KLF10. Therefore, curcumin might decrease the level of hsa-miR-6724-5p, leading to increased KLF10 expression, resulting in cell cycle arrest in curcumin-treated IPF fibroblasts. In conclusion, our findings might support the potential role of curcumin in the treatment of IPF, but further in-depth study is warranted to confirm our findings.Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive interstitial lung disease. Currently, therapeutic options are limited for this fatal disease. Curcumin, with its pleiotropic effects, has been studied for its potential therapeutic utilities in various diseases, including pulmonary fibrosis. However, the detailed mechanisms have not been studied comprehensively. We conducted a next-generation sequencing and bioinformatics study to investigate changes in the profiles of mRNA and microRNA after curcumin treatment in IPF fibroblasts. We identified 23 downregulated and 8 upregulated protein-coding genes in curcumin-treated IPF fibroblasts. Using STRING and IPA, we identified that suppression of cell cycle progression was the main cellular function associated with these differentially expressed genes. We also identified 13 downregulated and 57 upregulated microRNAs in curcumin-treated IPF fibroblasts. Further analysis identified a potential microRNA-mediated gene expression alteration in curcumin-treated IPF fibroblasts, namely, downregulated hsa-miR-6724-5p and upregulated  Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disease characterized by fibrosing interstitial pneumonitis ,5,6,7,8.Although the mechanisms underlying the pathogenesis of IPF remain largely unknown, fibroblasts are recognized as key players in the pathogenesis and progression of IPF ,6,7,11. The currently available treatment modalities for IPF are limited. Currently, only two medications, nintedanib and pirfenidone, are available to hamper disease progression and improve patients’ quality-of-life ,22,23,24Curcuma longa), a member of the ginger family (Zingiberaceae) [Curcumin, also known as diferuloylmethane, is the principle phenolic compound extracted from turmeric (eraceae) ,26. Besieraceae) ,27. Curceraceae) ,26,27. Deraceae) ,32,33,34Some in vitro and in vivo studies showed the effects of curcumin on fibroblasts, such as promoting apoptosis, inhibiting differentiation, modulating matrix metalloproteinase production, and enhancing antioxidant defense mechanisms ,37,38,39The IPF fibroblasts showed significantly increased cell proliferation/viability compared with normal human lung fibroblasts . Curcumiq-value < 0.05). A total of 23 downregulated and 8 upregulated protein-coding genes were identified in IPF fibroblasts treated with curcumin (The gene expression profiles in IPF fibroblasts treated with either curcumin (10 μM) or DMSO (control) were obtained by using NGS. As shown in the density plot a, the cucurcumin .p-value of <1.0 × 10−16 (p < 0.001).The 23 significantly downregulated and 8 significantly upregulated genes in curcumin-treated IPF fibroblasts were included in the analysis for protein–protein interactions and biological processes. We uploaded these differentially expressed genes into the Search Tool for the Retrieval of Interacting Genes (STRING) database for protein–protein interaction (PPI) network analysis. We set the minimum required interaction score to the high confidence (score = 0.700). This analysis obtained a highly interactive PPI network of 31 nodes (proteins) and 35 edges (protein-protein associations), with a PPI enrichment  × 10−16 . Gene on® Pathway Analysis (IPA) to outline the most enriched functional alteration associated with curcumin treatment in IPF fibroblasts. q-value = 0.00125); nine genes, including ANLN, BUB1, CENPF, CIT, KLF10, KNL1, PTX3, TOP2A, and TOX, were involved in this cellular function.Differentially expressed genes were further analyzed with IngenuityKLF10. Using TargetScan, the prediction of KLF10 as a target of hsa-miR-6724-5p was confirmed .Using NGS, we identified 13 significantly downregulated microRNAs and 57 significantly upregulated microRNAs associated with curcumin treatment in IPF fibroblasts . Using tPTX3, on both lists of dysregulated genes in IPF fibroblasts and altered genes in curcumin-treated IPF fibroblasts, which was upregulated in IPF fibroblasts compared to normal human lung fibroblasts (fold change = 2.19) and downregulated by curcumin (fold change = −5.26)  . We also= −5.26) b. ExcludKLF10 expression in IPF fibroblasts  is a major pathogenic mechanism of IPF, and fibroblasts are key effector cells of this process . The curKLF10) belongs to the Krüppel-like transcription factors (KLFs) family, which is made up of 15 zinc-finger proteins able to control cell proliferation and differentiation [TIEG or TIEG1), KLF10 is a primary response gene for TGF-β [KLF10 may mimic the antiproliferative and proapoptotic effects of TGF-β1 on various tumor cells [KLF10, which is induced by a nerve growth factor, suppressed cell cycle progression of pheochromocytoma cell line PC12 [KLF10 was also found to be an effective repressor of myoblast proliferation via repression of FGFR1 promotor activity [KLF10 was associated with vascular endothelial cell arrest in the G1 phase and promotion of apoptosis [KLF10 also plays a role in the protection against oxidative stress and in myofibroblast-like conversion in human skin fibroblasts [KLF10-deficient mice showed increased pulmonary inflammation, KLF10 might control fibrosis by negatively regulating inflammation [KLF10 in regulating the cellular physiology of pulmonary fibroblasts remain largely unknown, the curcumin-induced upregulation of KLF10 might provide potential targets to reverse pulmonary fibrosis, and therefore deserve further study.Krüppel-like factor 10  and downregulated by curcumin. PTX3 encodes pentraxin 3, also known as tumor necrosis factor-inducible gene 14 (TSG-14) protein, which regulates multiple aspects of inflammation. The increased serum level of pentraxin 3 was associated with the disease severity of systemic sclerosis [PTX3 gene expression [PTX3 in the effects of curcumin on IPF fibroblasts deserves further study.By combining the data of the current study with our previous study, we found a common gene, clerosis . In a miclerosis . In a clpression . The rolThe poor bioavailability of curcumin is always a major limitation to its wide application . In a moThis study demonstrated several limitations. Firstly, the cells used in this study were the primary cells from a single IPF patient. Because fibroblasts from several IPF patients may have marked heterogeneity in the baseline gene expression profiles and the response to stimuli, our findings might not be able to be generalized to other IPF patients. However, the findings of this exploratory study might provide a scientific basis for understanding the pathogenic mechanisms and developing novel treatments in the future. Secondly, the results of the bioinformatic analyses in the present study were not validated. Further in vitro and in vivo studies are warranted to confirm our findings. Thirdly, the number of differentially expressed genes was relatively low. Although the numbers of differentially expressed genes and interesting pathways could have been increased by lowering the stringency, we selected the most significant findings in this study. Fourthly, we only assessed the transcriptomic changes in IPF fibroblasts treated with a single concentration of curcumin, which may not reflect the changes in normal human lung fibroblasts treated with curcumin. While curcumin showed similar, yet milder, inhibitory effects on cell viability/proliferation in a dose-dependent manner in normal human lung fibroblasts as those observed in IPF fibroblasts, the curcumin-related transcriptomic changes might be similar in pulmonary fibroblasts to either IPF patients or normal subjects. Finally, in addition to fibroblasts, airway epithelial cells, especially type II alveolar epithelial cells, are also key drivers in the pathogenesis of IPF . Whether® Pathway Analysis (IPA) database [database , to invedatabase  for targdatabase .5 cells/well) and incubated for 24 h, and were then treated with either curcumin  or the carrier solvent (0.1% DMSO) for 48 h.Primary human lung fibroblasts from an 83-year-old male Caucasian IPF patient  and normal human lung fibroblasts  from a 67-year-old Caucasian man were incubated at 37 °C in a Fibroblast Growth Medium-2 (FGM™-2) Bulletkit™  containing 10 mL fetal bovine serum, 0.5 mL human fibroblast growth factor-basic (hFGF-B), 0.5 mL insulin, and 0.5 mL GA-1000, as performed in our previous studies [p-value of <0.05 was considered significant.The cell proliferation/viability was assessed using Premixed WST-1 Cell Proliferation Assay , as in our previous studies . Data we® Reagent , according to the manufacturer’s instructions. Purified RNA was quantified at OD260nm by an ND-1000 spectrophotometer  and qualitated using the Bioanalyzer 2100  with an RNA 6000 LabChip® kit (Agilent Technologies). Before library preparation, the quality of RNA was ensured based on the following criteria: amount ≥ 5 μg (for transcriptome) or ≥ 2 μg ; concentration ≥ 80 ng/μL (for transcriptome) or ≥ 200 ng/μL ; OD260nm/OD280nm ratio of 1.8–2.2; OD260nm/OD230nm ratio ≥ 1.5; RNA integrity number ≥ 7 (for transcriptome) or ≥ 8 . The RNA library preparation and deep sequencing were carried out in Welgene Biotechnology Company , according to Illumina’s official protocol.The expression profiles of microRNAs and mRNAs were assessed using an NGS platform, as described in our previous studies ,80,81,82p-values for differential expression were calculated by Cuffdiff (Cufflinks version 2.2.1), with the non-grouped sample using “blind mode”, whereby all samples were treated as replicates of a single global “condition” and used to build one model for a statistical test [q-values were p-values adjusted with false discovery rate (FDR) using the method by Benjamini and Hochberg [q-values of <0.05  > 1.3) were considered to be significantly differentially expressed genes.For transcriptome sequencing, the Agilent’s SureSelect Strand Specific RNA Library Preparation Kit was used to prepare the libraries, followed by AMPure XP Beads size selection. The sequence was directly determined using Illumina’s sequencing-by-synthesis (SBS) technology on the NovaSeq 6000 Sequencing System . Sequencing data were generated by Welgene’s pipeline based on Illumina’s basecalling program bcl2fastq v2.2.0. Reads with low quality were trimmed and removed using the Trimmomatic (version 0.36) . Qualifical test . The q-vHochberg . Genes wFor small RNA sequencing, sample libraries were prepared using the TruSeq Small RNA Library Preparation Kit (Illumina) according to the manufacturer’s protocols. The 3′- and 5′-adaptors were ligated to the microRNAs. Subsequently, universal cDNA synthesis with unique molecular identifier (UMI) assignment, cDNA cleanup, library amplification, and library cleanup were performed. Libraries were sequenced on the NextSeq500 Sequencer  . Sequencing data were processed with the Illumina software BCL2FASTQ v2.20. Trimmomatics (version 0.36)  was usedhttps://string-db.org/) collects and integrates this information by consolidating known and predicted protein–protein association data for many different organisms. STRING offers the information of the associations including direct  and indirect  interactions, which are derived from five main sources: Conserved co-expressions, high-throughput lab experiment, automated text-mining, genomic context predictions, and previous knowledge in the database [p-values (FDR-p) < 0.015 were selected.The functional interactions between proteins are very important and complicated in cells. The STRING database  of <0.5 and available activation z-scores (representing the magnitude of significant activation) were selected.To investigate the systems biology as a whole integrated system, network-based analysis is important. The IPA software  contains a large database with detailed and structured findings reviewed by experts. This IPA database is derived from thousands of biological, chemical, and medical researches, and provides researchers with quick searching. The IPA can also survey the analysis, integration, and recognition of genes, single nucleotide polymorphism (SNP) arrays, RNA and small RNA sequencing, proteomics, and many other biological experiments. Furthermore, IPA provides identification of associated signaling pathways, molecular interactions, upstream regulators, disease processes, and candidate biomarkers . All sighttp://mirmap.ezlab.org/) is an open-source software library which provides predictions of comprehensive microRNA targets [microRNAs can repress the expression of protein-coding genes. The miRmap  that predicts the targets of microRNAs by searching for the presence of conserved 8mer-, 7mer-, and 6mer- sites matching the seed region of each microRNA. The prediction results are ranked by the probability of conserved targeting or by the predicted efficacy of targeting [The TargetScan is an online database (argeting . The TarKLF10 expression, resulting in cell cycle arrest in curcumin-treated IPF fibroblasts. The current findings might support the potential role of curcumin in the treatment of IPF, but further in-depth study is warranted to confirm our findings.In this study using an NGS and bioinformatics approach, we found that curcumin might decrease the level of hsa-miR-6724-5p, leading to increased


The most stable adsorption configuration, the adsorption energy, the density of states, and the charge transfer are discussed to research the adsorption properties of intrinsic graphene (IG), Ni-doped graphene (Ni–G), vacancy defect graphene (DG), and graphene oxide (G–OH) for H2S and CH4. The weak adsorption and charge transfer of IG achieved different degrees of promotion by doping the Ni atom, setting a single vacancy defect, and adding oxygen-containing functional groups. It can be found that a single vacancy defect significantly enhances the strength of interaction between graphene and adsorbed molecules. DG peculiarly shows excellent adsorption performance for H2S, which is of great significance for the study of a promising sensor for H2S gas.In this study, the adsorption performances of graphene before and after modification to H These studies may provide some theoretical basis for the search of sensor materials with high sensitivity to H2S or CH4 gas, and they may also provide direction for the research of modified graphene-based sensors.In this study, Ni was selected to form Ni-doped graphene. As a cheap and promising metal catalyst, Ni attracted more and more attention in recent years, and it was widely used in many kinds of materials to achieve better results ,35. Howe−6 Ha, 0.002 Ha/Å, and 0.005 Å, respectively. Spin unrestricted was chosen to neglect the effect of spin polarization. The double numerical plus polarization (DNP) was selected, and the DFT semi-core pseudopotential (DSPP) was applied for core treatment because of its relativistic effects. The k point of the Brillouin zone integration and the self-consistent (SCF) field tolerance were set to 6 × 6 × 1 and 1 × 10−5 Ha in this study. The direct inversion of iterative subspace (DIIS) was set to 6 to accelerate the convergence of SCF charge density.All calculations in the paper were carried out using the Dmol3 module of Material Studio (MS) ,37. The adsE) of gas molecules adsorbed on four kinds of graphene-based materials was calculated using the following equation:(gas-substrate)E, (substrate)E, and (gas)E are the total energies of the graphene system after gas molecule adsorption, the graphene system before gas molecule adsorption, and a single gas molecule, respectively [In this paper, the adsorption energy  with an Ni atom. DG was obtained by removing C43. G–OH was formed by adding a hydrocarbon functional group to C43. The four optimized graphene models are shown in The structure of Hterature ,43. IG w3sp configuration. The charge transfer between the graphene monolayer and hydrocarbon group was 0.233 e. The former lost electrons while the latter gained electrons. However, the graphene oxide system was non-magnetic. Similarly, the Fermi level moved into the valence band, but the strength was between that of the DG and Ni–G systems, as shown in The C–C bond length and the bond angle of IG after optimization were 1.420 Å and 120.020°, respectively. The values are consistent with the theoretical values in the literature, which indicates that the IG model can be used for further calculations . There i2S is a kind of polar molecule; thus, three typical adsorption configurations were selected in this study: (1) the H2S molecule was perpendicular to IG, Ni–G, DG, or G–OH, and the S atom was close to the C, Ni, C, or H atom ; (2) the H2S molecule was parallel to IG, Ni–G, DG, or G–OH, and the S atom was above the C, Ni, C, or H atom ; (3) the H2S molecule was perpendicular to IG, Ni–G, DG, or G–OH, and the S atom was far away from to the C, Ni, C, or H atom . Initial adsorption distance was set to 2 Å. The 12 initial configurations of H2S molecule adsorbed on IG, Ni–G, DG, and G–OH are shown in 2S and the four graphene systems; thus, four adsorption models  were chosen for further study. In order to simply describe the four systems, we use H2S–IG, H2S–Ni–G, H2S–DG, and H2S–G–OH instead.H2S, and Adsorption energy, the final adsorption distance, and charge transfer are three important aspects for describing the strength of adsorption between gas molecule and the graphene system. A lower adsorption energy results in a shorter final adsorption distance, a larger charge transfer amount, and a stronger adsorption effect. 2S, which was further than the initial distance. Furthermore, the charge transfer was 0.004 e, which is very poor. Moreover, the absolute value of the adsorption energy was extremely small. The lengthy final adsorption distance, the poor charge transfer, and the low absolute value of the adsorption energy indicates that intrinsic graphene had a weak adsorption capacity for H2S. Compared with intrinsic graphene, the adsorption distance reduced from 3.811 Å to 2.426 Å, showing a stronger interaction between Ni–G and H2S. In addition, the absolute value of adsorption energy was significantly increased, but Ni–G did not form a strong chemical adsorption for H2S. A strong charge transfer existed between Ni–G and the H2S molecule, resulting in a value of 0.233 e, which illustrates that the doping of Ni significantly improved the electron transfer ability. Meanwhile, it can be seen that graphene obtained electrons and the H2S molecule lost electrons during the adsorption process. Therefore, Ni doping could slightly improve the adsorption ability of graphene with respect to H2S. Analogously, for the system of G–OH adsorption of H2S, the absolute value of adsorption energy increased evidently, which showed a strong chemical adsorption. The adsorption distance was 2.412 Å, which was smaller than that of IG and Ni–G systems. The charge transfer was 0.054 e, which illustrates that the hydrocarbon functional group acted as the bridge of charge transfer between graphene and H2S. However, the increase was far less than that of Ni–G. The absolute value of adsorption energy was 2.934 eV in the system of G–OH adsorbed to H2S, which was significantly increased compared with the other three systems. The adsorption distance decreased, and the charge transfer was 0.172 e. It can be seen that the increasing range of charge transfer was smaller than that of adsorption energy and adsorption distance, which was probably due to the electron transfer in DG interfering with the electron transfer between H2S and DG. The extremely low adsorption energy, the short adsorption distance, and the good charge transfer amount showed the extremely strong chemical adsorption between DG and the H2S molecule, which shows that graphene is an excellent material for H2S adsorption. In addition, Ni–G and G–OH can also be viable options.From 2S molecule, the DOS and the PDOS are shown in 2S on IG changed little from 2p orbital had almost no overlapping peak with H 1s and S 3p orbitals. In addition, there was no effective contact of charge density between IG and H2S molecule from 2S. There was almost no change in DOS at the Fermi level after the adsorption of H2S by Ni–G, but the overall DOS slightly shifted to the left from 3d, H 1s, and S 3p orbitals was analyzed. The Ni 3d, H 1s, and S 3p orbitals had overlapped peaks surrounding −8 eV, −4 eV, −12 eV, 1 eV, and 2.5 eV. Fewer overlapping areas result in insignificant orbital effects. Moreover, the charge density of Ni–G was similar to that of the H2S molecule from 2S was relatively strong, showing quite strong polarization. The whole curve of DOS moved to the right, and the DOS at the Fermi level showed a little decline from 2p, H 1s, and S 3p orbitals was obvious, as reflected in the overlapped peaks from −7.5 eV to 3 eV. 2S showed varying degrees of increase surrounding −14 eV, −8 eV, −6.5 eV, −4.8 eV, and −2 eV. According to the PDOS from 2p, S 3p, O 2p, and H 1s orbitals. There were obvious overlapping peaks near −14 eV, −8 eV, −6.5 eV, −4.8 eV, and −2 eV, which indicates the strong hybridization and bonding between the orbitals. Meanwhile, there was no effective contact of charge density between G–OH and the H2S molecule from 2S molecule showed an excellent orbital effect and a strong polarization effect. Therefore, it is obvious that DG graphene exhibited the best adsorption performance for H2S molecules. The strong chemisorption, charge transfer, and orbital hybridization suggest that the DG-based sensor may be used to detect H2S gas.For further study of the adsorption between the four graphene systems and the H4 molecule, two initial adsorption configurations were designed. As shown in 4–IG–O) in 4–IG–T) in 4–IG–T, CH4–Ni–G–T, CH4–DG–O, CH4–G–OH–T) were chosen for the further study. Similarly, CH4–IG, CH4–Ni–G, CH4–DG, and CH4–G–OH were the terms used henceforth. It can be seen that different adsorption configurations had little effect on the adsorption of CH4, which may have been caused by the non-polarity of the CH4 molecule.According to the structure of the CH4 molecule, it can be found that the intuitive structures of both IG and CH4 remained substantially unchanged. The adsorption distance after optimization was 3.865 Å, which was much greater than the initial distance. The adsorption energy was −0.022 eV, and the charge transfer was −0.002 e. The long distance, the bad adsorption energy, and little charge transfer indicate that the adsorption capacity of intrinsic graphene for CH4 was extremely poor. After Ni–G adsorbing CH4, the Ni–C bond length of Ni–G increased, but the form of CH4 was basically unchanged. The distance was 3.186 Å, which was shortened. The value of adsorption energy and charge transfer increased. This shows that the adsorption of this system was much stronger, and the doping of Ni improved the adsorption capacity of IG. The charge transfer between Ni–G and CH4 was negative, which shows that the Ni–G system lost electrons and the CH4 molecule gained electrons. The value of adsorption energy greatly increased, and the distance effectively shortened. The defect of graphene brought about excellent adsorption effects. However, due to the influence of electron transfer in the defective graphene system, the value of charge transfer was 0.004 e. Finally, for the system of G–OH adsorbing CH4, the adsorption distance was greatly reduced, which allowed improving the adsorption effect. The small adsorption energy and weak charge transfer showed that the effect of adsorption of CH4 by G–OH was not good. Speaking generally, the adsorption capacity of the three graphene systems was enhanced compared with that of intrinsic graphene. Among them, the adsorption performance of DG was the strongest, followed by Ni–G.4 molecule adsorbed on IG, Ni–G, DG, and G–OH, and the electron density difference of the four adsorption systems are shown in 4 showed almost no difference, but there was a significant increase at −12 eV and −4 eV. By comparing 2p, C 2p (the C atom of CH4), and H 1s orbitals, and the PDOS at −12 eV was composed of C 2p and H 1s orbitals. The C 2p (CH4) and H 1s orbitals had overlapped peaks surrounding −4 eV, and a small peak of the H 1s orbital was at −14 eV, which was the reason for the increase in DOS at these two points. As shown in 1s, C 2p (CH4), and C 2p (Ni–G). From 4, the DOS changed at the Fermi level and increased substantially at −12 eV and −4.5 eV, as shown in 2p, C 2p (the C atom of CH4), and H 1s orbitals. It can be found from 2p, O 2p, H 1s, C 2p (the C atom of CH4), and H 1s (the H atom of CH4) orbitals, and there were overlapping peaks from −10 eV and −2.5 eV, showing strong hybridization. It can be seen from 4 molecule and the four graphene systems; thus, the charge transfer of the four adsorption systems was not strong. In general, the three kinds of graphene shortened the adsorption distance, and they strengthened the adsorption energy and charge transfer. Among them, DG showed the best adsorption performance to CH4. However, the three modified graphene specimens did not produce strong chemical adsorption for CH4; thus, we think that they may be not suitable for CH4 gas detection.In order to analyze the adsorption performance of the systems, the DOS, PDOS, and the electron density difference are discussed. 2S and CH4. It can be clearly seen that different modification methods can improve the adsorption capacity of intrinsic graphene to different degrees. However, compared with CH4, the three modified graphene specimens had better adsorption capacity for H2S, with DG particularly showing excellent adsorption performance for H2S, beyond that of IG, Ni–G, and G–OH. Therefore, we carried out a comparative analysis of the research on the adsorption of H2S by Ni–G, DG, and G–OH with other published research work, as shown in 2S, which is reflected in its stronger adsorption energy, short adsorption distance, and large charge transfer value. Therefore, DG is a promising material for the detection of H2S, which can be used in further experimental research.Above, we analyzed the adsorption systems of three kinds of modified graphene for H2S and CH4 molecules on intrinsic graphene, Ni–doped graphene, single vacancy defect graphene, and graphene oxide was investigated using first principles to study the adsorption performance of functionalized graphene with respect to gas molecules. The adsorption energy, density of states, charge transfer, and electron density difference were discussed in terms of their effect on the adsorption of H2S and CH4 in different orientations. The results revealed that H2S and CH4 both show weak physical adsorption on intrinsic graphene. The three kinds of modified graphene improved the adsorption capacity of intrinsic graphene to varying degrees. Among them, DG showed the best adsorption performance for H2S and CH4. However, compared with the adsorption of CH4, DG had excellent adsorption capacity for the H2S molecule, with an adsorption energy value of −2.934 eV, an adsorption distance of 1.797 Å, a transfer charge of 0.172 e, and strong orbital hybridization in PDOS. Therefore, DG is a promising material for H2S detection which can be used for further experimental research.The adsorption of H


A highly conductive three-dimensional (3D) graphene network (GN) was fabricated by chemical vapor deposition on a 3D nickel fiber network and subsequent etching process. Then a lightweight and flexible polydimethylsiloxane (PDMS)/GN composite was prepared by a vacuum infiltration method by using the graphene network as a template. The composite showed the superior electrical conductivity of 6100 S/m even at a very low loading level of graphene (1.2 wt %). As a result, an outstanding electromagnetic interference (EMI) shielding effectiveness (SE) of around 40 and 90 dB can be achieved in the X-band at thicknesses of 0.25 and 0.75 mm, respectively, which are much higher than most of the conductive polymers filled with carbon. The 3D graphene network can also act as a mechanical enhancer for PDMS. With a loading level of 1.2 wt %, the composite shows a significant increase by 256% in tensile strength.  With the booming of electronic devices and equipment over past decades, severe electromagnetic interference (EMI) and radiation pollution are brought out ,2,3, whiThe loading level of carbon materials in CPCs is usually high to achieve the required electrical conductivity for EMI applications, because of the poor dispersion and connectivity of separated carbon. For examples, Zeng et al. reported that 76.2 wt % multiwall carbon nanotubes (MWCNT)-loaded porous PDMS (polydimethylsiloxane) composites has EMI shielding effectiveness (SE) of 21 dB in the X-band frequency range . The higIn order to balance the EMI SE and mechanical properties, the reduced loading level of CPCs is necessary. An effective way is to use three-dimensional (3D) connective carbons  as the filler. The stable and high-quality 3D connective carbons can act as the electron channels to improve the electrical conductivity of CPCs. Two types of 3D connective carbon have recently been intensely developed ,28,29,30Herein, a highly conductive 3D graphene network (GN) was prepared on nickel networks with a porosity of ~50% using a CVD method. With this low porosity, GN grown on the template is stable and highly conductive after nickel was etched. The PDMS was infiltrated into the GN in a vacuum to prepare the composites with excellent conductivity (6100 S/m) and good flexibility. An outstanding EMI SE of about 90 dB in the X-band was achieved for the composite with a thickness of 0.75 mm at a low graphene content of 1.2 wt %. For the composite with a thickness of 0.25 mm, the EMI SE is still higher than 40 dB. In addition to the superior EMI shielding performance, the composite also shows a significant increase in mechanical performance compared with pristine PDMS.The PDMS was obtained from Dow Corning Corporation . The argon, hydrogen, and methane gases with 99.999% purity were obtained from the National Institute of Measurement and Testing Technology . The nickel fibers with a diameter of 8 μm were purchased from Xi’an Filter Metal Materials Corporation . The preparation process of the composite is illustrated in 2 (10 sccm). Then, CH4 with a flow rate of 5 sccm was introduced into the quartz tube for 20 minutes to grow graphene on the surface of nickel fibers. Finally, the nickel network was cooled to room temperature in the ambience of Ar (300 sccm) and H2 (10 sccm). Thereafter, the nickel template was etched away in 20% HCl at 80 °C for 2 days. Finally, the connective freestanding 3D graphene network (GN) with duplicated structure of nickel network was produced, as shown in The nickel network was placed in a ceramic crucible inside a quartz tube for the growth of graphene by a CVD method under ambient pressure. In brief, the nickel network was heated up to 1100 °C and kept for 20 min in mixed gas of Ar (300 sccm) and H2 in size was used to hold the nanocomposite. The holders were connected to two coaxial to waveguide adaptors, which were connected to VNA with two coaxial cables. The incident power was set to be 1 mW.A field-emission scanning electron microscopy  was employed to analyze the morphology of the prepared products. A Raman spectrometer  equipped with a 488 nm laser was used to analyze the graphene structure. A transmission electron microscope (TEM) was used for microstructural analysis with a Double Cs-corrector FEI Titan Themis G2 60–300 microscope . A four-probe meter  was used to measure the conductivity by. An electric universal testing machine  was used to analyze the composite’s tensile strength. The S parameters of the PDMS/GN nanocomposites in the X-band frequency range were measured using a vector network analyzer . A pair of holders 22.9 × 10.2 mm3) of the GN. The GN obtained will inherit the good connectivity of the nickel template, which results in the excellent conductivity of the GN.−1 and 2700 cm−1, which can be indexed as the G band and 2D band of graphene, respectively. The 2D peak is asymmetric and the intensity of the 2D peak is much lower than that of the G peak, indicating that multilayer graphene was obtained [D band, which is related to structural defects and graphene edges, is almost negligible, indicating the high quality of graphene. The TEM results  in the stress required to deform it to this strain level at the 100th cycle b, indicaS-parameter of the composite according the following equation [iP and tP are the incident and transmitted power, respectively. As shown in With the excellent electrical conductivity and mechanical properties, the PDMS/GN composite has good potential application in EMI Shielding. The EMI SE is an evaluation criterion of material’s ability to attenuate the power of electromagnetic waves during the electromagnetic waves transmitted through. The EMI SE of PDMS/GN composites and pristine PDMS in X-band (8–12 GHz) can be extracted from the measured the equation ,37,38,39The thickness of the shielding materials plays an important role in the EMI shielding performance. To investigate the influence of the thickness on the EMI shielding performance of PDMS/GN composite, 2 and 3 pieces of PDMS/GN composites with the thickness of 0.25 mm (1.2 wt % GN loading) were stacked and tested. The result in I) reaching the shielding material shall be decomposed into three parts, i.e., the reflection (R) part, absorption (A) part and transmitted (T) part. R, A, T can be calculated by S-parameters [The EMI shielding of a material’s mechanisms depends mainly on two electromagnetic attenuation mechanisms, i.e., the wave reflection and absorption. The reflection relates to the reflected electromagnetic wave power on interface between the air and shield material, which results from the mismatched characteristic impedance; the absorption relates to the dissipative electromagnetic wave in the shield material caused by the interaction of the electromagnetic field and electric dipoles or magnetic dipoles ,44,45,47R, T and A powers as a function of the thickness at 11.5 GHz are presented in TSE) and contribution of absorption loss (ASE) and reflection loss (RSE) can be calculated as follows:For the prepared PDMS/GN composites, the calculated ASE and RSE increase with the increasing in electrical conductivity. It is worth noting that the contribution of absorption to the EMI shielding effectiveness is much larger than that of reflection. For the 0.75 mm composite, TSE, ASE, and RSE are ~85, 73 and 12 dB, respectively. After comparing the results in A flexible PDMS/GN composite was developed with an outstanding EMI shielding effectiveness and tensile strength. The 3D graphene network was prepared by a CVD method using a 3D nickel network as the template and subsequent etching process. The prepared 3D graphene network has a high electrical conductivity of 6300 S/m. The PDMS/GN composite produced with a vacuum infiltration method inherits the high electrical conductivity at a very low loading level of graphene (1.2 wt %). With this superior electrical conductivity, outstanding EMI shielding effectiveness of around 40, 60 and 90 dB can be achieved in the X-band when thicknesses of the composite are 0.25, 0.5 and 0.75 mm, respectively. In addition to the superior EMI SE, the 3D graphene tube network can also act as a mechanical enhancer for PDMS.


Mitochondria are the energy center of cell operations and are involved in physiological functions and maintenance of metabolic balance and homeostasis in the body. Alterations of mitochondrial function are associated with a variety of degenerative and acute diseases. As mitochondria age in cells, they gradually become inefficient and potentially toxic. Acute injury can trigger the permeability of mitochondrial membranes, which can lead to apoptosis or necrosis. Transactive response DNA-binding protein 43 kDa (TDP-43) is a protein widely present in cells. It can bind to RNA, regulate a variety of RNA processes, and play a role in the formation of multi-protein/RNA complexes. Thus, the normal physiological functions of TDP-43 are particularly important for cell survival. Normal TDP-43 is located in various subcellular structures including mitochondria, mitochondrial-associated membrane, RNA particles and stress granules to regulate the endoplasmic reticulum–mitochondrial binding, mitochondrial protein translation, and mRNA transport and translation. Importantly, TDP-43 is associated with a variety of neurodegenerative diseases, including amyotrophic lateral sclerosis, frontotemporal dementia and Alzheimer's disease, which are characterized by abnormal phosphorylation, ubiquitination, lysis or nuclear depletion of TDP-43 in neurons and glial cells. Although the pathogenesis of TDP-43 proteinopathy remains unknown, the presence of pathological TDP-43 inside or outside of mitochondria and the functional involvement of TDP-43 in the regulation of mitochondrial morphology, transport, and function suggest that mitochondria are associated with TDP-43-related diseases. Autophagy is a basic physiological process that maintains the homeostasis of cells, including targeted clearance of abnormally aggregated proteins and damaged organelles in the cytoplasm; therefore, it is considered protective against neurodegenerative diseases. However, the combination of abnormal TDP-43 aggregation, mitochondrial dysfunction, and insufficient autophagy can lead to a variety of aging-related pathologies. In this review, we describe the current knowledge on the associations of mitochondria with TDP-43 and the role of autophagy in the clearance of abnormally aggregated TDP-43 and dysfunctional mitochondria. Finally, we discuss a novel approach for neurodegenerative treatment based on the knowledge. Neurodegenerative diseases are a group of clinically heterogeneous diseases characterized by the progressive loss or dysfunction of neurons in the central nervous system (CNS) or the peripheral nervous system, including Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD) . AlthougTARDBP gene, was revealed as a key player in the pathogenesis of various neurodegenerative diseases [The discovery of transactive response DNA-binding protein 43 kDa (TDP-43) as the major component of ubiquitin-positive neuronal inclusion bodies is a milestone in understanding the pathogenesis of ALS [diseases , 6. Sincdiseases . For exadiseases –10. The diseases . Increasdiseases –17. Althdiseases –20, the diseases , 17.Mitochondria play a vital role in cell bioenergy and apoptosis . Damage Autophagy is a basic physiological process that maintains the homeostasis of cells  by clearIn summary, the combination of abnormal TDP-43 aggregation, mitochondrial dysfunction, and insufficient autophagy may lead to a variety of aging-related pathologies. In this review, we first summarize current knowledge on the associations between TDP-43 and mitochondria. Then, we describe the role of autophagy in clearing abnormally aggregated TDP-43 and dysfunctional or damaged mitochondria. Finally, we briefly discuss how the studies of TDP-43 proteinopathy, mitochondrial dysfunction, and autophagy may provide a novel approach for treatment of neurodegenerative diseases Fig. .Fig. 1TTARDBP gene (chromosome 1p36.2), and has a relative molecular mass of approximately 43000 daltons [TDP-43 is a DNA- and RNA-binding protein that belongs to the heterogeneous nuclear ribonucleoprotein family . It cons daltons . TDP-43  daltons  Fig. 2)TARDBP ge daltons , 42.FigTDP-43 was originally identified as a transcriptional factor that suppresses the transcription of human immunodeficiency virus 1 . Under nTDP-43 protein is the main component of tau-negative and ubiquitin-positive inclusion bodies in cortical neurons of frontotemporal lobar degeneration and spinal motor neurons of ALS , 56. TDPAt present, the pathogenesis of TDP-43-associated pathology is still unclear, but its importance in neurodegenerative diseases is self-evident and needs further investigation , 60. TheMitochondria have been extensively proven to participate in the basic processes of the nervous system such as energy and intermediate metabolism, calcium homeostasis, and apoptosis . The CNSIn addition to the role in chronic aging, the mitochondria also mediate acute cell death. As mitochondria age in the cells, they gradually become inefficient and potentially toxic, and an acute injury can trigger the permeability of mitochondrial membrane, which causes apoptosis or necrosis . MitochoThe pathological TDP-43 interferes with multiple mitochondrial pathways, including mitochondrial fission and fusion dynamics, mitochondrial transport, bioenergetics, and mitochondrial quality control, all being critical to the survival of nerve cells . The TDPet al. [The connection between TDP-43 and mitochondria has been proved . It has et al.  have demet al. . The inhet al. . In a heet al. . Intereset al. .However, controversy arose in a subsequent study, which did not found any defects in mitochondrial bioenergy function tested in TDP-43 mutants or a correlation between TDP-43 and respiratory chain dysfunction . In contThe co-localization of TDP-43 and mitochondria has been demonstrated using different approaches, and suggests that TDP-43 plays a vital role in the damage of the structure and function of mitochondria. Accordingly, understanding how TDP-43 co-localizes with mitochondria and results in abnormal mitochondrial structure and function has become a particularly critical issue.TDP-43 has various isoforms generated by caspases, which can recognize the endogenous cleavage site in the protein and generate two C-terminal fragments, namely, 25-kDa and 35-kDa fragments . Unlike Although the pathological mechanism of TDP-43 proteinopathy is unclear, pathologically related TDP-43 has been shown to exist inside and outside the mitochondria, and participate in the regulation of mitochondrial morphology, transport, and function, suggesting that mitochondria may be a target of TDP-43 proteinopathy. Numerous studies have demonstrated that pathological TDP-43 interferes with multiple mitochondrial pathways including mitochondrial fission and fusion kinetics, mitochondrial transport, bioenergetics, and mitochondrial quality control .2+ homeostasis [Studies have shown that the mitochondrial fission and fusion dynamics are essential for almost all aspects of mitochondrial function including respiratory complex assembly , ATP proeostasis , and ROSeostasis . Mitochoeostasis , Mfn1, Meostasis . It is weostasis , 80. Alteostasis . ConsistIn addition to the neurofibrillary tangles and senile plaques, cytoplasmic TDP-43 inclusions are also considered to be a possible proteinopathy in AD patients . AlthougSynaptic loss is an important pathological feature preceding neurodegeneration. The failure of correct positioning of mitochondria at the end of dendrites or axons has long been considered to be associated with neurodegenerative diseases and to be a potential cause of synaptic loss . In addi2+ homeostasis, lipid metabolism, autophagy, and even protein transport. This evidence shows that there is an inextricable relationship between TDP-43 and mitochondria, which provides necessary information for treating TDP-related neurodegenerative diseases. Considering the ability of TDP-43 to regulate mitochondrial function, clearing abnormal TDP-43 or blocking the interaction between TDP-43 and mitochondria may lead to unpredictable benefits to the disease. Simultaneously, mitochondria have a regulatory function on TDP-43 toxicity, which suggests that dealing with mitochondrial dysfunction or abnormal mitochondria is one of the strategies for treating TDP-43-related diseases.TDP-43 has unpredictable effects on mitochondrial function. In experimental models related to TDP-43, mitochondrial OXPHOS defects have been widely reported. In NSC-34 cells overexpressing wild-type or mutant TDP-43, decreases in mitochondrial complex I activity and mitochondrial transmembrane potential have been observed, accompanied by the increased expression of mitochondrial uncoupling protein 2, followed by a decrease in ATP synthesis , 86. Furatg) [Autophagy is a catabolic process that acts on all cells of the body and removes toxic and damaged substances through the degradation process. The main regulatory event in the process of autophagy induction is the triggering of the interaction of the complex with nutrient-sensitive mTOR kinase and energy-sensitive AMP-activated protein kinase (AMPK) and involves more than 35 autophagy-related genes (atg) , 91. Speatg) . These patg) . Conversatg) . Therefoatg) . TherefoAtg5 and Atg7) inhibits autophagy and promotes the neurodegenerative phenotype, which is characterized by axonal degeneration and accumulation of aggregation-prone proteins in the neuronal cytoplasm [Autophagy is necessary to maintain the normal function of the CNS to avoid accumulation of misfolded and aggregated proteins . Extraceytoplasm , 96.In addition to the removal of abnormally aggregated proteins, the most relevant function of autophagy is to clean up damaged organelles, such as dysfunctional mitochondria or damaged mitochondria , 35, 97.Numerous evidence has indicated that autophagy has a clearing effect on TDP-43. Studies have shown that the autophagy-related proteins such as LC3 and p62/SQSTM1 co-localize with TPD-43 aggregates, proving that autophagy is necessary for preventing accumulation of TDP-43 aggregates , 104. InDrosophila model of TDP-43 deficiency [TDP-43 loss-of-function increases the activity of transcription factor EB (TFEB), a major regulator of lysosomal biogenesis and autophagy, and prevents the autophagosome-lysosomal fusion, while inhibition of mTORC1 signaling by rapamycin exacerbates the neurodegenerative phenotype in the ficiency . Furtherficiency . Similartfeb, p62/sqstm1 and lc3, indicating that HspB8 may activate autophagy [The level of TDP-43 protein controlled by autophagy can be jointly regulated by heat shock protein (HSP). For instance, induction of autophagy by downregulating HSP-90 or cell division cycle 37 (CDC37) promotes the degradation of HSP-90, CDC37 and TDP-43 protein complexes . Same reutophagy . Previouutophagy . The difutophagy .in vitro and in vivo, and the control of TDP-43 protein level is critically essential. Autophagy is crucial for the occurrence and development of TDP-43-related neurodegenerative diseases. Regulation of autophagy toward the degeneration of TDP-43 protein will become a potential strategy for the treatment of these diseases.Together, these studies show that autophagy affects the aggregation of TDP-43 Autophagy regulates the accumulation of TDP-43, and TDP-43 is involved in the regulation of autophagy. In the mRNA hybridization experiment, researchers found that TDP-43 bond to atg7 mRNA through its RRM1 domain . The dowlamp1, lamp2, atg5, beclin-1, cathepsinL and other autophagy-related genes [In addition, TDP-43 is required to maintain the mTOR activity by stabilizing the mRNA level of RAPTOR, and mTOR further maintains the cytoplasmic position of TFEB by phosphorylating TFEB . Therefoed genes . Furthered genes . In shorLatest research shows that the chaperone-mediated autophagy (CMA) degrades TDP-43 protein and can be affected by TDP-43 aggregation . The aggIn summary, the binding of TDP-43 aggregates results in the loss of TDP-43 function , 116, anMitochondria are not only energy generators necessary for tissue homeostasis, but also channels for programmed apoptosis and necrotic cell death. Strict control of the quality and quantity of mitochondria is essential for normal functions of these organelles . In neurPINK1 and PARK2 contribute to obvious mitochondrial dysfunctions, leading to degeneration of muscles and neurons [PARK6 (coding PINK1) and PARK2 (coding Parkin) gene mutations will cause 5% of familial PD [The most studied mitophagy pathway is mediated by the PTEN-induced putative protein kinase 1 (PINK1) and Parkin. Mutations in  neurons . Abnorma neurons . In PD, ilial PD . In addiilial PD –126. Morilial PD . In summDamaged mitochondria are cleared by autophagy. In contrast, normal mitochondria have an extremely important regulatory effect on the induction of autophagy. Mitochondrial energy deprivation is the hub of autophagy induction . In star2O2 in mitochondria through mitochondrial PI3K, which is essential for the induction of autophagy [2O2 treatment in malignant glioma cells can activate autophagy, and these cells exhibit reduced BCL2 expression and increased BAX levels, leading to the loss of mitochondrial membrane potential and the release of cytochrome c [2O2 treatment results in increased levels of mammalian Atg6 homologue beclin-1 and decreased mTOR activity, which contribute to the induction of autophagy [2- produced in mitochondria in the regulation of autophagy cannot be ignored [2- participates in the signal transmission for autophagy activation [ROS produced in mitochondria were initially considered to be harmful byproducts of oxidative metabolism , 131, buutophagy . Exogenochrome c . Concomiutophagy . The rol ignored . For exativation . Taken tIt is clear that abnormally aggregated TDP-43, damaged mitochondria, and impaired autophagy play important roles in neurodegenerative diseases. Abnormal accumulation of TDP-43 can damage mitochondria and autophagy. Furthermore, regulation of the autophagy pathway by mitochondria is so important that the dysfunction of mitochondria may block normal autophagy. Similarly, damaged mitochondria can interfere with normal TDP-43, while dysfunctional autophagy cannot clean up the accumulated TDP-43 aggregates and the damaged mitochondria. Therefore, it has been widely recognized that targeting TDP-43 , 137, miSeveral small-molecule drugs have been reported to target TDP-43, mitochondria, and autophagy Table , adding + replenishment [in vitro and in vivo models, lithium prevents most of the pathological changes of ALS through mechanisms such as mitochondrial protection, autophagy induction, mitochondrial autophagy, and mitochondrial biogenesis, all being evidence for targeting both mitochondria and autophagy [In addition to a single target, there are drugs that act on multiple targets at the same time, such as melatonin, which can resist oxidative stress and prevent the breakdown of mitochondrial membrane potential , and cannishment , accompanishment . In bothutophagy , 180–182In this paper, we review crucial roles of TDP-43, mitochondria, and autophagy in neurodegenerative diseases. TDP-43 plays an important role in various DNA/RNA processes. Unfortunately, the accumulation of abnormal TDP-43 can cause severe damage to the mitochondrial morphology, structure and function, as well as autophagy, leading to a variety of neurodegenerative diseases . SubsequThese correlations have been confirmed in various studies from multiple angles. The most direct evidence is that the pathologically relevant TDP-43 has been repeatedly proven to exist inside or outside the mitochondria, and is functionally involved in the regulation of mitochondrial morphology, transport, and function . In addiInterestingly, autophagy/mitophagy clears abnormally aggregated proteins and impaired organs, including TDP-43 and abnormal mitochondria –35, whicAt present, the TDP-43-targetting drugs mainly work by regulating TDP-43 levels and modifying TDP-43 status (e.g. phosphorylation) to reduce the accumulation of abnormal TDP-43 aggregates . Five brAlthough accumulating evidence has shown the protection of regulation of TDP-43 or the mitochondrial or autophagy process against neurodegenerative diseases, it remains to be verified whether multi-step treatment is superior to the single-target treatment. In addition, the mechanism underlying the balance among TDP-43, mitochondria and autophagy remains to be studied. On the one hand, we can clarify the internal connections between TDP-43, mitochondria, and autophagy/mitophagy by establishing more advantageous models such as pig and non-human primate models of neurodegeneration , 186. OnIn conclusion, it is evident that TDP-43 protein pathology, mitochondrial disorders, and impaired autophagy are common prominent pathological features of major neurodegenerative diseases including ALS, FTD, and AD. Inhibition of mitochondrial localization of TDP-43 is sufficient to alleviate mitochondrial dynamic abnormalities, neuronal loss, and behavioral defects in transgenic mice with different mutant forms of TDP-43. Due to the close relationship between mitochondrial and autophagy functions and TDP-43, and their contributions to the progression of neurodegenerative diseases, we believe that maintaining the balance among TDP-43, mitochondria, and autophagy is a promising strategy for the treatment of neurodegenerative diseases.


The diagnostic utility is assessed by immuno‐positron emission tomography (immunoPET) imaging in ATC models. To facilitate total surgical removal of orthotopic ATCs, a near‐infrared fluorescent imaging probe IRDye 800CW‐ALT‐836 is designed. As the therapeutic component, 131I‐ALT‐836 is further developed and the radioimmunotherapy (RIT) efficacy of this agent is interrogated in orthotopic ATC models. The results demonstrate that TF is highly expressed on the ATC cell line THJ‐16T. 64Cu‐NOTA‐ALT‐836 immunoPET imaging clearly delineates both subcutaneous and orthotopic ATCs, with a peak tumor uptake of 19.93 ± 2.17% ID per g (n = 3) and 37.20 ± 1.71% ID per g (n = 3), respectively. Fluorescent imaging with IRDye 800CW‐ALT‐836 facilitates the total resection of orthotopic ATCs. Moreover, 131I‐ALT‐836 RIT prolongs the survival of ATC‐bearing mice. Taken together, TF is a promising marker for ATC and successive use of 64Cu‐NOTA‐ALT‐836 and 131I‐ALT‐836 can realize precise management of ATC.Anaplastic thyroid cancer (ATC) is the most aggressive subtype of thyroid cancers with a dismal prognosis. It is aimed to explore a new biomarker and devise a marker‐dependent theranostic pair for ATC. Flow cytometry is used to determine tissue factor (TF) expression in thyroid cancer cell lines. ALT‐836, a TF‐specific monoclonal antibody, is radiolabeled with  64Cu‐NOTA‐ALT‐836 and 131I‐ALT‐836. While 64Cu‐NOTA‐ALT‐836 immunoPET readily detects and stratifies ATCs, 131I‐ALT‐836 radioimmunotherapy efficiently inhibits the growth of the tumors.Anaplastic thyroid cancer (ATC) is a lethal disease. This study reports tissue factor (TF) as a potential biomarker for ATC and develops a TF‐targeted toolbox consisting of  However, adverse effects and drug resistance associated with these regimens limit their broad clinical applications. Immunotherapy using immune checkpoint inhibitors showed therapeutic effect in sparse cases, so the net benefit needs to be carefully characterized. Unlike well‐differentiated thyroid cancers, ATCs are virtually refractory to radioiodine therapy due to loss of sodium iodide symporter (NIS). Along with the above progress, continuous attempts have been made to re‐differentiate ATC to enhance NIS uptake, but the clinical effect of these agents remains to be determined.Anaplastic thyroid cancer (ATC) is a rare but deadly disease with a dismal prognosis. The median overall survival (OS) of ATC patients receiving multimodal therapy and palliative treatment was 21 months and 3.9 months, respectively. Despite the therapeutic effect of multimodal therapy, the median OS of patients with ATC was far from satisfactory.1 Rece Although therapeutic antibodies are among the mainstream of cancer therapy, practical challenges lie in how to efficiently stratify patients before drug administration and how to precisely monitor the therapeutic responses of antibody drugs. Immuno‐positron emission tomography (immunoPET), which integrates the specificity of the antibody vector and the sensitivity of the PET technique, emerges as a paradigm‐shifting molecular imaging modality. Various targets have been exploited to develop immunoPET probes, including receptor tyrosine kinases, and T cell markers. Besides its role in detecting heterogeneous tumors, immunoPET can select candidates who may benefit from antibody‐based therapies, such as radioimmunotherapy (RIT). RIT is developed by conjugating therapeutic radioisotopes with tumor‐targeting antibodies and allows delivery of a high dose of therapeutic radioactivity to the tumor cells. Traditionally, RIT was used for radiosensitive tumors, such as lymphomas and leukemia. Two CD20‐targeting RIT agents, 131I‐tositumomab (Bexxar), and 90Y‐ibritumomab tiuxetan , are among the FDA‐approved drugs for treating B cell lymphomas and have yielded considerable efficacy. For leukemia, several RIT agents targeting CD33, CD45, or CD66 have been developed. RIT agents labeled with α‐emitters also hold great promise. More recently, RIT has been extended to treat solid tumors such as neuroblastoma, and colorectal cancer.In recent years, antibody therapeutics are increasingly being approved for cancer treatment.12 Alt Tissue factor (TF) plays a pivotal role in mediating hemostasis and inflammatory diseases. Recent evidence has elucidated that TF serves as an alternative target for cancer therapy. Several antibody therapeutics targeting TF have been proposed, including a chimeric monoclonal antibody (mAb) ALT‐836. We reported that molecular imaging probes derived from ALT‐836 have shown great potential in diagnosing pancreatic cancer and breast cancer. More recently, we demonstrated the feasibility of pancreatic cancer theranostics using 86/90Y‐labeled ALT‐836. In the current study, we first elucidate whether TF serves as a tumor marker for advanced thyroid cancers and then investigate the diagnostic utility of TF‐specific 64Cu‐NOTA‐ALT‐836 in subcutaneous and orthotopic ATC models. We further develop a fluorescent probe IRDye 800CW‐ALT‐836 and investigate the feasibility of image‐guided surgery with this probe. Finally, 131I‐ALT‐836 is conjugated and the therapeutic efficacy of 131I‐ALT‐836 RIT will be explored in orthotopic ATC models.Bearing the unsatisfactory management status of ATC in mind, we turned to seek biomarkers that can be manipulated to develop theranostic probes for ATCs.26 Tis22.1Figure We first screened six thyroid cancer cell lines by flow cytometry. The results showed five of the six included cell lines were TF‐positive, including two ATC cell lines (THJ‐16T and 8505C) and three follicular thyroid cancer cell lines . TPC‐1, a papillary thyroid cancer cell line, was TF‐negative Figure 1. Consi2.264Cu‐NOTA‐ALT‐836 was developed with high decay‐corrected radiochemical yield (>80%) and excellent radiochemical purity (>99%). ImmunoPET imaging in subcutaneous ATC models showed a prominent accumulation of 64Cu‐NOTA‐ALT‐836 in tumors than in mesenchymal tissues. Both maximum intensity projection  achieved at 48 h after radiotracer administration. In comparison, uptake in other organs was low except that in the liver . The in vivo imaging results were corroborated by the ex vivo biodistribution data  but a higher tumor uptake .a Figure , which r64Cu‐labeled nonspecific human IgG  was able to delineate subcutaneous THJ‐16T tumors. As shown in Figure 64Cu‐NOTA‐IgG in the tumor area, the uptake was comparable to or lower than the liver uptake. Quantitatively, tumor uptake of 64Cu‐NOTA‐IgG plateaued at 48 h with a value of 5.30 ± 0.62% ID per g (n = 3), which was significantly lower than 13.20 ± 2.67% ID per g (n = 4) achieved by 64Cu‐NOTA‐ALT‐836 (p = 0.0044). From the region of interest (ROI) and biodistribution data  probe we previously described. Two weeks after tumor inoculation, serial fluorescent imaging demonstrated focal signals in the neck areas, indicating rapid formation and growth of the orthotopic tumors . 64Cu‐NOTA‐ALT‐836 immunoPET imaging of these mice another two weeks later showed dramatic uptake of the tracer in the thyroid areas . In comparison, uptake in the blood pool, liver, spleen, and kidney decreased over the imaging course and the organ with the highest uptake at 48 h was the liver . The tumor‐targeting ability of 64Cu‐NOTA‐ALT‐836 was further confirmed by the biodistribution study, which revealed an average tumor uptake of 37.20 ± 1.71% ID per g (n = 3) with the uptake in other organs less than 10% ID per g . As detected by instant thin‐layer chromatography (iTLC), the radiochemical purity of 131I‐ALT‐836 was 99.06 ± 0.01% . Protein integrity according to the high‐performance liquid chromatography (HPLC) test was 100% with a retention time of 13 min for the main peak  . Bioluminescence imaging before initiation of treatment showed comparable tumor burden in the control group and 131I‐ALT‐836 treatment group . Treatment with 131I‐ALT‐836 was safe because no weight loss was observed. Since it was unable to measure the volume of the orthotopic tumors longitudinally, we performed fluorescent imaging with IRDye 800CW‐ALT‐836 26 days after 131I‐ALT‐836 injection to monitor the tumor burden. When compared to the control group, 131I‐ALT‐836 treatment significantly reduced the tumor uptake of IRDye 800CW‐ALT‐836 , indicating a single administration of 131I‐ALT‐836 substantially saturated and/or downregulated TF expression in the tumors. Repeated IRDye 800CW‐ALT‐836 fluorescent imaging 41 days after the therapy further consolidated the above observation . RIT with a single injection of 131I‐ALT‐836 significantly extended the survival of the tumor‐bearing mice, whereas no other treatment options including ALT‐836 monotherapy showed any therapeutic benefit , 131I‐IgG , ALT‐836 , or free 131I .Motivated by the prominent tumor uptake of t Figure 6A. More 131I‐ALT‐836 treatment showed either no tumor or just small tumors at necropsy. Hematoxylin and eosin (H&E) staining showed no morphologic features of follicular cells but obvious fibrosis inside the small tumor of the 131I‐ALT‐836 treatment group , but this novel treatment did not cause any obvious damage to other major organs . We also stained the normal mouse tissues and found that expression of TF is quite low in organs such as heart, liver, kidney, and muscle . There was positive staining in the lung and spleen, which was largely localized to the blood cells. Finally, we performed biodistribution studies to compare the overall distribution and tumor‐targeting capacity of 131I‐ALT‐836 and 131I‐IgG . The results showed a significantly higher tumor uptake of 131I‐ALT‐836 than that of 131I‐IgG . Although the uptake of 131I‐IgG in the parenchymal organs  was higher than the corresponding uptake of 131I‐ALT‐836, there was no statistical difference in the uptake value in these organs. Both 131I‐ALT‐836 and 131I‐IgG were stable within two days since there was no abnormal accumulation of free 131I in the stomach.Mice receiving p Figure ,C. In cop Figure –F. H&E s131I‐ALT‐836 could specifically target THJ‐16T tumors and 131I‐ALT‐836 RIT could induce tumor regression and prolong the survival of the tumor‐bearing mice.These results together demonstrated that regardless of the differentiation status, 3 receptor tyrosine kinases, and enzymes. Recent advances have shown that TF is a promising diagnostic marker for several categories of malignancies, including pancreatic cancer, and breast cancer. In this work, we first reported the abundant expression of TF in advanced thyroid cancer cell lines, indicating the potential of TF as a biomarker for advanced thyroid cancers. We then developed and characterized the value of 64Cu‐NOTA‐ALT‐836 and 131I‐ALT‐836 in diagnosing and treating ATCs in preclinical mouse models.In developing antibody‐based theranostic approaches for human malignancies, two crucial factors are the binding affinity of the antibody moiety and the antigen expression level. Most of the antibody‐derived probes explored in clinical practice are targeting cluster of differentiation antigens,36 rec64Cu‐NOTA‐ALT‐836 immunoPET is its ability to detect advanced thyroid cancers irrespective of NIS expression. TF‐positive cases as selected by 64Cu‐NOTA‐ALT‐836 immunoPET will presumably benefit from TF‐targeted therapies, including antibody and antibody‐derived therapies. In our case, 131I‐ALT‐836 showed preliminary therapeutic effects in orthotopic ATC models. The preclinical evidence warrants further clinical investigation of 64Cu‐NOTA‐ALT‐836 in the first place. If clinical 64Cu‐NOTA‐ALT‐836 immunoPET could diagnose advanced thyroid cancers and the immunoPET signal correlates well with TF expression revealed by immunohistochemistry, then it is rational to investigate the therapeutic value of 131I‐ALT‐836 in the selected cases. Currently, combinatorial treatment incorporating surgery (when feasible) and chemoradiotherapy is the standard of care for ATC patients. Complete surgical resection of the tumors has been shown to improve the survival of ATC patients. Our preclinical results also showed that NIR imaging with IRDye 800CW‐ALT‐838 is a robust method to facilitate the total resection of the orthotopic tumors. However, assessing the impact of the dye‐to‐mAb ratio on the circulation profile of the developed fluorescent probe is very necessary before clinical translation. In several clinical trials, a dye‐to‐mAb molar ratio of 2.0–2.3:1 was used in developing the fluorescent probes.One merit of patients.38 Com131I and 90Y . We also used 131I in this preclinical study. However, 131I‐labeled mAbs are usually catabolized and disconnected after internalization, as a result, 131I‐tyrosine and free 131I will be released into the blood circulation which may subject family members and the public under unnecessary radiation exposure. Future studies may develop radiometal‐labeled ALT‐836 which may retain inside the tumor cells after antigen‐antibody complex internalization. Of the various radiometals, 90Y and 177Lu are of choice. For micrometastases or diseases significantly involving bone marrow, α‐particle‐emitting radionuclides, such as 211At (t1/2 = 7.2 h) and 225Ac (t1/2 = 10 d), are attractive choices for their high linear energy transfer within a few diameters (50–90 µm) and high potency in inducing DNA double‐strand breaks. Additionally, pretargeted RIT may be harnessed to relieve the side effects and maximize the therapeutic index.Most of the ongoing clinical RIT trials are using lization,45 as 464Cu‐NOTA‐ALT‐836 and 131I‐ALT‐836 could optimize the diagnosis and treatment of ATCs in preclinical settings.TF is a promising biomarker for ATCs. TF‐targeted theranostics using 5 To better monitor the tumor burden, the THJ‐16TLuc cell line was constructed using a pGMLV‐CMV‐Lu lentivirus luciferase reporter (Genomeditech). All the cell lines were cultured following the recommended protocols. To detect cell surface expression of TF by flow cytometry, 1 × 106 cells for each sample were collected and washed with cold phosphate‐buffered saline . The cells were then resuspended in flow cytometry staining buffer (Invitrogen) and incubated with primary antibodies (5 or 10 µg mL−1 of ALT‐836) on ice for 45 min, followed by washing with PBS and incubation with Alexa Fluor 488‐labeled goat anti‐human IgG . The samples were washed again, resuspended in PBS, and analyzed using a BD LSR Fortessa flow cytometer (BD Biosciences). The results were analyzed with the FlowJo software (FlowJo LLC).Six thyroid cancer cell lines  used in this study were kindly provided by Dr. Heather Hardin .26 To  we chose this cell line to establish subcutaneous and orthotopic ATC models in nude mice . Orthotopic ATC models were also established using the newly‐developed ultra‐immunodeficient R2G2 mice . For subcutaneous ATC models, 5 × 106 THJ‐16T cells (per mouse) were mixed in sterile PBS and matrigel matrix (Corning) with a ratio of 1:1 and injected into the right posterior flank of the experimental mice. Whereas for orthotopic ATC models, 0.5–1 × 106 THJ‐16T or THJ‐16TLuc cells or (per mouse) suspended in sterile PBS were gently injected into the left thyroid bed after surgical exposure of the thyroid.All animal experimental procedures and protocols had been approved by the Institutional Animal Care and Use Committees . Since the ATC cell line THJ‐16T has an excellent tumor take rate of 100%,26 we  IRDye 800CW‐ALT‐836 was developed in a similar way with a dye‐to‐mAb molar ratio of 10. Serial IRDye 800CW‐ALT‐836 fluorescent imaging (λex/em: 745/800 nm) was carried out using an IVIS Spectrum (Perkin Elmer Inc.), followed by image‐guided resection of the orthotopic ATCs using a handheld Fluobeam (Fluoptics). To detect the baseline burden of the orthotopic ATCs, D‐luciferin (3 mg per mouse) was injected into the THJ‐16TLuc‐bearing nude mice and bioluminescence imaging was acquired with the IVIS Spectrum 10 min later. The data were analyzed using the Living Image 4.5.5 (IVIS Imaging Systems) software.The burden of orthotopic tumors was monitored by fluorescent imaging with IRDye 800CW‐pertuzumab, an NIR probe that was previously described.26 IRDp‐SCN‐Bn‐NOTA; Macrocyclics) as previously described. For radiolabeling, 50–100 µg of NOTA‐mAb was reacted with 74–148 MBq of 64CuCl2 in 300 µL of sodium acetate buffer  at 37 °C for 1 h under constant shaking (500 rpm). The final 64Cu‐NOTA‐mAb was separated from the free activity using equilibrated PD‐10 columns  with PBS as the mobile phase. On average, 3.7–7.4 MBq of filtered 64Cu‐NOTA‐ALT‐836 or 64Cu‐NOTA‐IgG was injected to each mouse and PET imaging was acquired with Inveon PET/CT scanner (Siemens). PET data were reconstructed on the Inveon Acquisition Workplace and analyzed using the Inveon Research Workplace. MIP and coronal PET images were given to showcase the overall and regional distribution of the radiotracers, respectively. After analyzing the ROI on decay corrected PET images, quantitative data reflecting the dynamic change of the radiotracers in the blood circulation and other major organs were given in terms of percent of injected dose per gram of tissue (% ID per g).The TF‐specific mAb ALT‐836 and a nonspecific human IgG (Invitrogen) were used to conjugate TF‐targeted probe and isotype control probe, respectively. The antibodies were first linked to 2‐S‐(4‐isothiocyanatobenzyl)‐1,4,7‐triazacyclononane‐1,4,7‐triacetic acid , 131I‐ALT‐836 treatment group (n = 7), 131I‐IgG treatment group (n = 5), ALT‐836 treatment group (n = 5), and free 131I treatment group (n = 5). 131I‐ALT‐836 and 131I‐ALT‐IgG were prepared according to a previously reported protocol. Briefly, to a glass vial pre‐coated with 50 µg of IODO‐GEN (Pierce), 50 µL 0.5 m Na2HPO4, 400 µL antibody solution (400–600 µg of mAb in 0.1 m Na2HPO4), and 148–185 MBq of 131I solution  were successively added. The mixture was incubated on a constant shaker for 4 min at room temperature, followed by addition of 100 µL of ascorbic acid  and incubation for another 5 min. Free 131I was removed by PD10 columns with 0.9% NaCl/ascorbic acid  as the mobile phase. Radiolabeling efficiency and radiochemical purity of the collected radiopharmaceutical were assessed by iTLC  and HPLC (Agilent).To explore the therapeutic effect of TF‐targeted regimens, treatment studies were further designed. Nude mice bearing orthotopic THJ‐16T or THJ‐16Tprotocol.55 Bri131I‐ALT‐836 RIT with that of other counterparts. Mice in the control group were treated with tail vein injection of sterile saline (100 µL per mouse). Mice in the mAb treatment group were treated with a single tail vein injection ALT‐836 (125 µg per mouse). ATC‐bearing mice were given 0.1% potassium iodide‐containing drinking water two days prior to the administration of radioactive regimens. For the two RIT groups, mice were intravenously injected with 131I‐ALT‐836  or 131I‐IgG , respectively. Whereas only free 131I  was intravenously injected to the mice in the 131I treatment group. Toxicity was monitored by measuring body weight, and the endpoint criterion was defined as the presence of severe dyspnea. Treatment efficacy was evaluated by performing fluorescent imaging and constructing survival curves.Treatment was initiated two weeks after tumor cell inoculation. Five groups were set up to compare the treatment efficacy of γ‐counter (Perkin Elmer Inc.) and tissue uptake was calculated and given in terms of % ID per g. To examine the in vivo circulation, stability, and tumor‐targeting efficacy of 131I‐ALT‐836 and 131I‐IgG, a lower dose of the agents  was injected and biodistribution studies were performed two days post‐injection.After termination of the PET imaging, experimental mice were sacrificed and major tissues (including blood) were collected and weighed. The activity (counts per minute) was counted with a calibrated −1 of ALT‐836 and 10 µg mL−1 of CD31/PECAM‐1 antibody , respectively. The washed sections were then stained with 5 µg mL−1 of Alexa Fluor 488‐labeled goat anti‐human IgG (Invitrogen) and 5 µg mL−1 of Cy3‐labeled donkey anti‐rat IgG . After washing, the sections were mounted with the UltraCruz Hard‐set Mounting Medium containing 1.5 µg mL−1 of DAPI (Santa Cruz Biotechnology). All the fluorescent images were acquired using a Nikon A1R confocal microscope. To detect the involvement of the adjacent structures, orthotopic tumors together with thyroid, trachea, muscle, and larynx were carefully resected at necropsy and used for H&E staining as previously described.To detect TF expression and vasculature, tumor tissues were fixed in 10% paraformaldehyde and sliced tumor sections (10 µm) were stained with 20 µg mLcroscope.54 To t‐test, and multiple comparisons of grouped data were calculated using two‐way ANOVA. Survival was calculated using Kaplan–Meier curves. p < 0.05 was considered statistically significant.Statistical analyses were performed using the GraphPad software. All data are presented as the mean value ± SD or value ± SEM. Group data were compared using the two‐tailed Student's The authors declare no conflict of interest.Supporting InformationClick here for additional data file.


E. coli. Full-length TDP-43 could be prevented from aggregation during purification using a detergent. Crystals grown from an N-terminal construct (1-270) revealed only the N-terminal domain (residues 1-80) with molecules arranged as parallel spirals with neighboring molecules arranged in head-to-tail fashion. To obtain detergent-free, full-length TDP-43 we mutated all six tryptophan residues to alanine. This provided sufficient soluble protein to collect small-angle X-ray scattering data. Refining relative positions of individual domains and intrinsically disordered regions against this data yielded a model of full-length TDP-43.Mislocalization, cleavage, and aggregation of the human protein TDP-43 is found in many neurodegenerative diseases. As is the case with many other proteins that are completely or partially structurally disordered, production of full-length recombinant TDP-43 in the quantities necessary for structural characterization has proved difficult. We show that the full-length TDP-43 protein and two truncated N-terminal constructs 1-270 and 1-263 can be heterologously expressed in   •Purification of full-length TDP-43 with all tryptophan residues mutated to alanine•Crystallographic structure determination of the TDP-43 N-terminal domain•Small-angle X-ray scattering analysis of full-length TDP-43•Potential structural model of full-length TDP-43 Molecular Neuroscience; Structural Biology; Protein Folding The deposition of intracellular TDP-43 inclusions is the hallmark of TDP-43 pathology. Initially observed in neural tissues from individuals with frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) , TDP-43 TDP-43 protein has several functions, and its modular structure facilitates this multitasking. Two centrally located RNA recognition motifs (RRM) strongly bind UG-rich RNA  or TG-riin vitro characterization difficult, particularly using structural techniques that require monodisperse samples at relatively high concentrations. The modularity of TDP-43 means that individual domains can be produced recombinantly to shed light on their organization and structure-property relationship. This approach has been well utilized to gain insight on dimerization (WtoA), we were able prevent recombinant TDP-43 aggregation during protein preparation in each of these cases. We produced sufficient quantities of detergent-free TDP-43WtoA for small-angle X-ray scattering (SAXS) analysis allowing us to create a model of full-length TDP-43.In addition to the domains described earlier, TDP-43 also contains nuclear export and import sequences that flank the RRM domains. In the full-length form, this enables shuttling of the protein between the nucleus and cytoplasm . Observarization , RNA binrization , and therization . HoweverE. coli followed by cell lysis and centrifugation, we found the protein in the insoluble fraction as has previously been described  were prepared and evaluated. The N-terminal 1-270 wild-type construct provided stable purified protein  and was WtoA). Cell lysis and protein purification with standard immobilized metal ion chromatography buffers without any detergent facilitated production of full-length TDP-43WtoA that was stable for 3 days at 4°C  was visible. Analysis of several crystallization drops by SDS-PAGE indicated the 30-kDa original protein to have shifted to approximately 23 kDa, which would indicate cleavage of RRM2. During 1 week of crystallization the protein appears to have auto-cleaved. The TDP-43 NTD crystallized in space group P2solution . The NTDce group . A compace group A–4C, andThe head-to-tail interfaces between the molecules differ from those reported previously . A sulfaSolvent-accessible tryptophan residues within intrinsically disordered regions are unlikely to contribute to individual domain structures. It is, however, important to understand the perturbations caused by mutation of tryptophans within the NTD and RRM1 domain. Available RRM1 structures  show TrpWtoA to perform chromatographic SAXS experiments. Size exclusion chromatograms of full-length TDP-43 (WtoA elution indicates a decrease in mean radius of gyration (Rg) from 41.8 ± 3.5 Å to 38.9 ± 4.0 Å  was elucidated at 2.55 Å resolution. The protein appears to have been auto-cleaved at N terminal to RRM2 during the 7 days required for crystallization to produce diffraction-quality crystals, whereas RRM1 is conformationally mobile The ability to obtain full-length TDP-43 in a stable form without denaturing conditions opens possibilities for extensive biophysical studies on both the wild-type and C-terminal mutants, which are known to exert greater aggregation propensity . We propA population of intrinsically disordered sequences are, by their nature, very unlikely to simultaneously occupy the same conformation. Although the model presented in s.s.hasnain@liverpool.ac.uk).Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, S. Samar Hasnain (All unique and stable reagents generated in this study are available from the Lead Contact without restriction.http://www.rcsb.org/) under the accession code 6T4B.The atomic coordinate and structure factor of the NTD (NTD) of TDP-43 have been deposited in the Protein DataBank (Experimental SAXS data and TDP-43 models are available from the corresponding author on request.All methods can be found in the accompanying


A Multiple-Temperature Model is proposed to describe the flash laser irradiation of a single layer of graphene. Zhukovsky’s mathematical approach is applied to solve the Fourier heat equations based upon quantum concepts, including heat operators. Easy solutions were inferred with respect to classical mathematics. Thus, simple equations were set for the electrons and phonon temperatures in the case of flash laser treatment of a single layer of graphene. Our method avoids the difficulties and extensive time-consuming nonequilibrium green function method or quantum field theories when applied in a condensed matter. Simple expressions were deduced that could prove useful for researchers. Graphene is a two-dimensional 2D) material, consisting of a hexagonally arranged lattice of carbon atoms. The term “graphene” is fully functional for a solitary deposit of graphite. Most of the thermal properties of Graphene are derivatives of graphite, and allow the inscription of the exceedingly anisotropic nature of this material . For appD materiaElectron–Phonon (E–P) coupling is the main phenomenon that intermediates Joule heating, laser–material interactions, and hot electron relaxation. A complete understanding of E–P coupling phenomena is critical to understanding the material properties and improving electronic devices ,7,8,9,10In this study, the MTM for flash laser experiments in the case of a Single Layer of Graphene (SLG) is combined with Zhukovsky’s approach to heat equations. Simple expressions for the electron and phonon temperatures during flash laser experiments on single-layer graphene are developed.The Two-Temperature Model (TTM) was developed by Anisimov and collaborators . From a The classical TTM formalism can be expressed aseT and pT are the electrons and phonons temperature; t is time; x, y and z are spatial coordinates; and G is the coupling factor between electrons and phonons. aP is the heat source that is induced through laser–SLG interaction. The interaction could be of classical or steady-state quantum mechanical type. A is the electron heat capacity, K is the electrons thermal conductivity of the graphene, and pC is electron heat capacity of the graphene. MTM in our formalism has the following analytical form :ppGi, is the p-p coupling factor between phonon branch i and the scattering lattice reservoir, and latT is the lattice reservoir’s temperature, defined as to ensure the energy transfer among phonon branches conservation. Until now, we have presented the general formalism of thermal effects in laser-graphene interaction. Our main goal was to solve analytically the general formalism described above in order to predict the thermal field in the laser–graphene interaction, as accurately as possible.Here, To the best of our knowledge, there are several articles available on “Zhukovsky’s formalism and MTM” approach ,21,22, bi denotes different kinds of phonon branches:i (i) isIn case of flash laser experiments, one may write for the heat sourcebranches:i={ LA, TCe of 3.6 × 102 J/m3K and thermal conductivity eK of 50 W/mK , the 3D graphic, simulated via Mathematica software, is presented in T stands for temperature variation rather than the absolute temperature. The input parameters given in Refs. ,20 have eK and eC values, which are higher or similar (eK) to and much smaller (eC) than the phonon ones. The current simulation has also the advantage that it accurately indicates the thermal decrease of acoustic phonons, which is not very pronounced in Ref. [i, the better the concordance between current model and the one presented in Refs. [The output of current model is in good agreement with the one presented in Refs. ,20. The  in Ref. . The coo in Ref. ,20. It cin Refs. ,20. Whenin Refs. ,20. Thesin Refs. ,20, beca1 fs. If simulations are carried out for as pulse durations, the laser wavelength is quite close to the separation distance between nucleus and first electron and the concept of the temperature distribution becomes meaningless.The irradiation time/pulse duration should be superior to The target size is confined to the (20–100) nm range. If the particle size is smaller than 20 nm, the Fourier law collapses and cannot provide reliable results. For more than 100 nm, the restriction to nano-object is infringed.In current study, single-layer absorption has been used, thus limiting our study to single layer graphene. For multilayer graphene, the analysis becomes intricate and difficult to calculate since the absorption law and heat-transfer coefficients, in real-time, should be reconsidered for each layer, demanding a quantum-field theory in solid-state treatment. This subject is laborious to handle in simulations.The equations used in the present model have a “classical form”. Nevertheless, the solutions were figured out under quantum mechanical manner. For example, the heat operator is used to solve the heat equation.The following limitations were identified with respect to the application of this approach:21 W/cm2 [A new model for MTM applied to flash laser experiments on single-layer graphene is proposed. The results are found to be in good agreement with those reported in the Refs. ,20. The 21 W/cm2 . Further