The thermal conductivity of unwrinkled regions was measured to be, κ ∼ 165 W m−1 K−1. Measurements at the wrinkle sites revealed local enhancement of thermal conductivity, with κ ∼ 225 W m−1 K−1. Furthermore, the total interface conductance of wrinkled regions decreased by more than an order of magnitude compared to that of the unwrinkled regions. The physical origin of these observations is discussed based on wrinkle mediated decoupling of the stacked crystallites and partial suspension of the film. Wrinkles are ubiquitous in layered 2D materials, and our work demonstrates their strong influence on thermal transport.Wrinkles significantly influence the physical properties of layered 2D materials, including graphene. In this work, we examined thermal transport across wrinkles in vertical assemblies of few-layer graphene crystallites using the Raman optothermal technique supported by finite-element analysis simulations. A high density of randomly oriented uniaxial wrinkles were frequently observed in the few-layer graphene stacks which were grown by chemical vapor deposition and transferred on Si/SiO Thermal transport is strongly influenced by wrinkles in films of few-layer graphene crystallites with rotational stacking faults, as revealed from Raman optothermal measurements. The ultrahigh thermal conductivity of graphene stems from its unique phonon structure, where low energy acoustic phonon modes play a dominant role.12,13 It has been experimentally observed that the presence of vacancies, isotopic impurities, stacking faults, and different edges reduces the thermal conductivity of graphene. In one study, κ of graphene was probed under low energy electron beam irradiation. It was found that κ decreases from 1800 W m−1 K−1 to 400 W m−1 K−1 with an increase in defect density Nd by one order of magnitude.14 The presence of isotope impurity 13C was found to adversely affect the thermal conductivity of graphene. The value of κ decreased from ∼2800 W m−1 K−1 for Nisot = 0.01% to κ ∼ 1600 W m−1 K−1 for Nisot = 50%.15 Li et al. observed a decrease in thermal conductivity of twisted bilayer graphene (T-BLG) by a factor of ∼1.4 from the value of its parent AB-stacked or Bernal BLG.16 Apart from defects, deviation from flat morphology associated with the local curvature in graphene also influences the thermal transport. Ripples and wrinkles in graphene are ubiquitous and are formed due to the residual stress caused by various factors such as thermal expansion mismatch with the substrate, presence of impurities, release or application of external stress, edge stresses, surface tension of transferring liquid and random thermal fluctuations. Individual wrinkles are typically uniaxial, whereas the distribution of a set of wrinkles can be either quasi-periodic or random in orientation, depending on the nature of competing effects responsible for their generation.17 In some cases, wrinkles can span over the entire graphene sample. These are greatly stressed along the wrinkling direction and are compressed along the texture direction.18 Recently, heat conduction across graphene wrinkles was studied theoretically.18–20 The important observations included a decrease in thermal conductivity upon an increase of the degree of wrinkling and anisotropic heat flow between texture and wrinkling directions. In these calculations, the decrease in κ was attributed to phonon mode localization, phonon mode softening and increased phonon scattering rate corresponding to a reduced phonon mean free path. Similarly, a more relaxed structure was observed to possess higher thermal conductivity. Experimentally, Chen et al. studied the thermal conductivity of wrinkled suspended single-layer graphene. Based on statistics of data from 12 samples probed with Raman mapping, the authors inferred a 27% average decrease in thermal conductivity compared to the unwrinkled case.21 Enhancement of chemical activity is also observed in corrugated graphene.22 While supported graphene can be engineered with different wrinkle geometries, the subject of phonon transport through them has not received adequate attention. Wrinkles may be found in supported graphene layers either when graphene is a sandwich thermal management layer or an active over-layer. The study of the effects of local curvature on heat flow in graphene and related systems, therefore, becomes quite important.Deviations from the ideal chemical structure or morphology can have a significant bearing on heat transport. In the case of three-dimensional nanostructured systems, the porosity and architecture significantly influence the thermal properties and thermal transport.2 substrate and upon examination were found to possess a random network of uniaxial wrinkles with significantly variable density. Thermal transport measurements were performed using the Raman optothermal technique, both on unwrinkled and wrinkled locations. Local enhancement of thermal conductivity accompanied by a decrease in interfacial thermal conductance is observed at the wrinkle sites. The physical origin of these observations is discussed.In this paper, we discuss thermal transport across wrinkles in vertical assemblies of few-layer graphene crystallites probed using the Raman optothermal technique, complemented with finite element analysis (FEA). Graphene samples prepared using the chemical vapor deposition (CVD) technique were found to consist of vertical assemblies of few-layer graphene crystallites with rotational stacking faults. The graphene layers were transferred onto a Si/SiO210H16O) was utilized as a green solid precursor. Detachment of the methyl groups attached to hexagonal and pentagonal carbon rings of camphor is followed by fusion of these rings to form the graphene layers on the Ni substrate. 6 mg of the solid precursor was flash heated at a temperature of 180 °C upstream of the main heating zone. At this temperature, the solid precursor sublimated in approximately 3 min and was carried into the main furnace zone with the help of Ar carrier gas, which was maintained at 100 sccm throughout the experiment. Growth was allowed to happen for 2 min followed by rapid quench cooling of the substrate using a magnetic rod arrangement. Similar growth conditions have been reported in the literature involving upstream sublimation of camphor that is carried to the main furnace zone by Ar carrier gas, which is followed by pyrolysis in the main furnace at 800 °C for 3 min and quench cooling of the Ni substrate.23 Note that the somewhat longer total camphor exposure times in our work resulted in higher graphene film thickness. After the growth, graphene was dry-transferred onto the Si/SiO2 substrate using thermal release tape for further studies. The topography of the obtained graphene samples was studied using a Park systems NX-10 atomic force microscopy setup in non-contact mode. Raman spectroscopy of the samples along with power-dependent Raman studies were carried out using a WITec alpha300 R Raman spectrometer at a laser wavelength of 532 nm (2.33 eV) along with 1800 grooves per mm grating. Variable temperature Raman scattering measurements were carried out using a Linkam THMS-600 micro-thermoelectric cell and Horiba HR-800-UV Raman spectrometer with an Argon ion laser of 488 nm (2.54 eV) excitation wavelength. The incident laser power is maintained below 250 μW to avoid temperature rise of the sample due to intrinsic contribution. The thermal transport measurements and analysis procedures were validated using mechanically exfoliated graphene control samples having comparable thickness.Few-layer graphene samples were grown on a nickel substrate using the chemical vapor deposition (CVD) technique. The main furnace zone was kept at 850 °C and the substrate was initially annealed for 15 min at an inert Ar gas flow rate of 100 sccm. In place of conventional gaseous precursors like methane and hydrogen, camphor  and 2D2 (higher wavevector) deconvoluted peaks are of comparable intensity in few-layer graphene, while the 2D2 intensity becomes twice as strong as 2D1 in multi-layer graphene or graphite.25 The 2D peak for mechanically exfoliated multi-layer graphene was deconvoluted and was consistent with the fitting of 2 Lorentzian peaks, 2D1 and 2D2 as shown in 1 is ∼2681.96 cm−1 with the corresponding FWHM ∼51.11 cm−1 and the position of 2D2 is ∼2720.36 cm−1 with the corresponding FWHM ∼33.83 cm−1. The 2D-peak of mechanically exfoliated graphene shows a clear signature of multi-layer graphene or graphite, consistent with the literature.25,26 The 2D peak for wrinkled regions of CVD graphene was deconvoluted and it was consistent with the fitting of 2 Lorentzian peaks, 2D1 and 2D2 as shown in 1 is ∼2694.09 cm−1 with the corresponding FWHM ∼64.88 cm−1 and the position of 2D2 is ∼2726.58 cm−1 with the corresponding FWHM ∼41.72 cm−1. Raman signatures from CVD grown graphene can be ascribed to those of few-layer graphene, despite the overall film thickness, and these signatures point to the existence of stacking faults between the vertically assembled few-layer crystallites. The 2D peak profile of our wrinkled sample is consistent with that of 5-layered graphene, as studied rigorously in the literature.16,25–28 More details are provided in the ESI  is calculated to be ζ > 10.17,29 The wrinkle line profile is well described with a Gaussian curve with wavelength λ ∼98.44 nm and amplitude A ∼ 8.66 nm. Hence the wrinkling level19γ = [A/λ] × 100% is calculated to be 8.79%. While the wrinkle morphology bears a strong resemblance to similar morphologies noted in single-layer graphene,24 it is intriguing that these samples should show any wrinkling at all for this thickness which is 30-fold higher, given that the bending rigidity of a thin film increases as the cube of the film thickness.30 For comparison, mechanically exfoliated graphene samples with even lesser thickness and which lack stacking disorder and are cleaved on identical Si/SiO2 substrates show a complete lack of wrinkling as observed from the AFM topography image and height profile image shown in et al., who have demonstrated that pyrolysis of camphor results in the formation of rotational stacking faults in few-layer CVD graphene on a Ni substrate.23 In that work, the authors had synthesized multi-layer graphene films using growth conditions quite comparable to the one used by us, as discussed in the Experimental Section above. Their work emphasized the formation of rotational stacking faults between graphene layers as evidenced from relative rotations by variable angles of the set of hexagonal diffraction spots observed in high resolution transmission electron microscopy (HRTEM), considered together with the results from Raman spectroscopy.23 See ESI Fig. S5We first examine the morphology and thickness of these samples using AFM topography imaging and micro-Raman spectroscopy. Throughout our studies, mechanically exfoliated samples with thickness comparable to the CVD grown samples are used as control samples. The AFM topography image of mechanically exfoliated multi-layer graphene is shown in 1,2,8 Towards this objective, the G peak shift was measured as a function of the incident laser power for all the sample types using two different objectives, 100× (with NA – 0.9 and spot radius r0 ∼ 0.360 μm) and 50× (with NA – 0.55 and r0 ∼ 0.590 μm). Details of the calibration procedures are presented in the ESI (see ESI Section VI).ID/IG < 0.09 for the lowest and highest laser power used; such small intensities for the defect peak are also reported for pristine graphene on Si/SiO2.31The Raman optothermal technique has been one of the most effective methods to investigate thermal transport in graphene and related systems.versus incident laser power at both objectives for mechanically exfoliated multi-layer graphene. The G peak shows a red shift with increase in incident laser power. This can be attributed to intrinsic contributions such as thermal expansion, anharmonicity and extrinsic contribution from thermal expansion coefficient mismatch induced strain.32,33 The slope of the G peak shift with incident laser power for 50× objective was found to be χ50× = −0.2547 ± 0.0046 cm−1 mW−1, and for 100× objective χ100× = −0.3547 ± 0.0039 cm−1 mW−1. The power dependent G-peak shift measurements for unwrinkled locations of CVD grown samples yield a slope of χ50× = −0.5076 ± 0.0132 cm−1 mW−1 and χ100× = −0.8642 ± 0.0265 cm−1 mW−1 for 50× and 100× objectives respectively  = −0.0217 ± 0.0038 cm−1 K−1 (exfoliated graphene), −0.0301 ± 0.0039 cm−1 K−1 (unwrinkled site of CVD graphene) −0.0167 ± 0.0021 cm−1 K−1 (wrinkled site of CVD graphene). Since the temperature rises for two different incident laser powers are known, the estimates of thermal conductivity can be made from the solutions of the heat diffusion equation, as described below.ectively . For wriT(r), in the examined film upon laser irradiation can be obtained by solving the following set of heat diffusion equations as extensively discussed in the literature:2,34The steady-state temperature distribution, κ is the thermal conductivity of graphene, and h is the corresponding film thickness. g is the total interface conductance and Ts is the temperature of the substrate at the interface. z = 0 is taken as the interface between the film and the substrate. The last term in the heat diffusion equation represents the source term and r0 is the incident laser spot radius which depends upon the numerical aperture (NA) of the objective. The incident laser power distribution is taken to be Gaussian, which is represented by the source term in the equation. The average temperature rise of the film upon irradiation is calculated from T(r), following the equation:With the following boundary conditions:−1 with increasing incident laser power in our experiments. Hence the temperature rise of the Si/SiO2 interface is considered to be negligible, even as the temperature rise in SiO2 was considered. As the experiments were carried out under ambient conditions and the thermal conductivity of air is very low ∼0.025 W m−1 K−1, losses due to convection are negligible and radiation contribution is also negligible.34,35 The calculated temperature distribution of the graphene film is a function of material and optical parameters. Equation parameters such as NA, r0, Q and z can be obtained easily as they are system/optical parameters whereas κ and g are material parameters and these need to be extracted by solving for two different temperature rises associated with the respective incident laser energy densities. In our case, we obtained two different temperature rises by utilizing objective lenses with different magnification power.2,34We did not observe any shift in the fundamental Raman peak of silicon at 520.7 cmQ = nαP represents the total power absorbed by the film where α is the absorption coefficient of single-layer graphene and n is the layer number. Here P is chosen as 1 mW for our calculations and total absorption is calculated based on the consideration that each layer behaves as an independent atomic sheet. Hence the total absorption is the superposition of absorption by individual layers with αSLG ∼ 2.3%.36κ and g are considered in our model for characterizing the heat transport in the wrinkled region, to allow for extraction of these two unknown parameters. It may be noted that this scenario is not unique to our heat transport experiment, and these considerations equally apply, for example, to thermal transport studies in suspended single-layer graphene where quasi-periodic wrinkle arrays are frequently observed due to residual strain. We performed finite element analysis (FEA) to evaluate the thermal conductivity and total interface conductance of the films. Details of the modelling are discussed in the ESI Fig. S6.†g which corresponds to improved interface, the value of ΔT decreases, thereby showing the self-consistency of the calculations. The ratio of temperature rise T50×/T100× for mechanically exfoliated graphene is shown in κ and g uniquely, the intersection of experimentally inferred contours is taken in the κ–g plane. One of these contours is the temperature rise obtained with 100× objective, while the other is the ratio (T50×/T100×) contour, as shown in κ = 562.07 ± 77.54 W m−1 K−1 and the obtained interface conductance is g = 9.14 ± 1.74 MW m−2 K−1. These values are in very good agreement with the literature for mechanically exfoliated multi-layer graphene.3,8We next discuss the estimation of the thermal conductivity, with the help of FEA simulations. Individual models are made for each sample, factoring the layer number in each case. We first validate the procedure by estimating the thermal conductivity and interface thermal conductance of mechanically exfoliated multi-layer graphene. The computed temperature rise for the exfoliated sample for 100× objective is shown in T50×/T100×) respectively for unwrinkled sites of CVD graphene. The thermal conductivity of unwrinkled regions was obtained as ≈165 W m−1 K−1 with an interface conductance of ≈26 MW m−2 K−1. Unlike the case of mechanically exfoliated graphene, the FEA simulations only yielded approximate values since the curve intersections in the κ–g plane were only obtained within error bars of the experimentally measured temperature rises, rather than for the experimental average value of temperature rises. We concur that a slight laser power dependence of the κ and/or g in the unwrinkled sites of CVD graphene may be responsible for the same. κ and g. The corresponding simulated ratio of temperature rise (T50×/T100×) is plotted in T50×/T100×) are obtained from κ = 223.4 ± 20.0 W m−1 K−1 and g = 1.83 ± 1.60 MW m−2 K−1. To summarize the observations, the thermal conductivity measured for unwrinkled sites of CVD graphene was found to be 70% lower than the estimated value for mechanically exfoliated multi-layer graphene. However, the wrinkle sites of CVD graphene showed a locally enhanced thermal conductivity, which was higher by ≈35% when compared to regions devoid of wrinkles. At the same time, the wrinkle sites were associated with very low interface thermal conductance when compared to the other samples.We now consider the case of CVD graphene samples for unwrinkled and wrinkled sites. κ for CVD graphene as compared to that for exfoliated graphene can arise from defects in the former. CVD graphene is typically associated with atomic-scale defects and grain boundaries introduced during the growth process and these defects serve as phonon scatterers. Usually, a large decrease in the thermal conductivity of graphitic systems is associated with a decrease in grain size.37 In all our samples, however, the defect peak is small, ID/IG < 0.09. Attributing the defects to be edge-type, the grain-size, La, is obtained using the expression, La = 2.4 × 10−10λ (ID/IG)−1.38 For unwrinkled regions of CVD graphene, ID/IG (unwrinkled) = (3.36 ± 0.14) × 10−2, hence the calculated value of grain size, La = (572.02 ± 24.99) nm. Similarly, for wrinkled regions ID/IG (wrinkled) = (8.47 ± 0.30) × 10−2, hence the calculated value of grain size, La = (226.96 ± 7.94) nm. This grain size is large enough and introduces relatively smaller defect density by the grain boundaries so that it may not serve as the heat transport limiting factor. This brings up the question, is there a factor different from defects that is responsible for the lower thermal conductivity of unwrinkled regions in CVD graphene? The observed decrease in thermal conductivity for unwrinkled regions of CVD graphene samples can have important contributions from stacking faults present in the sample, which can lead to an increase in the phonon scattering rate.16,39 Considering the larger grain size in our samples, enhanced phonon scattering can arise if the relative orientation of the few-layer stacks remains intact over relatively larger distances allowing for supercell level periodicity. With stacking faults the size of the unit cell increases leading to a consequent decrease in the size of the first Brillouin zone in the Fourier space. As a consequence, several folded phonon-branches appear in the reduced Brillouin zone. This can cause an increase in the phonon–phonon scattering and hence causes a decrease in thermal conductivity in the CVD samples, which comprise vertical assembly of few-layer crystallites with rotational stacking faults.16,39We now discuss the physical origin of the thermal conductivity in our CVD graphene samples, with particular reference to the wrinkle-mediated enhancement. A significantly lower value of 18,19,21 This effect arises from phonon localization and is directly related to the curvature of the wrinkle. Thus, depending on the local curvature and on the stacking of layers, wrinkles can influence thermal transport in quite different ways. It is thus important to consider the height aspect ratio of the wrinkle along the shorter direction. This aspect ratio for wrinkles in our samples was found to be ≈11.36. In comparison, the theoretical literature which predicts phonon localization effects at the wrinkle sites of single-layer graphene had considered a smaller wrinkle aspect ratio of ≈2.80.19 Since our sample has a comparatively lower wrinkling level (<10%) and lower associated stress, any reduction in thermal conductivity from the pristine value may be correspondingly smaller based on the curvature of the wrinkle. The significant decrease in the total interface conductance value compared to that of mechanically exfoliated multi-layer graphene is in agreement with the fact that the wrinkle location is in poor contact with the substrate/base layer. Depending on the bending rigidity and film thickness, 2D materials tend to release the residual stresses by bending in the out-of-plane direction. These residual stresses in the film are caused by various factors including thermal expansion coefficient mismatch between the film and substrate, transfer procedures, edge stresses etc. In this way, partial and local detachment of the film from the substrate can take place. Again wrinkles by themselves are greatly stressed/compressed along the texture direction where as they are stretched along the wrinkling direction. This curvature in the film at the wrinkle sites or the local departure of the flexible sheets of graphene from equilibrium configuration can lead to local modulation of thermal properties. In our CVD graphene samples, the height of the wrinkle is itself comparable to the film thickness and the width is also in the order of few hundred nano-meters. Hence a decrease in the total interface conductance of the wrinkled location is in reconciliation with the fact that the contact between the substrate and the film at the wrinkle location has substantially reduced. This leads to a consequent decrease of heat transfer between the film and substrate. Both the model data and experimental results are in good agreement with this fact. Concomitant with the partial suspension of the film at the wrinkle sites are local and partial lifting of the stacking faults16 In other words, we suggest that the vertical crystallites get spatially decoupled when the film is locally delaminated. This partially restores the thermal conductivity whose original decrease had an important contribution from rotational stacking faults between the crystallites. Indeed, κ for suspended 5-layer graphene was estimated to be high, ∼1500 W m−1 K−1 by Ghosh et al.8 We thus suggest that the plausible explanation of wrinkle-mediated enhanced thermal conductivity lies in the local delamination and decoupling of the vertical crystallite assemblies.Finally, we discuss the interesting observation of wrinkle-mediated local enhancement of thermal conductivity in our CVD graphene samples. In the literature, wrinkles have been predicted to suppress rather than enhance the thermal conductivity of single-layer graphene.4Altered morphologies like wrinkling and stacking faults are ubiquitous in layered 2D materials. They can be deliberately induced or sometimes they result from synthesis and processing conditions. Understanding of their effects on thermal transport is useful for practical devices. In the literature, curvature or wrinkling is suggested to suppress thermal conductivity in single-layer graphene by localizing phonons. However, a remarkable wrinkle-mediated enhancement of thermal conductivity is observed in our CVD graphene films, where the associated wrinkle curvatures were relatively smaller. The CVD graphene films were characterized by vertical assemblies of few-layer graphene crystallites with stacking disorder, leading to a decrease in thermal conductivity. However, thermal conductivity at the wrinkle sites showed local enhancement, and we attribute this to wrinkle-mediated uncoupling or detachment of the mis-stacked layers together with partial suspension of the film. The total interface conductance at wrinkle locations was also decreased compared to that of their unwrinkled and mechanically exfoliated counterparts which clearly reiterates the departure of the film from local mechanical equilibrium and reduction of the interface contact. We demonstrate that the interplay of rotational stacking faults and wrinkling can substantially tune thermal transport in a graphene-based system. Our results are useful towards understanding heat transport in thermal coatings based on layered 2D materials, where departures from ideal morphologies can be commonly observed.The authors declare no competing financial interests.NA-003-D0NA00944J-s001


Designing intervention and implementation strategies with careful consideration of context is essential for successful implementation science projects. Although the importance of context has been emphasized and methodology for its analysis is emerging, researchers have little guidance on how to plan, perform, and report contextual analysis. Therefore, our aim was to describe the Basel Approach for coNtextual ANAlysis (BANANA) and to demonstrate its application on an ongoing multi-site, multiphase implementation science project to develop/adapt, implement, and evaluate an integrated care model in allogeneic SteM cell transplantatIon facILitated by eHealth (the SMILe project).BANANA builds on guidance for assessing context by Stange and Glasgow . Based on a literature review, BANANA was developed in ten discussion sessions with implementation science experts and a medical anthropologist to guide the SMILe project’s contextual analysis. BANANA’s theoretical basis is the Context and Implementation of Complex Interventions (CICI) framework. Working from an ecological perspective, CICI acknowledges contextual dynamics and distinguishes between context and setting .BANANA entails six components: (1) choose a theory, model, or framework (TMF) to guide the contextual analysis; (2) use empirical evidence derived from primary and/or secondary data to identify relevant contextual factors; (3) involve stakeholders throughout contextual analysis; (4) choose a study design to assess context; (5) determine contextual factors’ relevance to implementation strategies/outcomes and intervention co-design; and (6) report findings of contextual analysis following appropriate reporting guidelines. Partly run simultaneously, the first three components form a basis both for the identification of relevant contextual factors and for the next components of the BANANA approach.Understanding of context is indispensable for a successful implementation science project. BANANA provides much-needed methodological guidance for contextual analysis. In subsequent phases, it helps researchers apply the results to intervention development/adaption and choices of contextually tailored implementation strategies. For future implementation science projects, BANANA’s principles will guide researchers first to gather relevant information on their target context, then to inform all subsequent phases of their implementation science project to strengthen every part of their work and fulfill their implementation goals.The online version contains supplementary material available at 10.1186/s43058-022-00354-7. Contributions to the literatureWe provide a comprehensive, multi-component approach to guide contextual analysis in implementation science, i.e., the Basel Approach for coNtextual ANAlysis (BANANA).BANANA specifically provides guidance on how to combine theories, models, and frameworks; how to use empirical evidence in contextual analysis; how to choose a study design for assessing context and setting; how to use findings from the contextual analysis to inform subsequent phases of an implementation science project ; and how to report contextual analyses.Using a case example, we demonstrate a successful application of BANANA in an ongoing implementation science research project.The importance of contextConceptual inconsistencies between the applied methods and approaches hamper the development of a standardized approach . In theiTMFs provide guidance on which contextual factors to study, but not on how to study context per se –19. CommAs part of a series of guidelines commissioned by the Agency for Healthcare Research and Quality, Stange and Glasgow  providedTo develop BANANA, we used a multiphase approach. First, we conducted a literature review, focusing on methodological IS papers available via major electronic data bases . To identify existing methodological approaches for contextual analysis, we screened the identified papers’ reference lists. The only authors to provide a methodological description of an entire contextual analysis were Stange and Glasgow ; others Second, research group members  consulted with IS experts  and a medical anthropologist (SS) in iterative discussion sessions about the identified literature and how to elaborate Stange and Glasgow’s approach in view of its application for IS projects. We used the three-step approach as a basis, adapted it to IS , and complemented it with identified evidence on context and its assessment  as well as other relevant aspects   choIn general, the use of TMFs is essential to inform and guide all phases of IS projects and increase the findings’ generalizability , 41–43. The selection of a framework is often perceived as difficult, as a large number of IS and other TMFs are available , 44, 45.Resources that provide an overview of TMFs or support the identification, selection, and combining of TMFs include key IS papers , 44, 47 1, we suggest the combination of a context- and setting-specific TMF, as such combinations enhance the granularity of contextual analysis. While context-specific TMFs provide an overview of factors that may influence implementation , setting-specific TMFs indicate factors that influence a specific intervention’s implementation in a specific setting . A broad variety of TMFs are available for specific settings and/or interventions, e.g., the Chronic Care Model ., 35.1, 3TMFs provide a comprehensive overview of how context is conceptualized and which contextual factors are relevant for implementation. However, not all aspects mentioned in the TMFs are relevant to each IS project. Therefore, using available empirical evidence can help to determine what is already known about the specific implementation context and relevant contextual factors. Four sources of evidence exist, i.e., 1) local data and information, (2) professional knowledge/clinical experience, (3) patient experiences and preferences, and (4) research . The fir local daAdditional file Empirical evidence can shed light on gaps in our understanding of context and can point to aspects of context that need further inquiry. It thus informs the focus and extent of the primary data collection in component 4 or even unravels in certain circumstances that no further primary data collection is required to inform further steps of the implementation project .In order to optimally inform the SMILe contextual analysis, all sources of evidence were used. First, a literature review revealed limited evidence on follow-up practice patterns in allogeneic stem cell transplantation (research evidence). Other identified studies reported on challenges with eHealth implementation  –57, inclSecond, in addition to the literature review, our studies in allogeneic stem cell transplantation as well as clinical experience of the SMILe team and patient feedback highlighted the challenges patients face in trying to improve their self-management behavior, e.g., medication non-adherence or physical activity , 65. BasFurthermore, the SMILe project leader’s knowledge among others about the organizational culture, leadership, work processes, and structures in the implementation setting, helped (1) to specify factors relevant to consider in the contextual analysis ; (2) to identify relevant stakeholders in the clinical setting and to rate their influence on the implementation; (3) to provide a basic understanding of the setting’s readiness for change and their openness towards eHealth technology; and (4) to inform the study design  and selection of appropriate methods . Finally, information on the setting’s resources  and operability of the IT system were gained in individual, informal stakeholder meetings . Those meetings were essential for the context-based development and adaptation of the SMILe-ICM, to identify potential hindering factors for its implementation in the specific setting and plan ahead for its sustainability.Stakeholder involvement is essential in every component of a contextual analysis. Stakeholders are “those individuals [or organizations] without whose support and feedback an organization, or a project within an organization [or beyond] cannot subsist” . They caThe matter of which stakeholders to involve in contextual analysis always depends on the project’s specific focus . To helpSince no specific guidance is available regarding stakeholder involvement in IS projects, general guidelines such as INVOLVE  or the PThe SMILe project involves stakeholders at multiple levels throughout the project and thus also in the contextual analysis . PotentiAs a contextual analysis in our view is a foundational phase, and thus functions as a separate study within IS projects, it requires additional considerations regarding research methods and study design. Data collection concerning relevant contextual factors is informed by components 1–3 of BANANA, i.e., theory, empirical evidence, and stakeholder input. The choice of methods is driven by the research questions. In addition, considering that available resources  for contextual analysis are usually constrained, researchers need to strike a “balance between speed and rigor” . This baTo deepen the research team’s understanding of the context, a combination of quantitative and qualitative methods is typically used , 76, 77.Quantitative methods include numerous types of surveys , systematic interviews, direct observations, or routine data. Quantitatively assessed contextual factors include, e.g., implementation climate, organizational culture and climate, available resources, and readiness for change. Several reviews provide overviews of current measurement tools and their psychometric properties , 83–88. To explore qualitative contextual factors, interviews (unstructured or semi-structured), focus groups, observations, or document analysisSome of these quantitative and qualitative approaches have been criticized for focusing only on specific levels  or “only provid[ing] a cursory view of complex and dynamic contexts” . HoweverConsidering the limited resources available for contextual analysis, the current trend is increasingly toward rapid qualitative or rapid ethnographic approaches. For example, the Rapid Assessment Procedure-Informed Clinical Ethnography (RAPICE) method combines rapid assessment procedures with ethnography , 98. IniBANANA focuses on the prospective assessment of context; however, as context is dynamic and evolves further, timepoints for considering context should be planned through the IS project. This does not mean starting over with component 1 of BANANA, but rather “keeping the thermometers in the system.” This can be achieved, for instance, through repeated assessments  or other methods  as well as through regular exchanges or informal conversations with stakeholders . CurrentFor the SMILe contextual analysis, an explanatory mixed-methods  design was applied based on the research aims formulated . SpecifiThe questionnaires cover the eCCM’s five building blocks and have been applied by the research team to previous work in heart and solid organ transplantation , 106–109Second, to support our understanding of the quantitative findings and allow a deeper understanding of further aspects relevant to the development/adaptation and implementation of the SMILe-ICM , we conducted focus groups with clinicians as well as focus groups and individual interviews with patients. In both cases, our interview guides followed the eCCM’s building blocks . In the Quantitative and qualitative data were collected over a 1-year period. Ongoing changes in context  were noted by the SMILe project leaders and documented. The team also had a regular exchange with stakeholders via informal conversations and official stakeholder meetings. The data analysis followed three eCCM-guided steps: (1) analysis of quantitative (descriptive tables) and qualitative results (meta-maps), (2) mapping of quantitative and qualitative findings in a joint display, and (3) reflection on findings and their implications for intervention development and choices of implementation strategies (again in a joint display) .Implementation success and sustainment of an intervention and the implementation strategy depend heavily on how well they align to the target context , 110–114Numerous frameworks/guidelines help researchers develop interventions and select implementation strategies. One of these is the Medical Research Council (MRC) guidance for the development and evaluation of complex interventions in healthcare. Whereas previously context was mainly considered during process evaluation, i.e., retrospectively, the MRC guidance now recommends examining interactions between the intervention and context across all phases of intervention development, implementation, and evaluationAnother guidance focusing on both intervention development and implementation strategies is Bartholomew, Parcel, and Kok’s “Intervention Mapping”—a five-step process, the foundation of which is a contextual analysis . Other mFurthermore, originally designed to facilitate implementation strategy choices, the CFIR–ERIC Implementation Strategy Matching Tool speeds the identification of implementation strategies available in the Expert Recommendations for Implementing Change (ERIC) compilation. The ERIC compilation’s strategies address specific constructs described in the CFIR framework . Just asEven where proven intervention or implementation strategies are available, adaptions are usually required to ensure their effectiveness in a new context , 117. HoAn intervention’s likely effects will vary across contexts and settings . The finTo describe how and why a specific intervention leads to its expected effects, as well as to trace causal pathways between intervention components, implementation strategies, and contextual factors, it will be necessary to develop a program theory , 121.Contextual analysis guided the development/adaption of the SMILe-ICM and the selection of implementation strategies. All quantitative and qualitative findings were synthesized in a joint display and the intervention’s implications summarized. Identified gaps both in self-management  and in delivery system design  highlight a need to re-engineer the current acute care model towards an ICM . FollowiAs contextual analysis informs subsequent phases of an IS project—affecting, for example, intervention development—it is a critical component of that project and needs to be reported as such , 125. HoThe SMILe project’s contextual analysis findings for its first study site were published in a separate paper. The same paper described the research team’s implementation strategies and outlined their findings’ implications for re-engineering stem cell transplant follow-up care . A seconContextual analysis should be the foundation of every IS project in our view. As noted above, contextual analysis results inform all subsequent project phases, enhancing interventions’ implementation and sustainability in real-world settings. In comparison to previous studies on facilitators and barriers, the BANANA approach does not only describe individual methods to study context , it provides methodological guidance on planning and conducting contextual analyses in IS projects. Furthermore, BANANA describes how contextual information can be reported and used to inform further project phases . While we have described BANANA in terms of six individual components, neither all components have to be performed in every contextual analysis, nor do they always operate sequentially Fig. . ParticuWhen presenting BANANA at conferences or in workshops, participants often ask us for a checklist they can apply to their project’s contextual analysis. However, we have deliberately avoided a “checklist approach”: the aspects to be studied in context and the methods chosen always depend on the individual research project and its research questions. Applying a checklist risks oversimplifying the context and undervaluing the complex interconnections of contextual factors, many of which differ from one setting to the next , 129. InIn addition to the project and research questions, however, pre-existing contextual knowledge and the researcher’s role influences planning and conducting of a more targeted contextual analysis , 131. ThSecond, although the importance of context in IS projects has been widely emphasized, funding agencies remain hesitant to fund contextual analyses as they are not yet recognized as a crucial part of an IS project. A contextual analysis’ rigor and thoroughness both reflect the available resources such as time, personnel, and especially funding , 75. TheBANANA was developed based on evidence and expert discussion and successfully applied within the SMILe project to guide intervention development  as well Improving researchers’ consideration of context and their reporting of it in IS studies will clearly require conceptual and methodological developments; however, further measures are also required. First, coupled with the acknowledgment of contextual analysis as the foundational first phase of every IS project, its relevance to implementation success requires funding agencies to rethink how to support this phase. That is, adequately resourcing contextual analyses will require specific funding schemes . Within Contextual analysis is a foundational phase of every IS project, providing essential information to all subsequent phases. The BANANA approach successfully guided the SMILe project’s contextual analysis. To help researchers make sense of their target contexts, and to strengthen every part of their work, this approach’s principles can also be applied to other IS projects. However, further adaption and testing of BANANA in other projects are required. Equally importantly, considering the vast heterogeneity of the studies we reviewed, a coordinated campaign will be required to unify and enhance IS researchers’ efforts to conduct and report on contextual analyses. As a first step, a common set of analysis and reporting guidelines will do much to improve the success and quality of implementation efforts.Additional file 1. The Basel Approach for coNtextual ANAlysis (BANANA): Overview of its development process and theoretical underpinning.Additional file 2. Key resources for each component of the Basel Approach for coNtextual ANAlysis (BANANA).Additional file 3. Overview of contextual factors most commonly reported in empirical evidence to influence implementation.Additional file 4. Overview of variables assessed and themes explored in the SMILe project.


TARBDP) are known to result in both familial and sporadic ALS. In ALS, disruption of neuromuscular junctions (NMJs) constitutes a critical event in disease pathogenesis, leading to denervation atrophy, motor impairments and disability. Morphological defects and impaired synaptic transmission at NMJs have been reported in several TDP-43 animal models and in vitro, linking TDP-43 dysregulation to the loss of NMJ integrity in ALS. Through the lens of the dying-back and dying-forward hypotheses of ALS, this review discusses the roles of TDP-43 related to synaptic function, with a focus on the potential molecular mechanisms occurring within MNs, skeletal muscles and glial cells that may contribute to NMJ disruption in ALS.Amyotrophic lateral sclerosis (ALS) is a disease characterized by upper and lower motor neuron (MN) loss with a signature feature of cytoplasmic aggregates containing TDP-43, which are detected in nearly all patients. Mutations in the gene that encodes TDP-43 ( C9ORF72), superoxide dismutase 1 (SOD1), fused in sarcoma (FUS) and transactive-response DNA-binding protein (TARDBP) [TARDBP encodes TDP-43, a ubiquitously expressed DNA/RNA-binding protein involved in multiple steps of RNA metabolism. Heterozygous missense mutations in TARDBP are found in 3% and 1.5% of familial and sporadic ALS cases, respectively [TARDBP [Amyotrophic lateral sclerosis (ALS) is an adult-onset degenerative disorder characterized by loss of upper and lower motor neurons (MNs) and progressive muscle atrophy . Its pro(TARDBP) . TARDBP ectively –18. Althectively –21. Inte [TARDBP –21, suggA longstanding debate in ALS research is the primary site of disease onset, which opposes the “dying-back” and “dying-forward” hypotheses. The dying-back hypothesis posits that the disease process is initiated distally at the neuromuscular junction (NMJ) and progresses in a retrograde fashion to affect the axons and MN cell bodies. In support of this theory, studies have described early muscle denervation before the appearance of motor deficits in both patients and mouse models –25. AnalTARDBP have been linked to axonopathy and NMJ pathology in several animal and cellular models , resulting in impairments of synaptic transmission, locomotive deficits and reduced lifespan [Several reports using various TDP-43 models have linked TDP-43 dysfunction to NMJ abnormalities Table . Earlierlifespan –47. Similifespan –52. Theslifespan , 53, 54.lifespan –66, consG298S co-cultured with 3D skeletal muscle bundles form fewer thick neural fibers and NMJs compared to control motor units, resulting in reduced muscle contraction force [G298S iPSCs exhibit a decreased area of innervated NMJs compared with isogenic controls [Recently, the development of in vitro NMJ models using induced pluripotent stem cells (iPSCs) allowed exploration of the impact of ALS TDP-43 variants at this synapse in a human model. Patient-derived MN spheroids expressing TDP-43on force . Sensoricontrols . Taken tIn healthy cells, TDP-43 is predominantly localized in the nucleus where it regulates multiple steps of gene expression including transcription  and spliTARDBP have been shown to cause various RNA abnormalities such as changes in gene expression, mis-splicing and reduced transcript stability [In ALS, it is hypothesized that the loss of nuclear localization of TDP-43 may alter RNA processing that usually occurs in the nucleus, which may lead to dysfunction of cellular pathways critical for neuron health and NMJ integrity. In fact, mutations in tability , 86–89. tability , widesprtability , 87–94. tability –94, and tability , suggesttability . Incorretability  and in tGria2/3, Grik2, Grin1, Grin2a/b), ion channels  and synaptic vesicle proteins neurexin 1 to 3 (Nrxn1/2/3) and neuroligin 1 (Nlgn1) [VAMP1), synaptotagmins  and synaptosomal-associated protein 25 (SNAP25) [UNC13A in iPSC-derived motor and cortical neurons and post-mortem brain neuronal nuclei, resulting in depletion of the UNC13A transcript and protein [UNC13A  risk through genome-wide association studies) were found to promote this incorrect splicing in patient brain tissues [The first studies characterizing RNA targets of TDP-43 using cross-linking immunoprecipitation combined with high-throughput RNA sequencing revealed that TDP-43 binds to thousands of transcripts derived from genes implicated in RNA metabolism, neurodevelopment, neuronal survival and synaptic function , 95–97.  (Nlgn1) . Similar(SNAP25) . Recentl protein , 99. Fur tissues .AGRN transcript encoding agrin [AGRN was shown to undergo cryptic splicing upon TDP-43 depletion [MAP1B transcript [MAP1B transcripts has been described in spinal cord specimens of ALS patients [MAP1B ortholog futsch have been repeatedly shown to be decreased with TBPH/TDP-43 loss-of-function in flies [futsch phenocopy several pathogenic changes observed with TBPH/TDP-43 depletion [ACHE have been reported in ALS spinal cord tissue sections related to the site of symptom onset [Differential expression of synaptic transcripts was also observed in several cellular and animal TDP-43 models , 60, 100ng agrin , a key rng agrin . AGRN waepletion , 94. Lowepletion . Additioanscript , which epatients . Levels in flies , 46, 104epletion , supportepletion . AChE, cepletion –109. TDPepletion . Moreoveom onset , highligOverall, these findings strengthen the hypothesis that dysregulated TDP-43 may lead to synaptic destabilization through altered gene expression. Given the thousands of RNA targets regulated by TDP-43, the challenge now is to identify the transcriptomic changes most relevant to the development and progression of ALS.In addition to impaired RNA processing, TDP-43 dysfunction has been linked to defective DNA damage response (DDR) , 72. In A315T and TDP-43Q331K in multiple cellular models lead to higher levels of the DSB marker γH2AX, indicating a loss of DNA repair function induced by ALS mutations [Q331K [TARDBP mutations encoding TDP-43M337V also display increased levels of DNA damage and impaired NHEJ, implying that failure of DNA repair mechanisms by TDP-43 may occur early in the disease course [Hence, it is hypothesized that the loss of TDP-43 nuclear functions in ALS may cause persistent DNA repair defects and genome instability. In fact, TDP-43 nuclear clearing correlates with DNA damage and activation of DDR in sporadic ALS spinal cord tissues . Similarutations , 114. Ins [Q331K . Interese course .FUS phenotypes in iPSC-derived MNs and reported early DNA damage, followed by defects in axonal trafficking of organelles, axonal degeneration, and finally death of MNs [Focussing here on potential mechanisms of NMJ disruption, it could be hypothesized that persistent DNA damage can provoke MN death , therebyh of MNs . UnfortuMitochondria are the main producers of reactive oxygen species (ROS), which cause oxidative stress and lead to cell death through apoptosis at excessive amounts . MitochoA315T mice [Mitochondrial dysfunction has been repeatedly described in cellular models expressing human wild-type TDP-43 or ALS variants , including increased levels of mitochondrial ROS , activat15T mice .Mitochondria have also been detected within large TDP-43 aggregates in TDP-43 transgenic mice , 134, leΔNLS [A315T [Abnormalities in mitochondrial morphology and distribution are a prominent TDP-43 phenotype , 136–138S [A315T , 140.In summary, mitochondrial dysfunction is commonly linked to TDP-43 dysregulation. In ALS, aggregation and enhanced mitochondrial localization of TDP-43 along with abnormal distribution of mitochondria may induce the loss of MNs and NMJs.In the cytoplasm, TDP-43 associates with RNA and other effector proteins to form transport ribonucleoproteins (RNPs) responsible for RNA transport along microtubules in both anterograde and retrograde trajectories –76. ThisTARDBP  have been shown to decrease anterograde transport and enhance accumulation of transport RNPs in the cell body [A315T variant [One hypothesis is that impairment in anterograde transport  may prevent adequate maintenance of distal axons and presynaptic membranes, leading to denervation and neuronal cell death. ALS-associated mutations in ell body . As a reell body . Similar variant , 149. ThTDP-43 is detected at presynaptic membranes of NMJs , 150, suMoreover, Nagano and colleagues recently showed that TDP-43 binds and transports along axons the mRNAs of ribosomal proteins (RPs) that are locally translated and assembled into ribosomes which, in turn, participate in local protein synthesis themselves . Using iM337V variant was recently found to impair the retrograde axonal transport of neurotrophin-containing signaling endosomes in mice, preceding NMJ dismantling and motor symptoms [Another proposed mechanism for NMJ disruption in ALS is the abnormalities of retrograde transport that may prevent the delivery of factors supporting neuron survival back to the cell body, such as neurotrophin-containing signaling endosomes . Neurotrsymptoms .+ early endosomes to Rab11+ recycling endosomes at motor terminals [In addition to neurotrophins, other pathways that initiate at the NMJ are crucial for regulation of the formation and function of this synapse, including the bone morphogenetic protein (BMP) signaling pathway , 160. Muerminals . These rerminals . There ierminals . Taken tSARM1) has been identified as a key initiator of programmed axon death, as depletion of this gene confers long-term resistance to degeneration [SARM1 locus has been associated with an increased susceptibility to sporadic ALS [G348C mice exhibit sustained cytoplasmic mislocalization of TDP-43 and impaired recovery after nerve crush injury, as shown by fewer regenerating axons and persistent motility impairments compared with control animals [Axonal fragmentation is a prominent feature of neurodegeneration. According to the dying-back theory, degeneration originates distally at nerve terminals and progresses in a retrograde fashion to sequentially affect the axons and cell bodies, eventually leading to MN loss , 25. Thineration –169. Theadic ALS  and consadic ALS , 172. ALadic ALS –177), isadic ALS , 178–180adic ALS , 141, raadic ALS –183. Fur animals .SARM1 resulting in improvement of disease phenotypes in TARDBP models [C. elegans expressing TDP-43A315T, loss-of-function mutation in the SARM1 ortholog tir-1 improves motility deficits and MN survival [SARM1 knockout mitigates axonal degeneration and MN loss in TDP-43Q331K mice [+ hydrolase activity [+ depletion (and consequently ATP depletion), axonal degeneration, NMJ denervation and MN loss. It is worthy of note, however, that SARM1 deletion does not mitigate neurodegenerative phenotypes in the SOD1G93A mouse model, suggesting distinct mechanisms in SOD1-ALS [More direct evidence implicating TDP-43 in the Wallerian pathway was demonstrated by genetic ablation of P models , 185. In31K mice . Importa31K mice . These r31K mice . Recentlactivity , 172. InSOD1-ALS , 187.SARM1 depletion [A315T mice in the early symptomatic phase of the disease, preceding NMJ morphological defects [TDP-43 is also associated with other mediators of the Wallerian pathway, namely PHR1   and statepletion . Paradoxepletion –194. PHRepletion , 194. PH defects . FurtherSTMN2) [SARM1 [stathmin, or expression of a loss-of-function mutant, causes a reduction of bouton number and axonal retractions at the NMJ [Stathmin mutant or knockout mice develop a late-onset axonopathy and NMJ denervation, leading to muscle atrophy and severe motor impairments [Two studies have clearly shown that TDP-43 regulates expression of stathmin-2 (STMN2) , 190, anSTMN2) , 196. It) [SARM1 . Stathmi) [SARM1 , 190. Me) [SARM1 , 190. St) [SARM1 , 190, co the NMJ , 198. Siairments , 200.In summary, TDP-43 is functionally linked to factors involved in the Wallerian degeneration pathway, with dual roles in axonal outgrowth and NMJ maintenance. Disturbances in TDP-43 homeostasis in ALS may affect the expression levels of these factors, which in turn may contribute to defects at the NMJ, axonal degeneration and MN loss that characterize this disease.TARDBP, TDP-43 has an increased propensity to aggregate and is capable of interacting with the wild-type protein, recruiting it into further aggregates [TARDBP encoding the protein’s glycine-rich C-terminal domain, which has been proposed to mediate solubility and oligomerization [TDP-43 aggregation is a core feature of ALS . These igregates , 201. Thrization . This imG298S variant can sequester transcripts of the chaperone Hsc-70-4/HSPA8, resulting in decreased expression of the protein at the NMJ in transgenic Drosophila and mice [C9ORF72 or TARDBP mutations, confirming a post-transcriptional mechanism of expression inhibition [Cox4il and ATP5A1 are directly bound by TDP-43 and sequestrated within axonal condensates, resulting in decreased levels of the proteins [2O2-induced TDP-43 aggregates also sequester specific microRNAs in mouse neuroblastoma-derived N2a cells, leading to upregulation of their corresponding targets [M337V enhances the capture of the microRNAs compared with the wild-type protein, supporting a gain-of-function mechanism. Furthermore, TDP-43 co-aggregates with other RNA-binding proteins , raising the possibility that RNA sequestration within aggregates may not be limited to direct TDP-43 targets. Taken together, these studies support the hypothesis that TDP-43 aggregates may negatively impact NMJs by interfering with the expression of essential proteins for NMJ maintenance and function.It has been proposed that aggregates can sequester RNA from the translational machinery, thereby depleting MNs of critical proteins for NMJ maintenance. Indeed, an emerging property of pathologically altered TDP-43 is sequestration of mRNA into insoluble complexes , 203. Cohibition . Under phibition –76 and shibition –84, highhibition , 136. Sp targets . RNA immWhile MN dysfunction and degeneration has traditionally been the focus of ALS research, a growing body of evidence recognizes non-cell autonomous mechanisms exerted by cells interacting with MN cell bodies or presynaptic terminals. Here, we describe some of the studies focused on TDP-43 dysregulation in non-neuronal cell types which have been hypothesized to influence NMJ integrity in ALS.The hypothesis that skeletal muscle may play an active role in disease initiation and progression emerged from studies describing muscle-specific pathogenic changes in human ALS specimens, such as mitochondrial morphology defects –207 and MyoD, MYOG and Acta1) to regulate their expression and initiate the differentiation program [TDP-43 is essential for muscle regeneration . Vogler  program . Militel program . In zebr program –227. The program , 229. ItBET1L as a commonly downregulated gene in iPSC-derived myocytes from TARDBP, SOD1, C9ORF72 and sporadic ALS patients [G93A rats [TDP-43 expression in muscle has been shown to promote NMJ assembly . In fruipatients . In the 93A rats .C. elegans, miR-1 regulates the expression of acetylcholine receptor subunits and modulates ACh release from motor terminals via retrograde signaling [Other groups have proposed that misregulation of non-coding RNAs in skeletal muscle may promote NMJ disruption. King and colleagues showed that TDP-43 negatively regulates the activity of muscle-enriched microRNAs of the miR-1 family (namely miR-1 and miR-206)  with funignaling . MiR-206ignaling . TDP-43 ignaling . While hignaling , HDAC4 wignaling , 241. Inignaling .A315T [A315T and several other ALS-linked mutations [A315T and SOD1G93A mouse models [Co-culture of ALS myocytes with control MNs has provided additional evidence for a potentially toxic role of skeletal muscle via dysregulated TDP-43 , 243. WäA315T . Similare models . DepletiTARDBP were repeatedly shown to increase activation of astrocytes and microglia in mice [Ptp61f, a gene implicated in inflammation and ER stress signaling pathways [Ptp61f knockdown in TDP-43-overexpressing flies suppressed inflammatory cytokines secretion and rescued reduced synaptic button number at NMJs, climbing deficits and lifespan.Non-cell autonomous mechanisms of disease involving glial cells have also become important areas of ALS research. TDP-43 aggregates have been described in astrocytes, microglia and oligodendrocytes of patient specimens –21, 244, in mice , 248–253 in mice , 254. MeIn contrast, astrocyte-specific TDP-43 depletion in mice does not cause NMJ denervation or MN loss, but induces an A1-like reactive astrocyte molecular profile, upregulation of C1 complement expression in microglia (a marker of reactive microglia), and reduces the number of mature oligodendrocytes, referred by the authors as “triglial dysfunction” . SimilarEAAT2 transcript in the human brain [EAAT1 and EAAT2 transcript levels in the fruit fly [M337V in astrocytes results in a progressive decrease in EAAT1 and EAAT2 immunoreactivity in the spinal cord, and causes denervation atrophy of muscles and MN death [Aside from neuroinflammation, impaired glutamate clearing is another proposed mechanism implicating astrocytic dysfunction in ALS. In physiological conditions, astrocytes take up glutamate from excitatory synapses via excitatory amino acid transporter 1 and 2 (EAAT1/2). When uptake is impaired, accumulation of glutamate in the synaptic cleft causes excessive neuronal stimulation, calcium overload and intracellular damage leading to neurodegeneration. This process, termed excitotoxicity, has been proposed to play a role in ALS pathogenesis as per the dying-forward hypothesis. Motor cortex and spinal cord specimens of patients display a significant decrease in EAAT2 . Intriguan brain . Moreoveruit fly . In tranMN death . AlthougMN death ), notablMN death . In thisTARDBP knockout in myelin-forming Schwann cells causes severe motor deficits due to impaired formation of paranodal junctions, which maximize nerve conduction velocity in MNs [Although the involvement of glia from the CNS has been investigated more extensively, there is also evidence for a contribution of glial cells from the peripheral nervous system in ALS, namely the myelin-forming and terminal Schwann cells (TSCs). Myelin-forming Schwann cells produce the myelin sheath that wraps the axons of MNs to enable rapid saltatory conduction of action potentials. Peripheral nerves of ALS patients often show signs of demyelination , 264. Iny in MNs . Two recy in MNs , 266. Ity in MNs . CurrentSOD1 mice prior to onset of denervation [2+ response that allows for modulation of synaptic transmission [G37R mice [TSCs, also known as perisynaptic Schwann cells, are considered the third cellular component of the tripartite synapse as they cap motor terminals and provide trophic support to the NMJ  Fig. 2)2). The Tervation . Other servation , 277. TSsmission . TSC swi37R mice , 277. CoALS is a complex disease, most likely caused by a combination of genetic and environmental factors as well as age-related changes. As summarized in this review, disturbances of several cellular pathways have been suggested to play a role, making it difficult to reconcile the many fundamental features of ALS into one disease model. This challenge is further complicated by the heterogeneity of the clinical manifestations of ALS (including its close association with FTD) and the presence of multisystem impairments.In the sections above, we provided an overview of the potential mechanisms underlying NMJ disruption in ALS mediated by dysregulated TDP-43 in different cell types. Some of these studies argued that defects may originate distally, such as the evidence for a toxic role of skeletal muscle –222, theAt present, beyond the old dichotomy of “dying-back” versus “dying-forward”, we can speculate that several pathologies may occur simultaneously early in the disease course. For instance, excitotoxic effects exerted by upper MNs may be potentiated by lower MNs’ inability to cope with additional stressors  and accelerate the dismantling of already-vulnerable NMJs . An additional consideration is how the primary site of onset is defined, which can lead to different interpretations with regards to the direction of disease progression. For instance, let us consider the suggestion that dysregulation of key pathways in MN cell bodies triggers the dismantling of distal structures. In this scenario, the primary dysfunction originates from cell bodies and propagates anterogradely to affect the NMJs (dying-forward), but denervation and axonal fragmentation may occur before the MN cell bodies degenerate and symptoms become apparent (dying-back). This example highlights the importance of considering the multiple scales at which pathogenic changes can take place . Finally, we can hypothesize that different genetic or clinical subtypes of ALS patients may present distinct predominant dying-back or dying-forward patterns of neurodegeneration, with pathology being more pronounced in the motor cortex or in distal structures at early stages of disease. This integrated view could partially explain the heterogeneity of clinical presentations of ALS patients, including the variability in the site of symptom onset  and the presence of extra-motor manifestations .ALS is a complex and heterogeneous disease, most likely caused by a combination of factors. It is becoming clear that pathological dysregulation of TDP-43, not only in MNs but also in non-neuronal cells such as skeletal muscle and glial cells, plays an important role in disease pathogenesis. Abnormalities in RNA processing, DNA repair, mitochondrial function, axonal transport and protein aggregation are TDP-43-mediated changes that may be important contributors to NMJ disruption and MN loss. The detection of aggregates in several types of cells showing pathogenic changes in ALS  may indicate that the impact of TDP-43 dysregulation in ALS is underestimated. While TDP-43 is expressed ubiquitously, little is known about the consequences ALS-linked TDP-43 variants in non-neuronal cells, particularly those forming the NMJ. A better understanding of when and how NMJ defects arise in ALS will be critical for the development of therapies that can meaningfully delay or halt functional decline. In particular, therapeutics targeting NMJs would avoid the need for molecules that cross the blood-brain barrier or having patients undergo invasive drug delivery procedures. Combination therapies targeting several pathways, rather than individual targets, may also be a promising avenue given the multifactorial nature of this disease.


The results showed that the sensor had a good response recovery performance in the test concentration range, and the frequency offset was positively correlated with methane concentration. The 90% average response time and recovery times were 41.2 s and 57 s, respectively. The sensor sensitivity was 809.4 ± 6.93 Hz/(1% CH4). This study provides a good theoretical basis for the development of surface acoustic-wave methane sensors.Surface Acoustic Wave (SAW) methane-sensing technology is a new way to detect methane at room temperature. However, the material and structure of the sensitive film are the important factors affecting the detection performance of the sensor. In this paper—with a SAW methane sensor using graphene–nickel cavitation—a composite film is proposed, which can work at room temperature. A delay linear dual-channel differential oscillator with center frequency of 204.3 MHz and insertion loss of −5.658 dB was designed; Cryptophane-A material was prepared by the “three-step method”. The composite sensitive film was synthesized by a drop coating method, electrochemical deposition method and electroplating method. The composite film was characterized by SEM. The sensor performance test system and gas sensitivity test system were constructed to determine the response performance of the sensor at concentrations of 0~5% CH China is a country with high levels of coal output and consumption. However, the poor storage conditions of coal resources and the lack of effective monitoring and prevention measures have led to frequent coal-mine gas accidents. Therefore, it is necessary to develop an efficient and reliable gas sensor to conduct gas monitoring. About 83–89% of the gas is comprised of methane. Methane is colorless and tasteless, concentrations of more than 5% are prone to explosion, and its stable properties make it difficult to detect at room temperature. Methane sensors in China mainly include the infrared absorption type ,2,3, catIn recent years, surface acoustic wave (SAW) sensors have been widely employed due to the advantages of their small size, low power consumption, high sensitivity and flexible application ,13. The 3 resonator. The performance of a SAW sensor is better because the graphene surface has a good adsorption capacity of water molecules and mass-loading effect. Nash [Graphene has a high specific surface area and electrostatic adsorption performance, which can adsorb methane molecules. Its good physical properties and application potential are conducive to the integration of surface acoustic wave devices ,27,28,29ct. Nash  proved tct. Nash  carried ct. Nash  study alNickel is often used as a catalyst due to its excellent electrical conductivity and catalytic performance . The comMethane is a stable non-polar molecule, which is difficult to be adsorbed by conventional materials. Studies have found that cave fan is a caged supramolecular compound with a special encapsulation effect on methane molecules  which caIn this study, we combined graphene, nickel nanoparticles and Cryptophane materials with SAW technology, and deposited graphene–nickel–Cryptophane-A sensitive films on a SAW resonator with a central frequency of 204.3 MHz. A two-channel methane SAW sensor was developed and its performance was measured at room temperature.The offset of the center frequency of the traditional acoustic surface gas sensor can be expressed as :(1)Δff0=For ST-cut quartz crystal, when the sensitive film is an organic material, the viscoelastic effect of the gas to be measured on the sensitive film can be ignored due to the small shear modulus; that is, the second term on the right-hand side of Equation (1) can be ignored.For traditional SAW gas sensors, the electrostatic capacity of the device is usually regarded as a constant value. When the sensitive film of the SAW gas sensor adsorbs the measured gas t, the surface conductivity  becomes:Since the width of the transducer of the SAW device is much larger than its thickness the value of Formula (2) becomes:VST–x = 3158 m/s [W = 2.5 mm, finger width The research group completed the design of sensor devices , and ST-3158 m/s . Center Cryptophane-A was synthesized by a three-step method by using vanillin as the raw material, and the synthesis path is shown in The graphene, nickel nanoparticles and Cryptophane-A were combined with SAW technology, and deposited the graphene–nickel–Cryptophane-A sensitive film on the SAW resonator with a central frequency of 204.3 MHz. The deposition process is shown in Firstly, 3.0 mg Cryptophane-A, 0.3 mg polyvinyl chloride and 0.6 mg dioctyl sebacate were dissolved in 2 mL tetrahydrofuran to prepare the sensitive film solution. The 0.3 mL solution was drip-coated onto the SAW device with a micro syringe, and then it was baked in an oven at 80 ℃ for 1 h. After the solvent was completely volatilized, Cryptophane-A film was obtained on the surface of the device. Ni nanoparticles were then electrodeposited by cyclic voltammetry, and graphene films were prepared by direct current electroplating to form the graphene–nickel–Cryptophane-A composite film. The film was characterized by an electron microscope of FEI Quanta 250  as shown in The SAW device is the main component of the SAW sensor and its characteristics will significantly affect the performance of the SAW sensor. The performance testing system of the SAW device is shown in On the premise of ensuring the air tightness of the SAW device-testing chamber, first turn on the integrated test equipment to make the temperature sensor and humidity sensor work normally. Then, open the valve door of the nitrogen cylinder and inject nitrogen. When the oxygen concentration tester shows the oxygen concentration is zero, close the two-way valve. Finally, the SAW device is tested by a network analyzer. The center frequency, insertion loss and other related data of the SAW device were recorded and analyzed.The consistency of two signals of the delay linear sensor directly affects the performance of the sensor. The center frequency , is an important parameter in SAW device design. The minimum insertion loss in the frequency band is regarded as the insertion loss of the device. In general, the insertion loss of SAW devices refers to working attenuation. The expression of insertion loss is:The gas sensitivity test system is shown in The test process is as follows: first of all, check the air tightness of the entire pathway, and then turn on the DC-regulated power supply and frequency counter under the premise of ensuring no air leakage. Finally, methane gas was injected, and a personal computer was used to record and analyze the frequency value of the difference frequency signal.The relationship between the frequency offset .According to the figure analysis, the test results of the device have a good concentration–frequency linear relationship for 0–5% concentration of methane, and the frequency response of the device is 809.4 Hz ± 6.93 Hz/(1% CH4 concentration (0–5%).The fitting formula of response consistency of the device is as follows:Under the test temperature of 22 °C and humidity of 46%, the sensor operating voltage was set to 12 V, and the response recovery curves of methane concentration from 1% to 5% were measured by controlling the concentration of the incoming methane gas, as shown in 2, C2H6 and H2. It is necessary to consider the response sensitivity of the sensor to the background gases. Under the test temperature of 22 °C, the humidity of 46% and sensor power supply of 12 V, the sensitivity of 1 wt.% MWCNTs modified sensor to four common interfering gases at 1% CH4 volume concentration was measured, as shown in 4; the sensitivity of ethane is about 120 Hz/1% C2H6; the sensitivity of CO is about 200 Hz/1% CO; the sensitivity of CO2 is about 170 Hz/1% CO2; Hydrogen has the lowest sensitivity, only 80 Hz/1% H2. Experiments show that the selectivity of the sensor for methane is significantly better than that of the other four gases.In addition to methane, coal mine gas often contains background gases such as CO, CO4. The experiment shows that the humidity has a certain influence on the output of the sensor, but the overall influence is still within the required range of measurement accuracy (0.1%).An underground coal mine is an environment where humidity changes, with the maximum humidity exceeding 90%, sometimes reaching 100%. Here, we measured the sensor output frequency values, at 32.8%, 46%, 57.6%, 75.3%, and 93.6% humidity, at 22 ℃ ambient temperature, as shown in 4). The average response time of 90% was 41.2 s, and the average recovery time of 90% was 57 s. Meanwhile, the sensor has good selectivity for methane and is less affected by humidity.In this study, 0–5% concentration of methane gas was taken as the detection target under the test temperature of 22 ℃ and humidity of 46%. By depositing the graphene–nickel–Cryptophane-A composite sensitive film, we designed an acoustic surface wave methane sensor coupled with mass load effect, and obtained the following conclusions: The center frequency of sensor consistency is about 204.3 MHz. The return loss and insertion loss are −9.955 dB and −5.658 dB, respectively. The SAW methane sensor was prepared by the “three-step method” and was combined with graphene and nickel nanoparticles. The graphene–nickel–Cryptophane-A composite sensitive film was deposited on the surface acoustic wave device. In the range of 0–5% methane concentration, the response frequency of the methane sensor based on graphene–nickel– Cryptophane-A composite sensitive film has a good linear relationship with methane concentration, and the average response sensitivity is 809.4 Hz ± 6.93 Hz/(1% CH


Owing to the outstanding physical properties of graphene, its biosensing applications implemented by the terahertz metasurface are widely concerned and studied. Here, we present a novel design of the graphene metasurface, which consists of an individual graphene ring and an H-shaped graphene structure. The graphene metasurface exhibits a dual-resonance response, whose resonance frequency strongly varies with the geometrical parameters of the proposed metasurface, the carrier density of graphene, and the analyte composition. The transparency window, including width and position, can be artificially controlled by adjusting the geometrical parameters or the Fermi energy. Furthermore, the sensing parameters of the graphene metasurface for cancerous and normal cells are investigated, focusing on two factors, namely cell quantity and position on the metasurface. The simulated results clearly show that the theoretical sensitivity, figure of merit, and quantity of the graphene metasurface for breast cells reach 1.21 THz/RIU, 2.75 RIU Cancer ranks as one of the leading causes of human morbidity and death globally ,2. It haTerahertz (THz) spectroscopy, electromagnetic waves within the frequency range 0.1–10 THz, has attracted increasing attention from scientists for its promising applications in biomedical analytics, detecting, imaging, trapping, and sensing due to its advantages of being label-free, non-contact, and non-destructive ,19,20,21Graphene, as the most promising carbon-based nanomaterial for realistic application, has been applied favorably as optical imaging or sensing elements because of its high specific surface area , tunableIn this work, we propose a planar graphene metasurface, which is composed of a circular graphene ring and an H-shaped graphene structure. A feature of dual-resonance response is found in the transmission spectra, being expected for biosensors with high sensing performance. Such a feature can be controlled by adjusting the spacing size. The transparency window is dependent on the structural parameters of the metasurface and the carrier density of graphene. Moreover, the sensing performance of our proposed graphene metasurface for breast cells is also evaluated in our work. The results present that the parameters of breast cells, including cell types, number of cells, and position of cells on the graphene metasurface, have an obvious influence on the transmission spectra and sensing parameters of the proposed graphene metasurface.n = 1.956 [x-y plane, as depicted in P represents the periodicity of the array patterns. The spacing distance of the concentric inner and outer graphene rings and the corresponding ring width of the inner and outer rings are designated as The schematic structure of the graphene metasurface is illustrated in  = 1.956 . The gray-polarization propagates vertically in a direction paralleling to the z-axis to the graphene metasurface. The transmission spectrum as a function of the incident wavelengths and the electric field distributions at resonance peaks are calculated in the full-wave electromagnetic simulator, COMSOL Multiphysics. In order to obtain the response characteristic of the graphene metasurface in the THz domain, we utilize an effective surface conductivity approach to characterize the graphene monolayer. Theoretically, the surface conductivity of the graphene can be predicted within the Kubo approximation, being composed of the intra-band and inter-band contribution of the electron transitions. According to the Kubo formula, the corresponding surface conductivity and electron transition contributions are as follows [v = 299,792,458 m/s is the velocity of light in vacuum, ℏ = h/2e = 1.602176565 In our work, the thickness of monolayer graphene is set as 0.35 nm . The inc follows :(1)σ and breast cancer cell (MCF7), are chosen as the target analytes. In actual experiments, analytes are not evenly covered on the sensing region. Thus, in this simulation, MCF10A and MCF7 breast cells are supposed to be in discrete hemispheres, where their diameters are set to 18.7 ectively , and theectively , respectx axis, oblique direction . As the important elements of the biosensors, sensitivity (S), figure of merit (FOM), and quality-factor (Q) can effectively characterize the sensing quality. These elements are defined as (5)S=△f△Ultimately, to better evaluate the sensing performance of our proposed graphene metasurface for normal and cancerous breast cells, its sensing parameters are compared to that reported in previous studies, as listed in In summary, we numerically and theoretically investigate the graphene metasurface, which consists of an individual graphene ring and a H-shaped graphene microstructure. It is found that this metasurface presents a behavior of dual-resonance response, which may be favorable for improving the sensing capacity of human cancers. The dual-resonance feature is controlled by adjusting the spacing size, but is unchanged by the gap size of the H-shaped microstructure and the carrier density of graphene. Moreover, we notice that the size and position of the transparency window are highly sensitive to the geometrical parameters of the proposed metasurface and the carrier density of graphene. Thus, the transparency window can be adjusted over a broad frequency range. The sensing performance is also studied in depth, and it is clearly shown that the sensing parameters are dependent on the cell type, cell count, and the position of cells on the graphene metasurface. Such a graphene metasurface achieves an acceptable S, FOM, and Q, and the corresponding values are 1.21 THz/RIU, 2.75 RIU