Aiming in the development of printed electronic devices, a flexible polyimide substrate was used for the structures’ formation. A direct comparison between the f-rGO ink dispersion and a commercial graphene inkjet ink is also presented. Extensive droplet formation analysis was performed in order to evaluate the repeatable and reliable jetting from an inkjet printer under study. Electrical characterization was conducted and the electrical characteristics were assessed under different temperatures, showing that the water dispersion of the f-rGO is an excellent candidate for application in printed thermal sensors and microheaters. It was observed that the proposed f-rGO ink presents a tenfold increased temperature coefficient of resistance compared to the commercial graphene ink (G). A successful direct interconnection implementation of both materials with commercial Ag-nanoparticle ink lines was also demonstrated, thus allowing the efficient electrical interfacing of the printed structures. The investigated ink can be complementary utilized for developing fully printed devices with various characteristics, all on flexible substrates with cost-effective, few-step processes.The present work reports on the detailed electro-thermal evaluation of a highly water dispersible, functionalized reduced graphene oxide ( Most inkjet inks exhibit Newtonian behavior, with some examples of inks also showing a degree of viscoelasticity [−1 and μ = 1 mPa s), in this study, we demonstrate the printability of such dispersions with the appropriate nozzle settings.Inkjet-printing for flexible electronics poses several unique advantages over other printing processes in terms of developing direct patterning devices with good feature size and very low material waste . Being aasticity . Althoug−1 [There are various studies reported in the literature regarding inkjet-printed sensors on flexible substrate for detecting and measuring relative humidity ,12,13,14−1 ; it is a−1 , and dec−1 .Graphene is a promising candidate for printed electronics device formation. It is highly conductive, chemically stable and flexible, having a very high mechanical durability. Various methods have been developed to prepare graphene, which can be generally separated into mechanical exfoliation, solution-based and chemically assisted exfoliation, chemical synthesis, epitaxial growth through sublimated SiC surface and pyrolysis of hydrocarbons on metal surfaces ,28. GrapGraphite oxide, extensively exfoliated in water  is a non-conductive and oxidized derivative of graphite produced under strong acidic conditions . In ordef-rGO) via sulfonated aromatic diamine, which has been proven to restore an extensive sp2 carbon network, thus posing good electrical properties coupled with outstanding water solubility, allowing for use in printed electronics [f-rGO ink and a commercial graphene ink for compatibility with inkjet printing in order to be used for the implementation of various printed electronic devices. Jetting capabilities of both materials have been studied with an inkjet system, while electrical characterization of both materials (as printed and long term) has been performed. Moreover, electrical interfacing with a commonly utilized Ag-nanoparticle ink has been demonstrated, thus enabling the transition to multi-material device development. The materials have been thermally evaluated for applications in temperature sensing.This work utilizes a one-pot reduced and functionalized GO  and 2,4-diaminobenzenesulfonic acid (≥98%) were purchased from Merck, KGaA . Sulfuric acid (95–97%) and potassium chlorate (purum >99.0%) were purchased also from Merck, KGaA,  and nitric acid (65%) from Riedel-de Haen . All solvents were of analytical grade and were used as received. A commercial graphene ink (code 793663) with 2.4 wt% solids (graphene and ethyl cellulose) in cyclohexanone and terpineol and a silver nanoparticle ink (code798738), a 30 wt % dispersion in ethylene glycol, were used for inkjet printing. The silver ink was utilized for printing the testing interconnection pads.Graphite oxide was synthesized by the modified Staudenmaier method . A totalf-rGO was prepared according to [f-rGO was dispersed in deionized water in order to obtain a 3.5 wt% f-rGO ink.The rding to . In brieA Thetametrisis FR-DEPOSIT  drop-on-demand piezoelectric inkjet printer equipped with a Microdrop MD-K-140 nozzle  was treated with 1 M NaOH for 7 min for increasing wettability; afterward, the substrate was successively rinsed with acetone, deionized water, and isopropyl alcohol. The samples were loaded to the printer hotplate, which was kept at 34 °C at all printing sessions, in order to enhance the process repeatability and slightly assist with solvent evaporation. Moreover, this technique prevents the samples from absorbing environmental humidity during printing.To acquire the 3D topographies of the printed samples, a Filmetrics Profilm3D  white light interferometer-phase shift interferometer was utilized. The device incorporates a broadband white-light source and Mirau interference objective lenses. Information about the sample coloring was also acquired as a function of topographic height through Filmetrics TotalFocus™. A FLIR A655SC IR,  camera was used for monitoring the samples’ temperature during all temperature-related experiments.f-rGO inks exhibited a different spreading behavior as a direct implication of different solvent use; graphene ink contains cyclohexanone and terpineol while f-rGO ink is purely water dispersed. Therefore, the inter-droplet spacing was adjusted accordingly, after observing the printed structures via optical microscopy. The design consisted of 500 × 8 pixels parallel lines as well as a 500 × 500 pixel square.The test designs were loaded in the printer software, where the user can adjust the distance per pixel (droplet), which determines the droplet overlapping in the actual printed geometry. Given the fact that droplets have a diameter of approximately 80 μm mid-flight, a distance of droplet centers in the range of 60–70 μm in both axes yielded the optimal experimental results, depending on the ink. It should be noted that the G and f-rGO is a highly dispersible material in various solvents (water and organic) without the addition of surfactants or other stabilizers. This is due to the presence of sulfonated aromatic diamines, which are covalent bonded in the rGO sheets as derived from the characterization of similar samples with FTIR spectroscopy and x-ray photoelectron spectroscopy in our previous research work [f-rGO water dispersion remains stable for at least one month and is ready to be used after shaking of a few seconds. The commercial graphene ink is produced by the dispersion of graphene and ethyl cellulose powder in cyclohexanone and terpineol [The rch work . The samrch work ,48  are responsible for the completely different results when pulses of the same amplitude with opposite signs are driving the nozzle, as indicated in f-rGO inkjet-printed lines, respectively. The corresponding printed areas are shown in f-rGO photographs were taken with both the dark (4d) and bright filter (4e).Next to the droplet formation analysis, printing of the test pattern took place. Specifically, a line of 500 × 8 pixels and an area of 500 × 500 pixels with a droplet overlap of 65 μm in both axes were formed in single-pass printing, followed by a sintering at 240 °C for 1 h. It was observed that uniform, continuous lines were formed. Some areas (where the lines overlapped) exhibited edges that correspond to each printed line due to slow printing speed; moreover, the ink’s solvent that was deposited as first line needs some seconds to start drying, leading to printing of the successive line onto a semi-dry area, thus height is partially added. f-rGO line, respectively. The graphene line height was 149 nm ± 21 nm. Water-based f-rGO geometries exhibited a typical coffee ring pattern . This was attributed to faster evaporation on the line edges (assisted by the drying temperature), which induces a flow that moves the nanostructures from the droplet center toward the edges [f-rGO line height was 50 ± 31 nm.The 3D topography results highlight the impact of the solvent utilized and the effect of droplet overlapping: for graphene ink, whose solvent is cyclohexanone and terpineol, evaporation was relatively uniform across the sample line, therefore a coffee ring distinguishing pattern was absent in the line edges. he edges . On the he edges . The f-rf-rGO samples exhibited a mean resistance of 10.34 kΩ and 81.10 kΩ, respectively. The samples’ actual printed width and height differed, as explained in the previous paragraph, which had an impact on the extracted absolute electrical resistance value. Nevertheless, the sizes were comparable and these measurements should be taken into account to grasp the magnitude of each ink’s resistance; on the other hand, both inks showed good compatibility with the inkjet, thus enabling the development of multi-material graphene-based devices. As has already been presented in the literature, printed materials with different resistance values can substitute traditional resistors of various values [A series of several printed samples (designed as 5 mm × 0.5 mm) of each ink were characterized using a custom prober setup connected to a Keithley 2612 source-meter, in order to extract the corresponding electrical resistance. s values ; by usinf-rGO inks were ρG = 0.0247 Ω∙cm and ρf-rGO = 0.0649 Ω∙cm.To extract the mean resistivity for each ink, the topological data alongside the electrical resistance measurements were combined using Equation (1).f-rGO. The corresponding I–V curve  was printed onto Kapton and sintered at 140 °C for 2 h, forming 450 μm-wide lines  contact, the second ink was contained between the boundaries defined by the AgNP printed line, leading to a uniform line width throughout the sample, in contrast to the AgNP-G contact, where the G ink was spread over the AgNP line. It can be observed from the 3D interferometry images that AgNPs as a printing substrate can provide adequate wettability for overprinting. Nevertheless, as observed by the electrical measurements, in both cases, electrical interface was achieved.The 3D topographical measurements for the contacts between AgNPs and nterface . More spf-rGO with inkjet-printed AgNP lines, two needle probes were utilized to engage close to the junction (f-rGO)–AgNP interface had a mean resistance of 2.09 kΩ. Nevertheless, for both inks, it is evident that standard deviation is high, while both junctions exhibited an ohmic behavior. The specific experiment demonstrates the efficient coupling of both inks under study with Ag-based printed lines, thus rendering the electric interfacing of G and f-rGO printed devices feasible.To assess the electrical interface of printed G and junction ciii. Forjunction . Measuref-rGO; ongoing work includes a throughout investigation of contact resistance extraction alongside topology characterization of the junctions for a detailed characterization of the material interface. Nevertheless, AgNP-(f-rGO) lower contact resistance could be attributed to the absence of additional organic materials, which is not the case for graphene ink; ethyl cellulose could cause partial insulation between the sintered Ag nanoparticles and graphene sheets, therefore contributing in an increased contact resistance between the two printed structures.The extracted resistance values that are reported above are indicative, since in each case, the addition of an extra contact resistance value should be taken into consideration. It should be noted that these measurements were performed for a preliminary evaluation of the electrical interface of printed AgNP and G/f-rGO exhibited a negative temperature coefficient of resistance (TCR), therefore, the materials’ electrical conductivity rises with temperature. To measure the response of the printed samples to various heating conditions, an experimental setup was developed, which allows for selective heating of the substrate’s back plane, where the samples were printed onto  with the same dimensions (15 mm × 5 mm) were placed in the experimental setup of R2 = 0.9889 for f-rGO and R2 = 0.9855 for graphene).In the first experiment presented in The A, B, C parameters from fitting with Steinhart–Hart are presented in f-rGO in terms of response to temperature variations, exhibiting a negative TCR of −1.94 × 10−3 °C−1, with f-rGO presenting −1.64 × 10−2 °C−1. For more precise data approximation, f-rGO measurements can be fitted in two linear regions , as indicated in −2 °C−1 and −1.05 × 10−2 °C−1, respectively.Each samples’ TCR can be extracted from the corresponding results, as indicated in f-rGO was assessed. The two samples underwent several heating steps in successively higher temperatures up to 50 °C. After each heating step, the samples were allowed to reach the reference room temperature (27 °C). The results are presented in In the next stage, the transient thermal response of both graphene and The detailed graphene response and recovery behavior to three successive thermal pulses  is illustrated in f-rGO case is presented in f-rGO samples in each thermal pulse were calculated as 2.94 and 5.28 min, respectively.The corresponding transient thermal response for the In both samples and all the evaluated temperatures, the response phase was considerable faster than the recovery as expected, since the temperature increase was caused by the direct power application, while the recovery was dominated by natural convection. Comparing the two materials, graphene exhibited a slightly faster response and recovery time. This can be attributed to the differences in the combination of thermal conductivity and thermal capacitance of the two materials, but there are no solid scientific results yet to support the specific assumption. Future work will include both geometry optimization and detailed study of the response and recovery time in the full working range and the extraction of the main thermal parameters of each material.The vision of this work is the development of fully printed graphene-based electronics on flexible substrates, which requires the design and implementation of various interfaces between printed materials alongside electrical and topographical characterization, in order to describe in detail, the appropriate combinations. In addition, the group is actively working on the evaluation of the herein presented materials for printed micro-heater applications, keeping in mind that the exhibited TCRs alongside the measured electrical resistance strongly indicates that these materials are suitable candidates for such application.f-rGO water-based ink were evaluated for printability and compatibility with inkjet printing. Electrical, optical, and 3D topographical measurements were conducted for characterization of the printed structures; both inks successfully formed continuous, electrically conductive tracks on the Kapton substrate. Afterward, an initial approach on using these inks for the development of multi-material inkjet-printed electronics was presented, where electrical interface between both graphene-based materials with a commercial silver nanoparticle-based conductive ink was investigated. This type of ink is commonly used for the development of electrodes on various devices such as sensors and printed FETs; the results highlighted that such fully printed, multi-layer devices based on graphene can be developed, with electrical contacts between the printed materials adding a considerable ohmic resistance. Finally, an application for temperature sensing was designed and implemented with a custom setup for the measurement of electrical response in the range of 30–82 °C. Both materials exhibited a negative TCR, while the custom f-rGO ink outperformed graphene ink in terms of TCR value by approximately an order of magnitude. The two materials’ response and recovery time were found to be similar in the order of some minutes, with graphene exhibiting a mean faster recovery and response time.This work studied various aspects for the development of graphene-based printed electronics on flexible substrates. A commercial graphene and ethyl cellulose ink in cyclohexanone–terpineol and an


Building verticalvan der Waals heterojunctions between two-dimensionallayered materials has become a promising strategy for modulating theproperties of two-dimensional materials. Herein, we investigate theelectronic structures of non-twisted/twisted bilayer InSe/InSe andheterobilayer graphene/InSe (Gr/InSe) by employing density functionaltheory calculations. For twisted bilayer InSe/InSes, their interlayerdistances and band gaps are almost identical but a bit larger thanthose of the AB-stacking one due to the spontaneous polarization.Differently, the band gaps of twisted Gr/InSe are found to vary withthe rotation angles. Our results provide an effective way to tunethe electronic properties of two-dimensional materials. Remarkably, the physical andchemical properties of 2D materials are manifested to be stronglydependent on their thickness. For example, linear band dispersionis found for monolayer graphene6 whereas it is changedto be quadratic in the bilayer system,7 making the band gap of the latter tunable by applying an externalelectric field.8 Similarly, the interlayerhopping in bilayer phosphorus creates a significant difference inthe band gap with the monolayer.9 Correspondingto monolayer MoS2, a direct semiconductor, it is changedto be an indirect semiconductor for its bilayer isomer.10 In addition to band gap manipulation, it isrevealed that the magnetic properties can be tuned by interlayer coupling.For example, in-plane magnetic orders of mono- and few-layer CrS2 can be switched between striped antiferromagnetic and ferromagneticorders upon manipulating charge transfer between Cr t2g and eg orbitals.11 Interestingly,a tremendous amount of work has demonstrated that the electronic propertiesof bilayer 2D materials are largely dependent on their twist angles.14 For example, a flat band exhibiting insulating states at half-filling17 was revealed to appear near the Fermi level for the twisted bilayergraphene with “magic” angles. Moreover, the band gapsof twisted bilayer MoS2 can be tuned up to 5% when thetwist angle varies from 21.8 to 0°.18 As for bilayer BP, it is found that the anisotropy of its electronicstructure and optical transitions can be tuned by gating with an interlayertwist angle of 90°.19Todate, two-dimensional (2D) layered materials, such as graphene(Gr),2 (TM = Mo and W) heterobilayers,20h-BN,21 borophene,22 and so on, which makes it a potential candidatefor electronic devices. Theoretical and experimental explorationsdemonstrated that the heterostructures composed of MoX2 and WX2  have type-II band alignments.24 Theoretical studies indicated that twisted bilayers of Gr/MoS2 show significant differences in band structures from thenon-twisted ones with the appearance of the crossover between directand indirect band gap and gap variations.26 In addition, when coupling graphene with BP, on one hand, theirrespective properties are preserved in the composed heterostructure;on the other hand, the band structure of the two-sided monolayer materialcan be tuned by the application of an external electrical field perpendicularto the 2D plane.28Anothereffective way to expand the applications of 2D materialsis to construct van der Waals (vdW) heterostructures by coupling hetero2D materials. For example, the band gap of graphene can be openedby coupling it with other 2D monolayer systems like TMS31 InSe, displaysa hexagonal honey structure. Bulk InSe is a directband gap semiconductor with a gap of 1.25 eV,32 while the band gap of monolayer InSe is enlarged due to the quantumconfinement effect.33 The high electronmobility, quantum Hall effect, and anomalous optical response36 of monolayer InSe enable it to be a competitive choice in electronicapplications. Moreover, heterostructures combining InSe with other2D materials like graphene, MoS2, black phosphorus, h-BN, etc., have been successfully synthesized experimentally.42 However, efforts to explore the physical properties of layer-coupledInSe are limited. In this work, we investigate the structural andelectronic properties of InSe/InSe and graphene/InSe (Gr/InSe) vdWheterostructures with different rotation angles by first-principlescalculations. Our results show that, for twisted bilayer InSe/InSe,the interlayer distances and band gaps are almost insensitive to thetwist angles but a bit larger than those of the AB-stacking one. Meanwhile,for Gr/InSe, their electronic structures are found to vary with differentrotation angles.Analogue to graphene, thenewly emerging 2D indium selenium monolayer,2ab initio simulation package (VASP) under the framework of plane-wave pseudopotentialdensity functional theory (DFT) methods.44 The energy cutoff for the plane-wave expansion was set to 400 eV.The ion–electron interactions and exchange–correlationinteractions were described by the projector-augmented wave (PAW)method45 and the generalized gradient approximation(GGA) with the Perdew–Burke–Ernzerhof (PBE) functional,46 respectively. To consider the interlayer vander Waals (vdW) interactions, the DFT-D2 method47 was employed. All the atoms were allowed to be fully relaxeduntil the Hellmann–Feynman forces on each atom were smallerthan 0.01 eV/Å. The Brillouin zone integration was sampled bya k-point mesh of ∼0.06 Å–1 within the Monkhorst–Pack scheme.48 The vacuum region was set larger than 10 Å to eliminate artificialinteractions in adjacent unit cells. Spin polarization was taken intoaccount in all the calculations.All the calculations were performed in a Vienna 49 For a hexagonal lattice with lattice vectorsof a1 and a2, its correspondent deflective supercell has abasis vector of an1 + am2 and a rotation angle (θ), as defined in Figure S1 in the Supporting Informationr) for the twisted InSe/InSe andGr/InSe bilayer are definedas θr = θInSe-up –θInSe-down and θr = θGr – θInSe, respectively. We denotedthe twisted heterobilayer Gr/InSe with a notation of p:q, where p and q are the periodicities of graphene and InSe layers, respectively.The configurations of twistedbilayer InSe/InSe and heterobilayerGr/InSe were constructed according to accidental angular commensurations.50 In our calculations, the lattice constants ofGr/InSe supercells took the average values of the graphene and InSemonolayer, with the compressive and tensile strains on graphene orInSe are in the same magnitude and are smaller than 1%. We adoptedthree types of twisted Gr/InSe heterobilayers in this study, namely,6:√13, √19:√7, and √43:4.The lattice constants of graphene and InSe are 2.47 and 4.09 Å,respectively, which are consistent with a previous study.33.1r = 0, 21.8, 32.2, and 13.2°are considered  configuration without rotation betweentwo InSe layers, which is much stable than the AA-stacking one  by about15.36 meV lower in the binding energy (Eb), according to the following equation /Inatom (Se–In) or on the hollow site (Se–H) in the bottomlayer . Our results indicate that the energies and band structuresare almost insensitive to the configurations with a maximum energydifference of only 3 meV in the supercell, wherein the Se–Inconfiguration has a relatively lower energy; therefore, we use theSe–In configuration model to explore the properties of twistedbilayer InSe/InSes below.Four types of bilayerInSe/InSe with θered see 1. The syequation 11where Er = 0°), the twisted bilayer InSe/InSe is less energeticallyfavorable with the binding energies reduced by about 26–29%;as a result, the interlayer distances are increased by about 13–17%.Differently, no significant differences are found for the bindingenergies and the interlayer distances for the twisted InSe/InSes withvarious θr values. The charge density difference(CDD) of these bilayer InSe/InSes is calculated based on the followingequationFigure S4 in the SupportingInformation. Clearly, the charges for all the bilayer InSe/InSes areaccumulated between two InSe layers and depleted from the innermostSe atoms.The interlayer distances and the binding energies of the four studiedsystems are listed in 51 and almost insensitiveto the twist angles. Such an electronic feather makes the InSe/InSebilayer a stable candidate for data storing, optical catalysts, etc. 2 bilayer,52 a spontaneouspolarization between the two InSe monolayers is found for AB-stackingbilayer InSe/InSe  forAB-stacking and twisted InSe/InSes are shown in InSe see 2a, and t one see 1f–h. Acc one see 2b–d.3.2r = 42.5, 52.4, and 13.9° are considered. For the non-twistedstructure, the rhombus lattice parameters along the a/b vector direction are 12.30 Å, which is composedof the 5 × 5 graphene supercell and 3 × 3 InSe supercellwith lattice mismatches for the graphene and InSe layer of only 0.28and 0.29%, respectively . It is found thatalmost no differences are found for the structures and electronicproperties of these three configurations. First, the energy differencesof these three isomers are very small (<3.3 meV per unit cell)and the interlayer distances of them are around 3.39 Å. Comparedto freestanding graphene and InSe monolayers, the electronic structureof Gr/InSe can be regarded as a superposition of the energy statesfrom each constituent , and basedon this, we only used the Se–C configuration to explore thestructural and electronic properties of these Gr/InSe heterostructures.Similarly, to determine the influenceof the stacking order betweenthe graphene and InSe layer, three types of configurations are considered:(i) Gr/InSe with one Se atom sitting on top of one C atom in graphene(Se–C), (ii) Gr/InSe having one In atom locating atop of oneC atom in graphene (In–C), and (iii) Gr/InSe with one Se atomabove the hollow site of graphene (Se–H) (see Eb between differentconfigurations based on the following formulaEG/InSe, EG, EInSe, and NC are the energies ofGr/InSe, monolayer graphene, InSe, and the number of C atoms per unitcell, respectively. The calculated Eb fornon-twisted Gr/InSe is 36.5 meV per C atom (as listed in 54 indicating the weak van der Waals interaction between the grapheneand InSe monolayer.To evaluate the coupling interaction between graphene and InSe,we calculated the p:q values of 6:√13, √19:√7,and √43:4, respectively. The interlayer rotation angles, latticemismatches, binding energies, and distances between graphene and InSeare listed in Eb ranges from33.0 to 38.9 meV per C atom and d is in the rangeof 3.32–3.38 Å, which is in comparison with the interlayerdistances of other InSe van der Waals heterostructures56 and independent on the interlayerrotation angles.The top view of the three optimized twisted Gr/InSebilayers withtwist angles of 42.5, 52.4, and 13.9° are shown in 12 Comparisons of the CDDs atthe interface of non-twisted and twisted Gr/InSes are shown in –4 e per C atom for the non-twisted one, which is decreased to be 6.4× 10–4 e, 5.7 × 10–4 e, and 5.7 × 10–4 e per C atom for twistedGr/InSe with misorientation angles of 13.9, 42.5, and 52.4°,respectively. The other reason is the electric potential differencebetween layers when rotation occurs. This can be verified by the electrostaticpotential of the Gr/InSe heterostructure along the z direction (r = 13.9, 42.5, and 52.4°, respectively, indicating that twistingis an effective way to tune the electronic properties of Gr/InSe.One reason for the electronic property variation of the rotatedbilayer Gr/InSe is that the interlayer coupling varies with differentrotation angles as the states coupled in the two layers occur at differentmomentum.irection 5, and a 4In summary, we systematicallystudied the structures and electronicproperties of twisted bilayer InSe/InSe and heterobilayer Gr/InSeby using first-principles calculations. For bilayer InSe/InSe, theband structures of AB-stacking InSe/InSe are different from theirtwisted isomers due to the spontaneous polarization between two InSelayers, while for the twisted systems, almost identical band gapsare found. As for the twisted Gr/InSe heterostructure, its band gapsare found to vary with the interlayer misorientation angles. Our resultspropose an effective way to tune the electronic properties of InSe,supplying useful information for designing novel electronic or opticaldevices based on InSe van der Waals structures.


TDP-43 is an RNA-binding protein that has been robustly linked to the pathogenesis of a number of neurodegenerative disorders, including amyotrophic lateral sclerosis and frontotemporal dementia. While mutations in the TARDBP gene that codes for the protein have been identified as causing disease in a small subset of patients, TDP-43 proteinopathy is present in the majority of cases regardless of mutation status. This raises key questions regarding the mechanisms by which TDP-43 proteinopathy arises and spreads throughout the central nervous system. Numerous studies have explored the role of a variety of cellular functions on the disease process, and nucleocytoplasmic transport, protein homeostasis, RNA interactions and cellular stress have all risen to the forefront as possible contributors to the initiation of TDP-43 pathogenesis. There is also a small but growing body of evidence suggesting that aggregation-prone TDP-43 can recruit physiological TDP-43, and be transmitted intercellularly, providing a mechanism whereby small-scale proteinopathy spreads from cell to cell, reflecting the spread of clinical symptoms observed in patients. This review will discuss the potential role of the aforementioned cellular functions in TDP-43 pathogenesis, and explore how aberrant pathology may spread, and result in a feed-forward cascade effect, leading to robust TDP-43 proteinopathy and disease. RNA-binding proteins (RBPs) are heavily implicated in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD) and Alzheimer’s disease (AD). Mutations in RBPs such as TDP-43, FUS, MATR3, TIA1 and ATXN2 are linked to inherited forms of ALS, and aberrant aggregation of TDP-43 or FUS is observed in affected neurons in the majority of ALS and FTD cases . There aIdentified in 1995 as a repressor which binds to the trans-active response element DNA sequence of the HIV-1 viral genome, TDP-43 is a ubiquitously expressed DNA/RNA-binding protein that is predominantly localised to the nucleus but can shuttle between the nucleus and cytoplasm ,7. It biDisease-associated mutations in TDP-43 account for roughly 3% of familial ALS . HoweverTDP-43 contains both a nuclear localisation signal (NLS) and nuclear export signal (NES) which are recognised by importin and exportin proteins respectively, allowing TDP-43 to shuttle between the nucleus and cytoplasm. Nuclear import of TDP-43 is mediated by importin-α , whereasElevated cytotoxicity is observed in cells or animal models expressing cytoplasmic TDP-43 ,30, wherTDP-43 is an aggregation-prone protein under strict autoregulation where TDP-43 protein binds to the 3’UTR of its transcript to prevent it being translated, thereby maintaining a stable intracellular protein concentration ,32. DiseWhile TDP-43 protein production is regulated via an autoregulatory mechanism, other cellular machineries are involved in maintaining TDP-43 protein homeostasis. In particular, chaperone proteins including HSP70, DNAJB8 and DNAJB2a are known to interact with intracellular TDP-43 to facilitate its folding, refolding and the formation of reversible stress-induced granules ,42,43 F. ConsistThe final safeguard of protein homeostasis regulation is via protein degradation. It is known that TDP-43 can be degraded via three pathways: the ubiquitin proteasome system (UPS), the autophagy lysosome pathway, and chaperone mediated autophagy (CMA). The UPS has been shown to mediate the degradation of soluble full length TDP-43 as well as C-terminal fragments ,45,46,47To further support the importance of altered function of the protein degradation machineries in TDP-43 proteinopathy development, a number of TDP-43 proteinopathy causative mutations have been identified in genes linked to autophagy or the UPS, including p62, optineurin (another autophagy receptor) ,57, TANKCompared to other cell types, neurons are particularly susceptible to the build-up of aberrant protein aggregates due to their highly specified structures, often with axon and dendritic terminals some distance from the main cell body, particularly in the case of motor neurons. This increased susceptibility means these cells are likely to place an increased demand on chaperone proteins and protein degradation machineries. However, motor neurons have a higher threshold for chaperone activation , which, Although RBPs have been implicated in multiple neurodegenerative diseases, and the RNA splicing, regulation and trafficking mediated by these RBPs are demonstrated to play important roles in neuronal health, direct evidence linking TDP-43 RNA-binding to proteinopathy and disease development is scarce and ambiguous. It has been shown that in addition to protein chaperones, nucleic acids can also act as chaperones to prevent nucleic acid-binding proteins from aggregating and to enhance protein solubility . As a DNCurrently, there are only three ALS/FTD-linked mutations identified in or around TDP-43 RNA binding motifs (RRMs) that abolish the RNA interaction ,76. PatiFollowing the link between RBPs and neurodegenerative disease, an array of cellular studies have demonstrated that many RBPs including TDP-43 can be recruited to SGs in response to a variety of stressors . This leStress granules form when translation initiation is impaired, typically in response to a variety of stressors, such as heat, ER stress, proteasome inhibition, oxidative stressors such as arsenite, and osmotic stressors such as sorbitol (for review see ). A numbCell and stress type variability may be of key importance in unpicking the role of the cellular stress response and SGs in the formation of pathogenic TDP-43 aggregates as profound differences in SG dynamics are observed, dependent on the cell and stress type used. Specifically, cortical neurons appear to be somewhat resistant to SG formation compared to other non-neuronal cell types. However, once granules form, they persist for much longer . If SGs Recent studies however have thrown doubt on the role of SGs in TDP-43 pathogenesis. Stress-induced liquid-liquid phase separation (LLPS) of TDP-43 has been observed in both the nucleus and cytoplasm independently of classic SG makers . As discTo date, the bulk of this work has been conducted in isolated cellular systems, with evidence from in vivo studies being very limited. However, traumatic brain injury in drosophila has been shown to induce the formation of TDP-43 positive SGs, although TDP-43 recruitment to these granules appear to dissipate after 24 hours, suggesting a transient association . In contClearly there is a wealth of evidence demonstrating a possible role for cellular stress in TDP-43 pathogenesis. What is less clear at present, is whether this is linked to the formation of classic SGs, and potentially their persistence beyond the short term, or whether it is actually associated with the stress driven phase separation of non-RNA bound TDP-43, that explicitly does not get recruited to SGs. A recent study has demonstrated that impairment of SG assembly impairs the formation of TDP-43 foci in both acute and chronic stress paradigms, but crucially, did not completely prevent their formation, particularly in the chronic model, suggesting that while SGs may facilitate TDP-43 aggregation, TDP-43 proteinopathy can still develop through a SG-independent mechanism ,110.There is also still a dearth of evidence detailing the cellular stress response in vivo. The time course of granule formation and dissolution reported in mice in response to brain injury is markedly longer than that reported in drosophila, or indeed in cell models, suggesting that there may be distinct differences between the cellular stress response in mammals in vivo and that reported in cells, hence further research is still required.Once TDP-43 pathogenesis has been initiated, there are still a number of questions relating to how this pathology propagates and results in cellular dysfunction and death. Symptom onset in ALS patients tends to be asymmetrical and localised to a specific region of the body. As the disease progresses, symptoms appear to spread outwards from this initial site to ultimately affect the whole body . A similIn vitro studies have demonstrated that both wild-type and disease mutant TDP-43 and its C-terminal fragments can oligomerise ,119,120 Cellular studies have suggested that TDP-43, including oligomeric forms, can be transmitted between cells, providing a route for pathology spread ,132,133.It is important to note however, that not all cell studies support the notion of TDP-43 propagation. Two reports have shown that while cells may release TDP-43 into the media, there is no evidence of uptake of this protein into recipient cells treated with the conditioned media ,137. TheGlial cells provide important supportive functions to motor neurons and are increasingly being recognised as playing key roles in neurodegenerative diseases generally. Astrocytes have been shown to contribute to glutamate excitotoxicity, which has long been implicated in ALS, although more recent studies suggest a more general altered inflammatory response . OligodeIn vivo studies have demonstrated a protective role for microglia in TDP-43 proteinopathy in an inducible model of disease. Microglia were relatively unaffected by aberrant TDP-43 expression and were able to rapidly clear neuronal accumulations once the aberrant protein expression was halted . StudiesAn assessment of patient tissue has suggested that oligodendrocytes do not play a major role in the transmission of TDP-43 pathology. HyperphosphorylatedTDP-43 inclusions were only found in the white matter in subcortical regions directly associated with the motor and sensory cortices, with an absence of any overt pathology in axonal pathways . HoweverInvestigations into the role of non-neuronal cells in the pathogenesis and progression of TDP-43 linked disease are still comparatively in their infancy, but clearly they have the capacity to play a crucial role in the disease course, and further study will elucidate exactly how these cells interact with both TDP-43 and neurons to impact on disease pathogenesis.Since its identification 15 years ago, we have made significant progress in understanding the importance of TDP-43 proteinopathy in neurodegeneration, and the factors contributing to TDP-43 aggregation in cells. It is now clear that this is a tightly controlled system where the disruption of a small part could lead to TDP-43 dysregulation, resulting in a feed-forward cascade effect, further impairing the cellular machinery responsible for maintaining TDP-43 function and homeostasis. Accumulation of excess or aberrantly localised TDP-43 leads to protein aggregation which can spread from cell to cell, and also results in further mislocalisation of TDP-43 from the nucleus. This process is likely to involve not only neuronal cells, but at least in part be influenced by a concomitant dysfunction in supporting glial cells, particularly astrocytes and microglia. As TDP-43 proteinopathy is a major hallmark for age-dependent neurodegenerative diseases, it is not surprising that the activity of many important players involved in maintaining normal TDP-43 function are found to be reduced in aged neurons. Since multiple pathways are functionally interlinked and appear to contribute to TDP-43 proteinopathy, they may provide various potential therapeutic intervention angles to rectify TDP-43 aggregation and thereby halt or even rescue disease progression.


The proposed sensor recorded sensitivity and FoM higher 38% and 747% respectively than a similar structure composed of graphene sheets and porous silicon.In this study, the optical Tamm state is excited for the first time using gyroidal graphene/porous silicon one-dimensional photonic crystal terminated by a gyroidal graphene layer. The gyroidal graphene and porous silicon are used to enhance the figure of merit and sensitivity of the based Tamm resonance photonic crystal sensor. By tuning different parameters like the angle of incidence, the thickness of the sample layer, and the thickness of the gyroidal graphene layer, we have reached the optimized sensor. The observation of resonant dips in the reflectance spectra is strong evidence that Tamm plasmon-polaritons exist with higher sensitivity (188.8 THz/RIU) and figure of merit (355,384 RIU Contrary to the conventional surface plasmon resonance, TPP can be excited for both electromagnetic polarizations, at any incident angle, and without using grating or a coupling prism10. Besides the fact that the incident electric field is localized within the defected 1D-PC of the structure15, porous layers filled with analyte samples will be proposed in this study16. Recently, we proposed the excitation of TPP by porous silicon PC (PSi-PC) using graphene sheets17 or silver16. Porous silicon multilayers can be experimentally prepared by electrochemical etching with hydrogen fluoride as an electrolyte that is considered a simple one-step fabrication19.Tamm plasmon-polariton (TPP) is an optical resonance excited at the interface between metal and a periodic one-dimensional photonic crystal (1D-PC)20. In 2020, Sun et al. fabricated a gyroid structure of bioinspired Au–CuS to excite SPR21. In these calculations, we will use gyroidal graphene (GGr)/porous silicon array for the first time to excite TPP. The gyroidal structure possesses a surface with fixed mean curvature within a volumetric fill fractions range22. Gyroidal structures can be experimentally realized using inorganic templates from the butterfly nanostructure or self-assembly23, chemical vapor deposition24, self-assembly of a triblock copolymer25, selective laser melting26, solvent-free method27, light-based 3D printing process28, and controlled phase separation29. Graphene has very distinguished optical, electrical, and mechanical properties30. Chemical doping or the use of an external gate voltage can change the carrier concentration of graphene layers31. The negative conductivity of graphene at a certain frequencies makes it behave as a metalic layer36. So, the gyroidal graphene layer will be used to excite Tamm states.In 2017, Bikbaev et al. investigated a TPP excited by metallic gyroid layer1/PSi)N/cavity/GGr2/ substrate as clear in Fig. 38:The proposed structure will be GGr/PSiN/cavR, 40:F, e, ω, τ are the graphene sheets’ number, Fermi energy, the charge of the electron, angular frequency, and the relaxation time of the graphene sheet. Bruggeman’s effective medium equation is used to calculate the optical refractive index of the PSi layer (nPSi ) filled with sample (nsample ) with porosity (P)42:According to the approximate local random phase, the conductivity of monolayers of graphene (σ) in the THz frequency range is calculated as43. The transfer matrix method (TMM) is used to study the reflectance spectra of electromagnetic waves (TM mode) from the proposed structure as the following47:The refractive index of silicon . The unit cell size of gyroid (a) is 30 nm, The thicknesses of layers GGr2 possesses a photonic bandgap (PBG) with a bandwidth of 7.53 THz and 100% reflectance. By adding the GGr2 layer as a plasmonic layer, the TPP dip appears inside the frequency range of the PBG at 20.45 THz and 4.8% reflectance. Appearing of TPP dip is due to the trapping of light at the interface as a result of the PBG in the PSi-PC and strong attenuation of electromagnetic waves in graphene.As clear in Fig. 49:4 RIU−1. To enhance these values of parameters, the effect of the incident angle, the thickness of the sample layer, and the thickness of GGr1 on the performance will be numerically studied.As the position of the TPP dip is strongly dependent on the effective refractive index of the whole structure, the resonant dip can be tuned with any change in the refractive index of the analyte inside the pores of the layers. As clear in Fig. 17. The reflectance of experimentally PC was studied at the angle of incidence range from 0° to 90° in many works51. Within the proposed structure, the optical path length of the electromagnetic wave increases when the incidence angle increases. In addition, the confinement of the electromagnetic waves within the analyte increases16. Due to the previous results, the sensitivity increases from 13.19 THz/RIU to 136.71 THz/RIU with the increase of incident angle from 0° to 80° as clear in Fig. sample = 1.00 and nsample = 1.01). Even though the angle of 80° has a high average reflectance (89%), the angle of 80° will be the optimum angle to achieve the highest sensitivity.Changing the incident angle is an effective way to control and enhance the performance of the TPP sensors8 to 131,052 RIU−1 with increasing the thickness from 12,000 to 20,000 nm. Further increases in the sample thickness will have a slight effect on the FoM. For the average reflectance, it decreases from 89 to 50% with increasing the thickness of the sample layer from 12,000 nm to 20,000 nm. Then, average reflectance increases for further thickness increase, as clear in Fig. As clear in Fig. 1) is investigated, as clearly visible in Fig. −1, as clear in Fig. Herein, the effect of the gyroidal layer thickness (GGrsample from 1.01 to 1.10, the sensitivity and FoM decrease from 188.76 THz/RIU to 33.00 THz/RIU and from 635,984 to 7535 RIU−1, respectively. As clear in Fig. sample. Besides, with the increase of refractive index value, the reflectance of the TPP dips decrease. So, the proposed structure can be used in gas sensing and air pollution applications in refractive index range from 1.00 to 1.01.As clear in Table Finally, in Fig. 5. The effective index of refraction of the whole geometrical structure is the summation of the index of refraction of each layer multiplied by the volume fraction of this layer in the structure. So, we have tried to increase the volume fraction of the sample in the structure by using a wide sample layer. Besides, we used porous silicon and gyroidal graphene filled with sample analyte. This design helped make any tiny change in the sample refractive index strongly affect the value of the effective refractive index of the structure and increase the TPP dip shift.TPP is shifted to lower or higher frequencies with a tiny change in the effective index of refraction of the whole geometrical structure or the medium that surrounds the structureA novel structure of 1D-PC of porous silicon/gyroidal graphene terminated with a gyroidal graphene layer was proposed for sensing applications in the THz range. The effect of the incident angle and thickness of the gyroidal and sample layer was studied. The proposed sensor recorded sensitivity and FoM higher 38% and 747% respectively than a similar structure composed of graphene sheets and porous silicon. Another benefit of the suggested design is that it can be used at room temperature. Besides, our sensor has ultra-high performance comparing with other published papers. This study provides a new train of investigated methods for designing nanophotonic devices based on porous silicon and gyroidal graphene layers.


ZnO, which can exist in various dimensions such as bulk, thin films, nanorods, and quantum dots, has interesting physical properties depending on its dimensional structures. When a typical bulk wurtzite ZnO structure is thinned to an atomic level, it is converted into a hexagonal ZnO layer such as layered graphene. In this study, we report the atomic arrangement and structural merging behavior of graphene-like ZnO nanosheets transferred onto a monolayer graphene using aberration-corrected TEM. In the region to which an electron beam is continuously irradiated, it is confirmed that there is a directional tendency, which is that small-patched ZnO flakes are not only merging but also forming atomic migration of Zn and O atoms. This study suggests atomic alignments and rearrangements of the graphene-like ZnO, which are not considered in the wurtzite ZnO structure. In addition, this study also presents a new perspective on the atomic behavior when a bulk crystal structure, which is not an original layered structure, is converted into an atomic-thick layered two-dimensional structure. Bulk zinc oxide (ZnO) has a range of crystalline structures, namely wurtzite and zinc-blende structures . The wurWith the decreasing thickness of metal oxide semiconductor materials, unique electrical, mechanical, chemical, and optical properties are introduced, e.g., ZnO monolayer have increased band gap of ~4.0 eV comparing to bulk ZnO, which has a band gap of 3.37 eV, due to strong quantum confinement effects and graphene-like structure , while sWe report the atomic arrangements at the edge of the g-ZnO nanosheets using aberration-corrected transmission electron microscopy (ACTEM). We explain the atomic reconstructions when the sheets merge at the edge of the g-ZnO nano-flakes. To observe that in detail, we use the g-ZnO nanosheets synthesized using an adaptive ionic layer epitaxy (AILE) method ,25 and aCVD-synthesized monolayer graphene film on 35 μm thick copper foil was purchased from Graphene Square . Firstly, the graphene on copper foil was coated with ~200 nm poly methyl methacrylate (PMMA). Secondly, to release the graphene from the copper foil, the graphene on copper foil was floated onto copper etchant , then PMMA-coated graphene was transferred onto distilled water more than 3 times to remove the Cu etchant. The PMMA-coated graphene was then scooped up with TEM grids and annealed at 120 °C for 5 min. to firmly attach the PMMA-coated graphene onto the TEM grids. Lastly, the PMMA was dissolved with acetone for 1 day and the graphene on TEM grids were immersed into isopropyl alcohol several times to remove the acetone residue on the surface of the graphene. Synthesized ZnO nanosheets using the ILE ,25 metho−nm−2. The atomic images were taken using a white atom contrast to obtain the actual atom positions under the properly focused conditions needed for direct image interpretation. STEM HAADF images and EELS spectra were recorded with a monochromatic beam at 80 kV with a probe size of 1.5 nm and an energy resolution of 0.8 eV, as measured from the full-width-at-half-maximum of the zero-loss peak.Specimens were analyzed using an aberration-corrected FEI Titan Cubed TEM (FEI Titan3 G2 60–300), which was operated at an 80 kV acceleration voltage with a monochromator. The microscope provided a sub-Angstrom resolution at 80 kV and −13 ± 0.5 μm of spherical aberration (Cs). Typical electron beam densities were adjusted to ~6 × 105 eWe used synthesized g-ZnO nanosheets by an adaptive ionic layer epitaxy (AILE) method ,25. FirsFor understanding the atomic movements of the g-ZnO nanosheets, we used the monolayer graphene sheets as a substrate layer because the ZnO sheets were not fully formed as lateral planar sheets. Since the lattice parameter of the ZnO and diameter of Zn atom is larger than their graphene, atomic dynamics of the ZnO sheets can be observed on the graphene sheet. An atomic image shows the g-ZnO sheets on the monolayer graphene sheets  which were analogous to the graphene lattices. In addition, we observed the atomic migration by adatoms at the edge of the sheet. In terms of merging of the g-ZnO sheets, when the nano-flakes merge, they have the preferred directions depending on the growth direction. Once adatoms bond along the parallel direction the edge, the flakes expand toward the normal direction. Moreover, while the merging occurs, intermediate arrangements  form before the perfect merging into the hexagonal lattices. This work explains the atomic dynamics of the promising g-ZnO nanosheet, which suggests fundamental investigations of the 2D metal oxide materials.


TDP-43) gene have been reported in amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and other neuromuscular diseases. However, whether this modest elevation triggers neurodegeneration is not known. Although high levels of TDP-43 overexpression have been modeled in mice and shown to cause early death, models with low-level overexpression that mimic the human condition have not been established. In this study, transgenic mice overexpressing wild type TDP-43 at less than 60% above the endogenous CNS levels were constructed, and their phenotypes analyzed by a variety of techniques, including biochemical, molecular, histological, behavioral techniques and electromyography. The TDP-43 transgene was expressed in neurons, astrocytes, and oligodendrocytes in the cortex and predominantly in astrocytes and oligodendrocytes in the spinal cord. The mice developed a reproducible progressive weakness ending in paralysis in mid-life. Detailed analysis showed ~30% loss of large pyramidal neurons in the layer V motor cortex; in the spinal cord, severe demyelination was accompanied by oligodendrocyte injury, protein aggregation, astrogliosis and microgliosis, and elevation of neuroinflammation. Surprisingly, there was no loss of lower motor neurons in the lumbar spinal cord despite the complete paralysis of the hindlimbs. However, denervation was detected at the neuromuscular junction. These results demonstrate that low-level TDP-43 overexpression can cause diverse aspects of ALS, including late-onset and progressive motor dysfunction, neuroinflammation, and neurodegeneration. Our findings suggest that persistent modest elevations in TDP-43 expression can lead to ALS and other neurological disorders involving TDP-43 proteinopathy. Because of the predictable and progressive clinical paralytic phenotype, this transgenic mouse model will be useful in preclinical trial of therapeutics targeting neurological disorders associated with elevated levels of TDP-43.Modestly increased expression of transactive response DNA binding protein ( ALS is a neurodegenerative disease that causes relentless, progressive loss of upper and lower motor neurons leading to paralysis and death. Approximately 90 percent of ALS are sporadic, whereas ~10 percent are familial. The mechanism of motor neuron degeneration in ALS is not yet fully understood. Mutations in many genes can cause or enhance the risk of ALS. These genes function in multiple cellular mechanisms, including protein homeostasis, RNA processing, inflammation regulation, cytoskeleton dynamics, and intracellular trafficking, thus suggesting that abnormalities in diverse pathways may lead to motor neuron neurodegeneration in ALS .TDP-43 is an RNA binding protein whose intracellular inclusions are associated with >97% of ALS cases . PresentMutations in the TDP-43 gene cause familial ALS –16. ThesWhat drives wild type TDP-43 to proteinopathy is not understood. Several studies have suggested that modestly elevated levels of TDP-43 expression are associated with TDP-43 proteinopathy in ALS and other diseases such as FTD and inclusion body myopathy, Paget’s disease of the bone and frontotemporal dementia (IBMPFD) –30. ALS-To answer this question, a mammalian model with modestly elevated levels of TDP-43 should be informative, as such a model will closely mimic the modestly elevated levels in human ALS and create an opportunity to observe chronic TDP-43 toxicity. Previous mammalian models with TDP-43 overexpression have shown a wide range of phenotypes, including both motor and non-motor symptoms . In geneIn this study, we have constructed two TDP-43 transgenic lines with modest levels of TDP-43 overexpression. Both lines developed motor weakness after ~500 days. The weakness eventually progressed to paralysis in a few months. Conversion of one line into homozygotes accelerated the disease. The homozygous mice developed paralysis during a period of 300 to 430 days. Detailed analysis showed ~30% motor neuron loss in layer V cortex, oligodendrocyte degeneration, demyelination, and neuroinflammation, but no motor neuron loss, in the spinal cord. However, denervation at the neuromuscular junction was detected, indicating the presence of distal motor axon degeneration. Thus, this mouse model has replicated several key features of ALS, including progressive motor dysfunction, paralysis, loss of upper motor neurons, oligodendrocyte degeneration, loss of myelin and neuroinflammation. These results demonstrate that a modest elevation of TDP-43 can trigger late-onset neurodegeneration and motor dysfunction and thus may play a causative role in human ALS and other neuromuscular conditions involving TDP-43 proteinopathy.IRES) was generated by standard PCR using the full-length mouse TDP-43 cDNA and EGFP as templates. The resulting cDNA (mTDP43-IRES-GFP) was cloned into the XhoI site of the MoPrp.Xho plasmid (ATCC#JHU-2) [TGCTGCTGCCCGACAACCA and reverse ATAACCCCTCCCCCAGCCTAGA. The positive founders were bred with wild type FVB/NJ mice, and the offspring were sacrificed and characterized for their expression of GFP in the CNS. Their brain and spinal cord were examined under a fluorescence microscope. The lines were terminated if 3 to 5 animals from the line showed no detectable GFP fluorescence. The lines surviving this screen were further analyzed for their GFP expression levels by immunoblot. Two lines, 19 and 42, were selected for a further screen for motor phenotypes. Both lines developed incompletely penetrant, late-onset motor dysfunction and paralysis. Line 19 was successfully converted to a homozygous line and further analyzed in detail. This line has been deposited at The Jackson Laboratory and will be available as Stock No. 031609.A cDNA encoding mouse wild-type TDP-43 and EGFP linked by the internal ribosome entry site  facility. Each cage housed 1–5 animals Mice were observed daily on weekdays for their general health and motor behavior, and their body weights monitored biweekly. The disease stages were assigned as follows: pre-symptomatic (pre-sym), slightly weak (swk), weak (wk), and paralysis (par). At the pre-sym stage , the motor behavior was indistinguishable from the nTg mice. At the swk stage (at ~10 months), the mice showed slight foot-dragging in their gait. At the wk stage (~11–14 months), the foot-dragging became readily observable, and the movement became noticeably slowed. At the paralysis stage, two or more limbs became paralyzed, and the mouse was incapable of locomotion. The paralysis stage was the endpoint of the experiment, and the mouse was sacrificed for tissue harvesting.To monitor the mouse behavior in their home cage continuously, we used the HomeCageScan system as previously described , 45. BriTransgenic animals and age-matched controls were tested for time on accelerating rotarod from 12 to 72 RPM over three trials with a maximum time of 300s per trial at different age points. The longest time of three trials on the rotarod was recorded in seconds once the mice fell from the bar.Transgenic animals and age-matched controls were tested for loss of four-limb grip strength using a grip strength meter at different time points. Mice were allowed to grip on a horizontal metal wire grid with four limbs. They were gently pulled back by their tails with steady force until they release their grip from the grid. Peak tension was recorded from five consecutive trials.Mice under deep anesthesia were decapitated. The spinal cord, brain, and other tissues were quickly harvested, snap-frozen in liquid nitrogen, and stored at -80°C. For protein preparation, frozen tissues were homogenized in a homogenization solution containing 25 mM phosphate pH 7.2, 1 mM EGTA, 1% SDS, 0.5% Triton X-100 and protease inhibitor mixture (Thermo Scientific) and heated at 95°C for 5 min. After clearing by centrifugation, protein concentration was measured using BCA assay . The samples were heated in Laemmli buffer, and equal amounts of protein were loaded and resolved by SDS-PAGE. After transfer to nitrocellulose membranes, blots were blocked with 5% nonfat dry milk in PBST  for 1 h, and then incubated with primary antibodies overnight at 4°C and then again with horseradish peroxidase–linked secondary antibodies  in PBST with 5% dry milk for 1 hour at RT. The dilutions and source of primary antibodies were as follows: GFP , rabbit polyclonal antibody raised to amino acids 394–414 of human TDP-43 , α-Tubulin , CNPase , MBP, MCT1, NFkB-p65 , and phosphorylated NFkB-p65 . Membranes were washed three times, and proteins were visualized after ECL (Pierce) treatment and detected by the LAS-3000 imaging system (Fujifilm).3VO4, 5 mM EDTA) with protease inhibitor  and phosphatase inhibitor cocktails (Thermo Fisher). The homogenates (100 μL/sample) were centrifuged at 12,000g at 4˚C for 5 min. The supernatants were moved to new tubes and measured for protein concentration as described above. The pellets were rinsed 3 times with the lysis buffer and resuspended in 20 μL 1X Laemmli buffer. Ten micrograms of protein from the supernatant were mixed with 2X Laemmli buffer. The supernatant sample and an equivalent volume of pellet sample were heated at 95°C for 5 min, cleared by centrifugation, and then resolved by SDS-PAGE. The gel was then immunoblotted, as described above.Mouse lumbar spinal cords were homogenized using a handheld polytron for 20 sec in lysis buffer  following the manufacturer’s protocol. RNA was then reverse transcribed to cDNA using qScript cDNA SuperMix (Quanta BioSciences). For testing candidate splicing targets, RT-PCR amplification using between 33 and 37 cycles were performed from at least three nTg mice and three Tg mice. Products were separated on 2% agarose gels and visualized by staining with ethidium bromide and photographed. For qRT-PCR measurements of candidate gene targets, real-time PCR was performed on the cDNA using the primers for the targets. The PCR cycles were carried out in a Bio-Rad Real-Time PCR system , and the PCR product was detected using Sybr Green. The levels of target genes were standardized to the housekeeping gene GAPDH in individual animals and then further normalized to the mean ΔCT of the wild type mice.Mice under deep anesthesia were transcardially perfused with cold PBS, followed by 4% paraformaldehyde in PBS. The perfused mice were then immersed in the same fixative at 4°C for another 24-48h. After fixation, tissues were immersed in PBS containing 30% sucrose at 4°C for 2–3 days. Tissues were then frozen in OCT freezing media  and stored at -20°C. Frozen sections were cut at 20 μm using a cryostat. For immunostaining, sections were incubated in the blocking solution  for 1 hour at room temperature (RT) and then incubated with a primary antibody in the blocking solution overnight at 4°C. The dilutions and source of primary antibodies were as follows: NeuN , calbindin , GFAP , IBA1 , APC , GFP , ChAT , rabbit polyclonal antibody raised to amino acids 394–414 of human TDP-43 (custom made), TDP-43 , NF-L , CNPase , MBP, NFkB-p65 , activated caspase3 . Sections were then washed 3 times for 5 minutes each and incubated in the appropriate secondary antibody at room temperature for 90 minutes. For immunofluorescence, the sections were washed 3 times in PBS for 5 minutes each and mounted with Vectashield mounting medium containing 4,6-diamidino-2-phenylindole  and sealed with nail polish. Images of the brain and spinal cord sections were taken with a confocal microscope (Leica).For quantification of TDP-43 signal intensity in the nucleus and cytoplasm, sections were double-stained for TDP-43 and cellular markers. After staining, the cells were visualized and photographed using confocal microscopy. The cells in the ventral horn of the spinal cord were measured for their fluorescence intensity using the Nikon NIS Elements software. For each cell, the average fluorescence intensity was calculated. Cells on at least 5 different sections from each of the three or more mice per genotype were measured.For immunohistochemistry, sections were washed 3 times in PBS containing 0.25% Tween 20 and then stained following the manufacturer’s instructions for Vectastain ABC kit, Elite PK-6100 standard ImmPact tm DAB peroxidase Substrate kit SK-4105 (Vector Lab). The sections were then mounted on slides and dried overnight at 55°C. After soaking in Xylenen 2 times for 2 minutes each, the slides were sealed with Permount (Vector Lab).To detection of demyelination, mice were fixed by transcardial perfusion using 4% paraformaldehyde and 2.5% glutaraldehyde in 0.1M sodium phosphate (pH 7.6). Tissues were further fixed by soaking in the same fixative at 4°C for 24 hours. Luxol fast blue staining was performed on 10-μm spinal cord or brain paraffin-embedded sections for demyelination.Visualization and quantification of the cortical neurons were carried out as described previously {Aliaga, 2013 #1266}. Briefly, whole mouse brains were placed in 30% sucrose solution for 2 days, frozen, and sectioned sagittally at 50 μm thickness. Layer V pyramidal neurons were counted using every ninth section, with a total of nine sections per half brain. Sections were mounted onto gelatin coated slides and stained with CTIP2 or Cresyl violet. Stereological counting was performed using Stereo Investigator software . Counting was performed within the motor area of cortical layer V. Only pyramidal neurons with a soma greater than 15 μm in diameter were included. A single experimenter who was blinded to the genotype performed all counts.For visualization of ventral root axons, mice were fixed by transcardial perfusion using 4% paraformaldehyde and 2.5% glutaraldehyde in 0.1M cacodylate (pH 7.6). Tissues were further fixed by soaking in the same fixative at 4°C for 24 hours. L4 and L5 roots attached to dorsal root ganglia were dissected and postfixed with 2% osmium tetroxide in 0.1 M phosphate (pH 7.6), dehydrated in a graded ethanol series, and embedded in Epon-Araldite resin. One-micron sections were stained with toluidine blue and examined and photographed by light microscopy.For quantification of ventral horn motor neurons, lumbar enlargement of the fixed spinal cords was sectioned on a cryostat at 20μm thickness. Every other section was collected until a total of ten sections were collected from each spinal cord. The sections were stained with goat ChAT antibody at 4°C overnight. A secondary donkey anti-goat biotinylated antibody and a Vectastain ABC and DAB peroxidase Substrate kit (Vector Lab) were used to reveal motor neurons. Images of the spinal cord sections were taken using a Nikon microscope, and motor neuron numbers in the ventral horn region were counted manually from each section.For muscle histology, isopentane in a container was pre-chilled with liquid nitrogen until the isopentane started to solidify at the bottom of the container. A fresh specimen isolated from gastrocnemius muscle was placed on a cork disc with a drop of OCT, which kept the muscle in the desired orientation. The specimen was frozen by immersion into the isopentane for about 5 seconds and then stored at -20°C. The frozen tissue was sectioned using a cryostat and stained with Hematoxylin & Eosin (H&E).Mice were anesthetized by inhalation of isoflurane. Animals were placed immediately on a heating pad to maintain their core temperature at 37°C. Measurements were performed using a Cardinal Synergy electromyography (EMG) machine. A ground self-adhesive gelled surface electrode was placed over the tail. Potentials were recorded from several sites of the muscles of all four limbs with a concentric needle electrode (30G) using a gain of 50 μV/division and a bandpass filter with low and high cut-off frequency settings of 20 and 10,000 Hz, respectively. The entire recording process took 15–20 minutes per mouse, after which the mice were euthanized by isoflurane overdose or used for tissue collection.Animals are euthanized via an overdose of isoflurane, then transcardially perfused with PBS for 2 minutes, followed by 4% paraformaldehyde for 5 minutes. Gastrocnemius muscles were dissected out and placed in 1.5% paraformaldehyde for 24 hours at 4°C. The muscles were then washed with PBS for 30min at 4°C and placed in 25% sucrose overnight at 4°C. Muscles were embedded in OCT medium, frozen rapidly, and stored at -80°C. Sections were cut at 35nm thickness using a Leica Cryostat, placed on Superfrost Plus slides, and stored at -80°C. Slides were allowed to defrost for 30 minutes before use. Slides were washed 3 times with PBS for 5 minutes and then 3 times with 4% Triton X-100 for 5 minutes. Sections were blocked using 10% donkey serum in PBS for 3.5 hours at room temperature. A primary antibody solution of Rabbit anti-synaptophysin  and rabbit anti-Neuronal class III Beta-Tubulin  diluted in blocking solution was applied for 24 hours at 4°C. Slides were washed again with PBS. A secondary antibody solution of Alexa-488nm-labelled Donkey anti-Rabbit  and Alexa-555nm α-Bungarotoxin  diluted in PBS was applied overnight at 4°C in the dark. Slides were imaged on a Nikon microscope. Neuromuscular junctions were then counted and quantified as innervated or denervated Based on nerve occupancy of the endplates. Those with>50% occupancy was counted as innervated and those with <50% were counted as denervated.To determine the effects of an elevated level of TDP-43, we constructed a transgene in which the mouse prion promoter (Prp) . A smallTo facilitate the analysis of these mice, we sought to elevate the gene dosage by making homozygous lines. We were unable to generate homozygous mice from line 42 but succeeded from line 19. As anticipated, the homozygous mice developed motor symptoms earlier than the original hemizygous line and became paralyzed before 400 days . This phAs expected, the increased gene dosage in the homozygous mice increased the expression of the transgene compared with the hemizygous mice, as shown by the Western blots for TDP-43 . The hemTDP-43 is a gene-splicing modulator. To determine whether the modest elevation of TDP-43 level in the Tg mice impacted TDP-43 function, we measured the splicing patterns of some TDP-43-regulated mRNAs , 48. ForTo determine where the transgene is expressed in the CNS, we stained brain and spinal cord sections for GFP. We observed a broad expression pattern in all regions of the CNS . The eleThe increased TDP-43 expression may perturb the nuclear and cytoplasmic distribution of TDP-43. Because such perturbation may impact neurodegeneration , we quanTo determine the pathological basis of the clinical phenotype, we examined the spinal cord throughout the various disease progression stages in the Tg mice. The most conspicuous pathological feature was white matter degeneration. Luxol fast blue staining revealed normal myelination at age five months, but the staining became slightly pale at eight months. At ten months, the staining became paler . At the Myelin loss could be associated with injury in oligodendrocytes as a result of TDP-43 overexpression. Therefore, we doubly stained the spinal cord sections for oligodendrocyte marker APC and cell death marker activated caspase 3. We found an increasing association of activated caspase 3 and oligodendrocytes during the progression of the clinical phenotypes , and a rThe progressive clinical motor phenotype suggests the presence of motor neuron injury. By Nissl staining and choline acetyltransferase (ChAT) staining, we did not observe gross morphological deviations from the controls Figs  and 7A. Despite the preservation of motor neuron cell bodies and their proximal axons, the distal axons and neuromuscular junction could be affected. To test this possibility, we examined muscle morphology and physiology. We did not observe muscle fiber atrophy . By needDemyelination and gliosis are commonly associated with neuroinflammation . Therefophox, p22phox, and p67phox, were increased by ~600, ~80, and ~9 fold, respectively , which is involved in inflammation and oxidative stress, was dramatically increased. The three mRNAs of its subunits, gp91ectively . Two genectively . GLT-1, ectively . LCN2 anectively . Althougectively , the chaBecause the TDP-43 transgenic mice expressed an elevated level of TDP-43 in the forebrain Figs  and 3, iWe also surveyed gliosis in various regions of the brain in the paralyzed mice. We found elevated levels of astrogliosis and microgliosis in all regions, including the cerebral cortex, hippocampus, corpus callosum, internal capsule, cerebellum, and brainstem , suggestBecause TDP-43 was expressed in both neurons and glia in the cortex , we invein vivo and demonstrates that such low-level elevation of TDP-43, as observed in ALS and other neurodegenerative conditions, can trigger neurodegenerative clinical phenotypes.This study has established a line of TDP-43 transgenic mice that express exogenous TDP-43 protein at modestly elevated levels. Respectively for hemizygous and homozygous mice, the levels are ~10% and ~30% above the nTg level in the spinal cord and ~20 and ~40% increase frontal cortex . These lHow low-level overexpression of TDP-43 causes the late-onset neurotoxicity remains unknown. Pathological studies have consistently detected various forms of TDP-43 aggregates in ALS and FTD CNS tissues , suggestPrevious studies suggest that TDP-43 aggregation and nuclear depletion may lead to gain of toxicity as well as loss of function that ultimately cause cellular degeneration, including both neurons and oligodendrocytes , 57–61. An intriguing observation in this study is that different cell types may be responding to TDP-43 overexpression differently. In oligodendrocytes, TDP-43 overexpression elicited similar percentage of increases in nucleus and cytoplasm , therebyNon-cell autonomous mutant toxicity is a well-established phenomenon in motor neuron degeneration provoked by mutant SOD1 . ExpressHowever, whether this is the case for TDP-43 is less clear. Astrocyte-specific expression of mutant TDP-43 in rats induces motor neuron degeneration as the mutant expression in motor neurons , 74. Howin vivo study of chronic TDP-43 toxicity derived from the modest elevation of TDP-43 levels and in vivo preclinical tests of experimental drugs targeting chronic TDP-43 toxicity in the CNS.A unique feature of our TDP-43 mice, compared with other established TDP-43 mouse lines, is the development of a fully penetrant progressive disease course . The pheWe established a transgenic mouse model with a modestly elevated TDP-43 level. This model displays several ALS characteristics, including late-onset and progressive paralytic motor dysfunction ending in paralysis, neuroinflammation, and neurodegeneration. This study demonstrates that modest elevations in TDP-43 expression can trigger neurodegeneration and clinical phenotypes of ALS, suggesting that modestly elevated TDP-43 levels in humans could cause ALS and other neuromuscular disorders involving TDP-43 proteinopathy. Because of the easily observable, predictable, and progressive clinical paralytic phenotypes, this transgenic mouse model may be useful in preclinical trials of therapeutics targeting neurological disorders associated with elevated levels of TDP-43.S1 FigIRES) were inserted into the backbone of MoPrp.Xho plasmid to generate the Prp-mouse TDP-43 transgene. The construct was composed of the following elements in linear succession: the Prp promoter, mouse wild-type TDP-43, IRES, EGFP gene, and poly A signal. This construct (Prp-TDP-43) expresses TDP-43 and GFP separately. (B) Western blot showed that the transgene was expressed in all the CNS regions in transgenic lines 19 and 42. FC, frontal cortex; CSC, cervical spinal cord; LSC, lumbar spinal cord; BS, brainstem; CB, cerebellum. (C) A survey of the transgene expression in different organs in transgenic line 19 showed that the transgenes were predominately expressed in CNS. Low levels of expression were also detected in heart (Hrt), lung (Lg) and kidney (Kdn). Other tissues are muscle (Msl), liver (Lv), and spleen (Spl). (D) A survey of the transgene expression in different organs in transgenic line 42. Similar to line 19, the transgenes were predominately expressed in the CNS. The samples in B, C, and D were prepared from animals between 55 and 65 days old. (E) A mouse at the paralysis stage. Its limbs were paralyzed, and the mouse lost its local motion capability. (F) Monitoring small cohorts of mice from lines 19 and 42 showed late-onset paralysis but incomplete penetrance from both lines up to 750 days. (G) Progressive weight loss in the aged mice of the two transgenic lines. The animal numbers are 3 to 4 for line 42, 3 to 5 for line 19, and 3 to 15 for non-transgenic (nTg) control mice at different age points. For the TDP-43 mice, only animals that develop paralysis were included in the weight plots. Error bars are standard errors.(A) A cDNA encoding mouse TDP-43 and EGFP linked by internal ribosome entry site ((JPG)Click here for additional data file.S2 Fig(A) Western blot of detergent-soluble and insoluble TDP-43 and ubiquitinated proteins extracted from lumbar spinal cords of paralyzed Tg mice and age-matched nTg controls. Each lane was loaded with proteins from one animal. Arrows point to TDP-43 and its 35 KD and 25 KD fragments. Numbers on the right indicate molecular weights in kD. (B) Relative ratios of band intensity of the pellet over the supernatant. Bars represent averages of 7 nTg and 8 Tg animals in the TDP-43 quantification and of 4 animals in both nTg and Tg groups in the ubiquitin quantification. Student t test was used to compare between Tg and nTg mice. * indicates p<0.05 and *** p<0.001.(JPG)Click here for additional data file.S3 Fig cortical spinal track,  lateral funiculus,  ventral funiculus,  anterior commissure. (B) Electron microscopic images of ventral funiculus from a nTg mouse (left) and a Tg mouse (middle and right). Notice very few axons were wrapped by myelin in the Tg mice. (C) Immunohistochemistry staining for MBP in spinal cord ventral horn , ventral funiculus , and anterior commissure .(TIF)Click here for additional data file.S4 Fig(A) cyclooxygenase 1 (COX-1 or PTGS1). (B) lipocalin type of prostaglandin synthase (LPGDS). (C) prostaglandin D2 receptor (DP1). (D) prostaglandin D2 receptor 2 (DP2). (E) inducible nitric oxide synthase (iNOS). (G) neuronal nitric oxide synthase (nNOS). Student t tests with Bonferroni correction were used to compare between the Tg and nTg mice at different ages. n = 3–8 at each age for both groups. No significance was found (p > 0.05).(JPG)Click here for additional data file.S5 Fig(JPG)Click here for additional data file.S6 Fig(A) CTIP2 staining of the motor cortex in nTg and paralyzed Tg mice. The boxed areas in layer V are enlarged in the two panels on the right. Notice a substantial reduction in the CTIP2 staining intensity in the Tg mice. (B) quantification of CTIP2-positive cells in layer V. (C) Protein blot of CTIP2 in the motor cortex from nTg and Tg mice at different ages. P-value was derived from Student’s t test. n = 4 for nTg and 3 for Tg mice.(JPG)Click here for additional data file.S1 Table(JPG)Click here for additional data file.S1 VideoThe transgenic mouse was marked with red ink on its tail. The other mouse in this video was a control nTg mouse. Notice the Tg mouse goes through stages from hyperactive to final paralysis.(MP4)Click here for additional data file.S1 Raw images(PDF)Click here for additional data file.


The use of gamification elements has extended from being a complement for a product to being integrated into multiple public services to motivate the user. The first drawback for service designers is choosing which gamification elements are appropriate for the intended audience, in addition to the possible incompatibilities between gamification elements. This work proposes a clustering technique that enables mapping different user profiles in relation to their preferred gamification elements. Additionally, by mapping the best cluster for each gamification element, it is possible to determine the preferred game genre. The article answered the following research questions: What is the relationship between the genre of the game and the element of gamification? Different user groups (profiles) for each gamification element? Results indicate that there are cases where the users are divided between those who agree or disagree. However, other elements present a great heterogeneity in the number of groups and the levels of agreement. The use of gamification elements has gradually extended from being a mere complement to a given product or brand, to becoming an incentive in public services to motivate users to interact more frequently and generate more engagement.Gamification as a strategy has been proven partially successful in domains such as the business context, where different gamification features can be integrated into products, websites, or services . GamificAccording to ,6, to unMechanics–Dynamics–Aesthetics (MDA) by [(MDA) by  is a modSix D’s (Six Steps to Gamification) by [tion) by  is a modGAME by [GAME by  is a fraOctalysis by [lysis by  is a fraGamification Model Canvas by [anvas by : this moIn addition, there are different game/gamification frameworks that may help designers select the appropriate game elements, such as:The inclusion of gamification elements is gaining importance because technologies such as Augmented Reality (AR) technology, for example, have become an essential element in today’s digital life, changing the way users interact with different services. AR enables the superposition of virtual 3D objects in a real 3D environment thanks to devices equipped with a camera and a screen through which the citizen lives the immersive experience offered by the AR. AR aims to enrich the real environment with virtual information, creating the opportunity to offer real-time and collaborative input and it is increasingly common in public services and gamification elements can become an excellent addition to AR for tourism applications that require the user’s on-site visit, such as tours, shopping, and mapping different places of the city .What is the relationship between the genre of the game and the element of gamification?The decisions that designers take upon using some of these frameworks are normally based on the description and suppositions about the public of the service and their own expectations. Therefore, the first drawback encountered by the game designer is to match their expectations with the gamification elements which are appropriate for the target audience. In addition, the designer may be unaware of the existing interdependencies and synergies among different gamification elements. Through a questionnaire designed to determine the profile of the users of interest, we collected data to answer the following research questions:We wanted to examine whether users who prefer a particular type of game genre respond more positively to certain types of gamification elements. For example, we could expect that someone who typically plays adventure games will positively respond to missions and story elements.Different user groups (profiles) for each gamification element?We wanted to determine if for each type of gamification element, a similar number of user profiles are generated, or if there is a greater heterogeneity of profiles for some gamification elements than others. Additionally, what type of elements have a greater number of profiles? Are the profiles balanced, or is there a group that has a notable majority?Authors will first review existing research on gamification elements. Parts of this data have been used in previous publications , with mohttps://www.projectco3.eu/wp-content/gamificationformv4/ (accessed on 10 December 2021) that contained questions about the location, age, preferred games and the gamification questions to capture the participant’s profile (citizens). A screenshot of the questionnaire interface is shown in Data collection was carried out through an online form at After carrying out the data collection, we noticed that some users had not answered some important questions that were crucial for the intended, and thus were excluded from the sample. obtaining N = 138 complete answers to the more than 46 questions. Of these 46 questions, 39 are of the Likert scale type; that is, the answer ranges from a value of 1 to 5, with 1 being less in agreement with the question and 5 more in agreement.To carry out the analysis, we must associate the gamification elements with one key question, which is highly related to the concept. This step is critical since there is a human factor, and it is based on the interpretation from each of the question and the definition of each gamification element.Then, after determining the key question, we proceed with applying a correlation algorithm to find out other questions related to responses. Next, a correlation matrix is made, and those questions that get a value bigger than 0.5 (medium correlation) are selected.Once we have the list of key questions–elements, we can determine if people respond similarly to each gamification element. For this reason, we apply an unsupervised data analysis technique of the clustering type known as K-Means .The K-Means technique is a grouping technique in a certain number of groups (K). The elements in each group share similar characteristics (they respond similarly to the questions) and are, at the same time, separated from other groups that do not share characteristics. The K-Means algorithm determines whether they are similar or different by using a distance measure (Euclidean distance in our case). The algorithm has an iterative operation repeated as many times as there are elements to be classified/moved from the group. The technique needs to know in advance the number of groups that they want to generate (K).The first time, the K central points (centroids), indicated at the For each dot  the Euclidian distance is calculated to each centroid of each cluster, determined in step 1, then the dot cluster (color) is set in concordance to the nearest centroid .Recalculate the centroids based on the dots  that are clustered to each centroid. The calculation is performed by taking the average position between all the members of the current centroid-clusterRepeat from step 2.The K-Means algorithm repeats the following steps until there is no changes in terms of the dot cluster (color). It is important to determine well in advance the number of groups/clusters (K) for each gamification element to obtain the maximum detail about our data. To determine K, we used the technique known as “Elbow” . The “ElTo visualize the clustering results, we use a graphical tool such as the Boxplots . BoxplotThe simplicity of the box plot provides a quick way to visually compare the results between clusters (groups). Taking the Picking the best cluster for each gamification element and the statistics of the game genre played by the user, we can determine the preferred gamification element for each player type. To start the analysis, we have to pick up the best cluster. The best cluster is determined individually for each gamification element. The cluster with the highest average (mean) value is the selected one. Those scores are the responses rates facilitated by the users from the form questions. The best cluster is composed in theory, with the users that most agrees with the questions related to the current gamification element.Next, for each of those best clusters, we collected the statistics about the game genre that the players inside each cluster play. We were able to construct the relation between gamification element and game genre in the case of our population (dataset).We have divided this section in partial results, following the style of the previous sections with different subsections. Each of the steps have the explanation of the result found and how it has been used inside the study. The results are incremental, which means that the new partial result takes the results of the previous steps as required information.Starting with selecting the “key question”, as described in step 2, we have built the table  that relTo be able to understand the text inside column two and three, you must check the This section may be divided by subheadings. It should provide a concise and precise description of the experimental results, their interpretation, as well as the experimental conclusions that can be drawn.Continuing with the results on the clusters determined for each gamification element as indicated in point 3 of the methodology, we have found multiple amounts of groupings (clusters) of different sizes. Those differences in terms of sizes and number of clusters cannot be compared between gamification elements, because of the use of different questions for each gamification element. Next, we must analyze individually by observing the differences inside each category.Q6_enjoy_woking_with_others: Collaborating with two or more players to achieve a common goal ?Q22_helping_other: Helping other playersQ28_part_casual_guild: Being part of a friendly, casual guildQ25_player_vs._player: Competing with other playersQ23_getting_know_other: Getting to know other playersIn the case of teamwork, the results are based on the five related questions:Teamwork generated two clusters, as indicated in Q24_chatting: Chatting with other playersQ23_getting_know_other: Getting to know other playersQ32_meaningful_conversations: How often do you find yourself having conversations with other players?Q28_part_casual_guild: Being part of a friendly, casual guildQ22_helping_other: Helping other playersQ33_talk_personal_issues: How often do you talk to your online friends about your personal issues?Q29_part_serious_guild: Being part of a serious, raid/loot-oriented guildQ25_player_vs._player: Competing with other playersQ39_try_to_provoke_others: How often do you purposefully try to provoke or irritate other players?The social element generated more correlation between the questions, which could be because of the thin line that separates the collaboration (teamwork) with the socialization. There are a total of nine related questions:Q18_game_mechanics_rules: Knowing as much about the game mechanics and rules as possibleQ1_precise_game_mechanics: How interested are you in the precise numbers and percentages underlying the game mechanics?In the case of rules, there are only two related questions, which could be because of the design of the form questions that we modified from the original  documentIn this case see  we have Q16_becoming_powerful: Becoming powerfulQ17_accumulate_resources: Accumulating resources and itemsQ14_leveling_character_fast: Leveling up your character as fast as possibleQ19_self_sufficient_character: Having a self-sufficient characterProgression has four related questions, and is related to increment your impact inside the game by having more power and/or items to participate into the different missions. The related questions are:As seen in Q17_accumulate_resources: Accumulating resources and itemsQ16_becoming_powerful: Becoming powerfulQ15_acquiring_rare_items: Acquiring rare items that most players will never haveThere are no direct questions related to points elements in our form, instead there are three indirect related questions:The users’ answers to the points questions, generated a heterogeneity of opinions as show in Q21_escaping_real_worldQ20_immersed_fantasy_worldFor the case of narrative, there are two questions that are closely related to the term of narrative because it includes immersing the player:The narrative also generated a heterogeneity of opinions among the individuals that replied to our form. A total of 10 clusters have been generated, as indicated in Q6_enjoy_woking_with_others: Collaborating with two or more players to achieve a common goal ?Q22_helping_other: Helping other playersQ28_part_casual_guild: Being part of a friendly, casual guildQ25_player_vs._player: Competing with other playersQ23_getting_know_other: Getting to know other playersIn the case of missions, the related questions are:In the case of the missions, the questions are more related to teamwork rather than missions, but there is a small component to take into consideration, which is a common goal. Here the results are less relevant, because the average score is 3 as indicated in Q14_leveling_character_fast: Leveling up your character as fast as possibleQ16_becoming_powerful: Becoming powerfulLevel has two related questions. The first one, the key element, has a clear relation to leveling elements. The second element is a side-effect of leveling the character in the games:Q13_collecting_distinctive_objects: How much do you enjoy collecting distinctive objects or clothing that have no functional value in the game?Q15_acquiring_rare_items: Acquiring rare items that most players will never haveIn the case of goals, the results based on the three related questions:This time, eight clusters have been generated according to Q13_collecting_distinctive_objects: How much do you enjoy collecting distinctive objects or clothing that have no functional value in the game?Q15_acquiring_rare_items: Acquiring rare items that most players will never haveQ17_accumulate_resources: Accumulating resources and itemsIn the case of badges, the results are based on the three related questions:As depicted in Analyzing the data in a summarized way in the boxplots in Q25_player_vs._player: Competing with other playersQ26_dominate_kill_others: Dominating/killing other playersQ23_getting_know_other: Getting to know other playersQ29_part_serious_guild: Being part of a serious, raid/loot-oriented guildQ24_chatting: Chatting with other playersQ6_enjoy_woking_with_others: Collaborating with two or more players to achieve a common goal ?Q31_annoying_others: Doing things that annoy other playersIn the case of competition there are seven selected questions, variables, detailed as follows:We can observe in Q8_time_customizing_character: How much time do you spend customizing your character during character creation?Q3_use_character_builder: How often do you use a character builder? Q9_character_outfit_matches_style: How important is it to you that your character’s outfit matches in color and style?For the customizations there are only three related questions, as indicated in the following:Customization is composed by only two clusters as indicated in In this subsection we have condensed the information related to the preferred game genre for each specific cluster of each gamification element. Before conducting the study, we have surveyed the literature for tools to help us build the form with the different questions. After a wide search, we have found the study provided by . We haveAfter this, finding related questions, where users respond similarly, is relatively easy and straightforward through simple correlation. This process has produced cases with two cases and other cases with multiple related questions. We expected that the greater the number of related questions, the fewer groupings we would obtain as more information was available to the user. According to the results, there is no relationship between the number of questions available for each element and the number of users grouped.We can see how on some gamification elements, there is a generation of two or three groups where the users are polarized between a positive score, reflecting that they agree, and a negative or neutral one where disagreement or neutrality is shown. We have these cases in 6 out of 12, exactly in half of, the cases. However, we get more than 4 groups in the remaining elements, showing a diversity of opinions. Therefore, we could conclude that in those 6 cases, teamwork, social, rules, missions, competition, and personalization are elements that can be clearly described with the questions used in our study. We could also conclude that the users on whom the questionnaire has been made are easily polarized based on these elements. As far as those elements are concerned, it can keep a part of the users and make the other portion decide to stop using the gamified system with those elements.Analyzing the results of Teamwork: *action > shooter = strategy > adventureSocial: *strategy > *shooter > actionRules: *adventure > *strategy > actionMissions: *action > strategy = shooter > adventureCompetition: *strategy >action = massive multiplayer = shooterCustomization: *action > adventure = strategy > shooterIn the case of Social, we can see a general picture in the With the accumulated knowledge about the genres and the gamification elements, we can proceed straightforwardly with just one question about the game that the users play to decide which gamification element to get positive feedback on the gamification system. Another detail that we have noticed in the study is that we observe no clear relationship between a gamification element related to a game genre. In other words, there is not an element that is valued equally by the different people who play each of the genres, neither in the affirmative case (larger circle) nor in the negative case . However, we observe that for some game genres such as action, strategy, shooter, adventure, and MMO, all the gamification elements are represented to a greater extent than other genres, so it is easier to implement gamification in users that play any of these genres.We have been able to capture the different profiles of the users who have responded to the form. These profiles have been represented in different clusters of different sizes and numbers for each gamification element. In some cases, we obtain a cluster of uses who clearly agree with the questions representing a specific gamification element. In other cases, we obtain responses from profiles that do not agree with the use of mission elements or a slight indifference . On the other hand, we have been able to identify the preferred types of games according to the gamification element. In the near future, it would be necessary to know how recurrent these players are in the genres that indicate how much they play.