Non-small cell lung cancer (NSCLC) patients with idiopathic pulmonary fibrosis (IPF) show poor prognosis. Periostin is an extracellular matrix protein highly expressed in the lung tissues of IPF. This study aimed to investigate the possibility that periostin secreted by fibroblasts derived from IPF lung might affect proliferation of NSCLC cells. Periostin was more highly expressed and secreted by fibroblasts from diseased human lung with IPF (DIPF) than by normal human lung fibroblasts (NHLF). Cocultivation of NSCLC cells with conditioned media (CM) from DIPF increased proliferation of NSCLC cells through pErk signaling, with this proliferation attenuated by periostin-neutralizing antibodies. Knockdown of integrin β3, a subunit of the periostin receptor, in NSCLC cells suppressed proliferation of NSCLC cells promoted by recombinant human periostin and CM of DIPF. On in vivo examination, DIPF promoted tumor progression more than NHLF, and knockdown of integrin β3 in NSCLC cells suppressed tumor progression promoted by DIPF. Fibroblasts derived from surgical specimens from IPF patients also increased secretion of periostin compared to those from non-IPF patients. Periostin secreted from IPF-activated fibroblasts plays critical roles in the proliferation of NSCLC cells. The present study provides a solid basis for considering periostin-targeted therapy for NSCLC patients with IPF. IPF is a risk factor for the development of non-small cell lung cancer (NSCLC)2. The development of lung cancer (LC) is one of the main causes of death in IPF patients, along with respiratory failure and acute exacerbation (AE) due to the progression of the IPF itself3. NSCLC with IPF (LC-IPF) is more aggressive than NSCLC without IPF (LC-non-IPF)4. Indeed, patients with LC-IPF display even worse prognosis than patients with LC-non-IPF5. Whereas some reports have stated that the pathology of IPF relates to increasing malignancy of NSCLC in IPF patients, details of the underlying mechanisms remain unclear in many aspects. In addition, few treatment options are available for patients with LC-IPF in clinical practice. Major NSCLC therapies such as cytotoxic chemotherapy, molecularly targeted therapies, immune checkpoint inhibitors and radiotherapy are difficult to use for patients with LC-IPF because of the high risk of AE of IPF6. New treatments are therefore needed for NSCLC patients with IPF. Furthermore, the development of new targeted therapies requires elucidation of the relationships between IPF and NSCLC.Idiopathic pulmonary fibrosis (IPF) is a chronically progressive, intractable disease characterized by fibroblast activity in the lung parenchyma and showing a poor prognosis7. Several growth factors such as transforming growth factor β (TGF-β)8 and platelet-derived growth factor9 are chronically overexpressed in fibrosis and LC. Significant overexpression of the secreted mucin protein has been found in LC-IPF10. IPF-activated fibroblasts release molecules such as TGF-β and cytokines to advance the pathology of IPF, and many of these cytokines are also involved in the development of NSCLC11. The Wnt/β-catenin pathway and the TGF-β pathway activated in IPF-activated fibroblasts each play a role as signals promoting NSCLC progression and tissue infiltration13. Therapeutic agents targeting pathways that are activated in common for these two diseases have recently been attracting attention. We have reported that pirfenidone, an anti-fibrotic agent for IPF, inhibits not only fibroblast activity, but also the crosstalk between cancer cells and fibroblasts15. Nintedanib, an inhibitor of tyrosine kinase receptors, exerts antitumor effects as well as antifibrotic effects on IPF16. Identifying a brand-new common molecule for these two pathologies and clarifying the function of the molecule on cancer could thus be key to overcoming LC-IPF.A significant number of IPF patients display risk factors also associated with NSCLC, so multiple common genetic, molecular, and cellular processes appear to connect lung fibrosis with NSCLC17. Periostin is deeply involved in the abnormal activity of interstitial fibrosis, which is the main pathology of IPF18. Inhibition of periostin expression suppresses bleomycin-induced lung fibrosis in vivo19. Further, high expression of periostin is observed in many carcinomas, including NSCLC, and is a factor associated with poor prognosis21. Overexpression of periostin promotes proliferation of breast cancer cells both in vitro and in vivo22. Periostin thus offers a potential therapeutic target for LC-IPF. However, whether periostin expression in activated fibroblasts of IPF is involved in the tumor progression of LC-IPF remains unclear.Periostin is an extracellular matrix protein that binds to cells via integrin molecules to promote cell proliferationIn this study, we identified that periostin was highly expressed in IPF-activated fibroblasts apart from tumor lesions in LC-IPF. Here, we reveal the effects of periostin secreted by fibroblasts derived from IPF lung on NSCLC cell proliferation, and propose the possibility of a new therapeutic strategy targeting periostin for NSCLC patients with IPF.24, LC-IPF patients were more likely to be older men and heavy smokers. LC-IPF patients had larger tumor diameters and higher level of cytokeratin 19 fragment (Cyfra) than LC-non-IPF patients. In addition, LC-IPF had higher maximum standardized uptake value (SUVmax) in positron emission tomography-computed tomography (PET-CT). This value is clinically used to evaluate the metabolic activity and malignancy of tumors. Furthermore, western blot (WB) for pErk expression was performed using frozen specimens obtained from patients, which showed a high level of pErk expression in LC-IPF as compared to LC-non-IPF samples (n = 3 each), suggesting that cell proliferation signaling is more highly activated in tumors with IPF   and diseased human lung fibroblasts derived from idiopathic pulmonary fibrosis (DIPF) (one fibroblast cell line for each group). RNA sequence results revealed 761 genes that were more highly expressed in DIPF than in NHLF. From a total of 761 genes, 85  and TGF-β2 mRNA were significantly higher from DIPF than from NHLF, but no significant difference was observed in actin alpha 2 (ACTA2)  periostin. When cancer cells were cultured in serum-free media with or without rh-periostin, rh-periostin increased the growth of cancer cells under both 2D and 3D conditions  with NHLF or DIPF and injected these mixtures into the flanks of nude mice (n = 5). Tumor sizes of cancer cells with DIPF were significantly increased from day 17 on A549 cells and from day 24 on NCI-H520 compared to cancer cells with NHLF Fig. a. In botTo examine the effect on cancer cells of a loss of the periostin secreted by fibroblasts, we examined whether adding OC-20, a neutralizing antibody of periostin, to iCM would cancel the effects of periostin on NSCLC cells. Whereas OC-20 did not affect cell proliferation of A549 or NCI-H520 cells in serum-free culture media under 2D culture conditions, OC-20 significantly attenuated iCM-induced upregulation of NSCLC cell proliferation  RNA, constitutively reducing ITB3 expression  is a subunit of the periostin receptorThese results suggested that periostin stimulation played a major role in enhancing proliferation in iCM, and that iCM was a less suitable environment for cell proliferation than serum free culture media alone when periostin stimulation was eliminated.ITB3-depleted NSCLC cells were affected by DIPF in tumorigenesis in vivo, we injected mock or ITB3-depleted NSCLC cells with DIPF in a 5:1 ratio into the right flank of nude mice and NSCLC cells into the left flank (n = 5). No significant difference in tumor size or weight was seen between mock A549 and ITB3-depleted A549 cells. Mock A549 cells with DIPF were significantly larger and heavier from day 21 in tumor size and tumor weight as compared to those without DIPF  from the IPF patients  and LC-IPF patients . Fibroblasts from an IPF patient without LC  were used as a reference for comparison with the former two kinds of fibroblasts. The clinicopathological characteristics of the patients from whom fibroblasts were isolated are shown in Supplementary Table 27, the detailed molecular mechanisms remain unclear. IPF comprises a heterogeneous group of disorders characterized by lung damage as a result of lung fibroblast proliferation, interstitial inflammation, and fibrosis28. In this study, we identified periostin as a secreted protein involved in both IPF and NSCLC. Periostin is a matricellular protein secreted by fibroblasts that interacts with various cell-surface integrin molecules. This protein has been reported to increase cell survival, angiogenesis, invasion, metastasis, and chemoresistance in various cancers31, so we focused on periostin derived from IPF-activated fibroblasts.We showed that patients with LC-IPF had a poor prognosis and LC-IPF had higher SUVmax in FDG-PET, indicating that LC-IPF had higher malignant potential than LC-non-IPF. Although the risk of NSCLC is eight times higher in IPF patients than in the general population and IPF is associated with poor prognosis in IPF-LC patientsITB3, a subunit of the periostin receptor, indicating that periostin secreted from DIPF promoted tumorigenesis of NSCLC cells. In vivo examinations found that DIPF promoted tumor progression more than NHLF, and knockdown of ITB3 in NSCLC cells suppressed tumor progression promoted by DIPF. Our data showed that periostin affected NSCLC cell growth through pErk and pAkt signaling. This result is consistent with previous reports of functional analysis of periostin in a mouse NSCLC model31. Increased proliferative capacity is a major phenotype that characterizes malignant transformation of cancer. These data suggested that periostin secreted from activated fibroblasts in the IPF lung might correlate with carcinogenesis and progression of NSCLC.We showed that cocultivation of NSCLC cells with CM of DIPF increased proliferation of NSCLC cells, and these effects were attenuated by periostin-neutralizing antibodies as well as by knockdown of 32. Periostin seems to play a critical role in the pathogenesis of pulmonary fibrosis, and thus offers a promising therapeutic target for IPF. Nanri et al. found that cross-talk between the TGF-β and periostin axes is necessary for pulmonary fibrosis, and created an integrin αvβ3 receptor antagonist that attenuated fibroblast activation and has potential as a therapeutic agent for IPF33. Periostin is reportedly elevated during AE of IPF34, suggesting that periostin may contribute to such AE. These data suggest that periostin represents a potential target in IPF progression and AE as well as in NSCLC.Periostin was highly expressed in clinical samples from NSCLC patients with IPF as compared to those without IPF, especially in non-cancerous areas. We performed primary culture of fibroblasts from surgical specimens of patients and showed that fibroblasts from IPF lung also displayed greater secretion of periostin than those from non-IPF lung. Periostin is reportedly required for maximal proliferation of normal lung fibroblasts, and IPF lung fibroblasts retain this activity35. Although bone marrow-derived mesenchymal stem cells, hematopoietic stem cells, epithelial cells , and endothelial cells  are all considered possible predecessors of CAFs, a common theory of the origins of CAFs points to resident tissue fibroblasts36. Activated fibroblasts in IPF lung may thus represent one origin of CAFs in IPF-LC. On the other hand, periostin is reportedly found in serum37 and even in exosomes in vitro38. We also considered that periostin secreted by activated fibroblasts is most likely to be contained in exosomes and reach cancer cells hematogenously, but further experiments should be performed to elucidate the exact mechanisms of the interaction.We analyzed the function of periostin in NSCLC cells by stimulating NSCLC cells with CM of activated fibroblasts in vitro and co-injecting NSCLC cells and activated fibroblast into nude mice in vivo. These results indicated the effect of increased proliferation of NSCLC cells by periostin secreted from activated fibroblasts. Many researchers have demonstrated that cancer-associated fibroblasts (CAFs) secrete periostin, which promotes cancer initiation and progression39. Furthermore, eight splicing variants of human periostin have been reported and characterized40, and the application of blocking antibodies specifically targeting cancer-specific variants of periostin may be valid for cancer treatment41. An engineered peptide antagonist against periostin also binds to periostin at an integrin-binding site and inhibits periostin function, and thus is expected to see use in peptide therapies to reduce periostin-induced cancer progression and chemoresistance42. Whereas patients with LC-IPF have few treatment options because of the risk of IPF progression following cancer treatment, a periostin-targeted therapeutic agent for LC-IPF may be clinically beneficial for both IPF and NSCLC in the future.Periostin-targeted therapy for cancer has recently been developed. The periostin epitope recognized by monoclonal antibody OC-20 is a binding site for the integrins αvβ3 and αvβ5, localized in the second fasciclin-1 domain of the protein. An in vivo study found that this antibody significantly inhibited tumor growth and angiogenesisThis study has several limitations. First, it was not possible to statistically compare periostin expression between fibroblasts from IPF lungs derived from clinical samples of patients with LC and those from patients without LC because of ethical issues. On the other hand, periostin expression in cultured fibroblasts from IPF lung sections of a patient without LC was similar to that seen in those obtained from lungs of LC patients, thus we concluded that there is no significant difference between patients with and without lung cancer. Second, we were not able to clarify how periostin secreted by fibroblasts is actually transported from activated fibroblasts in non-cancerous areas to NSCLC cells in the human tumor microenvironment. Nevertheless, it is considered that periostin secreted by non-tumor stroma in IPF-LC plays an important role in tumor progression, especially in LC development, because LC is known to often arise de novo from an IPF lung.In conclusion, this is the first study to demonstrate that periostin derived from activated fibroblasts in IPF lung plays a critical role in the proliferation of NSCLC and that inhibition of periostin-receptor interactions attenuated the aggressiveness of NSCLC with IPF. Our findings provide novel insights into the roles of periostin in LC-IPF and indicate that periostin offers a potentially useful target for the treatment of both IPF and NSCLC.We analyzed clinical and pathological data from 538 patients who underwent lung resection to treat NSCLC at Osaka University Hospital between 2011 and 2016. Pathological diagnosis of the resected specimen determined the presence or absence of IPF. Tumors were staged according to the 7th edition of the Union for International Cancer Control TNM staging system. Resected specimens for histological examination were fixed in 10% formalin and routinely processed for paraffin embedding. Tissues were sectioned into slices with a thickness of 4 µm. The protocol for this study was approved by the ethics review board at Osaka University Hospital  in accordance with the Declaration of Helsinki, and written informed consent was waived because of the retrospective design. We also applied Opt-out method to obtain consent on this study which was approved by the ethics review board.2 at 37 °C. A549 and NCI-H520 cells were purchased from the American Type Culture Collection  and grown in Dulbecco's modified Eagle’s medium (DMEM)  with 10% fetal bovine serum (FBS) (Sigma-Aldrich) and 100 U/mL penicillin/streptomycin  under the same conditions as above.NHLF and DIPF were purchased from Lonza  and maintained using a Fibroblast Growth Media kit  in a humidified incubator with 5% CO® Mini Kit . Preparation of a next-generation sequencing library was performed using a SMARTer® Stranded Total RNA Sample Prep Kit–Pico Input Mammalian . Sequencing was performed on an Illumina HiSeq 2500 platform in 75-base single-end mode with Illumina Casava 1.8.2 software for base calling. Sequenced reads were mapped to the human reference genome sequence (hg19) using TopHat v.2.0.13 software, in combination with Bowtie 2 v.2.2.3 and SAMtools v.0.1.19. Fragments per kilobase of exon per million mapped fragments were calculated using Cufflinks v.2.2.1. Raw data files for RNA-sequencing have been deposited in the NCBI Gene Expression Omnibus (GEO) database under the accession code GSE185259. Of the 26,257 genes analyzed, 85 genes were upregulated with a more than twofold change in DIPF compared to NHLF. The Venn diagram shows genes related to cancer selected from ‘The Atlas of Genetics and Cytogenetics in Oncology and Haematology (http://atlasgeneticsoncology.org/index.html)’, genes related to extracellular protein, and genes related to IPF. Numbers in the Venn diagram represent the number of genes corresponding to that criterion.Total RNA was extracted from fibroblasts (NHLF and DIPF) using an RNeasy43. All sections stained with periostin were assessed as the percentage of the area positive for periostin compared to the total tissue area using ImageJ software. The labeling index (labeling frequency as a percentage) of Ki67 staining was calculated using the following formula:  × 100. All Results were quantified by counting more than 100 tumor cells in five randomly selected areas per specimen.Immunohistochemistry was performed as previously describedAntibodies used for Western blotting and immunofluorescence were as follows: anti-periostin ; anti-β-actin ; anti-integrin β-3 ; anti-phospho-Erk1/2 ; anti-Erk1/2 ; anti-phospho-Akt ; anti-Akt ; and anti-Ki67 . Reagents used in a cell proliferation assay were as follows: OC-20 , and recombinant human (rh-) periostin .Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an endogenous reference gene. The following primer sequences were used: for human periostin, F 5′-GTCTTTGAGACGCTGGAAGG-3′ and R 5′- AGATCCGTGAAGGTGGTTTG-3′; for human integrin β3 (ITB3), F 5′-AGATGCGAAAGCTCACCAGT-3′ and R 5′-TCCGTGACACACTCTGCTTC-3′, for human GAPDH, F 5′-ACCCAGAAGACTGTGGATGG-3′ and R 5′-TTCTAGACGGCAGGTCAGGT-3′. All experiments were performed three times independently. Data shown represent the averages of results from the three experiments.Total RNA was extracted from A549, NCI-H520, NHLF and DIPF cells using the RNeasy Mini Kit , and complementary DNAs were synthesized from 1.0 μg of total RNA with the PrimeScrip TM RT mastermix . RT-PCR was performed using Luna Universal qPCR Master Mix . 44. Quantification of immunoblots was performed using ImageJ. All experiments were performed three times independently.Monolayers of cultured cells were treated under the indicated conditions, and proteins were extracted with RIPA buffer . Cell extracts were resolved and immunoblotted as described previously5 cells/well). After 12 h of incubation, medium (10% FBS) was removed, and cells were fed with serum-free DMEM (2 ml). Conditioned media were collected after 72 h, and periostin was quantified using ELISA according to the protocol from the manufacturer . Absorbance was measured at 450 nm in a microplate spectrophotometer, and periostin levels were quantified with a calibration curve using a rh-periostin standard (ELISA kit). All experiments were performed three times independently.Cell lines and primary cultures of NHLF and DIPF, respectively, were seeded in 6-well plates (4 × 106 cells per 10-cm dish and incubated in DMEM (10% FBS) for 12 h. Attached cells were washed three times with phosphate-buffered saline, and the medium was replaced with serum-free DMEM. The supernatant was collected after centrifugation at 1000×g and stored at − 80 °C. Each CM was melted just before use in the experiment.CM were obtained from NHLF (nCM) and DIPF (iCM). Fibroblasts resulting from both cultures were replated at 3 × 102 and 1 × 103 cells/well, respectively. Cells were starved for 24 h, then used in cell proliferation assays. Periostin-neutralizing antibody (OC-20) was added into the culture medium at the same time cells were starved. The culture medium was changed on days 2 and 4. On days 0, 2, 4, and 6, according to the CyQUANT® NF protocol, culture medium was discarded and 100 μl of 1 × dye-binding solution was dispensed into each well. The fluorescence intensity of each sample was measured 30 min later using a fluorescence microplate reader . All experiments were performed three times independently.A549 and NCI-H520 cells were seeded in 96-well plates at 5 × 1045. Briefly, 40 μl of Matrigel was mounted on a round coverslip and incubated for 15 min at 37 °C to solidify the gel. A549 cells (5 × 103 cells) and NCI-H520 cells (1 × 104 cells) suspended in 1 ml of growth medium containing 2% Matrigel were incubated in DMEM (FBS10%) for 24 h. Cultured cells in the Matrigel were divided to 3 groups in which media were replaced with DMEM, DMEM + rh-periostin (200 ng/ml), or serum-free CM, and changed every 2 days. Seven days after medium was exchanged for DMEM + rh-periostin or CM, cells were fixed, photographed, and the area of the sphere was evaluated with ImageJ. All experiments were performed two times independently.The 3D Matrigel  cultures of A549 and NCI-H520 cells and were obtained as previously described4 cells/well). The CM was DMEM (FBS 10%). At 24 h after seeding, DMEM (10% FBS) was replaced with DMEM (FBS-) adjusted to 5 times the amount of virus of the seeded cells and polybrene (10 µg/ml), and centrifuged at 1200×g for 30 min. Four hours later, cells were replaced with DMEM (10% FBS). Cells were passaged at 90% confluency. From 24 h after passage, cells were selected in DMEM (FBS 10%) with puromycin (1 µg/ml). Finally, stable cells with decreased expression of ITB3 were established.A vesicular stomatitis virus G protein-pseudotyped lentivirus encoding shRNA targeting human integrin β3  and a nontargeting shRNA were purchased from Sigma-Aldrich. A549 and NCI-H520 cells were seeded in 12-well plates  mixed with DIPF or without (n = 5 mice per group). After injection, the same method described above was used. All animal studies were approved by the Osaka University Animal Experiment Committee  and performed in accordance with the Osaka University Regulations on Animal Experiments. All animal work was performed and reported according to the ARRIVE guidelines.A549 or NCI-H520 cells were mixed with NHLF or DIPF (5:1) co-injected subcutaneously into 4- to 5-week-old female BALB/cAJcl-nu/nu mice  (5 mice per group). Tumors were measured 2 times per week and tumor volume was calculated using the following equation: tumor volume = D/2 × dFrozen clinical specimens were obtained in an aseptic manner from patients undergoing a pulmonary resection procedure and stored in RNAlater™ Stabilization Solution  at 4 °C for 24 h followed by liquid nitrogen, then used for RT-PCR or WB analysis. Frozen clinical specimens from LC-non-IPF (n = 4), LC-IPF (n = 4), and cancer-free IPF patients (n = 1) were utilized in the present study, with both cancerous and non-cancerous portions used as specimens from patients with LC. After collection, the cancerous and non-cancerous tissues were clearly separated and stored separately. For RT-PCR, all samples were usable. However, for WB, one sample each from the LC-non-IPF and LC-IPF specimens could not be used because of lack of quantity. Thus, WB was investigated using tissues obtained from three LC-non-IPF and three LC-IPF n = 3) patients, as well as one patient without cancer. WB analysis of pErk was performed only using the cancerous part from the same cases. The clinicopathological characteristics of the patients are shown in Supplementary Table  patients44. All experiments using primary cultures of fibroblasts were performed between passages 2 and 7.Primary human fibroblasts were isolated from specimens of lung tissue surgically resected from NSCLC patients. Normal lung-derived fibroblasts (NLfs) were harvested from non-cancerous areas of LC-non-IPF patients (n = 5), and IPF-derived fibroblasts (IPLfs) were harvested from non-cancerous areas of LC-IPF patients (n = 6) and from a patient without NSCLC (n = 1), then isolated and cultured. Briefly, normal lung and lung with IPF obviously distant from cancerous tissue was obtained aseptically from patients with NSCLC undergoing pulmonary resection. The institutional review board at Osaka University Hospital approved the study protocol , and written informed consent for surgical intervention was obtained from each patient prior to resection. The method of isolating fibroblasts from resected lung and culturing them in primary culture has been described previouslyThe log-rank test and Mann–Whitney U test were used to compare results, as appropriate. Values of P < 0.05 were considered significant.Supplementary Information 1.Supplementary Information 2.


Graphene nanostructures are widely perceived as a promising material for fundamental components; their high-performance electronic properties offer the potential for the construction of graphene nanoelectronics. Numerous researchers have paid attention to the fabrication of graphene nanostructures, based on both top-down and bottom-up approaches. However, there are still some unavoidable challenges, such as smooth edges, uniform films without folds, and accurate dimension and location control. In this work, a direct writing method was reported for the in-situ preparation of a high-resolution graphene nanostructure of controllable size (the minimum feature size is about 15 nm), which combines the advantages of e-beam lithography and copper-catalyzed growth. By using the Fourier infrared absorption test, we found that the hydrogen and oxygen elements were disappearing due to knock-on displacement and the radiolysis effect. The graphene crystal is also formed via diffusion and the local heating effect between the e-beam and copper substrate, based on the Raman spectra test. This simple process for the in-situ synthesis of graphene nanostructures has many promising potential applications, including offering a way to make nanoelectrodes, NEMS cantilever resonant structures, nanophotonic devices and so on. Graphene nanostructures, forming a promising 2D nanomaterial with excellent properties, have been widely studied for electronic applications, sensing, biomedicine, and other fields ,2,3. DesGenerally, graphene nanostructures can be produced via the top-down method, which mainly includes micromechanical cleavage, mask patterning and graphene etching. Among these processes, the mask patterns on the graphene surface need to be acquired first, through various forms of lithography technology, synthetic block copolymer (BCP) and other nanomaterial structures ,8,9,10. 2/Si substrate, to construct a high-resolution graphitized nanostructure with the help of the post-annealing process [2/Si substrate. Compared with an insulating SiO2/Si substrate, a copper substrate is useful to synthesize single or small layers of graphene because of its catalytic effect. Meanwhile, the excellent conductivity of copper substrate is also beneficial as a way to avoid the proximity effect during the e-beam lithography process, which is another point in favor of high-resolution graphene fabrication.Apart from these techniques, e-beam irradiation in-situ enables the growth of a functional graphene nanostructure in a unique high-precision preparation method, representing an advanced fabrication method in this field. Duan et al. acquired a graphene nanoribbon from ultrathin electrospun poly (methyl methacrylate) PMMA nanofibers via the e-beam irradiation process, in which the graphene sheets with a honeycomb hexagonal crystal structure can be prepared during continuous e-beam irradiation . Chen et process . Further process . By adju−1 (D-mode), 1583 cm−1 (G-mode) and 2680 cm−1 (2D-mode). In conclusion, a nanoscale high-quality graphene nanostructure can be reliably fabricated with the help of a high-precision e-beam fabrication method using a high-temperature copper-catalyzed graphene growth approach.In this work, we adopted high-resolution PMMA organic resist for carbon-based precursors. Then, PMMA film on a copper substrate was selectively irradiated with a high-energy e-beam. By combining this with the subsequent high-temperature annealing process, various graphene nanostructures, including nanoribbon, dot arrays and other patterns, can be created. The minimum feature size of a graphene line is about 15 nm, and the irradiated products have a uniform and smooth morphological structure. The Fourier infrared absorption spectrum (FITR) test and Monte Carlo simulation also exhibit a molecular dynamic process between the incident e-beam and PMMA organic film. As a simple characterization tool, Raman spectroscopy is also used for characterizing the properties of graphene; the results were 1350 cm2 to 12,000 μC/cm2 exposure dose, and 320 pA beam current. The irradiated PMMA film was soaked in acetone solution for 120 s and then in deionized isopropyl alcohol for 60 s. Subsequently, the sample was put into a quartz tube at 900 °C for 20 min with a heating rate of 10 °C/min under vacuum conditions. The few-layers graphene nanostructure then formed on the copper surface with the temperature of the tube furnace cooling down to room temperature.Standard PMMA resist, with 950 k molecular weight and a concentration of 6% in chlorobenzene, was bought from EM RESIST LTD. A Zeiss scanning electron microscope (SEM) equipped with a pattern generator from the Raith company was used for high-resolution e-beam lithography. The irradiation conditions between the e-beam and PMMA organic film were: 10 kV to 30 kV EHT voltage, 5000 μC/cmAn SEM (Zeiss SUPRA-55) was used to produce a two-dimensional image and revealed information about the irradiated graphene nanostructures. A sub-nanometer resolution of the sample was presented under a 10 kV accelerating voltage and ~310 pA beam current.The sample roughness can be observed by using atomic force microscopy . The AFM test conditions were set to contact mode for clearer morphological characterization. The 50 μm × 50 μm AFM image was collected at a 1Hz scan rate in an air atmosphere with a silicon cantilever .−1. The XRD  characterization was adopted to study the crystalline structure of the irradiated PMMA sample. The X-ray diffractometers were used based on the θ/θ continuous scanning mode. Other working parameters, such as the 2.0000 deg/min scanning speed, 0.0500-degree sample tilt, 0° to 90° scanning angle and 1.5 s adjustment, were set to meet the test requirements.Raman characterization is a convenient and nondestructive testing technology for acquiring accurate information on molecular structure, material defects, surface dangling bonds, and so on. The Raman test was achieved with a low-powered 532-nm incident laser and a long integral time (10 s for one spectrum). In order to ensure the accuracy of the test data, 3~5 Raman spectra were collected from the same sample, avoiding accidental errors. All Raman tests were operated at room temperature, in an air atmosphere and dark-field mode. In the Fourier transform infrared spectroscopy  test, the radiation emitted by a light source was converted to interference light by an interferometer, and an infrared spectrum of the absorption of irradiated PMMA can be obtained, based on Fourier transform analysis using a computer. Here, the spectra were recorded between 4000 and 1000 cm2/Si substrate using magnetron sputtering equipment (step 1). Then, a PMMA layer of 50-nm thickness was spin-coated on the copper substrate (step 2). Subsequently, the Raith-150 two electron-beam lithography system was used for selective irradiation at a 30-kV acceleration voltage with an e-beam current of 320 pA. The PMMA was gradually driven into the carbonaceous material with an increasing e-beam irradiation dose (step 3). The graphic nanostructures could be obtained from the PMMA after soaking in acetone for two minutes. In order to further acquire high-quality graphene crystal film, the sample was then put into a tube furnace at 900 °C for 10 min. It would be useful to change the size thickness and properties of the produced graphene nanostructure with this in-situ process using an e-beam direct-writing and annealing step.PMMA is a typical high-resolution positive/negative e-beam resist for nanostructure fabrication. It has many advantages in terms of long-lasting results and a good sticking ability to the target substrate surface, and it is cost-effective and highly reproducible. Moreover, it can be transformed into a graphite-like material under e-beam irradiation. Considering the aim of this paper is to construct a graphene nanostructure, the copper substrate was used for fabrication, based on the Cu-catalyzed graphene growth mechanism. As shown in The radial energy density of incident electrons is closely related to organic PMMA film parameters, including resist thickness, surface roughness, EHT (extra-high tension) voltage, and so on. The PMMA film is 50-nm thick, for preparing ultrathin graphene. During the e-beam irradiation process, the incident electron-scattering effects, which mainly include forward-scattering electrons and back-scattering electrons, affecting the energy distribution in the PMMA film. In the electron-scattering process, the electrons that have interacted with PMMA valence electrons are the major factor that causes PMMA molecular structure changes. In addition, inelastic scattering between incoming electrons and atomic electrons in organic molecular conditions creates a large amount of energy transformed into heat, causing a locally high temperature for a chemical reaction. The higher local temperature is beneficial to driving copper-catalyzed graphene growth, based on a PMMA solid organic source ,20.Electron scattering in solids is a very complex physical problem. Generally, the Monte Carlo simulation . According to the formula, the smaller beam spot size required a higher acceleration voltage, which is also useful for the molecular structure transformation of PMMA. Eventually, graphene-like sheets can be obtained from PMMA organic film via the removal of the oxygen-containing functional groups by the e-beam irradiation process.With this simulation, the detailed motion trajectory and energy density distribution of the electron can be clearly calculated. By calculating the scattering process of each electron in the photoresist and recording and analyzing the energy deposition density in the photoresist, the physical and chemical change processes at different positions can be estimated. In this paper, we needed to identify graphene transformation and high-resolution patterns. The dimensional variation of the e-beam spot can be given empirically  test, the radiation emitted by a light source was converted to interference light by an interferometer, and an infrared spectrum of the absorption of irradiated PMMA can be obtained with a computer, based on Fourier transform analysis. Here, the spectra were recorded between 4000 and 1000 cm−1.We confirmed the changes in chemical elements and functional groups using FTIR spectra detection, which showed that the oxygen-containing groups gradually died out with the increasing irradiation doses. In the measurements, the interference light source was introduced into irradiated PMMA film for measuring the absorption spectra. The scanning was recorded across a 4000 and 1000 cm−1), -OH (3410 cm−1) and -OH groups of dimeric COOH groups (2927 cm−1), were obvious in the initial stages. The signal intensity of the oxygen-containing groups gradually disappeared with the increase of the e-beam dose. By the 10,000 μC/cm2 doses of irradiation, the oxygen-containing groups had almost disappeared. The C=C (1662 cm−1) and -CH2- (2927 cm−1) chemical bond of the PMMA molecular formula also cracked under high-dose irradiation [adiation ,22. The 2 substrate sample shown in −1 indicates that the sample has obvious defects and disordering. The G peak at 1580 cm−1 means that the sample has formed crystalline graphite. The D and G peaks may result from the charge-accumulation effect of the insulating SiO2 layer and limited carbon atom diffusion on the SiO2 surface. Different forms of SiO2 substrate and copper substrate have excellent electrical conductivity and catalytic effects. For the copper substrate sample (−1) in the spectrum is coming from the first-order scattering process of graphene crystal. The 2D and D bands (~2700 cm−1 and ~1350 cm−1) originate from a second-order scattering process, corresponding to two or one phonons near the K point and one defect. The prominent features that are shown, including the G band, D band and 2D band in Raman spectra, are compelling evidence to verify the existence of graphene, although it still has some defects because of the D peak and wide non-Gaussian distribution 2D peak. The value of ID/IG ≈0.4 implies that the lower defects were produced on the copper substrate. The FWHM of the 2D peak is about 123 cm−1, which is much higher than for 25–60 cm−1 of multilayer graphene. The annealing process is necessary for acquiring a single- or few-layer graphene sample. The statistical evaluation of the graphene parameters as 2D width, D/G intensity ratio and the 2D/G intensity ratio has also been provided in the Raman characterization is widely used in structural optical characterization. Much of the work using Raman tests of graphitic materials was reported, and it has gradually become a convenient and nondestructive testing technology for samples. The molecular material information can be obtained by the interaction between a 532-nm incident laser and molecular structural vibration ,24. For e sample c, the G During the XRD measurement process, the samples were irradiated with Cu Kα radiation (λ = 0.1545 nm). The 3c range and step sizes were 10–80° and 0.02°, respectively. The measurement time for the sample was 9 min. There is no obvious peak in the XRD spectrum of the pure PMMA sample. After e-beam irradiation, a weak and broad diffraction peak appeared at 21.3°, which can be attributed to the (002) reflection of graphene nanosheets. This peak indicates that the first minimum graphene/graphite structure was formed, which is consistent with the hexagonal structure . It alsoFor further optimizing the growth process and the ultimate graphene nanostructure properties, the high-temperature annealing process plays an essential role. Benefiting from the low-carbon-solubility metal, the high-quality monolayer or few-layer graphene nanostructure can be grown on the copper surface, due to the mechanism of dissolution and the precipitation of copper. The topographic AFM images of graphene before and after the annealing process are shown in As we know, high-temperature annealing can drive material cleavage and recombination, which is a useful technology to repair defects and remove impurities. In this work, by combining chemical reduction reaction with thermal annealing process, the quality of graphene has been further improved. At this point, the irradiated PMMA sample was put in a high-temperature furnace in a vacuum, increasing graphene crystallization and burning off the residual PMMA. In addition to Raman characterization for all steps, a photoluminescence characterization was also adopted. As we know, pure PMMA film and graphene cannot produce PL signals because of their zero bandgaps. However, the graphene with doping and defects may have PL singles ,28. Figu2 substrate and copper substrate. In 2/Si substrate has a flatter surface. We can clearly see that the widths of the nanoribbons prepared by e-beam lithography are close to the same size (~15 nm) in both SEM and AFM images , G peak (~1582 cm−1) and 2D peak (~2700 cm−1). Other optical characterizations, including XRD and FTIR spectra, also clearly exhibited the fabrication mechanism. In addition, e-beam lithography has played an important role in the fabrication of graphene nanostructures on the copper substrate. However, it is hard to further reduce the minimum feature size of the graphene nanostructure due to the disorderly scattering of electrons resulting from the rough surface of the copper film as prepared by magnetron sputtering technology. In short, the work provides some new ideas and possibilities for the preparation of high-resolution and high-quality graphene nanostructures, which will further the development of carbon-based electronics.As has been shown in this paper, we reviewed the fabrication of graphene nanostructures, as explained in the Introduction section. Facing the challenge of the reliable fabrication of a high-resolution and high-quality graphene nanostructure, we combined the advantages of the e-beam irradiation effect and the copper catalytic effect. The graphene array with a line width of 50 nm for the 150 nm pitch was fabricated by putting forward a simple one-step process for directly obtaining patterned carbonaceous nanostructures, and a high-temperature annealing process. The acquired graphene sample has an obvious Raman characteristic peak, such as the D peak (~1350 cm


TARDBP) is a multifunctional protein that regulates transcription and RNA metabolism by binding DNA or RNA. TDP-43 has been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS) because abnormal accumulation of cleaved and phosphorylated C-terminal fragments of TDP-43 in motor neurons is a pathological hallmark of ALS. Here, we cloned and analyzed the promoter region of the TARDBP gene. TARDBP upstream sequences and/or intron/luciferase constructs were generated, and their promoter activity was experimentally assessed. The upstream region predictably exhibited promoter activity and identified putative cis-acting elements, including the i-motif, was relevant for the regulation of TDP-43 expression. The cellular abundance of TDP-43 is strictly controlled, and its constancy is critically important for motor neuron survival. A machinery serving to maintain a constant level of TDP-43 is autoregulation via control of mRNA stability, a negative feedback system involving binding to the 3′ untranslated region of its own pre-mRNA. However, whether transcriptional mechanisms contribute to TDP-43 autoregulation is unclear. We further showed that TDP-43 negatively regulates the TARDBP promoter and, surprisingly, that disease-causing TDP-43 mutants lacked this regulatory activity. These results allowed the elucidation of a novel transcriptional autoregulatory mechanism of TDP-43.TAR DNA-binding protein (TDP-43, encoded by  Approximately 5–10% of ALS cases are familial , and more than 20 causative genes have been identified from fALS, such as superoxide dismutase 1 (SOD1), TAR DNA-binding protein 43 kDa (TDP-43), fused in sarcoma (FUS), optineurin and chromosome 9 open reading frame 72 (C9orf72)16. Some causative gene mutations are found not only in fALS but also in sporadic ALS (sALS). In fact, more than 30 disease-related mutations of TDP-43 have been found, and some TDP-43 mutations are found in both fALS and sALS11. A characteristic disease hallmark in ALS pathology is the frequent presence of insoluble ubiquitin-positive inclusions in MNs of ALS patients2. The neuronal inclusions located in the cytoplasm or nucleus include abnormally cleaved and phosphorylated C-terminal fragments of TDP-4323. In recent years, TDP-43 was shown to be present in the inclusions of more than 97% of ALS patients, including those with fALS and sALS, even if the patient did not harbor TDP-43 mutations24. Although whether TDP-43 affects MNs by loss of function or gain of toxic function remains controversial, these findings imply that TDP-43 plays a critical role in ALS onset and progression.Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that selectively affects motor neurons (MNs) in the brain motor cortex and spinal cord and is characterized by late-onset progressive muscle weakness and atrophy, and respiratory failure, leading to death typically within several yearsTARDBP, was initially identified as a transcriptional regulator that binds to a regulatory element in the human immunodeficiency virus type 1 (HIV-1) long terminal repeat known as TAR and suppresses HIV-1 gene expression25. TDP-43 binds not only DNA but also RNA, thus regulating aspects of RNA metabolism such as alternative splicing and mRNA stability28.TDP-43 includes two RNA recognition motifs (RRM) and binds to DNA/RNA targets. It is known that RRM1 is necessary and sufficient for DNA/RNA binding activity26.TDP-43, encoded by 29. In addition, MN-specific TDP-43 conditional KO mice developed motor dysfunctions such as abnormal clasping, decreased time on a rotarod, and late-onset loss of MNs30. These findings indicate that TDP-43 is essential for ontogeny and necessary for the survival of MNs. Furthermore, regarding the TDP-43 overexpression model, PrP promoter-mutant human TDP-43 transgenic (Tg) mice developed weak grip strength and spinal MN loss at 10 months of age31. Similarly, Thy-1 promoter-human wild-type (WT) TDP-43 Tg mice, in which the transgene is expressed virtually all neurons in the central nervous system (CNS), showed an abnormal hindlimb reflex response, MN loss and ubiquitin-positive aggregates that were also positive for phosphorylated TDP-43 in spinal MNs32. These findings indicate that excessive expression of mutant as well as WT TDP-43 leads to MN degeneration. Thus, inappropriate TDP-43 levels affect the homeostasis of MNs, and the level of TDP-43 should be strictly regulated within the appropriate range. A recent study showed that the envelope protein of human endogenous retrovirus K (HERV-K) is expressed in the motor cortex and spinal cord of patients with sALS but not in healthy controls33. Interestingly, TDP-43 interacts with consensus sequences in the HERV-K promoter and regulates its transcription. From these findings, although TDP-43 is often characterized as a regulator of RNA metabolism, TDP-43 as a transcriptional regulator may also have an important role in ALS pathophysiology.In organisms, tight control of TDP-43 expression is very important. TDP-43 gene knockout (KO) was embryonically lethal in mice at E3.5–12.5, and even heterozygotic animals appeared to develop motor impairment late in life35 of TDP-43 mRNA; however, whether the TDP-43 promoter is also involved in autoregulatory systems is unknown. The predicted TDP-43 promoter region was identified previously by using a promoter prediction database36, but no experimental study has been performed. In this study, we experimentally investigated whether the TARDBP upstream region shows promoter activity and demonstrated that the TARDBP promoter is involved in TDP-43 autoregulation.Previous reports have shown that the TDP-43 level is strictly regulated by a negative feedback mechanism via an alternative polyA site in the 3′ untranslated region (UTR)TARDBP were determined by the programs ElDorado and Promoter Scan in a previous study36. We cloned the TARDBP upstream sequence starting at nucleotide − 721, which included the predicted promoter region and the sequences described in Fig. TARDBP promoter region using the UCSC genome browser. In agreement with the previous prediction, the region upstream of the transcription start site (TSS), exon 1 and intron 1 exhibited high enrichment with epigenetic markers of active transcription  reporters containing the predicted region were generated on pcDNA3.1(−) backbone, and their promoter activities were determined  or with P1 were transfected into HeLaS3 cells. Although the level of TDP-43-Venus expression driven by P1 was lower than that driven by the CMV promoter, a moderate level of TDP-43-Venus expression was driven by P1  reporter construct showed promoter activity levels that were 30.2% (HeLaS3), 16.7% (HEK293T), and 25.7% (SH-SY5Y) that of P1   Fig. a. These TARDBP promoter  is sufficient to abolish DNA/RNA binding and TDP-43 function37. Our study demonstrated that TDP-43 (F147/149L) disrupted the negative effect for P16 activity   Fig. g–j. We c2T) Fig. f. Furtheter Fig. g. While ter Fig. h. We furter Fig. i. In addter Fig. j. Indeedter Fig. h,i. Thescis-acting elements important for regulatory factors are likely to be present in the region between nucleotides − 327 and − 223. We noted that this region includes C-rich sequences (− 327/− 309), which are highly conserved between humans and mice  in the c-MYC promoter contains five runs of cytosines that can form multiple i-motif structures and strongly control the transcriptional activity of the c-MYC oncogene. Interestingly, the TARDBP sequence upstream of P1 includes nine runs of cytosines (− 371/− 309), a predicted i-motif region; thus, we investigated whether the predicted i-motif region is important for P1 activation  which includes predicted i-motif. Since insufficiency of hnRNP K affect the activity of CMVp (38.2%), it is suggested that hnRNP K may also have a role in the general promoter  by ElDorado and in the − 577 to − 326 region by Promoter Scan. Our promoter prediction via the UCSC genome browser also indicated the existence of promoters in the TARDBP upstream region and intron 1  that presents the exact positions of TSSs in the genome49. We found that transcription actually starts from TSS2 in most TDP-43 transcripts, but only a minor population of TDP-43 transcripts in the human fetal brain have a TSS within exon 2 (data not shown). We also showed that the proximal half of TARDBP intron 1, the region that includes a critical element for promoter activity, is highly conserved in chimpanzees and macaques but not in mice, dogs, chickens or zebrafish , whose intron 1 was spliced out producing the transcript with exon 1  base pairing under acidic conditions in DNA sequences enriched in cytosine42. In fact, hnRNP K is reported to bind selectively to the i-motif and positively modulate KRAS transcription44. In our study, ChIP assay experiment revealed the interaction between hnRNP K and TARDBP promoter region . Our transient expression studies demonstrated that there are no significant differences in the subcellular localization pattern and the promoter binding ability among WT and mutant TDP-43s -derived neural stem cells (201B7) using TRIzol reagent (Invitrogen) following the manufacturer’s instructions. The cloned genome and luciferase (Luc2) gene liberated from the Luc2/pGL4 vector (Promega) were subcloned into the pcDNA3.1 (−) vector (Invitrogen). Promoter fragment/Luc2 constructs were produced using the primers described in Table TARDBP upstream region-TDP-43-Venus/pcDNA3.1(−) were constructed using the former plasmid. For the promoter assay, TDP-43  were cloned into the pFLAG-CMV2 vector (Sigma). All primer sequences used for plasmid construction are described in the Supplemental Information . The target and negative control siRNA sequences are described in Table For TDP-43 KD, double-stranded siRNA targeting TDP-43 was originally designed using siDirect software  supplemented with 2% B27 (Gibco), 20 ng/mL bFGF (Wako), 10 ng/mL LIF (Millipore), 10 ng/mL Y-27632 (Wako), 3 μM CHIR , 2 μM SB431542 (Sigma), 1 μM retinoic acid (Wako), and 1 μM purmorphamine .HEK293T and HeLaS3 cells were cultured in DMEM (Sigma) containing 10% FBS , nonessential amino acids (NEAA) (Gibco) and 1% penicillin–streptomycin (Wako). SH-SY5Y cells were maintained in DMEM/F12 (Sigma) containing 10% FBS, 1% penicillin–streptomycin and NEAA. hiPSCs (201B7) were purchased from RIKEN Cell Bank and maintained in StemFit medium (Ajinomoto). Neurospheres were produced and maintained according to a previous reportg for 2 min at 4 °C. The supernatants were collected for the conduction of subsequent experiments. Luminescence detection was performed using a WSE-6100H LuminoGraph I (Atto). The signal quantification was conducted ImageJ (Fiji) software 57.For plasmid transfection, cells were transfected with 2 μg of promoter/luciferase vector (or 1 μg of CMV promoter/luciferase vector) using Lipofectamine 3000 Reagent (Invitrogen) according to the manufacturer’s instructions. After transfection for 24 (HeLaS3 and HEK293T) or 48 h (SH-SY5Y), a promoter assay was performed using a luciferase assay system (Promega) according to the manufacturer’s instructions. Briefly, cells were collected with the extraction buffer provided in the kit and centrifuged at 12,000×For multiple plasmid cotransfection, HeLaS3 cells were transfected with 2 μg of promoter/luciferase vector; 0, 0.5, 1 or 2 μg of FLAG-hnRNP K or TDP-43 plasmid; and 2, 1.5, 1 or 0 μg of pFLAG-CMV2 empty vector to equal total 4 μg of plasmid DNA in each group. For siRNA and plasmid DNA cotransfection, HeLaS3 cells were transfected with 60 nM  siRNA using Lipofectamine RNAiMAX (Invitrogen), and after 24 h, additional transfection was performed using Lipofectamine 3000 with 2 μg of promoter/luciferase vector. After another 24 h, a luciferase assay was conducted.For immunocytochemistry, HeLaS3 cells transfected with the 1 μg of TDP-43  /pFLAG-CMV2 fixed with 4% paraformaldehyde (PFA) for 5 min at room temperature (RT) and washed with PBS after 24 h of transfection, and permeabilized with 0.01% Triton X-100 for 15 min at RT. After washing with PBS and incubation in 5% BSA for one hour at RT, cells were incubated with the primary antibodies, anti-DDDDK-tag (MBL M185-3 1:1000), for overnight at 4 °C. The cells were washed with PBS, then incubated with the secondary antibody Alexa 488-conjugated antibody  and Hoechst 33342 , for one hour at RT. After washing with PBS, fluorescence was observed using a confocal laser scanning microscope (Zeiss) with a Plan Apochromat 20×/0.8 objective. For promoter reporter overexpression and nuclear staining, HeLaS3 cells transfected with 1 μg of the Venus reporter vector (driven by CMV promoter or P1) were fixed with 4% PFA for 5 min RT and washed with PBS after 48 h of transfection. Incubation with Hoechst 33342 was performed for one hour. After washing with PBS, fluorescence was visualized using the confocal microscope with a Plan Apochromat 63×/1.4 oil immersion objective. Comparison images were acquired with the same laser intensity, exposure time, and filter. Details are described in each figure legend.Total RNA from HeLaS3 cells transfected P16 or P16 (Δintron 1) was extracted using TRIzol reagent and RNeasy Plus Mini Kit (Qiagen). cDNA synthesis and PCR were performed using ReverTra Ace α (TOYOBO) and EmeraldAmp PCR Master Mix (TaKaRa) following the manufacturer’s instructions. Primers for the detection of the transcript of exon 1/intron 1/luciferase are described in Table Cultured cells were washed with PBS, and protein was extracted with extraction buffer containing Tissue Extraction Reagent I (Wako), protease inhibitor cocktail (Roche) and a phosphatase inhibitor  on ice for 10 min. The extracted solutions were transferred into 1.5 mL tubes and centrifuged at 15,000 rpm for 15 min at 4 °C. Supernatants were collected in new tubes and used for subsequent experiments. Protein lysates were mixed with 2 × Laemmli sample buffer (Bio-Rad) and 2-mercaptoethanol (Sigma) and boiled for 3 min at 95 °C. Protein samples were separated by electrophoresis on 10% TGX FastCast Acrylamide gels (Bio Rad) and transferred to Immobilon-P membranes (Millipore). Nonspecific antibody reactions were blocked with 1% skim milk (Wako) for 30 min at RT. Membranes were incubated with primary antibody at 4 °C overnight. Membranes were then washed with TBST and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 h at RT. After a wash in TBST, protein signals were detected by Western BLoT Chemiluminescence HRP Substrate (Takara Bio) using the WSE-6100H LuminoGraph I (Atto). The following primary antibodies were used: anti-TDP-43 , anti-hnRNP K , anti-DDDDK (FLAG)-tag , anti-GAPDH , and anti-β-actin . The secondary antibodies are described below: HRP-conjugated goat anti-rabbit IgG  and HRP-conjugated goat ant-mouse IgG .6 cells were used for all immunoprecipitations. Immunoprecipitated chromatin was then incubated with protein G magnetic beads, washed and eluted. After reversal of the cross-links and purification, precipitated DNAs were analyzed by PCR with specific primers using EmeraldAmp PCR Master Mix (TaKaRa). The primers are described in Table ChIP assays were performed using SimpleChIP Enzymatic Chromatin IP Kit  as described by manufacturer’s protocol. Briefly, HEK293T cells were fixed in 1% formaldehyde for 10 min at RT, quenched, and enzymatically digested for 20 min at 37 °C. For overexpression analysis, the cell fixation was performed after 24 hr from transfection. Chromatin was briefly sheared by sonication , and was incubated overnight at 4 °C with specific antibody, anti-DDDDK (FLAG)-tag , anti-TDP-43  or anti-hnRNP K . Control IgG and Histone H3 antibody were provided with the ChIP kit as a negative or positive control for the immunoprecipitation. Approximately 4 × 10Statistical data were analyzed by GraphPad Prism 7.03 software (GraphPad Software). The significance level was set at 0.05, and Welch’s t test , one-way ANOVA  and two-way ANOVA  were used. After ANOVA, additional analysis was performed utilizing Tukey’s or Dunnett’s post hoc test, and details are provided in the figure legends.Supplementary Information 1.


For the reason that many studies have been inconclusive on the effect of humidity on respiratory disease, we examined the association between absolute humidity and respiratory disease mortality and quantified the mortality burden due to non-optimal absolute humidity in Guangzhou, China.Daily respiratory disease mortality including total 42,440 deaths from 1 February 2013 to 31 December 2018 and meteorological data of the same period in Guangzhou City were collected. The distributed lag non-linear model was used to determine the optimal absolute humidity of death and discuss their non-linear lagged effects. Attributable fraction and population attributable mortality were calculated based on the optimal absolute humidity, defined as the minimum mortality absolute humidity.The association between absolute humidity and total respiratory disease mortality showed an M-shaped non-linear curve. In total, 21.57% (95% CI 14.20 ~ 27.75%) of respiratory disease mortality (9154 deaths) was attributable to non-optimum absolute humidity. The attributable fractions due to high absolute humidity were 13.49% (95% CI 9.56 ~ 16.98%), while mortality burden of low absolute humidity were 8.08% (95% CI 0.89 ~ 13.93%), respectively. Extreme dry and moist absolute humidity accounted for total respiratory disease mortality fraction of 0.87% (95% CI − 0.09 ~ 1.58%) and 0.91% (95% CI 0.25 ~ 1.39%), respectively. There was no significant gender and age difference in the burden of attributable risk due to absolute humidity.Our study showed that both high and low absolute humidity are responsible for considerable respiratory disease mortality burden, the component attributed to the high absolute humidity effect is greater. Our results may have important implications for the development of public health measures to reduce respiratory disease mortality.The online version contains supplementary material available at 10.1186/s12199-021-01030-3. The health risks associated with respiratory diseases cause serious morbidity and mortality, especially in some developing countries , 2. ChanHumidity, as one of the meteorological factors, is not the first choice of researchers, and is often included in the confounding effects of relative humidity on the health effects of respiratory diseases , 17. StuThis research takes Guangzhou as the research object. As a subtropical city in China, Guangzhou, has relatively humid air every year with obvious seasonal changes. With the changes in humidity brought about by climate change, there is not enough evidence to show the impact of this changing environment on the risk of death in the respiratory system. In this study, the daily time series data of Guangzhou from 2013 to 2018 were selected to establish a distributed lag non-linear model (DLNM) using R software to estimate the influence of absolute humidity on respiratory diseases in Guangzhou population. In addition, this study further analyzed deaths from influenza and pneumonia (I&P) and chronic lower respiratory disease (CLRD), two major disease types, based on data from the 10th revision of the International Classification of Diseases (ICD10). Chronic lower respiratory diseases mainly include chronic obstructive pulmonary disease, asthma, and occupational pulmonary disease. Finally, the burden of death caused by non-optimal absolute humidity, i.e., the number of deaths and the proportion of deaths caused by non-optimal absolute humidity, were quantified. The results of this study can provide reference for local health authorities to take measures to reduce the mortality from respiratory diseases in the population.2. Guangzhou is mild in winter and hot in summer, with high humidity and obvious changes throughout the year.This study selected Guangzhou, China as the research area. Guangzhou , is the largest city in South China, located in the southeast of China, with a population of about 14.9 million and a population density of 1708 people/kmAccording to the 10th revision of the International Classification of Diseases (ICD10), daily data of deaths from respiratory disease (ICD10: J00-J99) in Guangzhou from 1 February 2013 to 31 December 2018 were obtained from the Guangzhou Center for Disease Control and Prevention, and influenza and pneumonia (ICD10:J09-J18) and chronic lower respiratory disease (ICD10: J40-J47) were further analyzed. We also stratified daily mortality by gender and age group .http://data.cma.cn/) and are authoritative and reliable. Absolute humidity is calculated from the collected temperature and relative humidity data, and the calculation formula is as follows [Daily meteorological data was collected from five meteorological stations  in Guangzhou Fig. . The met follows :\documen2.5, NO2, SO2, and O3, While O3 is the maximum 8-h average of daily ozone concentration. The data came from the China’s National City Air Quality real-time publishing platform (http://106.37.208.233:20035/) managed by the China’s National Environmental Monitoring Center.Air pollutants may alter the relationship between meteorological factors and mortality . This stA time series database of respiratory diseases, meteorological factors, and air pollution factors was established, and DLNM was used to examine the relationship between absolute humidity and death from respiratory diseases. DLNM is a regression model based on cross basis functions to study the exposure response relationship, and at the same time, it also takes into account the lag response of exposure-response factors and the non-linearity of the exposure-response relationship . Due to t is the day of observation; E(Yt) represents expected daily mortality from RESP; α is the intercept; cb refers to the cross basis function obtained by applying DLNM to absolute humidity; lag represents the days of lag; ns represents the function of natural spline; df means degrees of freedom; 7 df per year were selected to control trends and seasonality over long periods of time according to the minimum Akaike Information Criterion (AIC) value; Meteorology variables include mean precipitation and mean atmospheric pressure, with 3 df to control their trends; Pollution variables include PM2.5, NO2, SO2, and O3, which also use 3 df to control their confounding effects; day of week (Dow) and public holiday (Holiday) are included in the model as categorical variables; β1, β2 are the coefficient; ε is error term. Natural cubic spline function was used for exposure-response’s dimension fitting, the knot locations are set in the 10th, 75th, and 90th percentiles of absolute humidity [Where humidity . Naturalhumidity , 31, Morhumidity , 32. So Then, the relative risk (RR) of death from respiratory diseases due to absolute humidity was analyzed. DLNM could obtain the minimum mortality absolute humidity (MMAH) and the corresponding minimum mortality absolute humidity percentile (MMAHP) by the cumulative exposure-response relationship between absolute humidity and the number of deaths obtained by the best linear unbiased prediction of all results. Taking MMAH as baseline absolute humidity and adding the effect values of all days in DLNM, the total population attributable mortality (PAM) due to respiratory diseases caused by non-optimal absolute humidity can be calculated, and the corresponding attributable fraction (AF) can be calculated from the percentage of the population attributable mortality to the total mortality . The attSensitivity analysis was conducted in this study to verify the stability of the model. Therefore, we changed the degree of freedom of natural spline function of meteorological factors and air pollution in the model from 3 to 5 successively in order to control their confounding influence. The degree of freedom of the temporal natural spline function in the model is changed from 6 to 8 successively in order to control the trend of time . In the P < 0.05 is considered statistically significant.All the statistical analysis in this study was carried out in R 3.5.2 software, and the “dlnm” software package was used to build the DLNM. All statistical tests use two-tailed test, m3, 22.54 °C, 80.29%, 5.78 mm, 100.57 kPa and 2.67 m/s, respectively.Descriptive statistics of daily respiratory disease mortality and meteorological condition from 2013 to 2018 are shown in Table Mortality from respiratory diseases usually peak in winter, with an occasional small peak in summer, showing seasonal changes. Absolute humidity and mean temperature peak in summer, and atmospheric pressure peak in winter, both of which show seasonal changes. The relative humidity and wind speed fluctuated throughout the year, but the seasonal changes were not obvious Fig. .Fig. 2Thr = − 0.208, P < 0.01), temperature , and precipitation  and was positively correlated with atmospheric pressure , but has no significant correlation with relative humidity and wind speed , the average temperature is not included in the main model to avoid double calculation and collinearity. Instead, a sensitivity analysis of the influence of temperature on absolute humidity-respiratory disease death is done separately.Spearman correlation analysis showed that mortality from respiratory disease was negatively correlated with absolute humidity (m3 (47.5%). The relationship between absolute humidity and the relative risk of mortality from influenza and pneumonia presents a U-shaped curve with the minimum mortality absolute humidity of 14.58 g/m3 (36.4%), the relative risk increases first and then decreases at high humidity   of deaths from respiratory diseases presents a nonlinear trend in different lag days and absolute humidity, as shown in Fig. ig. Sm3 5.7% , among which the high absolute humidity was 15.92% (95% CI 4.25 ~ 23.99%) and the low absolute humidity was 5.97% (95% CI − 1.11 ~ 11.56%)  of deaths from influenza and pneumonia across lag 0-35 days  in Guangzhou and daily mean absolute humidity distribution. Figure S2. Overall cumulative relative risks (RRs) of deaths from chronic lower respiratory disease across lag 0-35 days  in Guangzhou and daily mean absolute humidity distribution. Figure S3. The residual variation scatter plots over time for main model in daily RESP deaths in Guangzhou. Figure S4. Sensitivity analyses of overall cumulative relative risks (RRs) of respiratory disease mortality due to absolute humidity by changing degrees of freedom (6 to 8) for time variables. Figure S5. Sensitivity analyses of overall cumulative relative risks (RRs) of respiratory disease mortality due to absolute humidity by changing degrees of freedom (3 to 5) for meteorological variables and air pollution variables. Figure S6. Sensitivity analyses of overall cumulative relative risks (RRs) of respiratory disease mortality due to absolute humidity by changing the lag parameters of the included temperature. Table S2. Sensitivity analysis results on the effects of df/parameter in DLNM on the associations between absolute humidity and respiratory diseases mortality burden. Table S3. Sensitivity analysis results on the effects after controlling for temperature at lag 0, 0-7 and 0-14 days in the model. Figure S7. Overall cumulative relative risks (RRs) of deaths from respiratory diseases across lag 0-35 days  in Guangzhou and daily mean temperature distribution. Table S4. Comparison of respiratory diseases mortality burden due to temperature models versus absolute humidity models.


The aim of this study was to investigate the effect of icaritin (ICT) on TAR DNA-binding protein 43 (TDP-43)-induced neuroblastoma (SH-SY5Y) cell damage and to further explore its underlying mechanisms.To investigate the possible mechanism, TDP-43 was used to induce SH-SY5Y cell injury. Cell viability was evaluated by the CCK-8 assay. The mitochondrial membrane potential (MMP) was determined with JC-1. The expression levels of TDP-43 and cytochrome C (CytC) were measuring by Western blotting. Changes in adenosine 5′-triphosphate (ATP) content, total antioxidative capacity (T-AOC), glutathione peroxidase (GSH-Px) activity, superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were detected with specific kits.The results showed that ICT reduced the cell damage induced by TDP-43. ICT reduced the expression level of TDP-43; increased ATP content and the MMP; decreased CytC expression; increased T-AOC and GSH-Px, total SOD (T-SOD), copper/zinc SOD (CuZn-SOD), and manganese SOD (Mn-SOD) activity; and decreased MDA content.The results suggest that ICT has a protective effect on TDP-43-transfected SH-SY5Y cells that is related to reductions in TDP-43 expression and mitochondrial damage and alleviation of oxidative stress. TAR DNA-binding protein 43 (TDP-43) is a multifunctional nucleic acid-binding protein composed of 414 amino acids and six exons. Under physiological conditions, it can regulate RNA splicing, transport and translation in a variety of ways and is involved in the biosynthesis of microRNAs. It is an important transcriptional regulator . This prEpimedium, is rich in flavonoids and easily crosses the blood–brain barrier (Icaritin (ICT), a component of  barrier . It impr barrier . Studies barrier . TherefoTherefore, to confirm our hypothesis, the protective effect of ICT against TDP-43-induced mitochondrial damage in SH-SY5Y cells   was evalReagent-grade ICT,  was obtained from Zhongke Quality Inspection Biotechnology Co., Ltd.  and dissolved in dimethyl sulfoxide (DMSO); the final concentration of DMSO in the medium was less than 0.1% (v/v). Human TDP-43 and polybrene were purchased from Hanbio Biotechnology Co., Ltd. , a TransZol Up Plus RNA Kit and EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix were purchased from TransGen Biotechnology Co., Ltd.  and NuHi Robustic SYBR Green Mix was purchased from Nuhigh Biotechnologies Co., Ltd. . High-Sig ECL Western Blotting Substrate was purchased from Tanon Technology Co., Ltd. . RIPA buffer (high) (R0010) was purchased from Solarbio Life Science . A GAPDH antibody, a TDP-43 antibody, a CytC antibody, and HRP-conjugated Affinipure goat anti-rabbit IgG (H+L) were purchased from Proteintech Group . A goat anti-mouse IgG (H+L) secondary antibody was purchased from Thermo Fisher Scientific .2 cell culture incubator at 37 °C. When the cells covered approximately 80% of the bottom of the bottle, they were collected in a single tube and centrifuged at 1000 rpm for 5 min after trypsin digestion. After discarding the supernatant, the cells were seeded in 96-well plates, 24-well plates, or 6-well plates at a density of 5  × 104/mL in complete medium, and the medium was changed every 2 days.SH-SY5Y human neuroblastoma cells were purchased from Wuhan University Collection Center. The cells were cultured in DMEM-F12 medium containing a mixture of 15% fetal bovine serum and 1% penicillin streptomycin in a 5% CO5 cells/mL) were plated in a 96-well plate at a density of 1 ×104 cells per well and maintained at 37 °C and 5% CO2 A total of 200 µL of fresh medium containing virus at an MOI of 0, 10, 20, 30, 40, 50, or 60 (including 1.2 µL of polybrene at a working concentration of 6 µg/mL). After 12 h, the transfection was terminated, and the optimal MOI was selected by observing and photographing the cells under a fluorescence microscope.Virus transduction construct, cloning, and production were carried out by Hanbio Biotechnology Co., Ltd. The detailed steps and more information are provided in the supplementary files. SH-SY5Y cells showing good growth were chosen. The SH-SY5Y cells (1 ×105 cells/mL) were seeded in each well of a 96-well plate for 24 h. After treatment, CCK-8 solution (10 µL) was added to each well of the 96-well plate, the cells were incubated at 37 °C for 2 h. The absorbance was measured at 450 nm with a Synergy HTX microplate reader .Cell viability was assessed by the CCK-8 assay ; in this assay, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5--2H-tetrazolium monosodium salt produces water-soluble maz dye after it is formed and undergoes biological reduction in the presence of electron carriers. Briefly, SH-SY5Y cells . The proteins were then heated at 100  °C for 5 min for denaturation. Equal amounts of total protein (25 µg per lane) were loaded on 12% SDS-PAGE gels and separated. After sample loading, the voltage was set to 90 V and then to 120 V until the sample reached the bottom of the gel. The proteins were then transferred to a nitrocellulose (NC) film by the sandwich method. The membrane was washed three times with TBST and then blocked with 5% skimmed milk. The membrane was then incubated for 12 h at 4 °C with a GAPDH antibody , TDP-43 antibody , and CytC antibody . The membrane was washed with TBST 3 times and incubated with HRP-conjugated Affinipure goat anti-rabbit lgG (H+L)  for 2 h at room temperature. The membranes were developed using hydrogen peroxide and Supersignal West Pico Luminol . Finally, High-Sig ECL Western Blotting Substrate  was used to visualize the membrane.A JC-1 MMP test kit  was used according to the manufacturer’s protocol. The culture medium of each group of cells was discarded, the cells were wash once with PBS, and the same amount of culture medium and JC-1 staining working solution was added and mixed well. Afterwards, the cells were placed in a 37 °C cell incubator and incubated for 20 min. Then, the supernatant was discarded, the cells were washed twice with prepared 1 × JC-1 staining buffer, and cell culture medium was added. After this, the cells were washed with PBS and observed under an inverted fluorescence microscope .We used a commercial ATP content test kit, GSH-Px test kit, SOD typing test kit, MDA test kit  and T-AOC test kit . A BCA protein quantitative kit (Beijing Solarbio) was used to determine the protein content.To measure ATP levels, reagents and the sample to be tested were added to tubes according to the manufacturer’s protocol. A blank tube, a standard tube, experimental tubes, and a control tube were analyzed, and the experiment was performed in triplicate. The absorbance value of each tube at a wavelength of 636 nm was measured .To determine T-AOC, cells were scraped off, lysed on ice, and centrifuged at 4 °C at 12,000 rpm in a low-temperature centrifuge for 5 min, and the supernatant was taken for determination. Reagents and the sample to be tested were added to each well according to the manufacturer’s instructions, the absorbance value of each well at a wavelength of 734 nm was measured, and the T-AOC of the sample was calculated according to the standard curve.To measure GSH-Px levels, prepared cell homogenates were centrifuged in a low-temperature centrifuge at 2000 rpm for 10 min, and the supernatant was centrifuged at 10,000 rpm in a low-temperature centrifuge for 15 min. After adding 0.5 mL of PBS and mixing, the prepared detection reagents were added, and the absorbance value of each sample was determined at a wavelength of 412 nm.via the hydroxylamine method according to the manufacturer’s protocol. SH-SY5Y cells were seeded into a 6-well plate, and the absorbance of the samples was measured at 550 nm using a microplate reader.To analyze SOD levels, T-SOD, CuZn-SOD, and Mn-SOD activity in SH-SY5Y cells was measured using a SOD typing assay kit To assess MDA levels, treated cells were washed with cold PBS and homogenized in PBS in a glass homogenizer to form a suspension. The supernatant was used for analysis according to the manufacturer’s protocol, and the absorbance of each sample was measured at a wavelength of 530 nm.t-test with correction. A value of P < 0.05 was considered statistically significant.All statistical analyses were performed with SPSS 22.0 . All results are expressed as the mean ± SD. The difference between the means of more than two groups was analyzed by one-way analysis of variance (ANOVA). When ANOVA showed significant differences, pairwise comparisons of means were made by Bonferroni’s post hoc TDP-43 in the TDP-43 group was higher than that in the control group (Cell were transfected with virus an MOI of 0, 10, 20, 30, 40, 50 or 60. Cells transfected at an MOI of 0 were in good condition and showed no green fluorescence under a fluorescence microscope. Cells transfected at an MOI of 30 were in good condition, showed clearly visible dendrites, fluoresced under a fluorescence microscope, and showed high transfection efficiency. The fluorescence and transfection efficiency of cells transfected at MOIs of 10 and 20 were lower than those transfected at an MOI of 30. The cells transfected at MOIs of 40 and 50 were in a poor state; there cell bodies were crumpled and collapsed, and some of the cells were dead. Cells transfected at an MOI of 60 were blurred or absent, and a large number of cells were dead. In conclusion, the optimal MOI was determined to be 30 . The morol group . Westernol group . In summAfter modeling, ICT at concentrations of 0.0, 0.1, 1.0 and 10.0 µmol/L was added, and the cell activity of each group at 12, 24 and 48 h were determined. The results showed that optimal cell viability was obtained at a drug concentration of 1.0 µmol/L and a treatment time of 48 h. Therefore, subsequent experiments were carried out under these conditions .Changes in cell activity in the control group, control + ICT group, TDP-43 group, and TDP-43 + ICT group were assessed after treatment with 1.0 µmol/L ICT for 48 h. The results showed that compared with that of the control group, the activity of the TDP-43 group was decreased, while the cell activity of the TDP-43 + ICT group was increased after ICT treatment .The Western blot results showed that compared with that in the Con group, the expression level of TDP-43 in the TDP-43 group was increased; however, after ICT treatment, the expression level of TDP-43 decreased .JC-1, which can quickly and sensitively detect MMP changes, was used a fluorescent probe in this experiment. In normal mitochondria, JC-1 aggregates in the mitochondrial matrix to form a polymer that emits strong red fluorescence. In unhealthy mitochondria, due to a decrease in the MMP, JC-1 can only exist in the cytoplasm as monomers, which produce green fluorescence. The results showed that compared with the control group and control + ICT group, the TDP-43 group showed reduced red fluorescence, enhanced green fluorescence and a decreased MMP. Green fluorescence was weakened, red fluorescence was enhanced, and the MMP was increased in the TDP-43 + ICT group compared with the TDP-43 group  and 4B.The results showed that there was no significant difference in ATP content between the control + ICT group and control group and that ATP content in the TDP-43 group was lower than that in the control group. Compared with that in the TDP-43 group, the ATP content in the TDP-43 + ICT group was increased .The Western blot results showed that compared with that in the control group, the expression level of CytC in the TDP-43 group was increased, while the expression level of CytC decreased after ICT treatment .T-AOC , GSH-Px TDP-43 is a highly conserved nuclear RNA/DNA-binding protein involved in the regulation of RNA processing . TDP-43 Abnormal accumulation of TDP-43 in mitochondria is an important cause of mitochondrial dysfunction and neurodegenerative diseases . PreventA large amount of TDP-43 is localized in mitochondria mainly because a long-term increase in oxidative stress increases the nuclear depletion and cytoplasmic accumulation of TDP-43 and promotes the entry of a large amount of this protein into mitochondria. The interaction between TDP-43 and OXPHOS-related mt-mRNA affects the mitochondrial respiratory chain, resulting in a decrease in ATP production, a decrease in the MMP and an increase in ROS levels. This increase in ROS levels promotes the TDP-43/OXPHOS-related mt-mRNA interaction, forming a vicious cycle . Lipid pHow does ICT reduce the level of TDP-43? According to our findings and previous results, we speculated that ICT promotes the degradation of TDP-43 by increasing the levels of LonP1 and ATP. We also speculate that the mechanism is related to the antioxidant capacity of ICT based on existing experiments and related research. A large number of studies have shown that ICT is a relatively good plant-based antioxidant and it has good antioxidant capacity in different cell and animal experiments . This exIt is well known that TDP-43 gene mutations are one of the recognized genetic causes of ALS, which is closely related to an imbalance of energy homeostasis and disease susceptibility and progression . Other rThe results suggests that the protective effect of ICT against TDP-induced SH-SY5Y cell injury is related to a reduction in TDP-43 expression, alleviation of mitochondrial injury and amelioration of oxidative stress.10.7717/peerj.11978/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.11978/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.11978/supp-3Supplemental Information 3Click here for additional data file.