The emergence of graphene and other two-dimensional materials overcomes the limitation in the characteristic size of silicon-based micro-resonators and paved the way in the realization of nano-mechanical resonators. In this paper, we review the progress to date of the research on the fabrication methods, resonant performance, and device applications of graphene-based nano-mechanical resonators, from theoretical simulation to experimental results, and summarize both the excitation and detection schemes of graphene resonators. In recent years, the applications of graphene resonators such as mass sensors, pressure sensors, and accelerometers gradually moved from theory to experiment, which are specially introduced in this review. To date, the resonance performance of graphene-based nano-mechanical resonators is widely studied by theoretical approaches, while the corresponding experiments are still in the preliminary stage. However, with the continuous progress of the device fabrication and detection technique, and with the improvement of the theoretical model, suspended graphene membranes will widen the potential for ultralow-loss and high-sensitivity mechanical resonators in the near future. A resonator measures the target parameters by detecting the frequency signal. It has the advantages of high resolution, good stability, and strong anti-interference ability , which iThe emergence of graphene and other two-dimensional materials brings new opportunities for the miniaturization of resonators. Graphene has a high elasticity modulus, low surface density, and high surface area-to-volume ratio, leading to a significant enhancement in resonant frequency and sensitivity to external parameters, such as force, pressure, and mass . HoweverHere, we review the state-of-the-art theory and experiments of graphene resonators. Firstly, we summarize and analyze the fabrication processes of graphene and devices. Then, the electrical, optical, and piezoelectric excitation and detection schemes of the resonator are described. From theoretical analysis and experimental verification, the resonant performance including frequency tuning, loss, quality factor, and the non-linear effect are briefly introduced. Finally, the device applications of graphene resonators in the sensor, pneumatic measurement, and sensitive detection of graphene intrinsic properties are discussed.2·V−1·s−1 at room temperature [Since graphene was discovered by mechanical exfoliation for the first time in 2004, a variety of preparation methods of graphene have been developed . Among tperature . In addiperature .In resonator devices, graphene is usually placed in two ways. The first is to etch and evaporate metal electrodes followed by covering graphene on the pre-fabricated substrate through exfoliation, transfer, or direct growth. The first graphene resonator was successfully realized by exfoliating graphene onto the substrate with preset channels and electrodes, and graphene was attached to the silicon oxide wafer by van der Waals force . This meThe second way is to place graphene on the target substrate by exfoliation, transfer, or growth firstly; then deposit metal electrodes to contact and fix the graphene; and finally suspend graphene by etching or other nano-fabrication techniques, as shown in The electrode of the graphene resonator can be divided into the gate electrode and source-drain electrode. The source and drain electrodes not only play the role of electrical contacts but also fix the suspended graphene film to retain the tensioned architecture. Au or other metals are deposited by plasma vapor deposition, thermal evaporation, or other means after ultraviolet lithography or electron-beam lithography. In addition, in order to improve the adhesion between the contact metal and graphene, Ti  or Cr 1 is usualWhen the resonator is excited by electrical input, the gate electrode is required to generate electrostatic force. A simple way is making by use of the substrate directly. In this case, the heavily doped Si substrate covered with silicon oxide on top is usually working as the gate ,15,26. HThe etching process, which can be divided into dry etching and wet etching, is the most common approach used to suspend graphene. Wet etching is a pure chemical process, in which buffered oxide etchant  is often used to etch silicon dioxide ,17,19. HCompared with wet etching, dry etching has advantages of high precision, anisotropy, and fast etching speed. Reactive ion etching (RIE) combining ion bombardment and chemical reaction is the most widely used method in the dry etching of silicon dioxide ,11,22. FSU-8 3005 (SU-8) is spun on the silicon oxide wafer and patterned by the electron beam (EB), and then the graphene is exfoliated to SU-8 followed by the SU-8 developed to suspend graphene ,24. JungThe excitation methods for graphene resonators can be summarized into electrostatic, optical, and piezoelectric excitation schemes. In the devices driven by electrostatic force, a voltage potential difference between the gate electrode and graphene is applied to generate the electrostatic force and to drive the resonance of the graphene ,29, as sThe gate voltage consists of two parts: a constant DC voltage maintains the tension of the graphene membrane and another RF AC voltage drives the graphene to a resonance frequency. Alternatively, when the gate electrode is grounded, the same effect can be achieved by applying the drive voltage to either the source or the drain electrode . Jung etIn an optically excited device, an intensity-modulated laser spot is focused on the surface of the graphene, regulating graphene temperature via light absorption of the graphene and causing periodic contraction/expansion, as well as thus vibrations of the graphene, as shown in In addition to direct excitation, parametric excitation is also widely used in electrical or optical excitation schemes ,38, whicAlthough pristine graphene is not a piezoelectric material, in recent years, piezoelectric excitation has been successfully applied to graphene resonators by indirect driving approaches ,41. The The small size of the graphene resonator makes the resonance signal weak and difficult to detect, thus the detection method needs to be carefully designed and improved. Electrical and optical detection methods are the most widely used schemes to detect the resonance signals.It is worth noting that the widely used frequency mixing technique will lead to the decrease of bandwidth and measurement speed of the graphene resonator. Alternatively, the direct RF readout technique has significant advantages in the high-speed detection for real-time applications. A common RF readout scheme for graphene resonators is to detect the RF current, combining the operation principles of graphene field-effect transistors and mechanical resonators . HoweverThe optical detection scheme, with advantages of high sensitivity and simplicity without electrical contacts, is also an important route for the effective detection of the resonance signals . InterfeCompared to conventional Micro-Electro-Mechanical System (MEMS) resonators, graphene can withstand very high strain, suggesting an extremely wide tunable range of the resonance frequency of graphene resonators. Electrostatic tuning of the frequency can be easily achieved by applying a dynamic gate voltage. Kang et al. investigated the effect of the initial strain-induced tension and deflection-induced tension generated by a gate voltage on the resonant frequency and tuning range by molecular dynamics simulation ,50,51. TDue to the excellent conductivity and thermal stability of graphene, Ye et al. achieved frequency tuning by Joule heating of graphene . The curQ value can be obtained by spectral measurements or time-domain measurement. It is worth mentioning that the time-domain measurement can effectively avoid the spectral spreading effect in spectral measurements [The loss is an important parameter in determining the performance of a resonator and is usually represented by quality factor urements . Table 1Q value increases accordingly [A number of mechanisms can lead to the reduction of the quality factor. Imboden et al. summarized the losses in graphene resonators into the extrinsic and the intrinsic losses according to their source . Extrinsordingly , which iQ factor, Eichler et al. reduced the driving force until the motion became almost undetectable and they eventually obtained a graphene resonator with an ultra-high quality factor of 100,000 at 90 mK [The source of non-linear damping is a complex problem. One explanation is that it mainly comes from two aspects: general loss path and geometric non-linearity . For exaat 90 mK .Due to the fact that graphene is an atomically thin membrane, the out-of-plane deflection of graphene is much larger than its thickness in a vibrating graphene sheet, which means that it is easy to trigger the non-linear effect in graphene resonators . Such no2rtε5/2) . As a reic range . Parmar ic range .Considering the non-linear effect, the Newtonian equation of the resonator can be expressed as follows :3)md2xmd2x(3)mdng force . If α issonators . This woGraphene has excellent mechanical properties, such as ultra-high Young’s modulus and high fracture strength, as well as ultra-low surface density. In addition, the resonance properties of graphene are very sensitive to external physical parameters and the environment, such as force, attached mass, and acceleration. These characteristics make suspended graphene very suitable for various kinds of resonant sensors.Resonant mass sensors are used to detect masses through the resonant frequency shift of graphene due to the attached mass. Based on the analysis methods of classical mechanics or nano-mechanics, a great deal of theoretical calculations and simulations of graphene resonant mass sensors are studied. For example, Dai et al. investigated the effect of non-linear vibration behavior and in-plane tension on the detection sensitivity of mass using the continuum elastic model . Their rThe pressure sensors based on graphene nano-mechanical resonators have been studied by finite element simulation . The simYet, the exploration of graphene resonant accelerometers is at the preliminary stage. Kang et al. studied the resonant accelerometers based on graphene nano-ribbons  and crosIn addition to measuring and sensing the external parameters, the graphene resonator can also be used to measure the intrinsic properties of graphene. Guan et al. fabricated a graphene-based strain sensor, in which the strain is adjusted by bending the substrate . The strGraphene resonators can also provide a feasible scheme for detecting fundamental physical properties. For example, Chen et al. controlled the electron energy in the Landau level under an external magnetic field through the graphene resonators. In this way, the energy gap between the adjacent Landau levels and the energy conversion between the energy gaps are measured . In addiinewidth .Recently, graphe0ne drum resonators have been used for pneumatic testing due to the flexibility and gas tightness of graphene membranes . As showThe high tensile strength and mechanical tunability of 2D materials such as graphene have attracted more and more attention to 2D phononic crystals in recent years. The feasibility of phononic crystals based on graphene electromechanical resonators is studied theoretically and experimentally ,90. In tThe Van der Waals heterostructure is a unique structure in two-dimensional materials. By stacking different types of two-dimensional (2D) structures, the mechanical, electrical, and thermal properties of heterojunctions can be actively regulated, which brings an endless potential of two-dimensional resonators.Inoue et al. fabricated graphene-molybdenum disulfide heterojunction resonators . By stacWith the continuous discovery of new two-dimensional materials, some of them have become new candidates for nano-electromechanical resonators, such as transition metal dichalcogenides (TMD) ,66,96, hGraphene is the first two-dimensional material to be discovered. It has atomic thickness, an extremely high Young’s modulus and fracture strength, and very low surface density, which make graphene an ideal material for the fabrication of nano-electromechanical resonators. The large surface-to-volume ratio induced by ultra-thin thickness also makes graphene resonators have great potential in the application of pressure, mass, acceleration, and other ultra-sensitive detectors. Since the first fabrication of graphene resonator in 2007, after more than ten years of development, great breakthroughs have been made in the fabrication technology and weak signal detection scheme, and the theory on the resonance characteristic also has been constantly updated. The application of graphene resonators is gradually moving from simulation to experiment and from the classical field to quantum field.However, there are still many challenging issues in graphene resonators to be solved in this stage. We summarize these challenges and the main directions of future research as follows.Fabrication of high-quality grapheneThe repeatability of graphene preparation is too weak due to the uncontrollable factors such as graphene strain, doping, defects, and so on. Additionally, the fabrication of graphene with large-scale, high-quality, uniform, and controllable thickness is still a challenge.Strain engineeringThe strain has a strong influence on the resonant performance of graphene resonators. In the process of fabrication, graphene is easy to produce residual strain, which is usually harmful. Thus, the approaches to actively control the strain of graphene is important.Device integrationAfter successful fabrication of graphene resonators, it is still a challenge to integrate graphene resonators into electronic systems and necessary to establish a set of integrated processes with a high efficiency, low cost, and high success rate.Loss source of graphene resonatorsThe loss of graphene resonators is very diverse but the source and mechanism of many losses are not clear; in particular, the relationship between loss and temperature still has a lack of accurate description. Therefore, it is necessary and urgent to study the loss source of graphene.Research of non-linear effectsSince the graphene resonator has a high level of non-linearity, resonant sensors based on the non-linear behavior are also widely concerning. In particular, the non-linear vibration has been proved to improve the sensitivity of sensors, which attracts attention to application in the non-linear regime.The expansion of the application fieldIt is very attractive to further expand the application field of graphene resonators from the classical field to quantum field. Considering the atomic thickness of graphene, the graphene-based resonator can reach an extremely high frequency, small mass, and dramatical large zero-point motion, which makes it an ideal device for quantum-electromechanical systems.In general, graphene resonators promote the development of micro-nano-electromechanical systems to miniaturization, ultra-low power consumption, and ultra-high sensitivity. With the continuous progress of the device fabrication and detection technique, and with the improvement of the theoretical model, graphene resonators will certainly open the potential for wide applications in various high-tech fields.


Background: Idiopathic pulmonary fibrosis (IPF) is a chronic progressive disease with unknown etiology and unfavorable prognosis. Ferroptosis is a form of regulated cell death with an iron-dependent way that is involved in the development of various diseases. Whereas the prognostic value of ferroptosis-related genes (FRGs) in IPF remains uncertain and needs to be further elucidated.Methods: The FerrDb database and the previous studies were screened to explore the FRGs. The data of patients with IPF were obtained from the GSE70866 dataset. Wilcoxon's test and univariate Cox regression analysis were applied to identify the FRGs that are differentially expressed between normal and patients with IPF and associated with prognosis. Next, a multigene signature was constructed by the least absolute shrinkage and selection operator (LASSO)-penalized Cox model in the training cohort and evaluated by using calibration and receiver operating characteristic (ROC) curves. Then, 30% of the dataset samples were randomly selected for internal validation. Finally, the potential function and pathways that might be affected by the risk score-related differently expressed genes (DEGs) were further explored.Results: A total of 183 FRGs were identified by the FerrDb database and the previous studies, and 19 of them were differentially expressed in bronchoalveolar lavage fluid (BALF) between IPF and healthy controls and associated with prognosis (p < 0.05). There were five FRGs  oncogene homolog [NRAS], Ectonucleotide pyrophosphatase/phosphodiesterase 2 [ENPP2], Mucin 1 [MUC1], and ZFP36 ring finger protein [ZFP36]) identified as risk signatures and stratified patients with IPF into the two risk groups. The overall survival rate in patients with high risk was significantly lower than that in patients with low risk (p < 0.001). The calibration and ROC curve analysis confirmed the predictive capacity of this signature, and the results were further verified in the validation group. Risk score-related DEGs were found enriched in ECM-receptor interaction and focal adhesion pathways.Conclusion: The five FRGs in BALF can be used for prognostic prediction in IPF, which may contribute to improving the management strategies of IPF. Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, age-related interstitial pulmonary disease (ILD) with unknown etiology, significant morbidity, and unfavorable survival , 2. The Ferroptosis, a newly identified form of iron-dependent cell death unlike other cell death forms, is primarily driven by the iron excessive accumulation, lipid peroxidation, and subsequent plasma membrane rupture, which ultimately caused cell death and is often accompanied by the disturbance of lipid and iron metabolism . FerroptHerein, we aimed to investigate whether FRGs in BALF are associated with the prognosis of IPF. First, we evaluated the expression levels of FRGs in BALF of IPF patients by downloading the mRNA expression profiles and corresponding clinical data from the Gene Expression Omnibus (GEO). Subsequently, a multigene prognostic model with FRGs was constructed and validated in the training and testing cohort, respectively. Finally, a functional enrichment analysis was performed to investigate the potential biological functions and signaling pathways affected by these risk-related differently expressed genes (DEGs). We hope to find some biomarkers that can contribute to the diagnosis, treatment, or prognosis of IPF in these ways.http://www.zhounan.org/ferrdb/) is the first ferroptosis-related database that integrates the latest makers and regulators of ferroptosis and ferroptosis-disease associations  and normalized using the R “limma” package. This study does not need to be granted by the local ethics committee as the data of GEO are publicly available, and we strictly adhere to the data access policies and publication guidelines of the GEO. The clinical information of the GSE 70866 cohort is shown in The GSE70866 dataset is the only one that simultaneously includes the prognostic data and mRNA expression profiles in BALF of IPF , which were obtained from GEO  <0.05 and p < 0.05 was confirmed as the filtering criteria. The results were visualized by the forest map. Next, a Venn diagram was performed to identify the intersection of DEGs of FRGs and prognostic FRGs.A univariate Cox regression of the “survival” package was conducted to investigate the FRGs associated with the prognosis of IPF in the GEO database. The value of https://www.string-db.org) (The protein–protein interaction (PPI) network for the overlapping genes was constructed using the STRING database (-db.org)  to exploThe patients with IPF from the GSE70866 dataset were randomly separated into two groups at a ratio of 7:3, the former for the training group and the latter for internal validation. The least absolute shrinkage and selection operator (LASSO)-penalized Cox regression analysis was utilized in the present study to establish a prognostic model in the training group to minimize the risk of overfitting by using the “glmnet” package of R , 34. FirThe “rms” package of R was utilized to visualize the risk score in the nomogram and generate the calibration curve to assess the prediction accuracy of the nomogram. Principal component analysis (PCA) and t-distributed stochastic neighbor embedding (t-SNE) were carried out with the “Rtsne” and “ggplot2” R package to explore the distribution of the two risk groups. Then, the “survival,” “suvminer,” and “timeROC” R package were utilized to generate Kaplan–Meier (K–M) survival and time-dependent receiver operational characteristic (ROC) curves to further evaluate the predictive capacity of the prognostic model, respectively. Additionally, the “heatmap” package was performed to visualize the gene expression levels in each sample in the two risk groups. Subsequently, the “survival” package was performed to evaluate whether clinical parameters and risk scores are independent prognostic factors for overall survival (OS). Finally, the results were further identified in the internal validation group.p < 0.05  as well as Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed by the “clusterProfiler” package to explore the biological functions and signaling pathways affected by the risk-related DEGs with the criteria of FDR <0.05 and p < 0.05 .p < 0.05 was considered statistically significant.The datasets containing the mRNA expression profiles of IPF lung tissue were retrieved from the GEO database. Four datasets  with a total of 28 healthy control and 84 patients with IPF were selected for analyzing and visualizing the expression of the prognostic model FRGs between the tissue of normal and IPF by using the “limma” and “beeswarm” package of R. The value of p < 0.05 was considered significant.The Perl, R (4.0.1), and SPSS 18.0 were used for data processing and statistical analysis. The Wilcoxon and univariate Cox regression tests were utilized to evaluate the expression levels of FRGs in BALF between IPF and healthy controls and identified the genes associated with prognosis, respectively. A prognostic model was generated by the LASSO-penalized Cox regression analysis and further evaluated the predictive capacity of which by K–M survival and ROC curve analysis. The univariate and multivariate Cox regression analysis were utilized to identify independent predictors of OS. The probability P < 0.05, A total of 176 patients with IPF and 20 healthy controls from the cohort of GSE70866 dataset were enrolled in this study, and 183 FRGs were explored from the FerrDb database and the literature report . The detThe PPI network based on the 19 prognostic ferroptosis-related DEGs was generated by the STRING online platform , which dThe 176 patients with IPF in the GSE70866 dataset were randomly categorized into training and testing cohorts at a ratio of 7:3, and the LASSO-penalized Cox regression analysis was used to set up a prognostic signature in the training group. The result showed that only five genes  oncogene homolog [NRAS], ectonucleotide pyrophosphatase/phosphodiesterase 2 [ENPP2], mucin 1 [MUC1], and ZFP36 ring finger protein [ZFP36]) remained in LASSO regression from the 19 prognostic FRGs , among wp < 0.001, p < 0.05).Then, the patients with IPF in the training cohort were categorized into a high- and low-risk group according to the median risk score , and thep < 0.001, The patients from the testing cohort were also separated in the two risk groups based on the median risk score to evaluate the robustness of the prognostic model constructed in the training cohort . SimilarThe univariate and multivariate Cox regression analyses were performed to determine whether the clinical parameters  and risk score are independent prognostic factors for OS in IPF. The results demonstrated that the risk score remained as an independent risk factor for the outcome of IPF in both the training  and testThere were 256 and 334 risk-related DEGs identified in the training and validation group by using Wilcoxon's test, respectively . The GO Idiopathic pulmonary fibrosis is a progressive and devastating disease with limited treatment options, unsatisfactory therapeutic effects, and poor prognosis , 2. DespFerroptosis is considered as an effective cancer treatment because it overcomes the resistance of malignant cells to chemotherapy and promotes the clearance of defective cells. Recently, various studies have illustrated that ferroptosis regulate the development of pulmonary fibrosis to some extent –27, 37, In our study, the predictive model was established based on the five FRGs . Cytosolic aconitase 1/iron regulatory protein 1 (ACO1/IRP1) is a bifunctional protein expressed in cytoplasm that performs its function as aconitase or regulating intracellular iron homeostasis according to iron availability , 39. Wanroptosis , 48 and roptosis . Herein roptosis , 51. Overoptosis . Interesroptosis , and wasIt is worth mentioning that we further evaluated the expression of the five FRGs in the tissue of IPF. The results demonstrated that three of the five FRGs  are dysregulated in the lung of IPF patients. Contrary to our findings, MUC1 and ZFP36 were found to be downregulated in the lung tissues of patients with IPF. The possible reasons are as follows: First, the authors of the IPF-BALF dataset (GSE70866) pointed that none of the patients received pirfenidone or nintedanib before BAL examination in their study . HoweverFunction analysis found that the risk score-related DEGs are mainly enriched in epithelial cell proliferation and extracellular matrix organization, etc. In addition, these DEGs were identified enriched in the cytokine–cytokine receptor interaction, TGF-β signaling pathways, focal adhesion, and ECM–receptor interaction signaling pathways, all of them are the most crucial pathways involved in the development of IPF –62. ThesThe advantage of this study is that the FRGs were explored more comprehensively by using the FerrDb database and the previous studies. In addition, the data of IPF from an online open database were systematically analyzed and the predicting value about the FRGs in IPF prognosis was evaluated and summarized for the first time. Meanwhile, there are some limitations present in this study. First, the sample size of normal control from the GEO database was not large enough. Second, only internal validation of the model was performed in the study due to the lack of similar datasets available for external validation. Third, molecular biology experiments were not carried out to further investigate the above results. Therefore, the prospective cohort study and molecular biology experiments need to be designed and performed to further verify the accuracy of the prognostic model and the mechanism of the five FRGs in the pathogenesis of IPF.To sum up, 19 FRGs associated with the OS were identified in BALF of IPF and a novel prognostic model was constructed based on the five FRGs in the present study. Then, the model was verified as an independent risk factor of OS of IPF patients in both the derivation and validation cohorts. These findings have potential reference value for guiding the treatment and prognosis evaluation of the patients with IPF.https://www.ncbi.nlm.nih.gov/geo/.Publicly available datasets were analyzed in this study. This data can be found here: HR, MC, and ML: conception and design. HR and MC: administrative support. ML, KW, YZ, MF, AL, JZ, TY, PS, DL, and GZ: provision of study materials or patients. ML, KW, YZ, MF, AL, JZ, and DL: experiments operations, collection, and assembly of data. ML, KW, YZ, AL, JZ, TY, PS, DL, GZ, MC, and HR: data analysis and interpretation. All authors: manuscript writing. All authors contributed to the article and approved the submitted version.This study was funded by the Key Research and Development Projects of Shaanxi Province , Fundamental Research Funds for the Central Universities in Xi'an Jiaotong University (Nos. xzy012021064 and 1191320149); the National Natural Science Foundation of China (No. 81802291), China Postdoctoral Science Foundation Grant (No. 2019M653666).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


We also report DFT data used to obtain hydrogen Gibbs free energies for adsorption on the MoS2 monolayer and on graphene of the heterostructure. The DFT data were calculated using the periodic DFT code CRYSTAL17, which employs Gaussian basis functions, under the hybrid functionals PBE0 and HSE06. Moreover, we also report the data used for Quantum Theory of Atoms in Molecules (QTAIM) and Non-covalent Interaction (NCI) analysis calculations. These data were obtained using the optimized unit cell configurations from the periodic DFT and inputted to Gamess program, thus generating files that could be read by the Multiwfn program used for QTAIM and NCI calculations.The data presented in this paper refer to the research article “Dry and Hydrated Defective Molybdenum Disulfide/Graphene Bilayer Heterojunction Under Strain for Hydrogen Evolution from Water Splitting: A First-principle Study”. Here, we present the Density Functional Theory (DFT) data used to generate optimal geometries and electronic structure for the MoS Defect MoS2/graphene heterostructures serve as hydrogen evolution catalysts (HER).We provide (a) structural and electronic information for dry and hydrated pristine and defect MoS•Electronic information shows a bandgap opening at the Dirac point region, which is affected by hydration and vacancies. Thus, this bandgap could be engineered for producing efficient HER electrocatalysts.•2-graphene interaction is var der Waals.The presence of QTAIM S-C bond critical points and NCI calculations show that MoS•2/graphene use as HER, when S and C defects are included in the lattice. These data can be further used by experimentalists to examine the needed concentration of S that produce an HER catalyst with hydrogen Gibbs energy of approximately zero.We also provide data that support the MoS12/graphene heterostructures with and without interacting waters on the S surface of the MoS2, as calculated by the PBE0 functional. We found no significant differences in the above optimal geometries for calculations using the HSE06 functional. D), minigaps (ΔE), and MoS2 bandgaps (Eg) for pristine and defect configurations of this work under the HSE06 calculations. Corresponding ED, ΔE, and Eg using the PBE0 functional are shown on 2 bandgaps Eg relative to the HSE06 calculations, as expected. H for H adsorption on MoS2 and graphene for each MoS2/graphene configuration under the PBE0 calculations. For H/MoS2, ΔGH is positive for adsorption on pristine MoS2 and negative, when S vacancies are present. 2/graphene using Multiwfn and plotted via VMD . Thus, for these calculations a molecular cluster has been used.The submitted data are grouped in five directories. Two directories contain CRYSTAL17 input and output files from electronic band structure and densities of states (DOS) calculations and from optimized geometries. Each of these directories contain information from HSE06 and PBE0 calculations, which are found in separate subdirectories. The other three directories contain HER calculations for the PBE0 functional (CRYSTAL 17 input and output files), Gamess input and output files, and QTAIM-NCI Multiwfn output files.22/graphene heterostructure under comprehensive strain by using a 2H-MoS2 three-layer 4 2 surfaces contain three water molecules per unit cell. These waters are placed on the S layer away from graphene. We built defective MoS2/graphene heterostructures by considering all combinations of single (VS) and non-adjacent double S vacancies (VS2) in the MoS2 with a single C vacancy (VC) in the graphene layer.We constructed the pristine dry MoS2/graphene configurations were obtained using the periodic DFT code CRYSTAL17 D appears at the Γ point.Optimized geometries and electronic structure of all MoS2. The Gibbs free energy Hydrogen adsorption energies QTAIM ,10 and NNone.Nicholas Dimakis: Conceptualization, Methodology, Supervision, Validation, Writing – review & editing. Sanju Gupta: Conceptualization, Methodology, Writing – review & editing. Razeen Wadud: Visualization, Investigation, Validation. Muhammad I. Bhatti: Writing – review & editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:


Mycobacterium tuberculosis infection and tuberculosis (TB) disease across these communities. We sought to clarify whether specific communities could have been more/less vulnerable to M. tuberculosis transmission as a result of sociological variety relevant to transmission efficiency.Selected Zambian communities formed part of a cluster randomised trial: the Zambia and South Africa TB and AIDS Reduction study (ZAMSTAR). There was wide variability in the prevalence of We conducted a mixed methods secondary analysis using existing data sets. First, we analysed qualitative data to categorise and synthesise patterns of socio-spatial engagement across communities. Second, we compared emergent sociological variables with a measure of transmission efficiency: the ratio of the annual risk of infection to TB prevalence.ZAMSTAR communities in urban and peri-urban Zambia, spanning five provinces.Fifteen communities, each served by a health facility offering TB treatment to a population of at least 25 000. TB notification rates were at least 400 per 100 000 per annum and HIV seroprevalence was estimated to be high.M. tuberculosis transmission. For example, watching video shows in poorly ventilated structures posed a presumptive risk in more socially open communities, while outdoor farming and/or fishing were particularly widespread in communities with lower transmission measures.Crowding, movement, livelihoods and participation in recreational activity differed across communities. Based on 12 socio-spatial indicators, communities were qualitatively classified as more/less spatially crowded and as more/less socially ‘open’ to contact with others, with implications for the presumptive risk of M. tuberculosis transmission efficiency. To reduce transmission, certain socio-spatial aspects could be adjusted , while more structural aspects are less malleable . We recommend integrating community level typologies with genome sequencing techniques to further explore the significance of ‘social permeability’.A dual dynamic of ‘social permeability’ and crowding appeared relevant to disparities in ISRCTN36729271. We employed a novel qualitative approach to investigate disparities in transmission efficiency.We combined population (community) level socio-spatial data with actual measurement of tuberculosis (TB) infection and disease.Data were effectively cross-sectional, focusing on interactions within localised geographical boundaries, and did not capture details of extra-community travel, nor of infrequent and/or seasonal events, potentially relevant to TB transmission.Results are context specific; therefore, further investigations in other settings would be valuable to assess the generalisability of key concepts.Measures of association should be interpreted with appropriate caution and the study regarded as hypothesis generating.Mycobacterium tuberculosis, within and outside households, is critical to developing alternative approaches that could reduce ongoing M. tuberculosis transmission at the community level.M. tuberculosis transmission dynamics. We used a novel mixed methods interdisciplinary approach to conduct a secondary analysis of Zambian data sets, collected during a cluster randomised trial (CRT)—the Zambia and South Africa TB and AIDS Reduction study (ZAMSTAR)—and as part of an ancillary study, named the Contact Observations of Daily Activities (CODA).M. tuberculosis transmission efficiency .Interactions between the tuberculosis (TB) and HIV epidemics have complicated and compromised TB containment efforts in Zambia and other high burden countries. There is evidence that as the HIV epidemic ages, an increasing burden of undiagnosed TB is emerging among older adults in Zambia, underscoring the resilience of epidemic TB in the region.M. tuberculosis transmission and TB prevalence across Zambia and South Africa at a population level. The two interventions—enhanced community TB case finding and household level combined TB–HIV care—were implemented from 2006 to 2009. In Zambia, 16 communities were selected by TB notification rates in excess of 400 per 100 000 per annum and a high HIV prevalence.M. tuberculosis infection, as measured by tuberculin skin test (TST) surveys in primary school children, was highly variable, ranging from 2% to 13% across the Zambian communities and allowed for the estimation of ARI.M. tuberculosis transmission dynamics. Towards the end of ZAMSTAR, CODA sought to measure patterns of social contacts relevant to M. tuberculosis transmission and model the implied ARIs in adult age groups.M. tuberculosis transmission.ZAMSTAR (2004–2011) compared two interventions to reduce This secondary analysis used whole communities, rather than the individual, as a unit for analysis and drew on four sources of data: qualitative data collected for the purpose of the CODA study; estimates of population density from the ZAMSTAR household enumeration; estimates of ARI from ZAMSTAR TST surveys; and TB prevalence data from the final ZAMSTAR TB prevalence survey. Details of ZAMSTAR household enumeration, TST data and implied ARI estimates, TB prevalence data and linked methods have been published.M. tuberculosis ‘transmission efficiency’.We focus on the CODA qualitative data collection and analysis used for the identification of socio-spatial variables; our use of quantitative ARI and prevalence data from ZAMSTAR; and the mixed method analysis approach that investigated relationships between socio-spatial variables and calculated Following the intervention period of ZAMSTAR, Broad Brush Surveys (BBS) were conducted in 2011 across Zambian ZAMSTAR communities as part of CODA.The qualitative BBS data indicated how public places and transport were used by different age groups and genders within each community. Our focus on the broader community is justified as we understand transmission is less likely to take place within households in this particular setting.We delineated the strongest variables as broadly linked to one or more of the following aspects: (a) levels of spatial crowding, pertaining to the amount of people within shared/confined spaces and the density of physical structures; (b) the range and degree of participation in predominantly indoor recreational activity; (c) the dominance of outdoor livelihoods, for example, widespread commitment to farming or fishing dramatically shaped socio-spatial engagement in certain communities; and (d) what we have termed as ‘social permeability’, that is, the level of exchange between, or movement of, local residents and non-residents across and within geographical boundaries, with some communities clearly more socially ‘open’ to extra-community contact. Eleven qualitative indicators were identified to represent assessments of socio-spatial variability, selected on the basis of being observable (tangible) and categorical, with perceived relevance for TB transmission. The indicators were general crowding in public spaces/places; the use of overloaded and poorly ventilated transport; local residents remaining in their residential community during the day; housing density; crowding at health centres; time spent at home during the day; watching video shows; going to bars/taverns; spending time farming/ fishing; the presence of non-residents; and evidence of residents travelling out of their residential community.M. tuberculosis transmission. Karel Styblo used this ratio to help describe historical patterns in European TB epidemiology. While the values that Karel Styblo found no longer apply, the ratio remains a useful metric.As part of ZAMSTAR, TST surveys were conducted in a cohort of primary school children to calculate ARI across communities, estimating community ARIs of between 0.25%/year and 1.49%/year (using a 15-millimetre cut-off for positivity).After completing the qualitative analysis and quantitative calculations described above, we first correlated the measure of transmission efficiency with the 11 qualitative variables. Each variable was titled according to one of the four social aspects to which it most strongly indicated. For example, the variable ‘presence of non-residents’ was regarded as a strong indicator of social permeability, given the manner in which it presented in the BBS. Qualitative variables (indicators) were also ranked by a priori expectations of increasing transmission risk. For example, ‘high’>‘low’ for housing density, or ‘WMGB’>‘M’ for women, men, girls and boys spending time in bars versus only men. Previous work on these communities combining the TB prevalence results, ARI estimates and quantitative social contact data suggested men are responsible for more infections than women.10.1136/bmjopen-2020-047136.supp1Supplementary data10.1136/bmjopen-2020-047136.supp2Supplementary dataCommunity Advisory Boards (CABs) were consulted throughout the ZAMSTAR trial and formed before the implementation of interventions. In the 16 Zambian communities, CABs were formed from pre-existing Neighbourhood Health Committees (NHCs) in each community. NHCs were already representative of different geographical ‘zones’ in the trial communities, endorsed by an Act of Parliament and democratically elected. NHCs were, therefore, asked if they would like to represent public (including patient) TB and HIV interests, and sought representation across interest groups, age and gender. Although not involved in the design of the ZAMSTAR trial, CABs were closely involved from ZAMSTAR baseline research onward. Zambian research work was conducted by Zambart, a research organisation housed by the University of Zambia’s School of Medicine and with links to the London School of Hygiene and Tropical Medicine. CAB members received training in good clinical practice, research protocols and research dissemination. They were involved in the public randomisation of the trial communities and all research dissemination. Other than the CABs, representative participants involved in different components of the trial were invited to community and district dissemination events. This included BBS disseminations, carried out in the communities, using informative flyers for community members. Being a substudy of ZAMSTAR, CODA used the same CAB structures and communication channels. The community engagement lead, Musonda Simwinga, used his experience with the ZAMSTAR CABs as a platform for his own PhD on community engagement using CABs in trials, including reflections on equitable partnerships and accountability, and to develop other CAB structures for subsequent trials.23Certain communities were classified as more ‘socially permeable’ than others: socially ‘open’ to exchange across geographical boundaries and orientated toward mixing across age and gender in congregate settings. In nine communities, adults from other regions—‘outsiders’—engaged more with local community life. The presence of ‘outsiders’ appeared particularly diverse in three of these nine communities, where men, women, girls and boys visited. In contrast, there was little to no evidence of non-residents operating in the remaining six communities. In places with a strong presence of ‘outsiders’, social mixing across age groups and genders appeared widespread, although where and how this contact took place varied. For example, in community 14, bars played a greater role in bringing people together than video shows. We also distinguished between communities where both adults and children travelled out and those where mostly adults travelled out. In four communities, only men travelled out regularly. This travel could be linked to livelihood pursuits, often of an agricultural nature. Involvement in seasonal farming or fishing was particularly widespread across age and gender in four communities; in an additional seven communities, there was evidence of engagement with outdoor farming or fishing, but this was notably less intense. Residents left communities for short periods to fish and/or buy fish or tend to fields in the rainy season. There was no evidence of farming or fishing in the remaining 4 (of the 15) communities.Levels of crowding varied at health centres and in other gathering places, although the majority of communities were classified as generally ‘crowded’ by fieldworkers. The same was true for housing density: only five communities were observed to have relatively low-density housing. Importantly, public transport was often overloaded and gauged as crowded, with extended durations of travel. Ventilation during travel was ordinarily poor given that it was common practice to keep windows shut to prevent dust. In one community, panel vans with no windows were being used. Although most communities were reliant on buses, minibuses and/or vans for travel, some were substantially less dependent on public transport; residents of one community mostly travelled by foot. When assessing who remained within the geographical boundaries of their residential community during the day, communities again differed. In six communities, we found little evidence of residents remaining. Children and/or women generally remained in six of the other nine communities, but men rarely did. In three communities, local men were significantly more present.Proportionally, women generally spent more time at home . In some communities, men appeared more engaged with recreational activity. Indoor recreational activities across communities were often similar, but contact patterns differed. Children came into contact with adults at drinking places and/or at pool table clubs in seven of the communities, while in the others this was far less evident. In three communities, only men were ever seen at bars. In certain communities, women were observed with babies strapped to their backs and boys were frequently trading in eggs and/or bottles, highlighting the multifunctionality of drinking places in these areas. Bars and taverns were generally crowded and poorly ventilated, with limited small windows designed to deter theft. Watching films in small makeshift tented ‘theatres’ was a popular leisure activity in 14 communities. Shows were held in small outdoor structures, erected using poles and tarpaulin, measuring approximately 4×2.5 m. These structures were darkened as much as possible to enhance viewing, by closing gaps in the tarpaulin, which in turn limited ventilation. Audiences were ordinarily seated on a few tightly packed benches. Children watched video shows during the day, and in the evening, adults and children often watched together. All communities showed widespread commitment to their local churches. Community members of all ages spent a substantial proportion of their time at services ranging between 2 and 6 hours, which often exceeded the time spent on many of the other contact activities identified. It should be noted that most community residents are Christian and the fieldwork was conducted in the period leading up to Easter.Population density and qualitative assessments of crowding were broadly concordant, given the small sample size (number of communities). Communities sharing higher transmission efficiency could generally be classified as more crowded, whereas communities with lower transmission efficiency tended to present in more of a mixed way (across lower and higher degrees of crowding). By tracing an individual community (number) through M. tuberculosis transmission in unique and sociologically complex ways. Communities with lower measures of transmission efficiency included, but were not limited to those in which fishing and/or farming were particularly dominant across gender and age; where public transport was more ventilated and less overloaded; where less crowding took place in public space; where fewer ‘outsiders’ were present; where only men mostly travelled out of their community; where there was negligible evidence of indoor recreational activity; and where population and housing density was lower. Links between some of these socio-spatial aspects and M. tuberculosis transmission risk have been shown, using different approaches, in other sub-Saharan settings. In Malawi, Odone et al identified that TB was more common among those working in the cash economy than those in subsistence economy,25–27This interdisciplinary analysis suggests that certain communities may have been protected from M. tuberculosis and local aerosol transmission in poorly ventilated spaces. ‘Social permeability’ not only reflects the presence of non-residents within a community, but it also captures the reach of ‘mixing’ across age and gender, and patterns of locals travelling out. The latter provides additional opportunity for extra-community M. tuberculosis exposure and infection (or re-infection), outside of the residential community. It seems that this dual dynamic of spatial ‘closeness’ and social ‘openness’ is relevant to increased M. tuberculosis transmission across the communities.From a distinctly qualitative angle, our work begins to unveil and define how the sociological topography of a particular community could play an important role in shaping transmission efficiency. Two meta-observations are significant in this regard. The first observation relates to the relative vulnerability of the communities with more outwardly orientated and socioeconomically diverse features in comparison to their more socially secluded and/or agriculturally focused counterparts. Certain communities exhibited a dual dynamic of crowding and ‘social permeability’, which could be providing increased opportunity for the importation of M. tuberculosis exposure. Routine variables refer to habits or patterns of behaviour, which, although related to socioeconomic factors, are not obviously reducible to them. Watching video shows is a good example; it is a form of routine leisure activity which carries a particularly high presumptive risk for M. tuberculosis transmission because of the darkened, poorly ventilated and contained space in which it is conducted. For health policy, our distinction between ‘structural’ and ‘routine’ variables is important because the latter would be more malleable to intervention. While structural dependency on the outside world cannot be altered easily, people can be encouraged to change the way in which they conduct routine leisure activities; for instance, a concerted effort could be made to increase ventilation in these places.28The second observation relates to a distinction that needs to be drawn between what we could call ‘structural’ and ‘routine’ variables. By structural variables, we refer to socioeconomic factors that are to a certain extent beyond people’s agency. For instance, communities that are more structurally dependent on exchange and interaction with outsiders could be more vulnerable to M. tuberculosis transmission risk in communitiesOur secondary analysis has limitations. Findings are context specific and data were effectively cross-sectional. We focused on interactions within localised geographical boundaries, and did not capture interactions on the far side of extra-community travel. Furthermore, fieldwork was conducted at a particular time of year and observations took place during the daytime, which limits the generalisability of our observations accordingly—not only across but also within the study communities. We anticipate relevant socio-spatial features and their role in mediating TB transmission will vary between contexts. However, the dual dynamic of spatial closeness (crowding) and social openness (permeability) may be relevant in other settings, with further investigations valuable. The addition of pathogen genomic data might help generate or eliminate hypotheses on transmission location or context, and recent research illustrates that quantitative measures of geographic mobility could be applied to studies of social permeability.M. tuberculosis transmission. Interactions between social permeability and crowding appear relevant to increased transmission efficiency, at least in these Zambian communities. A conceptual framework differentiating structural and routine elements of high-risk socio-spatial features could provide a valuable foundation for the development of contextualised public health interventions. For example, some socio-spatial aspects carrying transmission risk are easily adjusted , while more structural aspects are less malleable . The significance of social permeability could be further explored by integrating community level typologies with genome sequencing techniques able to map strain introduction events and community transmission chains.Localised sociological distinctions offered an explanation of why certain communities might have been more (or less) vulnerable to


Triticum aestivum) and barley. Both diseases are predominately caused by the pathogenic fungi, Fusarium graminearum and Rhizoctonia cerealis. The roles of the wheat-wall-associated kinases (WAKs) in defense against both F. graminearum and R. cerealis have remained largely unknown. This research reports the identification of TaWAK2A-800, a wheat WAK-coding gene located on chromosome 2A, and its functional roles in wheat resistance responses to FHB and sharp eyespot. TaWAK2A-800 transcript abundance was elevated by the early infection of R. cerealis and F. graminearum, or treatment with exogenous chitin. The gene transcript seemed to correspond to the resistance of wheat. Further functional analyses showed that silencing TaWAK2A-800 compromised the resistance of wheat to both FHB (F. graminearum) and sharp eyespot . Moreover, the silencing reduced the expression levels of six defense-related genes, including the chitin-triggering immune pathway-marker genes, TaCERK1, TaRLCK1B, and TaMPK3. Summarily, TaWAK2A-800 participates positively in the resistance responses to both F. graminearum and R. cerealis, possibly through a chitin-induced pathway in wheat. TaWAK2A-800 will be useful for breeding wheat varieties with resistance to both FHB and sharp eyespot.Fusarium head blight (FHB) and sharp eyespot are important diseases of the cereal plants, including bread wheat ( Triticum aestivum) is one of the most important staple food crops in the world. However, wheat production is threatened in many areas by devastating pests and pathogens [Fusarium fungi, which may be dominated by Fusarium graminearum [Fusarium species may contain high levels of mycotoxins, which threaten the health of human beings and animals [Rhizoctonia cerealis is a necrotrophic fungal pathogen responsible for the sharp eyespot, which is a devastating disease of wheat and other cereal plants in some countries of the world, especially in China [F. graminearum and R. cerealis, it is important to isolate the pivotal resistance QTL/genes.Bread wheat  domain, a transmembrane domain, and a carboxyl (C)-terminal cytosolic serine/threonine kinase domain [Arabidopsis thaliana, the WAK gene, AtWAKL22, participates in resistance to Fusarium oxysporm [Zea mays), qHSR1 and Htn1, encoding two distinct WAK proteins, mediate the resistance of corn to head-smut disease and northern corn leaf blight, respectively [Oryza sativa), the WAK-encoding gene, Xa4’s, high-expression improves resistance to bacterial blight disease [WAK-encoding gene Stb6, isolated by the map-based cloning method, confers wheat resistance to Septoria tritici blotch disease caused by Zymoseptoria tritici [TaWAK6, another wheat WAK gene located on chromosome 5B, participates in adult plant resistance against wheat leaf rust [TaWAK2A-800). The relative transcript level of the TaWAK2A-800 gene in FHB-resistant wheat lines was significantly higher than in the susceptible wheat lines. After inoculation with F. graminearum, three TILLING mutant lines  presented more severe FHB symptoms [TaWAK2A-800 might have potential broad-spectrum resistance traits in wheat against other pathogens.In plants, receptor-like kinases (RLKs) are involved in various life processes, such as growth, development, plant-pathogen interactions, and responses to environmental stimuli . As a cle domain . The exte domain . Increasoxysporm . In cornectively ,14. In r disease . In whea tritici . Additioeaf rust . Recentlsymptoms . HoweverTaWAK2A-800 participates in broad-spectrum resistance against both fungal pathogens, comparative transcriptomics and reverse transcription quantitative PCR (qRT-PCR) were used to test the TaWAK2A-800 gene transcript profiles in the wheat response to R. cerealis. Moreover, we further dissected the functional role of TaWAK2A-800 in wheat defense against R. cerealis and F. graminearum infection. After R. cerealis and F. graminearum infection, TaWAK2A-800 was significantly induced in the R. cerealis-resistant wheat cultivar, CI12633, and the F. graminearum-resistant cultivar, Sumai 3. The functional assay results show that the expressed TaWAK2A-800 is required not only for wheat resistance to both sharp eyespot and FHB, but also for the expression of the receptor-like kinase-coding gene, TaCERK1, the wheat receptor-like cytoplasmic kinase-coding TaRLCK1B, and the wheat mitogen-activated protein kinase-coding TaMPK3, defensin, chitinase3, and β-1,3-glucanase in wheat. This study reveals the function of TaWAK2A-800 in the broad-spectrum resistance of wheat to both sharp eyespot by R. cerealis, and FHB by F. graminearum.To explore if R. cerealis strain, RC207, at 4 and 10 days, and also mock, the gene, TaWAK2A-800 (sequence with ID TraesCS2A02G071800.1), was significantly higher in the resistant RIL pool (RIL-R) than in the susceptible RIL pool (RIL-S) at the same inoculation time points , inoculation with the e points A.TaWAK2A-800 in the wheat response to the pathogen, R. cerealis, by qRT-PCR. The analyses showed that the TaWAK2A-800 gene transcript was rapidly induced in the sharp-eyespot-resistant wheat cultivar, CI12633, at day 1, post inoculation (dpi) and 2 dpi, and reached a peak at 2 dpi   C. This s Sumai 3 D, similaTaWAK2A-800 genomic sequence was cloned from DNA taken from the stems of the wheat cultivar, Wenmai 6. According to a BLAST analysis against the hexaploid wheat genome sequence, the full-length cDNA coding sequence of TaWAK2A-800 contains a complete open reading frame (ORF) with 2151 bp, and the gene locates on the short arm of wheat chromosome 2A. A comparison of the cDNA and genomic sequences indicates that the TaWAK2A-800 genomic sequence contains four exons and three introns , with a 2139 bp ORF. A pairwise comparison showed that the ORF and the amino acid sequences of TaWAK2A-800 shared 93.18% and 89.42% identities, respectively, with those of TaWAK-2D-600.The  introns A. The de introns B. All thA. thaliana, cotton (Gossypium hirsutum), Craterostigma plantagineum, Capsicum annuum, and Solanum lycopersicum. The phylogenetic tree indicated that TaWAK2A-800 with Xa4 and OsWAK1 from rice, TaWAK5, TaWAK6, and TaWAK (Snn1) from wheat, and ZmWAK (encoded by qHSR1) from maize, were clustered on the same clade. Other WAKs were clustered into the second clade, including GhWAK7A from cotton, AtWAK1, AtWAKL10 and AtWAKL22 from A. thaliana, OsWAK91 and OsWAK25 from rice, CaWAKL20 from Capsicum annuum, CpWAK1 from C. plantagineum, TaWAK7D from wheat, and SIWAK1 from Solanum lycopersicum. Meanwhile, TaWAKL4 (Stb6) and ZmWAK-RLK (Htn1) were clustered into the third clade  in the resistant wheat cultivar, Sumai 3, via the barley yellow dwarf virus (BSMV)-mediated VIGS method, and then the disease severities of these plants were assayed. Briefly, Si-Fi software prediction showed that the 172 bp fragment specific to TaWAK2A-800 (To investigate whether the role of AK2A-800  was screCP) in the wheat plants transfected by BSMV:GFP, or BSMV:TaWAK2A-800, at 15 days could be detected , were injected into the spikelets of the spikes of the BSMV-transfected Sumai 3 using a syringe. At 20 dpi with F. graminearum, the FHB symptoms in the TaWAK2A-800-silenced Sumai 3 plants were more severe than in BSMV: GFP-transfected Sumai 3 (control) plants  plants D. AccordTaWAK2A-800 expression is also required for the innate immunity of the wheat plant to R. cerealis, TaWAK2A-800 transcript was knocked-down by VIGS in the R. cerealis-resistant wheat cv. CI12633. Fifteen days post the virus transfection into the wheat leaves, the mild chlorotic mosaic symptoms and CP of the BSMV transfection could be observed on newly emerged leaves  displayed on the sheaths and stems of the TaWAK2A-800-silenced CI12633 plants but displayed, to a lesser extent, on the sheaths and stems of the BSMV: GFP-treated (control) CI12633  on the stems of the TaWAK2A-800-silenced wheat cultivar CI12633 plants were between 2.50 and 2.89  wheat plants, the transcript levels of three wheat defense-marker genes, including defensin, chitinase3, and β-1,3-glucanase, were significantly reduced in TaWAK2A-800-silenced wheat plants after R. cerealis inoculation  plants  plants B. These TaWAK2A-800, with broad-spectrum resistance against R. cerealis and F. graminearum. TaWAK2A-800 belongs to the WAK subfamily, but its protein sequence is distinct from those of the five reported wheat WAKs, including TaWAK5, TaWAK6, TaWAK7D, TaWAK (Snn1), and TaWAKL4 (Stb6). The deduced protein sequence of TaWAK2A-800 contains one signal peptide, one galacturonan-binding GUB domain, one EGF-like domain, one EGF-CA domain, one transmembrane region, and a non-RD kinase domain. Among various types of RLKs, non-RD RLKs predominantly bind to native immune receptors, recognize conserved microbial characteristics, activate PTI, and even confer disease resistance [In Arabidopsis, rice, maize, cotton, and wheat, some WAK/WAKL proteins were reported to have an important function in resistance responses to the bacterial or fungal pathogens ,24,25,26sistance . For inssistance , ZmWAK [sistance , Xa4 [15sistance , all conTaWAK2A-800 in wheat was significantly upregulated by F. graminearum infection, or in the early infection stage (1–2d), of the pathogen R. cerealis. Intriguingly, after inoculation with R. cerealis for 2 d, the transcript level of TaWAK2A-800 was significantly higher in R. cerealis-resistant wheat cultivars, Shanhongmai and CI12633, than in the susceptible wheat cultivars, Yangmai 9 and Wenmai 6. Importantly, knocking-down TaWAK2A-800 impaired the resistance of wheat to both sharp eyespot caused by R. cerealis infection, and FHB caused by F. graminearum infection. A previous study reported that TaWAK2A-800 was responsible for FHB-resistance QTL in wheat [TaWAK2A-800 in the wheat immune response to sharp eyespot. Further, the current study reveals a potentially conserved mechanism of wheat resistance against F. graminearum and R. cerealis.Further, qRT-PCR analyses showed that, compared with nontreatment, the expression level of in wheat . Herein,PR-encoding genes, including Defensin, Chitinases, and β-1,3-Glucanase, contribute to the resistance of wheat to sharp eyespot [TaWAK2A-800 in the defense response against both pathogens, we examined the transcripts of Defensin, Chitinase 3, and β-1,3-Glucanase in BSMV: TaWAK2A-800-silenced wheat, and the BSMV:GFP-transfected wheat plants, inoculated with R. cerealis or F. graminearum. The results show that, compared with BSMV:GFP-transfected wheat plants, the transcript levels of the wheat, Defensin, Chitinase 3, and β-1,3-Glucanase were lower in the more susceptible TaWAK2A-800-silenced wheat plants after inoculation with R. cerealis or F. graminearum. The data suggest that TaWAK2A-800 might indirectly activate the expression of the above defense-marker genes in wheat resistance responses against R. cerealis or F. graminearum.Previous studies have shown that three types of  eyespot ,29. To uPBL27, the chitin-dependent activation of the MAPKs (MPK3 and MPK6) was markedly reduced [Verticillium dahliae and Fusarium oxysporum f.sp. vasinfectum [TaWAK2A-800 transcript level was significantly induced by chitin treatment. Moreover, compared to the control wheat plants, the transcript abundance of the chitin-induced immune pathway marker genes, TaCERK1, TaRLCK1B, and TaMPK3, were reduced in TaWAK2A-800-silenced wheat plants after inoculation with R. cerealis and F. graminearum. These results suggest that TaWAK2A-800 contributes to the wheat defense responses to R. cerealis and F. graminearum, possibly through a chitin-induced defense pathway. Similarly, the transcripts of rice OsWAK90/91 were upregulated after chitin treatment, and boosted resistance against rice blast caused by Magnaporthe oryzae [Chitin, a typical pathogen-associated molecular pattern (PAMP) on the fungal cell wall, can trigger the plant innate immune response by activating PAMP-triggered immunity (PTI) in many plant species . In Arab reduced . In cottinfectum . In thise oryzae .R. cerealis-resistant CI12633 , and Shanhongmai , R. cerealis-susceptible Yangmai 9 (Prof. Jizeng Jia), and Wenmai 6  [TaWAK2A-800. The sharp-eyespot-resistant and -susceptible recombinant RILs (derived from Shanhongmai and Wenmai 6 cross) were used for RNA-sequencing, and RNA-sequencing analyses were performed as described by Guo et al. [R. cerealis-resistant cultivar, CI12633, and the F. graminearum-resistant cultivar, Sumai 3 , were used to detect the defense role of TaWAK2A-800 against R. cerealis and F. graminearum by the virus-induced gene-silencing (VIGS) experiment, respectively. The fungal pathogenic R. cerealis isolate, WK207, and the F. graminearum isolate, F0609, were provided by Prof. Jinfeng Yu  and Prof. Miaoping Zhou , respectively.Four wheat cultivars/varieties, including , China) , were uso et al. . The R. R. cerealis. Then, the wheat plants were grown in a growth chamber at 22 °C and high humidity during the first 14 days. To prepare conidial suspensions for inoculation, F. graminearum mycelia were collected from potato agar plates, then transferred into liquid mung bean medium, and cultivated at 25 °C for 3 d with shaking at 150 rpm. The conidial suspension was filtered, centrifuged, resuspended, and adjusted to 100/μL in sterile water. At the anthesis of Sumai 3, the suspension was further injected into a spikelet of spike using a syringe. Then, these spikes of Sumai 3 were placed in a plastic moist chamber, with high humidity, at 22 °C for 2 days. The RNA-sequencing-based transcriptomics of TaWAK2A-800 (All wheat plants were grown in a glasshouse in 14-h light/10-h dark (22 °C/12 °C) conditions. At the tillering growth stage, these wheat plants  were further inoculated on each stem base with small toothpicks harboring the well-developed mycelia of AK2A-800  were subChitin was suspended in sterile water and used at a final concentration of 100 μg/mL . Wheat sTaWAK2A-800 gene sequence was cloned from the wheat cultivar Wenmai 6 leaves based on the means of two rounds of nest PCR reactions by gene-specific primer  following the above sequence. TraesCS2A02G071800.1 was downloaded from the Chinese Spring V1.0 genome . The deduced protein sequence of TaWAK2A-800 and the phylogenetic tree were obtained and constructed as described by Guo et al. [Total plant RNA extraction was performed following Zhang et al. . This Tao et al. .TaWAK2A-800, and its specificity and efficient siRNA hits, were predicted by the Si-Fi software . The specific fragment of TaWAK2A-800 was amplified from the cDNA sequence for TaWAK2A-800 using a gene-specific primer  from the CI12633, and inserted in an antisense orientation into the site of the RNAγ of the part of the BSMV to generate a BSMV: TaWAK2A-800 construct. Following Zhu et al. [F. graminearum isolate, F0609, or the R. cerealis isolate, WK207, at 15 days transfected with BSMV, and then scored for disease symptoms at 20 and 35 days post the fungal inoculation.The VIGS fragment (172 bp) of u et al. , the mixu et al. ,39, the TaWAK2A-800 , TaRLCK1B [TaCERK1 , TaMPK3 [Defensin [Chitinase 3 [β-1,3-Glucanase [−ΔΔCT method [TaActin). Three independent replications were performed in each test.The transcriptional levels of TaRLCK1B , TaCERK1, TaMPK3 , DefensiDefensin , Chitinatinase 3 , and β-1lucanase  in wheatlucanase , the qRTT method  was usedTaWAK2A-800 was expressed to a higher extent in the R. cerealis-resistant wheat accessions than in the susceptible wheat ones. The gene transcript abundance is induced after F. graminearum infection and chitin treatment. The expressed TaWAK2A-800 is required for the resistance of wheat to R. cerealis and F. graminearum by promoting the expression of TaCERK1, TaRLCK1B, TaMPK3, Defensin, Chitinase 3, and β-1,3-Glucanase. This work reveals a novel role for the wheat TaWAK2A-800 gene in wheat plant immunity against pathogens. TaWAK2A-800 is a potentially important gene for improving the broad-spectrum resistance of wheat to R. cerealis and F. graminearum.


Sarcoidosis is a chronic granulomatous disease of unknown etiology. A variety of studies have pointed out that almost every part of the body can be affected, but it most often affected the lungs and intrathoracic lymph nodes. However, cases of sarcoidosis involving multiple organs in one patient are rarely reported. We describe a unique case of sarcoidosis, which was characterized by multiorgan involvement, including leg ulcers, splenomegaly, pancytopenia, and polyserositis. Glucocorticoids were effective during the treatment of the above lesions. This case highlights the diversity of clinical manifestations of sarcoidosis and emphasizes the importance of its differential diagnosis and the periodical follow-up. These are crucial to physicians in the diagnosis and treatment of sarcoidosis.A 30-year-old male complained about intermittent fever 3 years ago. A computed tomographic scan of the chest showed lymphadenopathy in the mediastinum and hilar regions. Routine blood tests showed leukopenia and mild anemia. The pathologic result of mediastinal lymph node biopsy was granulomatous lesions; thus, the patient was diagnosed with type II sarcoidosis without glucocorticoid therapy. In the following 2 years, the patient suffered from intermittent fever accompanied by dyspnea, fatigue, occasional cough, less sputum, and apparent weight loss. Abnormal physical examinations included leg ulcers and splenomegaly. Laboratory and physical tests revealed pancytopenia, polyserositis, and enlargement of lymph nodes. The pathological findings of leg ulceration, pleura, and left supraclavicular lymph node all suggested granulomas.It strongly suggested sarcoidosis since tuberculosis, lymphoma, and connective tissue disease were all excluded. Due to severe conditions and multiorgan involvement, we tried to provide methylprednisolone for this patient. After 9 months of oral glucocorticoids therapy, his subjective symptoms as well as hematological and radiological findings were all improved. His leg skin ulceration and scab were also completely disappeared.Sarcoidosis has diverse clinical presentations, and many patients present with atypical symptoms. It needs to be timely identified by the clinician and carefully differentiated from other diseases with similar findings so as to make an accurate diagnosis. In this case, the patient had a poor clinical response to glucocorticoids in the early stage of treatment due to the severe condition and multi-organ involvement. It is worth noting that the patient had improved significantly after 9 months of treatment of corticosteroids, which suggested that follow-up is critical. Sarcoidosis results from non-caseating granuloma formation due to ongoing inflammation, the latter of which causes the accumulation of activated T cells and macrophages. There are three criteria for the diagnosis of sarcoidosis: 1) a compatible clinical and radiologic presentation, 2) pathological evidence of non-caseating granulomas, and 3) exclusion of other diseases with similar findings such as infections  and malignancy . Almost all organs can be affected by sarcoidosis, including the lung, eyes, lymph nodes, skin, spleen, liver, kidney, abdomen, heart, and bone marrow , and the spleen was larger than before; thus, we continued to maintain the current dosage (24 mg once daily). After another months' treatment, the right pleural effusion was decreased slightly, then the dose of methylprednisolone was reduced to 16 mg once daily. Six months later, a follow-up HRCT showed that the bilateral pleural effusion was significantly less than before . HepaticThe strength of our study is that it is the first case of sarcoidosis with systematic manifestations, including skin, spleen, pleura, lymph node, and abdomen manifestations, which were probably sensitive to glucocorticoid therapy. However, our study also had a limitation. We lack pathological evidence of abdominal organ involvement, such as the peritoneum, spleen, liver, and bladder. Furthermore, there was no significant improvement in splenomegaly, which may be related to the insufficient courses of treatment or the overlapping presentations. Glucocorticoids have been reported to be the first-line treatment for sarcoidosis. But our patient is at high risk of side effects with glucocorticoid therapy because of diabetes history. Conventional and biological immunosuppressive drugs such as methotrexate and tocilizumab have been studied for their glucocorticoid-sparing properties to reduce glucocorticoid exposure and maintain remission . TherefoPulmonary involvement can be seen in more than 90% of patients with sarcoidosis. Almost all organs can be affected by sarcoidoses, such as the spleen, liver, kidney, abdomen, heart, and bone marrow. However, the eyes, lymph nodes, and skin are the most commonly involved organs –8. Our sCutaneous involvement occurs in nearly one-third of sarcoidosis patients , and theCorticosteroids have been reported to improve the symptoms of sarcoidosis in all organs. Our patient showed a good response to methylprednisolone therapy for his skin ulcers. In our review of ulcerative sarcoidosis, many types of treatments have been reported, such as skin grafts, antimalarials (chloroquine or hydroxychloroquine), topical corticosteroids, anti-ulcer cream, immunosuppressive drugs (azathioprine or cyclosporine), and biologic agents . However, failures and side effects were also accompanied by the above treatments, including corticosteroid-induced hyperglycaemia and methotrexate- or infliximab-induced organ injury. Therefore, it is essential for us to pay attention to the response and the side effects of drug treatment depending on the patients' conditions.N = 3; fatigue N = 7; weight loss N = 9). Serum angiotensin-converting enzyme (ACE) and serum calcium levels were elevated in 68 and 23% of cases, respectively. In addition, sIL-2R and liver enzymes were elevated in several patients. Therefore, measurement of the above marker levels, even though not diagnostic, may be helpful to monitor activities and evaluate the therapeutic effect. Moreover, of the 23 cases, including our patient, 16 cases had haematologic abnormalities . Several putative mechanisms may explain haematologic abnormalities in sarcoidosis: hypersplenism, bone marrow involvement, and autoimmune destruction, such as immune thrombocytopenia (ITP) and lymphoma  for the publication of any potentially identifiable images or data included in this article.XQ and SH drafted the case report. AA, YY, and W-yL performed the biopsy and language modifying. JK, YY, and Q-yW revised the report. All authors contributed to the article and approved the submitted version.This study was supported by the National Clinical Key Specialty Project Foundation (Project Number: 2016YFC 1304103 and 2016YFC 1304500).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Transactivation response DNA binding protein 43 kDa (TDP-43) is known to be a pathologic protein in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). TDP-43 is normally a nuclear protein, but affected neurons of ALS or FTLD patients exhibit mislocalization of nuclear TDP-43 and cytoplasmic inclusions. Basic studies have suggested gain-of-neurotoxicity of aggregated TDP-43 or loss-of-function of intrinsic, nuclear TDP-43. It has also been hypothesized that the aggregated TDP-43 functions as a propagation seed of TDP-43 pathology. However, a mechanistic discrepancy between the TDP-43 pathology and neuronal dysfunctions remains. This article aims to review the observations of TDP-43 pathology in autopsied ALS and FTLD patients and address pathways of neuronal dysfunction related to the neuropathological findings, focusing on impaired clearance of TDP-43 and synaptic alterations in TDP-43-related ALS and FTLD. The former may be relevant to intraneuronal aggregation of TDP-43 and exocytosis of propagation seeds, whereas the latter may be related to neuronal dysfunction induced by TDP-43 pathology. Successful strategies of disease-modifying therapy might arise from further investigation of these subcellular alterations. Transactivation response DNA binding protein 43 kDa (TDP-43) is known to be a pathologic protein in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) ,2,3. TDPALS encompasses a sporadic or familial motor neuron disease, which is clinically characterized with upper and lower motor neuron signs and symptoms . The musFTLD is the pathological term corresponding to the clinical term of frontotemporal dementia (FTD) . FTD is There is a loss of upper and lower motor neurons in ALS patients, which results in regional atrophy. Atrophy of the anterior roots is the most informative finding upon macroscopic observations, whereas that of the primary motor cortex is usually mild . TDP-43 In FTLD, TDP-43 and tau each accounts for nearly 50% . FTLD-TDALS-TDP and FTLD-TDP may overlap clinically or pathologically . ALS-TDPThese ambivalent concepts may have arisen from the double face of TDP-43 pathology: cytoplasmic aggregation and mislocalization from the nucleus. It is broadly believed that the cytoplasmic inclusions of TDP-43 are neurotoxic. Neuronal death or axonal dysfunction has been observed in models with overexpression of TDP-43  and cellNeuropathologic analyses of autopsied patients with ALS-TDP and FTLD-TDP have indicated that TDP-43 pathology spreads along certain neurally connected anatomical systems rather than depending on spatial proximity. Direct or indirect corticofugal spreading of TDP-43 pathology from the primary motor cortex to the lower motor neurons has been suggested in ALS-TDP patients ,43. In FPremortem neurophysiological studies have supported a corticofugal manner in the spreading of ALS lesions. Studies of transcranial magnetic stimulation revealed that reduction in short-interval intracortical inhibition precedes lower motor neuron dysfunction among ALS patients, indicating early impairment of intracranial circuit within the primary motor cortex . StructuC9orf72 repeat expansion [Other patterns in the spreading of TDP-43 pathology have also been suggested. A neuropathological study suggested that the dentate gyrus of the hippocampus is a starting point for TDP-43 pathology in ALS-TDP and FTD-MND; the pathology may subsequently spread to the anterior olfactory nucleus, the periamygdaloid complex, the piriform cortex and eventually reach the orbital cortex and olfactory bulb ,54. Morexpansion .Molecular biological studies have revealed propagative properties of TDP-43 aggregates. Full-length TDP-43 contains NLSs, whereas the C-terminal fragments do not . TherefoUltrastructural observations of autopsied ALS patients revealed degradation of axon terminals in the motor neuron system, associated with alterations of pre-synaptic vesicles, mitochondria, and neurofilament bundles ,58. We hStudies using models of increased expression of TDP-43 have reported a loss of axon terminal or dendritic spines. It has also been suggested that TDP-43 is physiologically transported within the axons and contributes to axonal outgrowth . Mice ovAggregated TDP-43 is ubiquitylated and tagged with p62, which indicates a contribution of ubiquitin-proteasome and endosome-autophagy systems to process the aggregates. The role of p62 would be to guide the ubiquitylated proteins towards the autophagy system . A neuroSQSTM1 (p62), UBQLN2, VCP, GRN, and OPTN, are important players in the autophagy system [GRN gene cause neuronal ceroid lipofuscinosis, which is characterized by lysosomal storage of lipopigment [C9orf72 correlated to disruption of autophagy, particularly of endosomal trafficking [C9orf72-mutated patients frequently and broadly displayed granulovacuolar degeneration (GVD) containing an accumulation of CHMP2B (charged multivesicular body protein 2B) [CHMP2B gene is also known to cause familial FTD, although the postmortem findings remain unknown [C9orf72 mutation [ALS-TDP- or FTLD-TDP-related genes, including y system . Indeed,opigment . Haploinfficking . Upon outein 2B) . CHAMP2Btein 2B) ,79. A mu unknown . A recenmutation .It is well known that chaperone proteins are critical to regulating the folding or solubility of pathologic proteins in motor neuron diseases and other neurodegenerative disorders ,83. A coSOD-1G93A models and sporadic ALS patients, and drug-derived inhibition of cyclophilin-A reduced nuclear factor kappa B (NF-κB) activation, endoplasmic reticulum stress, and insoluble phosphorylated TDP-43 [In addition to the intraneuronal machinery, neuroinflammation toward TDP-43 aggregation has been emphasized. A study revealed high extracellular expression of a neuroinflammatory marker, cyclophilin-A, in CSF from d TDP-43 . From thd TDP-43 ,91. Impad TDP-43 , whereasLRRK-2 mutated Parkinson disease [Postmortem studies have demonstrated that TDP-43 pathology is observed in a subset of non-ALS/FTLD disorders, including Alzheimer disease (AD) ,95, PSP  disease , and pos disease . Subpopu disease .The prevalence of TDP-43 pathology is particularly high in AD patients and accounts for up to 75% ,95. ImpaC9orf72-repeat expansions tended to have a higher burden of tau aggregations in the temporal cortex and hippocampus than GRN-mutated patients [+1) is highly expressed in the hippocampus or cerebral cortices, and dyshomeostasis of the ubiquitin-proteasome system is suggested [Additionally, abnormalities in tau protein have also been discussed in ALS-TDP patients. Aggregation or hyperphosphorylation of tau has been observed in the motor neurons of ALS-TDP patients ,109. It patients . Howeverpatients . ALS/PDCpatients . Althouguggested .microtubule-associated protein tau (MAPT) gene [C9orf72 has recently been reported to be a risk factor for CBD [The coexistence of TDP-43 and tau pathologies suggests overlapped mechanisms between these two major groups among neurodegenerative disorders. Our observations revealed that the hippocampal and cortical neurons of autopsied patients with ALS/FTLD-TDP, ALS-FUS, PSP, and CBD showed aberrant interaction of two intranuclear proteins, namely, FUS and splicing factor proline/glutamine-rich proteins (SFPQ) ,115. FUSPT) gene . Genome PT) gene ,118. Int for CBD . Upon po for CBD ,120. It Recent studies have revealed correlations between TDP-43 abnormalities and impairment of some cellular substructures. We focused on the involvement of the endosome-autophagosome system and synaptic integrity as key actors in the pathogeneses of TDP-43 proteinopathy from the viewpoint of translational approaches across neuropathological and basic investigations. The concept of multiple proteinopathies, including interactions between TDP-43 and tau, suggests a pathophysiological link at a very early stage before protein aggregation. Although the whole pathway leading to neuronal dysfunction remains unclear, successful strategies of disease-modifying therapy may arise from those results.


PGRN (progranulin). To date, depletion of parkin E3 ubiquitin protein ligase, a key mitophagy regulator, has been reported in sporadic ALS patients and ALS mice models with TDP-43 proteinopathy. In this work, we show parkin downregulation also in fibroblasts derived from FTLD patients with four different PGRN pathogenic variants. We corroborate this finding in control fibroblasts upon PGRN silencing, demonstrating additionally the decrease of parkin downstream targets, mitofusin 2 (MFN2) and voltage dependent anion channel 1 (VDAC1). Importantly, we show that TDP-43 overexpression rescues PRKN levels upon transient PGRN silencing, but not in FTLD fibroblasts with PGRN pathogenic variants, despite upregulating PGRN levels in both cases. Further observation of PRKN downregulation upon TDP-43 silencing, suggests that TDP-43 loss-of-function contributes to PRKN decrease. Our results provide further evidence that parkin downregulation might be a common and systemic phenomenon in neurodegenerative diseases with TDP- 43 loss-of-function.Frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) are neurodegenerative diseases with TDP-43 mislocalization and aggregation. Genetic forms of FTLD and ALS are caused by pathogenic variants in various genes, such as  Mitochondrial dysfunction is one of the common mechanisms underlying the pathogenesis of many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) . The maivia (a) the ubiquitin-proteasome system which marks mitochondrial outer membrane proteins for disposal, and (b) autophagy, which eliminates mitochondria as whole organelles through lysosomal pathway  with mitochondrial dysfunction. Importantly, PD-related symptoms (parkinsonisms) are found in FTLD with PGRN pathogenic variants (FTLD-PGRN)  . On the ral FTLD . Interespatients  and cellpatients . The depinopathy . To dateinopathy .SOD1 vs. PGRN pathogenic variants. Indeed, PGRN pathogenic variants, apart from leading to TDP-43 pathology in affected brain tissue, confer a particular landscape of molecular changes including, among others, deregulation of the lysosomal pathway   . Two fibPGRN silencing was predesigned . The siRNA sequence (sense: 5′-GCAAAGCCAAGAUGAGCCU-3′) was used to silence human TDP-43  and Lipofectamine RNAiMAX (Thermofisher Scientific) as per the manufacturer’s instructions. siRNA used for n TDP-43 , while aFor overexpression experiments, 0.5–2 μg of plasmids  or pFlag-TDP-43 F4L, a mutant unable to bind RNA)  were tra® Reverse Transcription kit (Qiagen), respectively. Quantitative real time PCR analysis was done with RT HS-PCR Mix SYBR (A&A BIOTECHNOLOGY) (primers are listed in PPIB) or GAPDH gave similar results.RNA was extracted and reverse transcribed according to standard protocol with TriReagent (Ambion) and QuantiTectCultured primary fibroblasts was lysed in Cell Lysis Buffer , and 10–30 μg of proteins in Laemmli buffer were resolved by SDS-PAGE and electro-transferred to PVDF membranes. Western blot (WB) assays were performed using antibodies specific for MFN2 (1:300), VDAC1 (1:300), FLAG (1:500), TDP-43 (1:300) and Beta-actin  , TDP-43 C-terminal , PGRN , parkin , and GAPDH . Proteins on membranes were visualized by chemiluminescence reagent (Biorad) and autoradiography.n ≥ 3) were used to calculate mean, standard deviations (SD), and statistical significance in two-tailed t-test (GraphPad Prism 6.0). p < 0.05 was considered significant. For quantitative densitometric analysis of WB results, ImageJ software was used  and in control fibroblast line (C3), overexpression of Flag-TDP-43 WT for 24 h was not able to upregulate PRKN levels  while itHowever, when we overexpressed Flag-TDP-43 WT for longer times—48 h—we observed downregulation of PRKN levels compared to Flag-TDP-43 F4L mutant or GFP overexpression, both in fibroblast line with PGRN pathogenic variant (Q337X – P3) and in control line (C3) .PGRN pathogenic variants  and those with PGRN pathogenic variants , while PGRN levels remained unchanged , compared to controls. This result suggests that parkin depletion occurs early in PGRN deficiency, preceding symptomatic phase. However, this was not accompanied by changes in MFN2 and VDAC1, parkin dowstream targets.PGRN silencing for 48 h (SOD1 pathogenic variant) characterized by TDP-43 proteinopathy.We further confirmed that parkin protein and mRNA levels dropped upon for 48 h , suggestPGRN silencing in control, neurologically healthy, age-matched fibroblasts we observed downregulation of parkin partners such as MFN2 and VDAC1  could have masked existing differences.While upon transient nd VDAC1 , such chnd VDAC1 . These dC9orf72, TARDBP, FUS, and SOD1 genes) indicating that fibroblast model reflects the changes observed in human brain to some extent  without significantly modifying PGRN levels . We summOur observation that parkin downregulation occurs both upon TDP-43 overexpression (for 48 h) and silencing may seem apparently contradictory see . HoweverPGRN in control fibroblasts (see In summary, our project, carried out in two models of PGRN haploinsufficiency—fibroblasts from FTLD-PGRN patients and upon silencing of asts see , suggestasts see . The maiPARK2 pathogenic variants (In addition, our results indicate that parkin decrease associated with PGRN depletion reflects a systemic event, present in tissues not affected by degeneration, such as skin fibroblasts. This finding underlines the need to analyze the extend of parkin downregulation in neuronal and neuroglial models of PGRN deficiency along with its potential functional effects on mitophagy and other mitochondrial functions in the nervous system. In particular, it would be interesting to compare the consequences of parkin dysfunction between PD caused by variants  and FTLDOn the other hand, it is known from various animal/cellular models that PGRN deficiency leads to upregulation of lysosomal machinery, responsible for clearance of PINK1/parkin-primed mitochondria . InteresThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Bioethical Committee of the Central Clinical Hospital of the Ministry of Interior Affairs and Administration in Warsaw and the Bioethical Committee of Medical University of Gdańsk. The patients/participants provided their written informed consent to participate in this study.KG-W: conceptualization, methodology, project administration, supervision, and writing of original draft, review and editing. KG-W, EB, and DW: formal analysis. KG-W, DW, and MB: validation and investigation. CZ, DW, and MB: funding acquisition. KG-W, DW, CZ, MB, and EB: resources. KG-W, EB, DW, and CZ: writing – review and editing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.