L1 transposons occupy 17% of the human genome and are widely exapted for the regulation of human genes, particularly in breast cancer, where we have previously shown abundant cancer-specific transcription factor (TF) binding sites within the L1PA2 subfamily. In the current study, we performed a comprehensive analysis of TF binding activities in primate-specific L1 subfamilies and identified pervasive exaptation events amongst these evolutionarily related L1 transposons. By motif scanning, we predicted diverse and abundant TF binding potentials within the L1 transposons. We confirmed substantial TF binding activities in the L1 subfamilies using TF binding sites consolidated from an extensive collection of publicly available ChIP-seq datasets. Young L1 subfamilies  contributed abundant TF binding sites in MCF7 cells, primarily via their 5′ UTR. This is expected as the L1 5′ UTR hosts cis-regulatory elements that are crucial for L1 replication and mobilisation. Interestingly, the ancient L1 subfamilies, where 5′ truncation was common, displayed comparable TF binding capacity through their 3′ ends, suggesting an alternative exaptation mechanism in L1 transposons that was previously unnoticed. Overall, primate-specific L1 transposons were extensively exapted for TF binding in MCF7 breast cancer cells and are likely prominent genetic players modulating breast cancer transcriptional regulation. The normal growth and function of cells rely on the precise and coordinated expression of complex gene networks. Transcription is a primary step in gene expression and is regulated by functionally interacting transcription factors (TF), which recognise and bind specific cis-regulatory elements in genomic DNA ,2,3. OveMany human TFBSs are located within transposons, which in total occupy 45% of the human genome ,10. TranAIM2 and APOL1 [Exaptation is the process where transposons contribute cis-regulatory elements for the regulation of host genes and has been described as a common phenomenon in various biological and disease contexts in the human genome ,17,18. And APOL1 ,21. In mnd APOL1 . Furthernd APOL1 . The genThe most abundant class of transposons are the L1 transposons, with more than 500,000 copies colonising 17% of the human genome ,24,25. TL1 subfamilies that expanded during the primate radiation are denoted as “L1PA#”, where higher numbers indicate repeats of increasing age ,26. L1HSMET chimeric transcript, which is associated with enhanced malignancy [SYT1 (L1PA2), TYRP1 (L1PA3), REL (L1PA4) and MRE11A (L1PA5) [Generally, human transposons, including L1 transposons, are heavily suppressed by epigenetic mechanisms and post-transcriptional silencing in normal somatic tissues . The loslignancy ,44,45,46 (L1PA5) ,47.We previously investigated the TF binding and promoter activity of an individual L1PA2 transposon in triple-negative breast cancer cell lines . Upon fuGiven the relatedness and structural similarities between L1 subfamilies, we hypothesised that the capacity for genomic regulation observed in the L1PA2 subfamily was a conserved feature amongst primate-specific L1 transposons and that sequence variations amongst L1 subfamilies, resulting from mutations and truncation of the L1 5′ UTR , would lTFs exert regulatory roles by interacting with sequence features in genomic DNA or their binding motifs. Although putative binding motifs do not always define functional binding sites, they are indicative of potential TF binding activities. We scanned individual transposons of primate-specific L1 subfamilies for occurrences of known human TF binding motifs. A total of 114 out of 401 TFs from the HOCOMOCO core database were identified to have putative binding motifs in over 10% of transposons in each subfamily . HierarcUsing the previously established “TEprofiler” pipeline and consolidated TFBSs from the GTRD and ChIP-Atlas databases ,49,50, wWhile ESR1 binding was highly prevalent in L1 transposons of various evolutionary ages, the youngest L1 subfamilies  displayed a distinct TF binding repertoire compared to the more ancient L1 subfamilies (L1PA4-L1PA12), where the youngest subfamilies were frequently bound by cancer-associated TFs, including MYC, FOXA1 and E2F1, as well as the nuclear receptor NR2F2 and chromatin remodelling-associated CTCF . These TThe distinction in TF binding between young and more ancient L1 subfamilies was apparent when considering all L1 transposon copies in the human genome and could be attributed to varying structural integrity and sequence variations . When coWe next sought to investigate the distribution of TFBSs in different L1 subfamilies. As expected, TFBSs clustered in the 5′ UTRs of relatively young L1 subfamilies, and the TF-binding activity of the 5′ UTR was corroded by heavier 5′ truncation in the more ancient subfamilies. However, in the older L1 subfamilies, the 5′ UTR-lacking transposons contributed TF binding activities via their 3′ ends, which likely corresponded to their 3′ UTRs . SimilarWe next sought to explain the variability in TF binding observed in the intact L1 5′ UTRs. We hypothesised that the differential TF binding between young and more ancient L1 subfamilies was due to variations in the DNA sequence features that the TFs recognised and interacted with. To test that hypothesis, we scanned the L1 5′ UTRs for the known motifs of ESR1, which bound all L1 subfamilies at a similar frequency, as well as FOXA1 and E2F1, which frequently bound young L1 subfamilies but showed fewer binding activities in ancient L1 subfamilies. We used motif models from the JASPAR database , which iTo visualise differences between subfamilies at the locations of these known binding motifs, we used a multiple sequence alignment of the L1 subfamily consensus sequences. The consensus sequences were derived from full-length individual transposons in each subfamily . SequencFor ESR1, motif scanning in individual L1 5′ UTR identified several regions that could account for binding. These regions exhibited different degrees of sequence conservation amongst the L1 subfamilies but showed a general trend of increasing differences between subfamilies that were further apart in their evolutionary ages . For botThe replicative nature of transposons has made them an exceptional vector for dispersing TFBSs of functionally associated TFs. We thus examined the pattern of TFBS co-localisation within the primate-specific L1 subfamilies. The degree of TFBS co-localisation was inversely correlated with the evolutionary age of L1 subfamilies, where binding site co-localisation was a common event in young L1 subfamilies, yet gradually diminished as the evolutionary age of L1 subfamilies increased . Transposons are often heavily suppressed by epigenetic mechanisms in normal somatic tissues, which is loosened in epithelial cancers, allowing the transcriptional activation and functional exaptation of transposons ,54,55,56We next examined each TF individually for their dependency on L1-derived TFBSs. We identified a number of TFs, including CTCF, ESR1, MYC and JUN, to bind L1 transposons almost exclusively in the MCF7 cells while showing minimal L1-binding activity in the MCF10A cells . This caThere is growing evidence supporting the widespread exaptation of transposons as a means of human gene regulation. Transposons are ubiquitous in the human genome and contain cis-regulatory elements that can be recognised by human transcriptional regulatory proteins ,14,15,16L1 transposons are the most abundant family of human transposons, occupying 17% of the total human genomic sequences . There aStudies have shown extensive exaptation of L1 transposons for human genome regulation. They contribute abundant TF binding activities, primarily via their 5′ UTR, to a diverse range of TFs ,28,42. LWith a focus on breast cancer, we developed the “TEprofiler” analysis pipeline for identifying cell type- or tissue-specific transcriptional activities within transposons . Our anaIn our analysis, we showed while sequence truncation was a common event observed in all primate-specific L1 subfamilies, younger subfamilies generally contained more full-length elements, which harboured intact 5′ UTRs . We predThe rapid developments in high-throughput, parallel sequencing technologies have enabled the identification of genome-wide cis-regulatory elements. ChIP-seq technology has been widely applied to identify global TF binding activities, and an extensive collection of ChIP-seq-identified TFBSs has been compiled and made available by public databases, such as the GTRD and ChIP-Atlas databases ,50. UsinIn MCF7 cells, L1 transposons were found to be bound by a total of 99 TFs, including ESR1, MYC, FOXA1 and E2F1, which have well-established and critical roles in cell growth and division and have been linked to breast cancer development ,65,66,67Upon closer examination, it was discovered that the loss of 5′ UTR in the ancient L1 subfamilies was compensated by alternative binding sites in the 3′ end, possibly in the 3′ UTR . The seqIn contrast to ESR1, the other TFs, such as E2F1, MYC and FOXA1, showed a differential binding frequency between the young  and the more ancient L1 subfamilies, as their binding activity was more prevalent in young L1 transposons . This diCis-regulatory elements of functional association show a pattern of co-localisation in the genome, where their genomic locations are often found in close proximity to each other . InteresTranscriptional regulation occurs in a tissue-specific and context-specific fashion, where the interactions between regulatory TFs and the genomic DNA may change drastically from one cell state to another. One mechanism for exerting the context-specificity of transcriptional regulation in diseases is differential TF binding, observed as loss or gains of TF binding activities ,74. We pIn this study, we investigated the exaptation of primate-specific L1 subfamilies for TF binding events in a breast cancer model. Our analysis pipeline demonstrated broad utility and identified abundant TF binding activities in the L1 transposons. Despite having a shared origin, the remnants of L1 mobilisation in the human genome demonstrated great structural and sequence diversity, owing to 5′ truncation as well as sequence variations that resulted from ancestral recruitment of different 5′ UTRs and accumulated mutations. These sequence variations predisposed L1 transposons to diverse TF binding events, where L1 subfamilies of similar evolutionary age displayed resemblance in TF binding profiles. Younger L1 subfamilies harboured substantial and comparable TF binding activities in their 5′ UTRs and contributed to TFBS co-localisation and the combinatorial regulation by functionally interacting TFs. Interestingly, the TF binding capacity of ancient L1 subfamilies was not disadvantaged by 5′ truncation. Instead, these transposons contributed abundant TF binding activities via alternative binding sites in their 3′ ends, suggesting the presence of raw sequence materials in the L1 3′ UTR that could be exapted for TF binding over evolutionary time. Despite the structural differences, these L1 subfamilies showed highly consistent, cancer-specific TF binding activity, where they contributed to differential TF binding between the cancer cell model and the near-normal cells. Overall, L1 transposons exhibited widespread exaptation in breast cancer cells through more diverse mechanisms than previously anticipated. Given their abundance in the human genome and the conserved co-binding patterns of oncogenic TFs, these primate-specific L1 transposons likely play a prominent role in modulating global transcriptional regulation in cancer, leading to alternations in the cancer transcriptome.Transposons and TFBSs that mapped to alternate chromosomes were excluded from all analyses in this study. All genomic locations were in hg38 unless otherwise stated.The genomic locations of primate-specific L1 transposons (L1HS and L1PA2 to L1PA12) were retrieved from the UCSC RepeatMasker table ,75. FullThe GTRD and ChIP-Atlas databases contained a large collection of TFBSs generated by compiling publicly available ChIP-seq datasets ,50. TFBSThe L1HS, L1PA2 and L1PA3 transposons were investigated for their epigenetic states in MCF7 cells using publicly available ChIP-seq and DNAse-seq datasets  ). Brieflp-value < 1 × 10−4 [To predict the TF binding profiles of transposons, known TF binding motifs in the human core mononucleotide collection were acquired from the HOCOMOCO (version 11) database, which contains 401 motif models . Individ1 × 10−4 . For eacFor each cell line, TFBSs overlapping L1 transposons were identified using the “TEprofiler” pipeline . Brieflyp-value < 1 × 10−4 [L1 5′ UTRs bound by ESR1, FOXA1 and E2F1 were scanned for binding motifs. Briefly, TF motif models were acquired from the JASPAR 2020 database for ESR1 (MA0112.2), FOXA1 (MA0148.4) and E2F1 (MA0024.2) . DNA seq1 × 10−4 .The full-length consensus sequences of primate-specific L1 subfamilies were acquired from Khan et al. . Multiplp-value < 0.0005 We next investigated the events of binding site co-localisation for 10 frequently binding TFs  in individual L1 transposons using the “TEprofiler” pipeline . BrieflyExaptation of transposons for the regulation of human genomes has been described and studied in a diverse range of biological and disease contexts. In this study, we demonstrated that the L1 transposons from related evolutionary lineages were exapted to contribute substantial TF binding activities in a breast cancer model. Primate-specific L1 transposons contained abundant “raw” cis-regulatory sequences that predisposed them to TF binding and conferred functional TF binding events in MCF7 breast cancer cells. While intact L1 5′ UTRs remained a prominent reservoir of TF binding activities, 5′ truncated L1 transposons were found to contribute abundant TF binding activities through alternative binding sites in the 3′ ends. This suggests that the mechanisms of transposons exaptation are likely more dynamic than previously anticipated. Current methodology and bioinformatic resources available for investigating transposon-derived transcriptional regulation tend to focus more on young, structurally intact L1 transposons and will likely lead to underestimation of L1 regulatory potential. We find it peculiar that there is a large increase in the number of functional binding events within the youngest L1 subfamilies. This pattern suggests a potential selective advantage for pro-tumorigenic transcriptional events during recent L1 propagation, at least in this breast cancer model. Overall, our findings demonstrate a correlation between evolutionary relatedness and functional conservation in human transposons and reveal primate-specific L1 transposons as prominent genetic players in breast cancer transcriptional regulation.


The growing demand for lightweight and multifunctional products in numerous industrial fields has recently fuelled a growing interest in the development of materials based on polymer matrices including graphene-like particles, intrinsically characterized by outstanding mechanical, thermal, and electrical properties. Specifically, with regard to one of the main mass sectors, which is the automotive, there has been a significant increase in the use of reinforced polyamides for underhood applications and fuel systems thanks to their thermal and chemical resistance. In this frame, polyamide 6 (PA6) composites filled with graphene nanoplatelets (GNPs) were obtained by melt-compounding and compared in terms of thermal and mechanical properties with the neat matrix processed under the same condition. The results of the experimental tests have shown that the formulations studied so far offer slight improvements in terms of thermal stability but much more appreciable benefits regarding both tensile and flexural parameters with respect to the reference material. Among these effects, the influence of the filler content on the strength parameter is noteworthy. However, the predictable worsening of the graphene sheet dispersion for GNPs contents greater than 3%, as witnessed by scanning electron images of the tensile fractured sections of specimens, affected the ultimate performance of the more concentrated formulation. In the last decades, the addition of nanoscale fillers to enhance specific properties of polymers has triggered intensive research activities in science and engineering. Nanoscale fillers such as nanoclays, carbon nanotubes, and graphene appear ideal to develop new multi-functional materials ,2, but tAmong the aforementioned nanofillers, it is worth noting that graphene enjoys a marked competitiveness, especially in terms of cost-effectiveness. It can be derived at high purity from graphite, an abundant natural resource, using relatively convenient approaches; other graphitic carbon nanofillers such as carbon nanotubes and carbon nanofibers, instead, usually require expensive and complicated equipment, as well as high energy consumption. Over time, the increased availability of graphene has meant that many researchers, already engaged in studies on polymer nanocomposites containing nanoclays or nanotubes, have increasingly turned their attention to the graphene-based ones for which dispersion issues have been successfully faced through functionalization ,5, thermPolyamides 6 (PA6) are thermoplastic resins with interesting mechanical properties in terms of resistance and toughness such as to fully fall within the family of so-called techno polymers. Currently, they are widely used in a wide range of industrial sectors, among which the automotive one is noteworthy, where the potential offered by PA6-based materials are strongly fuelled by the continuous attempt to replace metal parts with plastic materials aimed to reduce weight and costs.The interest of experts has so far highlighted the need to further improve the performance of PA6 compounds to meet specific and particularly demanding requirements ,10,11, aIn this frame, special attention was paid to the development of new materials obtained by modifying PA6 by the inclusion of graphene-like fillers such as graphene nanoplatelets (GNPs) ,13,14,15For example, Mayoral et al.  investigGomez et al.  considerLee et al.  reportedThis contribution, as part of a larger project aimed at the development of novel materials potentially usable for the production of energy storage components intended for the growing market of electrical vehicles, reports the results of a preliminary study carried out on compounds based on an injection moulding grade of PA6 and lamellar graphene, supplied by an Italian start-up. To obtain easily scalable solutions, the examination focused on samples, including various amount of the carbonaceous filler (up to 5 wt%), prepared and potentially processable with technologies traditionally used in the reference mass sector.Melt compounded samples were transformed into plates by compression moulding from which specimens of suitable size were obtained for the subsequent static-mechanical tensile and flexural tests. The experimental evaluations revealed the effect of the GNPs content on the main mechanical parameters such as modulus and ultimate strengths and, therefore, highlighted their performances compared with the pure matrix processed under the same conditions. Scanning electron microscope observations of tensile-fractured surfaces of the specimens provided information about the effective dispersion level of the filler, actually achieved under the applied process conditions, supporting mechanical behaviour detected for studied materials.3, HDT: 50 °C) as the matrix and graphene nanoplatelets (GNPs) purchased as G2Nan at Nanesa S.r.l.  as the reinforcing phase. The latter, having a density of 2.2 mg/cm3, a Young’s modulus equal to 1 TPa and a tensile strength of 5 GPa, is available in the form of flakes with diameters between 10 and 20 µm and thickness between 5 and 20 nm.The research was focused on compounds constituted by a polyamide 6 (PA6) resin supplied by Ravago Group under the trade name Ravamid B NC  by setting the screw speed at 40 rpm and the screw temperature profile 25–240–250–250–230–220 °C from the hopper to the die.Two-millimetre-thick plates were prepared by compression moulding at 250 °C, using a Collin GmbH  model P400E press and applying the following pressure profile over time: 3 min at 0 bar, 1 min at 2 bar, and subsequent maintenance of the pressure during the cooling phase of the sample up to room temperature to prevent any shrinkage of the material, which, in this last phase, would generate, among other things, undesirable aesthetic defects of the products.The thermal analysis of the compounds and of the pure PA6 matrix, chosen as a reference, was carried out by differential scanning calorimetry using a DSC instrument from TA Instruments, model Q2000.The tests were carried out at a scanning speed of 10 °C/min on samples of 8–10 mg, applying a typical step-by-step procedure: heating from room temperature to 250 °C, isothermal stasis at 250 °C for 1 min, cooling from 250 °C to room temperature, and subsequent heating again up to 250 °C. A thermal rate of 10 °C/min was applied in all non-isothermal stages. The mean values and standard deviations of these parameters are the result of at least three tests performed on each compound. In this analysis, as in the following ones, the compounds are compared with the pure matrix processed under the same conditions.mT) and melting enthalpy . Furthermore, the average values of the melting enthalpies were used to estimate the degree of crystallinity of the materials studied in accordance with the following simple expression:cX is the crystallinity degree, fX is the weight fraction of the filler, and The processing of the thermograms was mainly limited to those relating to the second heating run to exclude any influences of the previous thermomechanical history (processing) of the material on the thermal parameters such as temperature , which allows the recording of the weight loss of the material examined as a function of temperature. In this regard, all the extruded samples were heated from 25 to 600 °C in nitrogen and from 600 to 800 °C in air, with a heating rate of 10 °C/min.5%) and to the maximum rate of thermal degradation (Td), the maximum rate of weight loss (R), and the percentage of residue at the end of the test. The repeatability of the results was verified by performing at least three measurements for each material.The processing of typical diagrams percentage of weight loss vs. temperature and their derivative graphs provided some characteristic data of the tested material such as temperatures corresponding to a weight loss of 5%  were loaded according to a three-point bending configuration. In both cases, at least 5 measurements were carried out for each material to verify the repeatability of the data (modulus and strength) that will be later provided as mean values and corresponding standard deviations.Morphological observations of sections obtained by cryogenic fracture of compression-moulded samples were collected with the aid of a field emission scanning electron microscope (SEM) (mod. FEI QUANTA 200 F)  operating in high vacuum conditions at the voltage of 20 kV. The observed sections were subjected to a preliminary metallization with a gold–palladium alloy in order to make them suitably conductive.Thermogravimetric curves of neat PA6 and its compounds are shown in (a)The temperature corresponding to the 5% weight reduction, reasonably associated with the thermal stability of the materials, is improved by increasing the filler content. This effect is usually attributed to the consolidated ability of graphene to prevent the emission of volatiles and, thanks to its lamellar structure, to hinder the permeation of oxygen (barrier effect) .(b)d) towards higher values, especially for GNP contents up to 3% by weight as well as by the progressive reduction of the peak height (PH) compared to the pure reference PA6 matrix.The inclusion of GNPs also promotes a slowing down of the degradation kinetics as the filler content increases. This effect, attributable to the organization of graphene flakes in rigid mesostructures hindering the progress of degradation phenomena, is manifested by the slight shift of the peak temperature (T(c)The amount of the residue (ashes) at 800 °C increases from approximately 2%, for the neat matrix, up to about 6%, measured for the compound with the highest GNPs content. This trend, although it does not faithfully reflect the added filler content, reasonably shows a monotonically increasing trend. Among other things, these data also depend on the degradation mechanisms of the GNP used, governed, in turn, by the possible presence of residues of surfactants used during the industrial synthesis of the product to favour its exfoliation , degradaGiven that all the materials examined show a one-step decomposition trend, from the mean values of the thermogravimetric parameters obtained from the processing of these curves and summarized in The tensile properties of the examined compounds are compared with those of the neat PA6 in In our case, given the reduction of the degree of crystallinity of the PA6 matrix in the presence of GNPs already discussed above, it can be assumed that the increase in Young’s modulus is mainly determined by the high stiffness of the added graphene flakes. As regards the strain at break, excluding chemical interfacial interactions between the GNP, hydrophilic and non-functionalized, and the hydrophobic PA6 matrix, the reduction of this parameter, very common in many particle-filled polymers, can be reasonably attributed to a lower probability of exfoliation of the graphene lamellae with increasing their content. The segregation of the graphene flakes generates macro-defects acting as stress concentration points, which induce cracks and favour the premature failure of the tested specimen.As regards the tensile strength, apparently unchanged at too low filler content (1% by weight), the results indicated that this parameter is increased for GNP contents up to 3 wt% but reverses its trend at the concentration of 5 wt%.In this regard, it is well known that it is difficult to predict the trend in tensile strength, because it not only depends on variables to which the modulus is also sensitive, such as the size of the aggregates of the dispersed phase and the crystallinity of the matrix, but also is strongly influenced by the polymer–filler interaction. In our case, as the filler content increases, alongside the reduction of the crystallinity of the matrix already discussed above, it is also reasonable to expect a worsening of the dispersion of GNPs with an increase in the size of the graphene aggregates. These considerations, together with the foreseeable poor interfacial interaction, justify the reduction of the tensile strength over the concentration range 3–5 wt% ,24.Representative flexural stress–strain curves of PA6/GNPs compounds and reference material (neat PA6) are shown in In short, the experimental tests have shown mean values of the flexural stiffness (modulus) visibly increasing with respect to the neat matrix at least up to a content of GNPs equal to 3% by weight; further increases in filler concentration do not seem to significantly affect this parameter.The flexural strength of compounds, on the other hand, is always higher than the neat PA6 one. In particular, this parameter shows a trend similar to that described for the tensile strength: a nearly constant value at low filler content (1 wt%), an increase of about 30% for the compound at 3 wt% of GNPs, and a subsequent reduction with the further increase in the concentration of graphene up to an average value of about 54 MPa, still 18% higher than the one characterizing the pure matrix.In flexural loading, the upper part of the specimen undergoes compressive stresses while the lower part of the same is subjected to tensile stresses. That said, for the 3%-by-weight GNP-filled system, the significant increase in both stiffness and tensile strength can justify the benefits found also in terms of flexural strength offered by the side of the specimen opposite to the one subjected to the concentrated bending load.For the more concentrated compound, despite the further increase in tensile stiffness, the reduction of both tensile and flexural strength was attributed to a worsening of the dispersion of graphene flakes with a higher probability of inclusion of voids in the sample.The microscopic observation of neat PA6  shows thRepresentative SEM micrographs of the tensile fracture surfaces of the compounds see , on the In summary, formulations based on a polyamide 6 matrix and containing 1, 3, and 5 weight percent of a commercial graphene nanoplatelets have been prepared by melt mixing and characterized in terms of thermal and mechanical properties as well as through morphological observations of the tensile fracture surfaces.The inclusion of GNP hinders the crystallization of the polyamide matrix, which shows an evident reduction in the degree of crystallinity, especially for contents up to 3% by weight of filler. Further additions of GNP seem to have no major repercussions on this parameter, which, however, does not reverse this trend.Graphene nanoplatelet compounds, in addition to confirming the improvement in thermal stability widely consolidated in the literature, slightly increase the maximum temperature of the derived signal and progressively reduce its height without changing its shape.Tensile and bending stiffness of compounds increase with the GNP content, but the strength parameter, for both types of loading, shows a non-monotonous trend. Specifically, a non-monotonous trend was detected for this parameter essentially dominated by the inevitable aggregation phenomena of graphene sheets for contents above a threshold value usually dependent on a multitude of factors such as aspect ratio of the filler, matrix viscosity, interfacial issues, and so on. This behaviour, also favoured by the foreseeable increase in viscosity of the host molten matrix, was supported by SEM micrographs of sections of specimens broken under tensile tests.In short, the main results obtained so far show that:


The selectivity of electrochemical sensors to ascorbic acid (AA), dopamine (DA), and uric acid (UA) remains an open challenge in the field of biosensing. In this study, the selective mechanisms for detecting AA, DA, and UA molecules on the graphene and graphene oxide substrates were illustrated through the charge population analysis from the density functional theory (DFT) calculation results. Our substrate models contained the 1:10 oxygen per carbon ratio of reduced graphene oxide, and the functionalized configurations were selected according to the formation energy. Geometry optimizations were performed for the AA, DA, and UA on the pristine graphene, epoxy-functionalized graphene, and hydroxyl-functionalized graphene at the DFT level with vdW-DF2 corrections. From the calculations, AA was bound to both epoxy and hydroxyl-functionalized GO with relatively low adsorption energy, while DA was adsorbed stronger to the electronegative epoxy groups. The strongest adsorption of UA to both functional groups corresponded to the largest amount of electron transfer through the pi orbitals. Local electron loss created local electric fields that opposed the electron transfer during an oxidation reaction. Our analysis agreed with the results from previous experimental studies and provided insight into other electrode modifications for electrochemical sensing. One of the open problems in the field of biosensing is the simultaneous detection of ascorbic acid (AA), dopamine (DA), and uric acid (UA) within body fluids. The lack of ascorbic acid (AA) can cause scurvy and other diseases , while a2, carbon nitride nanosheets (C3N4), polypyrrole, or ionic liquids that could extensively alter the redox potentials [Carbon-based 2D materials, e.g., graphene , graphententials ,26,27,28In this study, relatively simplified model systems were considered to address the fundamental mechanisms of the oxidation potential shift of AA, DA, and DA molecules. The binding of these analytes to graphene oxide models was elucidated through a series of density functional theory (DFT) calculations. Firstly, representative configurations of GO were selected by the lowest formation energy. Then, geometry optimizations were performed on AA, DA, and DA analyte molecules on pristine graphene, a graphene model functionalized by hydroxyl groups, and a graphene model functionalized by epoxy groups. After that, partial charge analysis was performed on the AA, DA, and DA analytes in cases for free analytes, oxidized forms, and the molecules bound to different substrates. Functionalization of a pristine graphene plane by epoxy and hydroxyl groups could result in the altered charge distribution patterns of the analyte and might affect the tendency of oxidation reactions.−6 Ry, and the convergence of geometry optimization loops was set with the force tolerance of 0.001 Ry/Å.All calculations in this study were based on density functional theory (DFT), using the Quantum Espresso 5.3 package . For theGO surface containing GO surface, the pristine graphene substrate, and a free epoxy (O atom) or a free hydroxyl (OH) group. Formation energy is the energy required for each structure to form a configuration. In order to estimate the most energy favorable of surface structures, Monolayer pristine graphene and graphene oxide supercells of the dimension 5 × 5 were created. The vertical slab distance was set up at 20 Å to prevent surface interaction from the above graphene layer under the periodic boundary condition. The formation energy of z-axis) so that the center of the ring was at distances 2.0 Å to 8.0 Å from the substrate, and the potential energy was calculated as a function of vertical distance. The potential energy Then, the optimized structure of each analyte was translated along the vertical axis 5-paraC . SimilarDFT calculations were performed for isolated AA, DA, and UA to understand the molecular basis of the interactions between the proposed graphene oxide surface with AA, DA, and UA analyte molecules. After each DFT calculation, Löwdin charge population on each atom, electron density isosurface, and electrostatic potential were extracted. 3 iso-density surfaces in the right panels of Now, consider the electrostatic potential of AA, DA, and UA molecules mapped onto the 0.005 e/Å3 iso-density surfaces that roughly represent the van der Waals surfaces. Then, the optimized structure of each analyte was translated along the vertical axis (z-axis), and the potential energy was calculated as a function of vertical distance. The potential energy curve was then fit to the Morse potential function, and the adsorption energy can be reproduced from the well depth   between According to our potential energy calculations, the adsorption of AA, DA, and UA on the pristine graphene surface a–c was sLocal partial charge transfer at each atom within an analyte molecule resulted in changes in charge distribution and local dipole moments. As a result, the partial charge around the oxidation sites was affected, which also affected the oxidation potentials. To further quantify this phenomena, partial charge differences of the analytes (i) in oxidized form, (ii) binding with pristine graphene, (iii) binding with GO5-para, and (iv) binding with G(OH)5-paraA were calculated compared to those of bare analytes for each ring member group and shown in Consider the partial charge difference between the atom groups in the analyte molecules before and after binding with the pristine graphene 5-paraA substrates to the charge distribution within the analyte molecules might affect the oxidation. As an analyte molecule became adsorbed on the functionalized substrate, electronegativity of oxygen atoms in the functional groups caused a small amount of electron to transfer from the analyte to the substrate via pi-orbitals of the analyte. This effect was the most prominent for the epoxidized substrates with a relatively uniform negative partial charge of the functional groups and relatively high electronegativity. The spontaneous electron loss from the analyte to the substrate via this physical adsorption resulted in (i) an electric field between the analyte and the substrate that opposed further electron transfer from an oxidation reaction, and (ii) changes in charge distribution and local electric fields that prevented oxidation reactions. According to the partial charge difference analysis between the neutral analyte molecules and their oxidized forms, UA already required the largest local charge transfer to the oxidation site. The largest amount of electron loss from adsorption would only cause a further positive shift for the oxidation of UA. Meanwhile, the lower amount of electron loss from DA and AA adsorption resulted in a greater difference in oxidation potential, which was in agreement with experimental studies using the electrodes modified by rGO.In this study, the mechanisms of oxidation potential shift of AA, DA, and DA molecules on the GO or rGO surfaces were illustrated through changes in the charge distribution within the analyte molecules at different oxidation and binding states. According to our DFT geometry optimizations, the strongest adsorption of UA to both types of functional groups corresponding to the largest amount of electron transfer through the pi orbitals. The local electric field created by the altered charge distribution within the analytes could prevent the oxidation and cause a further positive shift for the oxidation potential of UA from DA and AA. The more electronegative epoxy functional groups contributed to the charge transfer more than the hydroxyls. This understanding of this oxidation potential differentiation through the intrinsic properties of substrates and analyte molecules could be useful for other electrode modification designs of more reliable AA/DA/UA sensors by controlling the electron distribution of analytes via surface functionalization.


Sargassum ilicifolium, provides shelter and food for numerous coral reef species, but it can also be detrimental at high abundances where it outcompetes other benthic organisms for light and space. Here, we investigate the microbial communities associated with S. ilicifolium in Singapore, where it is an abundant and important member of coral reef communities. We collected eight complete S. ilicifolium thalli from eight island locations along an approximate 14 km east-to-west transect. Each thallus was dissected into three separate parts: holdfast, vesicles, and leaves. We then characterized the bacterial communities associated with each part via polymerase chain reaction (PCR) amplification of the 16S rRNA gene V4 region. We then inferred predicted metagenome functions using METAGENassist. Despite the comparatively short distances between sample sites, we show significant differences in microbial community composition, with communities further differentiated by part sampled. Holdfast, vesicles and leaves all harbor distinct microbial communities. Functional predictions reveal some separation between holdfast and leaf communities, with higher representation of sulphur cycling taxa in the holdfast and higher representation of nitrogen cycling taxa in the leaves. This study provides valuable baseline data that can be used to monitor microbial change, and helps lay the foundation upon which we can begin to understand the complexities of reef-associated microbial communities and the roles they play in the functioning and diversity of marine ecosystems.The large canopy-forming macroalga,  Sargassum ilicifolium (Phaeophyceae: Sargassaceae), is a large and abundant canopy-forming brown seaweed found throughout the coral reefs of Singapore. It provides habitat and food for numerous reef-associated species [The macroalga,  species ,2, and a species . Microbi species ,6,7,8,9. species . Bacteri species ,12,13.Sargassum ilicifolium dominates many reef flats in Singapore where it comes into frequent contact with corals [h corals ,14. Thish corals ,16, and h corals ,18,19. Th corals , which ih corals ,22,23,24h corals ,26, and h corals ,28.Sargassum spp., with differences in photosynthetic rates [Sargassum ilicifolium .Research examining the macrophyte-associated microbiome shows that microbial communities associated with different tissues or plant structures contain different communities, likely a consequence of the different roles different parts play in promoting host survival ,30. Thesic rates , phlorotic rates  and C:N ic rates  being reic rates . SimilarSargassum ilicifolium in the waters of Singapore, the acknowledged influence the microbiome has on host survival, and the growing interest in how species will interact and change under the projected climate change scenarios, characterizing microbial communities in marine environments is important. It provides a baseline on which we can monitor change and, importantly, it provides a framework on which we can build to understand the complex interactions that constitute the marine microbiome as a whole.There is rapidly increasing interest in the marine microbiome, particularly in how coral reefs and other coastal organisms such as mangroves and seagrasses interact with one another, and more broadly regarding how the entire marine microbiome can be influenced by the species present along with terrestrial and atmospheric inputs and interactions . ConsideSargassum ilicifolium thalli were collected over two days in January 2018 from each of the eight islands  at 8 m/s for 2 min, and DNA was extracted with a Qiagen DNeasy Powersoil kit following the manufacturer’s instructions. PCR amplification targeting the V4 region of the 16S rRNA gene was performed using the bacterial and archaeal primers 515F and 806R . Forward and reverse primers were modified to include Illumina adaptors, a linker and a unique barcode . Each rePCR products were visualized on a 1% TBE buffer agarose gel. The normalization and cleaning of PCR products were performed in SequalPrep normalization plates  and submitted for sequencing on the Illumina MiSeq platform  with a 30% PhiX spike (Macrogen Korea).https://benjjneb.github.io/dada2/ for a detailed explanation of filtering parameters, accessed on 22 April 2021).Sequences were demultiplexed, and barcodes and adaptors removed with Cutadapt . Reads wThe DADA2 algorithm was used to estimate error rates from all quality-filtered reads and to merge forward and reverse reads and infer amplicon sequence variants (ASVs). Chimeras were removed with de novo detection. Sequenced extraction negatives were used to identify and remove any possible contaminants using the prevalence method implemented in the decontam R package , and remAny ASVs assigned to mitochondrial or chloroplast genomes and those not present in at least 5% of samples were removed. Rarefaction curves were produced using the rarecurve function implemented in the vegan R package version 2.5.6 . SequencThe filtered ASV table was used to predict metagenome functions using METAGENassist . ASVs wiAll sequences associated with this work have been deposited at the National Center for Biotechnology Information under BioProject ID: PRJNA681720.R2 = 0.30, p < 0.001) and between island locations   . There wp > 0.05), while the holdfast is significantly different from the leaves and vesicles (p < 0.01) and contains the lowest diversity. Diversity at all islands is similar and not significantly different between any pairwise comparisons (p > 0.05) , with holdfasts appearing to have the most distinct community composition. These findings are in agreement with other work from the region that shows microbial community divergence at small spatial scales in a diverse range of marine taxa such as seagrasses [In this study, we use high-throughput amplicon sequencing to characterize the microbial communities associated with the commonly encountered reef macroalga, agrasses , mangrovagrasses  and coraagrasses .There are no obvious barriers to dispersal between the islands in this study, and the prevailing water currents run from east to west with semi-diurnal tidal oscillations . These oS. ilicifolium from different locations are not a consequence of seasonality, as all collections were made over a two-day period in January 2018. Rather, we suggest, much like P. acuta [The differences seen in the microbial communities associated with P. acuta , the sigP. acuta . It is aLocalized rainfall-created runoff does not affect the submerged reefs in this study; while salinity will be reduced in the short term in the waters over these reefs, it will quickly return to the same salinity as the surrounding water with tidal mixing. Conversely, precipitation that falls on islands requires time to reach the reef; while this means that drops in salinity are likely more gradual and less dramatic, they probably occur over a longer duration and take on the chemical characteristics of the substrate they travel over or through  ,66. Our S. ilicifolium structures have also been shown to harbor distinct fungal communities in Singapore [Our analysis shows a differentiation in the microbial communities associated with the structures sampled, with the holdfast harboring the most distinct community. This is in line with studies performed on other marine organisms from the region, which have shown microbial community divergence over small spatial scales in seagrasses, mangroves and corals ,33,34; tingapore . Like otingapore .Sargassum horneri collected in the Yellow Sea indicating that bacteria likely play important roles in nutrient cycling in Sargassum species [Sargassum communities, particularly under nutrient limitation [Sargassum-associated microbial communities in nutrient cycling on coral reefs.Metagenome functional predictions suggest that leaf-associated communities contain more taxa with the potential to perform nitrogen cycle functions, while the holdfast-associated communities contain more taxa with potential sulphur cycling roles, and these predictions are consistent with results from  species . Nitrogemitation ,71, and mitation . Sulphurmitation ,74, and Sargassum ilicifolium, in the highly urbanized marine environment of Singapore. Characterizing the microbes present in marine environments, living within hosts, in the reef matrix, or water column, is an important first step in understanding how the marine microbiome is structured and could respond to change, and this is especially crucial for some of the most commonly encountered species such as S. ilicifolium. Work such as this provides foundational information that can be further advanced with the development and testing of explicit hypotheses that facilitate a greater understanding of how species and their associated microorganisms interact and evolve as environments change.Our study characterizes the microbial communities associated with the reef macroalga,


Amyotrophic lateral sclerosis (ALS), Alzheimer’s disease, Parkinson’s disease and similar neurodegenerative disorders take their toll on patients, caregivers and society. A common denominator for these disorders is the accumulation of aggregated proteins in nerve cells, yet the triggers for these aggregation processes are currently unknown. In ALS, protein aggregation has been described for the SOD1, C9orf72, FUS and TDP-43 proteins. The latter is a nuclear protein normally binding to both DNA and RNA, contributing to gene expression and mRNA life cycle regulation. TDP-43 seems to have a specific role in ALS pathogenesis, and ubiquitinated and hyperphosphorylated cytoplasmic inclusions of aggregated TDP-43 are present in nerve cells in almost all sporadic ALS cases. ALS pathology appears to include metal imbalances, and environmental metal exposure is a known risk factor in ALS. However, studies on metal-to-TDP-43 interactions are scarce, even though this protein seems to have the capacity to bind to metals. This review discusses the possible role of metals in TDP-43 aggregation, with respect to ALS pathology. Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterised by a specific and progressive demise of upper and lower motor neurons in the motor cortex, brainstem and spinal cord ,2,3. DeaC9orf72 hexanucleotide repeat extension, develop frontotemporal lobar degeneration (FTLD) and progressive cognitive decline with behavioural changes subsequently leading to clinical frontotemporal dementia (FTD) 9+ with an approximate mass of 20,098 Da [Zn is so far also the only metal for which the binding to TDP-43 has been characterised. In a study by Garnier et al. , the RRM0,098 Da . In furt0,098 Da . Simulta0,098 Da . The oli0,098 Da ; further0,098 Da . Later, 0,098 Da .bis(thiosemicarbazonato complexes inhibited TDP-43 aggregation, likely via the inhibition of phosphorylation, suggesting a neuroprotective function of the compound.Fe and Cu have important roles in intracellular redox reactions ,131,132.In a recent case report study, Mitani et al.  describeElevated Mn concentrations have been observed in CSF from ALS patients  as well Aggregation of TDP-43 has been observed in the majority of all studied ALS cases  9,31,32,3231,32.Zn is the only metal where binding to TDP-43 has been characterised at the molecular level . RRM1/2 Full-length TDP-43 aggregates are predominantly detected in the spinal cord , and somIn this review, six metals have been discussed in relation to TDP-43, yet several other metals may correlate with ALS and/or other TDP-43 proteinopathies, such as Cd and Al . ApplyinThe roles of metal imbalances and metal exposure in neurodegenerative disorders remain to be clarified. Many of these disorders have been proposed to be metallopathies , as metaMetal imbalance and/or environmental exposure to neurotoxic metals may contribute to the harmful aggregation of the protein TDP-43 as seen in amyotrophic lateral sclerosis.


Graphene is a two-dimensional material, with exceptional mechanical, electrical, and thermal properties. Graphene-based materials are, therefore, excellent candidates for use in nanocomposites. We investigated reduced graphene oxide (rGO), which is produced easily by oxidizing and exfoliating graphite in calcium silicate hydrate (CSHs) composites, for use in cementitious materials. The density functional theory was used to study the binding of moieties, on the rGO surface , to CSH units, such as silicate tetrahedra, calcium ions, and OH groups. The simulations indicate complex interactions between OH/rGO and silicate tetrahedra, involving condensation reactions and selective repairing of the rGO lattice to reform pristine graphene. The condensation reactions even occurred in the presence of calcium ions and hydroxyl groups. In contrast, rGO/CSH interactions remained close to the initial structural models of the epoxy rGO surface. The simulations indicate that specific CSHs, containing rGO with different interfacial topologies, can be manufactured using coatings of either epoxide or hydroxyl groups. The results fill a knowledge gap, by establishing a connection between the chemical compositions of CSH units and rGO, and confirm that a wet chemical method can be used to produce pristine graphene by removing hydroxyl defects from rGO. The che devices ,22. TherThe most widely used technique for preparing graphite oxide by oxidizing graphite is known as Hummer’s method ,23,24,253 treatments with active solvated electrons as a strong reducing agent [1s X-ray photoelectron spectroscopy spectra. By considering Fourier transform infrared spectra, they found a marked decrease in the number of hydroxyl and epoxide groups. Pei et al. [2H4·H2O solution, and a 50 mM NaBH4 aqueous solution at room temperature. They found that after reducing the GO films with HI, most of the oxygen-containing groups from the GO film had been removed, causing the C–C bonds to become dominant, giving a C/O atom ratio ≥ 12 and an electrical conductivity of up to 298 S/cm. Much lower C/O atom ratios and electrical conductivities were found for higher GO films that were reduced using N2H4·H2O and NaBH4.Many recent studies have focused on manufacturing very pure rGO sheets  from GO . Feng etng agent . Liao etng agent  producedi et al.  fabricat9 t and it is currently increasing by 5% per year, and contributes 5–10% to global anthropogenic carbon dioxide emissions [Concrete- and cement-based materials are only second to water, in terms of their use around the world ,34,35,36missions ,38. The missions ,40. Thismissions ,41. CSH missions ,42,43, imissions ,45, a glmissions ,47,48, amissions ,50,51. Tmissions ,39,52, wmissions ,54,55,563H) groups, with a 10% ultra-confined coverage with the calcium silicate hydrate gel (CSH), as shown in The current main challenge in the field area lies in improving our understanding of the mechanisms involved in the interactions between the chemical components of cementitious CSH gel moieties  and prisTo our best knowledge, the mechanism involved in the interactions between rGO and monomers in CSH gels during the fabrication of cementitious composite materials c have noThe interactions between rGO and CSHs are studied by performing DFT electronic structure calculations . The Vie4 monomers, because these moieties occur in solution when the calcium silicates in cementitious clinkers are hydrated. SiO(OH)3− units that were produced in small amounts during the hydration process, causing the pH of cement, were also studied. The contributions of van der Waals interactions were not considered in all the configurations, because the differences in van der Waals potential energies were found to be small when calculated in some tests. The ΔE was calculated from the difference between the energies of the relaxed configuration and the ground-state structure. Further, rGO structures with oxygen-to-carbon reduction ratios (O:C) of 1:50 were used in the simulation models.  is high . Thus, sads) relates to interactions between the sorbent and substrate, and was calculated using Equation (1), as follows:Total is the total energy of the composite systems, ESub is the energy of the graphene or rGO plane, and EAb is the energy of the absorbed moiety [The adsorption energy 4 in two different positions relative to the hydroxyl/rGO sheets, as input geometric structures, were therefore first prepared. Two dissimilar Si(OH)4 unit configurations, with respect to the distance to the hydroxyl/rGO sheet, were found. There was a stable configuration in which the starting geometry did not change much after optimization, as shown in 3 unit, as shown in 4 deposited slightly further from the hydroxyl/rGO sheet than in the expected configuration.Feasible configurations of hydroxyl- and epoxide-reduced graphene with CSH gel moieties were calculated to allow the investigation of the mechanisms involved in the interactions between the different moieties. We were interested in investigating the mechanism involved in the interaction between Si(OH)3 and Si(OH)4 configurations were −1.683 eV and −0.094 eV , respectively. Therefore, the SiO(OH)3 unit in the ground-state structure had a lower adsorption energy than the other configurations, indicating that the system containing SiO(OH)3 with a water molecule next to a graphene plane was the most favorable configuration. Importantly, a condensation reaction occurs when a hydroxyl group at the GO interface becomes dissociated and combines with a hydrogen atom released by Si(OH)4.The calculated adsorption energies for the SiO(OH)3 unit on graphene were considered, to determine the optimum location of the water molecule. The geometric structure consisting of a system containing SiO(OH)3, a water molecule, and graphene, shown in 3, and, most importantly, it is near the graphene surface, implying an interaction with the graphene surface and an increase in the bonding energy.Then, several configurations of water molecules in a system with a SiO(OH)3 unit on hydroxyl/rGO, in a singly negatively charged system, was also considered. Perhaps surprisingly, we found that in the ground-state structure, hydrogen is dissociated from the hydroxyl/rGO surface and is being transferred to the SiO(OH)3 unit to saturate the dangling oxygen atom, as shown in 3 on hydroxyl/rGO in a neutral system was placed as another initial simulation model, and we also found that the hydrogen becomes dissociated from the hydroxyl/rGO surface and is transferred to the SiO(OH)3 unit. This indicates that a hydrogen atom becomes dissociated from the hydroxyl/rGO surface regardless of whether the system is charged or neutral. A dangling oxygen bond therefore points towards the graphene plane and is almost fully occupied through charge transfer from the graphene sheet.A SiO(OH)3 unit on the graphene surface. The unit on the graphene plane was placed at several distances in four different configurations, with the system being either neutral or singly negatively charged. All four configurations were optimized, and the ground-state structure was found for the configuration associated with the unit that was physisorbed to the graphene sheet. The configurations for the neutral cases are shown in 3 physisorbed to the graphene sheet, which has a lower energy state (−0.23 eV) than the chemisorbed configuration. When the system is singly negatively charged, both relaxed structures have the SiO(OH)3 unit physisorbed to the graphene sheet.Next, we considered the adsorption properties of a SiO(OH)3, for the physisorbed ground-state structures and the next chemisorbed configuration for the neutral system, are −1.59 and −1.36 eV, respectively, as shown in 3 to form SiO (OH)3− for the total charged system, so the initial energy state must change by the difference between the neutral and charged silicate units. We expect that the adsorption of SiO(OH)3 to the doped graphene sheet in the neutral and singly negatively charged state is almost the same, because the doping electron must be shared between a large number of carbon atoms. The corrected adsorption energy for the SiO (OH)3− unit, typical in a basic solution, was found to be exothermic by −1.4 eV, because charge fills the oxygen levels. It seems that the adsorption of silicate hydrate units onto rGO would remain favorable at basic pH values and with negatively charged units.The calculated adsorption energies for SiO(OH)4 and hydroxyl/rGO sheets ending in SiO(OH)3 were performed. Previously studied structures, including Si(OH)4 and SiO(OH)3 units with a hydroxyl/rGO substrate, were used, but in the presence of Ca ions. Two different Si(OH)4 and hydroxyl/rGO configurations are used with a Ca ion at different positions, one with the Ca ion close to Si(OH)4 and the other with the Ca ion far from Si(OH)4, as the initial geometric structures. The structures were optimized, and the ground-state energy was found to be 0.495 eV lower for the configuration with a Ca ion close to Si(OH)4 3. Therefore, in a system containing a Ca ion, the Ca ion interacts directly with SiO(OH)3. Some of the charge on the Ca ion is transferred to the 2p orbital of an oxygen atom in SiO(OH)3, to occupy the orbital fully, and the remaining charge is transferred to the graphene sheet. The structure of Si(OH)4 on a hydroxyl/rGO substrate was also optimized in the presence of a Ca ion, in a doubly positively charged system. The hydroxyl group dissociates from the hydroxyl/rGO substrate and a water molecule is formed, and the Ca ion also interacts with SiO(OH)3. The distance between the Ca ion and the oxygen atom in the ground-state structure is ~2.075 Å, as shown in Comprehensive investigations of systems containing Si(OH) Si(OH)4 a than fo3 in the four different configurations, with a water molecule on the graphene surface, vary between −1.43 and −1.69 eV; the latter is given as the ground state indicated in 3 model are slightly higher than the adsorption energies of −1.36 to −1.59 eV, when no water molecule is present on the graphene surface  surface . The enea Ca ion a,b. In o3 on the hydroxyl/rGO sheet in the initial simulation model, the hydroxyl group is found to not dissociate from the hydroxyl/rGO sheet. A lower energy, −1.93 eV, is found for the ground-state structure than in other configurations. However, the ground-state structure in the initial simulation model relates to the configuration with the Ca ion near SiO(OH)3. Optimization for the ground-state structure indicates that the hydroxyl group becomes dissociated from the hydroxyl/rGO, and the Ca ion is involved in bonding, so charge transfer from the Ca ion to two neighboring oxygen atoms occurs, to cause a nearly full occupation of the oxygen orbitals (When a Ca ion is far from SiO(OH)orbitals b.4 and SiO(OH)3, and the hydroxyl/rGO sheet, in the presence of a hydroxyl group and a Ca ion. The results indicate that the ground-state structure has the same configuration as the structure with a Ca ion close to the silicate monomer, which causes the hydroxyl group to dissociate from the hydroxyl/rGO sheet and the charge to be compensated by charge transfer from the Ca ion to the two neighboring oxygen atoms on two hydroxyl groups, as shown in 4 and SiO(OH)3 have energies that are almost 3 and 0.4 eV lower, respectively, than the next lowest energy configurations. In contrast, for the configuration with a Ca ion that is far from the silicate, the hydroxyl group is not dissociated from the sheet. The adsorption energy for SiO(OH)3 in the ground-state structure, in the presence of a hydroxyl group and a Ca ion on the graphene plane, is −2.63 eV, which indicates that stronger adsorption occurred than for the removal of only hydroxyl, as shown in 4 remain strongly bonded to the Si atom.We also studied the interactions between Si(OH)4 is on the epoxide/rGO surface, and the ground-state structure remains similar to the initial structure, with Eads~0.127 eV . The mechanism involved in the interaction between SiO(OH)3 and the surface of the epoxide/rGO sheet was investigated, and the results are shown in 3 on the epoxide/rGO surface of −0.853 eV, which is lower than the adsorption energy for a neutral Si(OH)4 silicate hydrate moiety. The SiO(OH)3 silicate unit moves away from the epoxide groups, as shown by the physisorption model described above. The nearest distance between the dangling oxygen atom and the epoxide/rGO sheet is 2.931 Å. Even longer distances were found in several previous studies. For example, Gao et al. [2S and CH4 on intrinsic graphene, which were −0.038 and −0.022 eV, respectively, and distances of 3.813 and 3.865 Å, which are larger than the distances included in 3 and the epoxide/rGO sheet was considered to be neutral in the presence of a Ca ion and two hydroxyl groups, as shown in 3, in the presence of a Ca ion and hydroxyl functional groups of hydroxyl, is therefore −1.52 eV lower 3 on the graphene plane, for the neutral and singly negatively charged systems, were assessed. The Bader charge analysis method indicates that the charge transfers between the graphene and SiO(OH)3 were similar to the charge transfers in the ground state on the rGO, as shown in The electronic properties of the ground-state structures given by the physisorption model of SiO(OH)3 on the graphene plane in the neutral system, with respect to the graphene sheet (200 electrons) and the SiO(OH)3 unit (31 electrons), are shown in 3 unit. The adsorption energy for SiO(OH)3 on graphene was calculated with respect to the negative unit, and was in the range of a few electron volts. This is typical for Coulomb interactions over a few angstroms distance, caused by charge transfer. Water molecules near the graphene increase the adsorption energy because they form hydrogen bonds with SiO(OH)3, and because the oxygen atom in a water molecule becomes more stable by interacting with the depleted positive charge in the graphene layer.We constructed the 3D charge density difference plots to determine the spatial distribution of the charge. The charge density differences for the ground-state structure of SiO(OH)We assessed the changes in the hydroxyl/rGO electronic properties, caused by adding CSH. The structures with the most favorable adsorption energies were analyzed. The DOS and Bader charge distributions are shown in 3 on the graphene plane in the neutral system are shown in 3 unit resembles the bond in the basic bonding model described in 3 occurs in the neutral system, to give an almost fully occupied oxygen 2p orbital.The charge density distribution of the neutral systems, determined by Bader charge analysis, and the charge density differences for the molecular orbital isosurfaces, are shown in A DFT method was used to study the mechanism involved in the interactions between hydroxyl or epoxide rGO and the CSH moieties, such as CSH gel in cement. The DFT calculations for silicate tetrahedra, Ca ions, and hydroxyl groups improve our understanding of the bonds between rGO and primary CSH moieties. The results led to the following conclusions. The interactions between hydroxyl/rGO and silicate tetrahedra can repair hydroxyl defects selectively in the rGO lattice, and cause graphene to re-form. The dissociation of defects in the graphene plane, and the formation of water, even occurs in the presence of Ca ions and hydroxyl groups. In fact, the main interactions between the graphene plane and CSH gel are Coulomb interactions, caused by charge transfer. In contrast, the ground-state structure remains similar to the initial structure model for interactions between epoxide/rGO and CSH gel. Consideration of the strong interactions in this way could allow improvements to be made in the design of composite materials.


TARDBP and FUS gene mutations. Pharmacological intervention with the apoptosis inducer staurosporine and mutation in a secondary gene (CYLD) also induced measurable cytoplasmic mislocalisation of endogenous FUS and TDP-43, respectively. These findings validate this methodology as a novel in vitro technique for the quantification of TDP-43 or FUS mislocalisation that can be used for initial prioritisation of predicted FTD/ALS-causative mutations.Identified genetic mutations cause 20% of frontotemporal dementia (FTD) and 5-10% of amyotrophic lateral sclerosis (ALS) cases: however, for the remainder of patients the origin of disease is uncertain. The overlap in genetic, clinical and pathological presentation of FTD and ALS suggests these two diseases are related. Post-mortem, ~ 95% of ALS and ~ 50% of FTD patients show redistribution of the nuclear protein TDP-43 to the cytoplasm within affected neurons, while ~ 5% ALS and ~ 10% FTD show mislocalisation of FUS protein. We exploited these neuropathological features to develop an unbiased method for the in vitro quantification of cytoplasmic TDP-43 and FUS. Utilising fluorescently-tagged cDNA constructs and immunocytochemistry, the fluorescence intensity of TDP-43 or FUS was measured in the nucleus and cytoplasm of cells, using the freely available software CellProfiler. Significant increases in the amount of cytoplasmic TDP-43 and FUS were detectable in cells expressing known FTD/ALS-causative  Increasing genetic evidence, neuropathological and clinical observations have identified a substantial overlap between these two disorders2. Several genes have been identified where mutations can cause either FTD or ALS (FTD-ALS genes) including C9orf72, VCP, OPTN, SQSTM1, TBK13 and, most recently, CYLD4. In addition, FTD and ALS share neuropathological similarities: ~ 95% of ALS and ~ 50% of FTD patients show cytoplasmic inclusions of TAR DNA-binding protein 43 (TDP-43) in the brain6, while ~ 5% of ALS and ~ 10% of FTD patients show cytoplasmic inclusions of fused in sarcoma protein (FUS)8. In healthy cells, TDP-43 and FUS are largely expressed within the nucleus. In FTD and ALS, affected neurons can display a redistribution of TDP-43 or FUS to the cytoplasm, as well as insoluble TDP-43 or FUS aggregates10. The importance of TDP-43 and FUS is further demonstrated by the fact that mutation of their encoding genes TARDBP and FUS is sufficient to cause ALS12 or, rarely, FTD13.Frontotemporal dementia (FTD) is one of the most common forms of presenile dementia and involves the progressive degeneration of the frontal and temporal lobes of the brain. Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder that affects the upper and lower motor neurons leading to muscle weakness and paralysis15; however, for the remainder of patients the origin of the disease is uncertain. Despite TDP-43 being a prominent feature in the majority of FTD and ALS cases, including all those carrying FTD-ALS gene mutations13, this fact has not been applied to the screening of FTD and ALS candidate gene variants on a large scale. In previous studies, TDP-43 and FUS mislocalisation has largely been assessed by subcellular fractionation, immunohistochemistry and manual analysis of confocal microscopy24 or high-content imaging27. Whilst studies utilising these techniques have been informative, these assays are labour intensive and low-throughput or, expensive and in some cases produce qualitative data. In addition, some microscopy-based techniques could be subject to bias due to manual selection of the cells to be analysed.Identified genetic mutations cause 20% of FTD and 5-10% of ALS cases28. We validated this technique using known genetic drivers of TDP-43 or FUS mislocalisation. We were able to detect increased cytoplasmic localisation of exogenously expressed FUS and TARDBP mutations relative to wild-type (WT) sequence. We also observed mislocalisation of endogenous FUS upon treatment with the apoptosis inducer staurosporine. Importantly, we could also detect more subtle changes in the cellular distribution of endogenous TDP-43, arising from mutation in a secondary gene (CYLD p.M719V). This validated methodology can now be utilised for rapidly prioritising newly discovered FTD and ALS gene variants by quantifying their effect on TDP-43 and/or FUS localisation.Here we report the development of a rapid, cheap and unbiased technique for the in vitro study of TDP-43 and FUS cytoplasmic mislocalisation. TDP-43 or FUS staining in nuclear and cytoplasmic subcellular compartments was measured in thousands of cells from confocal microscope images, using the freely available analysis software CellProfilerFUS mutations that drastically alter FUS subcellular localisation29. Expression of green fluorescent protein (GFP)-tagged FUS protein with missense mutation FUSR521C or truncation mutant FUSR495X in the neuronal-like neuroblastoma cell line, SH-SY5Y, led to a significant increase in the fluorescence intensity of cytoplasmic FUS  and FUSR495X , respectively, relative to FUSWT  and FUSR495X  mutants in human embryonic kidney (HEK293) cells , ImageJ36 and Huygens (RRID:SCR_014237). We chose CellProfiler based on its ease of use, the ability to perform batch analysis of images, cost and accessibility.Previous studies examining TDP-43 and FUS have quantified cytoplasmic mislocalisation in different ways. Many studies have reported the proportion of the cell population that display cytoplasmic TDP-43 or FUS expressionAnother general observation in our experiments was that SH-SY5Y cells often displayed a greater degree of cytoplasmic mislocalisation than that in HEK293 cells under the same conditions. This may be due to SH-SY5Y cells being a neuronal cell line and thus more disease relevant, or due to the longer transfection time required to achieve optimal transfection efficiency in SH-SY5Y cells , causing additional stress on the cells. It should also be noted that transfection efficiency also dictated the number of images required to reach the desired cell number per replicate (300 cells). SH-SY5Y cells, which had a lower transfection efficiency, required more images to achieve the same number of cells for quantification.FUS mutations has been widely established12. This pathology has been recapitulated by expression of FUS mutations in mammalian cells29. In this study, exogenous expression of the severe truncation mutation FUSR495X caused the majority of FUS to be mislocalised to the cytoplasm as previously demonstrated by Bosco et al.29 using live-cell confocal microscopy and 3D images (11–28 cells). Our quick and simple methodology was able to duplicate these results using 2D images and a larger number of cells . While we recognise the merits of quantifying the entire cell volume, measuring such a small number of selected cells is not representative of the population and may not be able to detect more subtle or variable effects in protein localisation.The cytoplasmic mislocalisation and/or aggregation of FUS in neurons and glia of post-mortem tissue from patients with ALS caused by R521C missense mutation and treatment with staurosporine (1–10 μM) also reproduced previous qualitative microscopy and subcellular fractionation results37 showing a significant increase in the FUS cytoplasmic/nuclear ratio. Confirmation of these results demonstrates the effectiveness of this new methodology in providing quantitative data where previously qualitative or labour-intensive experiments were required. Our ability to quantify FUS cytoplasmic mislocalisation following staurosporine treatment also demonstrates the possibility of this technique for use in testing the ability of novel FTD and ALS drug treatments to modulate FUS or TDP-43 mislocalisation.Expression of the FUSTARDBP are absent6. Mutations in TARDBP have been associated with both FTD and ALS and lead to TDP-43 cytoplasmic mislocalisation13. Unlike FUS, the expression of TARDBP mutations in primary cells and cell lines does not reproduce the dramatic cytoplasmic mislocalisation seen in post-mortem tissue40. In addition, overexpression of exogenous TDP-43 in vitro is sufficient to induce mislocalisation even for WT sequence23. Any differences between WT and mutant TDP-43 for this characteristic are therefore harder to discern and represent a significant limitation for all TDP-43 exogenous expression assays, including the one described here, and may be responsible for differing results being reported for the same mutations. Our technique was able to detect a significant increase in the TDP-43 cytoplasmic/nuclear ratio in TDP-43A315T and TDP-43A382T cells when compared to TDP-43WT. This confirms previous results for TDP-43A315T19 and TDP-43A382T42. The TDP-43M337V variant, which displayed a significant decrease in cytoplasmic TDP-43 in SH-SY5Y cells, has previously been shown to increase cytoplasmic TDP-43 in some24, but not all studies22. We note that TDP-43M337V has previously been shown to aggregate into nuclear puncta in SH-SY5Y cells21, which we observed in our experiments. As these puncta fluoresce at a higher intensity than diffuse TDP-43 expression, this may lead to an overall decrease in cytoplasmic/nuclear fluorescence ratio relative to TDP-43WT. Thus, in some cases quantification of nuclear and cytoplasmic puncta may be a useful adjunct to assess the pathogenicity of a given mutation. However, the disease relevance of nuclear inclusions in in vitro studies of TDP-43M337V is unclear: although both intranuclear and cytoplasmic inclusions were reported in a transgenic TDP-43M337V mouse model43 and it is reminiscent of intranuclear inclusions observed in post-mortem tissue from sporadic FTD patients9, such inclusions are rare in human disease. In the only neuropathological report to our knowledge that concerns a patient with a TARDBP M337V mutation, no mention was made of intranuclear inclusions44. We also note that mislocalisation of TDP-43 is not the only mechanism by which TARDBP mutations lead to disease: for example, changes in protein stability or interaction partners have also been described31. However, we envisage that the mislocalisation assay as implemented here would be a useful tool in a battery of assays to screen novel TARDBP variants.The majority (~ 95%) of ALS and ~ 50% of FTD cases are characterised by the abnormal accumulation of TDP-43 in the cytoplasm of neurons and glia, even though in most cases mutations in 4. In comparison to the other experiments used to validate this quantification method, the degree of change in the endogenous TDP-43 cytoplasmic/nuclear ratio between the empty vector, the CYLDWT and the CYLD mutations was very small: e.g., absolute increase in ratio of ~ 0.03–0.04 between CYLDWT and CYLDM719V 46, and TDP-43 forms homodimers via its N-terminus48. By tagging the protein at the opposite end to its NLS or dimer-binding region we hoped to minimise any effect on the normal localisation pattern of TDP-43 and FUS. Additionally, aggregates of TDP-43 contain C-terminal fragments49, which may have been undetectable had we used a N-terminal GFP tag. We note that the cytoplasmic/nuclear ratio of exogenous GFP-FUSWT  at the C-terminus aa497-526)645. Conv9 contribute to its mislocalisation and aggregation49. Antibodies raised against the extreme N-terminus of TDP-43 would therefore not be suitable for a mislocalisation assay, as they would not detect these cleavage products. In contrast, the anti-TDP-43 antibody we used  recognises all major truncated TDP-43 forms9.A parameter of equivalent importance for detection of endogenous FUS and TDP-43 mislocalisation is selection of an appropriate detection antibody. This is particularly important for TDP-43, where C-terminal TDP-43 fragments observed in FTD and ALS patientsIn summary, this study demonstrates a simple methodology for unbiased quantification of TDP-43 and FUS cytoplasmic mislocalisation in vitro. This technique can easily be added to studies utilising qualitative data to strengthen clearly noticeable phenotypes in an unbiased manner, as well as to highlight subtle changes that may have not been previously identified. Utilisation of this fast and cost-effective technique to aid in assessing pathogenicity of gene variants observed in patients with FTD or ALS will help to prioritise these variants for more intensive research efforts into how they cause disease.CYLD, TARDBP and FUS mutations were introduced into the pCMV6-CYLD, pCMV6-TARDBP and pCMV6-FUS constructs (Origene) by site-directed mutagenesis using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent). Mutated CYLD and TARDBP cDNA sequences were then subcloned, using AsiSI and MluI restriction sites, into the pCMV6-AC-GFP vector (Origene) for expression of C-terminal GFP-tagged CYLD and TDP-43 proteins, respectively. Mutated FUS cDNA sequences were subcloned similarly into the pCMV6-AN-GFP vector (Origene) for expression of N-terminal GFP-tagged FUS protein, to avoid interference of the GFP tag with the C-terminal NLS of the FUS protein45. All clones were verified by restriction digestion and sequence analysis.4 cells/well and HEK293 cells were seeded at 8 × 104 cells/well. After 24 h, cells were transfected with GFP constructs (250 ng/well) for 24 (HEK293) or 48 (SH-SY5Y) hours using Lipofectamine 3000  as per the manufacturer’s protocol. For the staurosporine experiment, cells were incubated for 48 h after seeding, then treated for 5 h with 0.1, 1 or 10 μM staurosporine, to induce apoptosis. Staurosporine dilutions were prepared from a 1 mM staurosporine stock solution dissolved in dimethyl sulfoxide . All vehicle and staurosporine treatment groups were adjusted to a final concentration of 1% DMSO.HEK293 and SH-SY5Y were purchased from ATCC and authenticated by STR analysis prior to use in this study. HEK293 cells were maintained in Eagle’s Minimum Essential Medium  and SH-SY5Y cells in Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12  each containing 10% heat-inactivated fetal calf serum (Sigma-Aldrich). For immunocytochemistry, 8-well chamber slides (Ibidi) were coated with 0.005% poly-L-lysine (Sigma-Aldrich) for 1 h and then washed twice with Dulbecco’s phosphate-buffered saline . SH-SY5Y cells were seeded at 5–7.5 × 10Transfected or drug-treated cells were fixed in 4% paraformaldehyde (PFA) in PBS for 20 min in the dark. For visualisation of exogenous TDP-43 and FUS, cells were then washed twice with DPBS and mounted using DAKO fluorescent mounting medium containing 4′,6-diamidino-2-phenylindole . For visualisation of endogenous TDP-43 and FUS, non-specific background staining was blocked after cell fixation using 1% bovine serum albumin (BSA) in PBS for 30 min. Cells were then incubated overnight at 4 °C with rabbit anti-TDP-43  or rabbit anti-FUS  antibodies. The next day, cells underwent 2 × 5-min washes with DPBS, incubation with rabbit Alexa Fluor-647 secondary antibody  for 1 h, followed by 2 × 5-min washes and mounting as above. Cells were imaged for fluorescence intensity quantification with a 40 × objective on a Nikon A1R confocal microscope. Images of representative cells were obtained with a 100 × objective for figure preparation. Laser intensity, gain and offset settings were kept constant and at least five random fields of view were imaged for each experimental replicate.28. To prepare images for analysis, ND2 microscope files were converted to TIFFs, blinded and split into individual wavelength channel images using ImageJ 36. In CellProfiler, the DAPI channel was analysed first using the Identify Primary Objects module and a global, three-class Otsu thresholding method. This allowed for identification and separation of each nucleus within an image. Strict filtering for cell diameter (30–60 pixels) eliminated any irregular or overlapped nuclei that were present. TDP-43- or FUS-positive cells were also identified using the Identify Primary Objects module. Cell diameter filtering for this step had a much larger range and was dependent on experimental conditions and cell type (20–150 pixels). The Relate and Filter modules were used to link filtered nuclei to TDP-43- or FUS-positive cells. Centroid distance filtering, which limits the distance between the centre of the nucleus and the centre of the cytoplasm (maximum 40 pixels) was also applied to remove any debris or abnormal cells that may be fluorescing in the TDP-43/FUS channel. For the experiments quantifying endogenous TDP-43 in cells expressing GFP-tagged CYLD constructs, additional steps in the analysis were required. GFP-positive CYLD cells were identified in the same way as TDP-43- or FUS-positive cells using the Identify Primary Objects module, described above. However, prior to this, TDP-43-positive cells were identified by association with their corresponding nuclei, using the Identify Secondary Objects module. The watershed-image method was used with global, three-class Otsu thresholding. Size filtering was also applied (maximum 3000 pixels) to remove any clumped cells. The Relate and Filter modules were used to select only TDP-43-positive cells that were also positive for GFP. TDP-43- and GFP-positive cells were then linked to their corresponding nuclei using the Relate and Filter modules described above. For all experiments, in each TDP-43/FUS-positive cell, the nucleus and cytoplasm were separated into individual objects using the Identify Tertiary Objects module. Integrated  fluorescence intensity for both cellular compartments were then measured from the TDP-43/FUS channel image using the Measure Object Intensity module. Using these measurements, the cytoplasmic to nuclear ratio of TDP-43 or FUS was calculated for 300 cells per group, for each of the six replicates of the experiment. All filtering and thresholding steps in each experimental pipeline were kept consistent throughout all experimental replicates. Filtering values were specific to each experiment and require adjustment for different cell types and microscope parameters.Quantification of changes in the cellular localisation of TDP-43 or FUS between the nucleus and the cytoplasm was done by utilising CellProfiler 3.1.9 software Data are presented as mean ± standard error of the mean (SEM) from at least six independent experiments of n = 300 cells. Mean fluorescent intensity of TDP-43 or FUS across six biological replicates was in most cases compared by repeated measures one-way ANOVA and Dunnett’s multiple comparisons test. The effect of CYLD on endogenous TDP-43 was analysed using repeated measures one-way ANOVA and Sidak’s multiple comparisons test. All statistical analyses were performed using GraphPad Prism 9. Significance for all tests was set at p < 0.05.Supplementary Figures.


Macrotermes species: termites preferentially deposit soil on the mound surface at the boundary of the high-humidity region characteristic of the mound interior, implying a coordination mechanism through environmental feedback where addition of wet soil influences the humidity profile and vice versa. Here we test this potential mechanism physically using a robotic system. Local humidity measurements provide a cue for material deposition. As the analogue of the termite's deposition of wet soil and corresponding local increase in humidity, the robot drips water onto an absorbent substrate as it moves. Results show that the robot extends a semi-enclosed area outward when air is undisturbed, but closes it off when air is disturbed by an external fan, consistent with termite building activity in still vs. windy conditions. This result demonstrates an example of adaptive construction patterns arising from the proposed coordination mechanism, and supports the hypothesis that such a mechanism operates in termites.Many species of termites build large, structurally complex mounds, and the mechanisms behind this coordinated construction have been a longstanding topic of investigation. Recent work has suggested that humidity may play a key role in the mound expansion of savannah-dwelling  Mound-building termites of several different genera are known for their prowess in collective construction: colonies of millions of insects construct mounds that can be several meters tall, with elaborate outer features and complex networks of internal tunnels . These eThe classic explanation for how termites coordinate their building activity is based on a putative cement pheromone: a hypothetical chemical added to pellets of soil deposited by workers, which attracts other workers to the same site and triggers further deposition Grassé, . HoweverMacrotermes michaelseni mounds in Namibia  and determine with certainty that their actions are solely due to humidity levels. To explore whether the humidity template mechanism on its own can produce the kinds of building patterns seen with the insects, in this work we test an artificial agent-based construction system in which humidity alone is the defining template characteristic. Simulations can indicate the feasibility of the basic mechanism , but simWe constructed an autonomous mobile robot equipped with humidity sensors Carey, , and an Initially, a pair of parallel walls provide a short tunnel; tiles between the walls are saturated, while those beyond the tunnel's end are left dry. A humidity bubble, like that observed during active building on the termite mounds  The external air is still, as is typical for termites building on the surface of their mound at night, or in protected areas on calm days. (2) A fan blows air across the arena outside the tunnel, corresponding to wind, typical especially during the day. (3) The robot's water reservoir is left empty, corresponding to conditions experienced by termites in the dry season.We performed experiments under three conditions, analogous to those experienced by These methods are described in detail in the t-test demonstrates a significant difference in deposition trigger location between the still high-humidity experiments vs. the disrupted and dry trials  from the mound interior and freshly deposited material, or  from the wet tiles. The second is the evolving physical structure itself, which provides the substrate to which more material is attached; the locations of previous depositions shape the possibilities for further growth of the mound.Macrotermes michaelseni building activity at the mound surface, expanding the mound, varies seasonally and with time of day. Almost all expansion occurs during or soon after the rainy season, when soil brought up by workers from below the nest is much wetter; in the dry season, when deep soils have much lower water content, little or no mound expansion occurs  with wide-field touch or force sensing may convey a richer, more salient landscape profile for such a construction algorithm. Nevertheless, the robot used here produced the observed building patterns despite its sensory limitations and imprecise navigation. Such robustness to unreliability of individual agents and actions is necessary to collaborative construction algorithms, for engineered systems no less than for social insect colonies, whose successful operation likewise cannot depend on unerring precision by each insect.A template-based feedback process like the one considered here could potentially provide a novel mechanism to help coordinate environmentally-responsive building in collective robots. It requires no direct communication between agents, nor do agents respond directly to perceived configurations of material depositions, as they do in many insect-inspired models (Theraulaz and Bonabeau, The original contributions presented in the study are included in the article/NC, PB, JW, and RN: conceptualization. NC, PB, and JW: methodology. NC: robot hardware, software development, robot experiments, and visualization. JW: simulations and project administration. NC and JW: analysis and writing. RN and JW: supervision and funding. All authors contributed to the article and approved the submitted version.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.