Both preventive measures and social restrictions became relatively stronger in Indigenous communities than in mestizo communities .Almost three quarters of communities had communication access  . Radiophones are typically available in some communities beyond the reach of cell phone coverage. When we consider these three modes separately, preventive measures at the baseline were stronger in communities with radiophone, but not internet or cell phone access, although social restrictions were stronger in communities with any of these three modes Fig. A. In conThe status of public river transportation changed between the baseline and follow-up surveys: it was inactivated (the restriction which had been relaxed before the baseline was reimplemented) and activated (the restriction which had been maintained at the baseline was relaxed) among 10% and 13% of communities, respectively, and remained active and inactive among 54% and 3%, respectively . These patterns were similar between Indigenous and mestizo communities .We examine the impacts of a change in preventive measures and social restrictions (indices), and activated and inactivated public river transportation (indicator variables) on the mitigation and spread (mainly resurgence) of COVID-19 between the baseline and follow-up surveys . This first difference specification addresses the endogeneity of social behaviors by controlling for time-invariant community heterogeneity including unobserved factors that could cause omitted variable bias.Inactivated public transportation reduced the probability of spread , but did not increase mitigation Fig. B. Thus, Both preventive measures and social restrictions increased mitigation but did not reduce spread  in communities with inactive public transportation, as found for initial spread, but was not related to distance from cities or district capital or to indigeneity .Government cash assistance served as an important safety net for poor households during the pandemicAt the time of the follow-up survey, people had recently received cash assistance in 12% of communities . Cash assistance was more common (by 9%) in Indigenous communities than mestizo communities; it was also more common in communities closer to cities, but was not related to public river transportation  suggests that community leaders with an information advantage could better coordinate people’s adoption of individual behaviors which they were not familiar with . Such information advantage may not have been significant for restricting community-level social behaviors which were common before the pandemic .Poor communication infrastructure is likely to have caused important delays and gaps in information, or misunderstandings about COVID-19The impacts of preventive measures  on the mitigation of contagion were stronger with greater social restrictions (preventive measures), indicating that these two sets of self-protective individual and community behaviors were complementary to each other . The effectiveness of self-protective behaviors was differentiated according to whether public river transportation was activated, but not to whether it was inactivated, as follows. On one hand, preventive measures and social restrictions increased mitigation only in communities with no activated transportation Fig. C, indicaWe conjecture that the impacts of transportation restrictions on contagion were stronger in remote communities because people there rely more on public river transportation for market access. Indeed, both the diminishing effects of activated public transportation on mitigation and those of inactivated transportation on spread were stronger in communities far from cities . Moreover, in remote communities, activated public transportation increased spread and inactivated transportation increased mitigation. We also conjecture that self-protective behaviors were effective to suppress contagion in remote communities because preventive measures and social restrictions were stronger there Fig. A. IndeedAlthough the effectiveness of preventive measures and social restrictions for mitigation was not differentiated by indigeneity, both were effective at reducing the spread in Indigenous communities only Fig. C. This iThree limitations in our study are to be noted. First, as in any survey, we cannot rule out the possibility of measurement errors, though community telephone surveys such as those we conducted better circumvent reporting bias  than standard household telephone surveys (‘Surveys’ in Methods). Second, our data do not allow us to examine the extent to which the different patterns found between Indigenous and mestizo communities may be underlain by contrasting cultural and social norms. Our community-level survey data also preclude us from exploring household-/individual-level factors. Finally, although our analysis sample captures well the dynamics of spatial contagion, it is not necessarily regionally representative (‘Surveys’ in Methods). Concern about external validity has been a common issue with telephone surveys during the pandemic.47.Community-level analysis on case incidence is effective for capturing not only the spatial spread of disease, but also its spatial evolution  and people’s self-protective behaviors. This approach is practical for rural research during pandemics when researchers must rely on telephone surveys to collect data. This approach can be applied also to understand rural people’s vaccination behaviorsThe virus can spread from cities to rural communities through limited river transportation driven by people’s actions for livelihood and coping. Remoteness delays the speed of contagion but does not prevent the spread to rural communities altogether.21. Balancing this health gain against associated economic costs of limited market access and various costs of reduced social connection 2 is a central tradeoff in policymaking in remote rural areas under pandemics.It is possible to significantly delay and suppress contagion across rural communities by restricting their transportation access to cities. Failing to employ such restrictions across cities led to the rapid spread of COVID-19 in Brazil43, who receives information and how it is used may shape the adoption of unfamiliar individual behaviors44. This applies also to communication for promoting COVID-19 vaccination49.Rural people’s self-protective behaviors are also key in suppressing contagion. Although improving communication access can promote such behaviors and facilitate data collection51.The effectiveness of self-protective behaviors will likely depend on the combination of individual and community behaviors, and government policies such as transportation restrictions. Policy makers need to design pandemic policies considering their potential complementarities and possible unintended side effects. Developing a digital payment platform in places where it does not yet exist is essential for the effective and timely distribution of cash assistance while avoiding negative public health externalities52.In pandemics, people in different socio-cultural groups may adopt self-protective behaviors including vaccination and respond to government policies in distinct ways. In Peru, increased vulnerability of Indigenous people is a major concern as found also in BrazilOur study on rural communities without road access in the Peruvian Amazon revealed that COVID-19 contagion during the early phase of the pandemic was shaped by river transportation and social behaviors. Our findings have the following implications for research and policy related to pandemics more broadly.This study was approved by the Research Ethics Board of McGill University (#290–114) and performed in accordance with relevant guidelines and regulations. We obtained informed consent from all participants.53. Iquitos can be reached only by riverboat or by air; Pucallpa has also been connected with Lima, the capital city of Peru, by road since the 1940s. Small towns  which function as district capitals and secondary markets and many smaller communities (100–300 inhabitants) with limited access to public and health services line the main rivers and tributaries. Forest peoples (both Indigenous and mestizo) practice agriculture, fishing, hunting, timber and non-timber forest product gathering, and small livestock raising for subsistence and cash earnings, sending produce to market by boat55.The Departments of Loreto and Ucayali cover about 85% of the area of the Peruvian Amazon Fig.  consistihttps://parlap.geog.mcgill.ca)56. We selected four major river basins—the Amazon, Napo, Pastaza, and Ucayali—to capture the diversity of ecological conditions, economic activities, history, and ethnicity of its peoples 53, maps from the Instituto del Bien Común (IBC) for their census of Indigenous communities58, and Google Earth imagery, supplemented by local enquiries by the teams to identify unmapped settlements. The community survey conducted from December 2012 through March 2014 reached a total of 919 communities , which we estimate represents 92% of all communities in the study area . Each community was geo-referenced using a handheld Garmin GPS unit. The survey collected information through a focus group-based in-person interview among community leaders and elders following a structured questionnaire. We use data from this community survey to construct some variables.Our COVID-19 surveys were part of the Peruvian Amazon Rural Livelihoods and Poverty (PARLAP) project . We subsequently conducted a follow-up telephone survey in August and early September 2020 that reached 435 of the 469 communities in the baseline sample (7% attrition). The analysis sample is the panel sample of 435 communities.Excluding district capitals and communities with a health center from 919 communities covered in the community survey, the remaining 893 communities were eligible for the COVID-19 surveyOur surveys sought information from community leaders following a structured questionnaire. With the suspension of public telephone service since November 2019 and an unreliable radiophone system, we relied mostly on cell phone contact. Our field teams visited ports and markets in Iquitos and Pucallpa to find people from the target communities. Some telephone interviews were arranged through an intermediary when people from the target communities visited a town where the intermediary lived. In these ways the surveys also contacted people in communities with no telephone access.The non-randomly sampled communities in the COVID-19 survey are not representative of the PARLAP study area: communities in the Napo basin  and Indigenous communities are more (less) likely to have been sampled in the baseline survey and be found in the follow-up survey (Supplementary Table 4). The panel sample was not correlated with distance from cities, access to public river transportation, telephone access, and the availability of health facility. These results suggest that the findings from the COVID-19 sample may not be generalizable to the whole PARLAP study area. The limitation of external validity is a common problem of telephone surveys during the pandemic.https://www.poverty-action.org/recovr/research-projects) have been conducted at the household (or firm) level and a small number of projects have included community questionnaires59. Our COVID-19 survey is the first to our knowledge to employ a large-scale community telephone survey.Most surveys in other extant and on-going research projects on COVID-19’s socio-economic impacts in low- and middle-income countries listed at Innovations for Poverty Action (lanchas), small river boats (colectivos), and speed boats (rápidos)36. Large river boats offer regular passenger and cargo service between cities and towns, stopping in selected towns and communities along main rivers, many of which function as intermediary hubs between the cities and other rural communities. Small river boats are more common within a day’s travel from cities and towns for both passengers and cargo; serving as river buses, they run along both main rivers and tributaries which are not accessible by large river boats. In the last decade, powerful speed boats have begun offering regular passenger service along main rivers between cities and towns within a 10-h trip. Travel is about 3–4 times faster and more comfortable than on a large or small river boat but the cost is prohibitive for most rural people. Local private river transportation consists of small personal boats powered by a small air-cooled engine attached to a long shaft and a small propeller (peque-peques).In the region, public river transportation consists of three modes: large river boats  and at the time of the follow-up survey. Our social restriction index is a z-score constructed by taking the first principal component of 7 indicator measures: primary and secondary school closure at the time of the surveys (which takes 1 if there was no school), not playing soccer and volleyball during the previous 7 days, and no gatherings for communal work, community meetings, and church services during the previous 7 days (which takes 1 if there was no church).Yict is outcome variables, such as an indicator variable for case incidence and self-protective index (z-score), of community i in basin c at period t; Xit is a vector of predictors, including interviewer fixed effects which capture interview heterogeneity including potential reporting bias (8 interviewers); ϕc is a vector of basin fixed effects which capture basin heterogeneity (6 basins); and εic is an error term. Inference is based on robust standard errors. Supplementary Fig. 6A shows the distribution of transportation variables used as predictors and Supplementary Table 5 shows the definition and descriptive statistics of other predictors (the construction of transportation variables and some of other predictors is provided below). Full regression results are reported in Supplementary Tables 1, 2, 3, 6, and 7. The number of observations for some outcome variables is slightly smaller than 435 due to missing values.We estimate predictors of outcomes using Ordinary Least Squares (OLS) regression specifications of the formYict is case incidence of community i in basin c at period t ; Zit is a vector of self-protective behavior indices—preventive measures and social restrictions (z-score); Xit is a vector of public river transportation; and εict is an error term. Controlling for time-invariant community heterogeneity—both observed and unobserved factors—this first difference specification allows us to capture the relationships of COVID-19 case with social behaviors and transportation restrictions within communities. The first difference of binary outcomes takes −1, 0, or 1. We consider two binary measures: (1) an indicator variable which takes 1 if the difference takes −1, that is, the original measure decreases from 1 to 0 ; and (2) an indicator variable which takes 1 if the difference takes 1, that is, the original measure increases from 0 to 1 . Analogously, we consider two binary measures for public river transportation: (1) an indicator variable which takes 1 if the original measure decreases from 1 to 0 ; and (2) an indicator variable which takes 1 if the original measure increases from 0 to 1 . Inference is based on robust standard errors. The number of observations is 367 communities with non-missing values in self-protective behavior indices at the baseline and follow-up.We estimate impacts of social behaviors and transportation restrictions on COVID-19 case using first difference specifications of the formWhen we examine complementarity of preventive measures and social restrictions, we add their interaction term to Eq. , estimatAccess to public river transportation—access to at least one public river boat  before the COVID-19 pandemic.Access to large river/small river/speed boat—access to at least one large river/small public river/speed boat before the COVID-19 pandemic.Active/inactive public river transportation—at least one/no large river boat, small public river boat, or speed boat during the previous 7 days at the time of interviews .Active/inactive large river/small river/speed boats—at least one/no large river/small public river/speed boat during the previous 7 days at the time of interviews .Active/frequent local private river transportation—at least one/five small personal boats during the previous day at the time of interviews .60 from Iquitos or Pucallpa (closer one) to each community.Distance from city—River network distance60 from district capital (including one outside the study area) to each community. The analysis sample covered 21 districts.Distance from district capital—River network distance60 from the nearest market town out of 13 (including ones outside the study area) to each community.Distance from nearest market town—River network distance60 from the nearest community out of all other communities covered in the community survey from each community.Distance from nearest community—River network distanceRiver order—A positive whole number to indicate the level of branching in a river system. We consider the rivers on which Iquitos and Pucallpa (the source of virus) are situated as order 1. Specifically, we consider the Amazon, Ucayali, and Marañón rivers as order 1  published by INGEMMET ; available on-line at: https://portal.ingemmet.gob.pe/web/guest/carta-geologica-nacional-escala-1-100-000. Simplified reclassification of INGEMMET’s ‘Soil_NAME’ variable, as soils being formed during the Holocene period (Qh), Pleistocene (Qp), or earlier (Tertiary).Floodplain soil—A proxy for young alluvial soils in floodplain. The proportion of land area in a 5 km buffer centered on the community that is underlain by Holocene parent material. Based on Since most market towns are district capitals, we use either distance from district capital or distance from nearest market town as a predictor. We report results using distance from district capital; those using distance from nearest market town are similar.Supplementary Information.