Astrocytes with cytoplasmic TDP-43 inclusions can cause motor neuron death, however, whether astroglial metabolism and metabolic support of neurons is altered in astrocytes with TDP-43 inclusions, is unclear. We measured lipid droplet and glucose metabolisms in astrocytes expressing the inclusion-forming C-terminal fragment of TDP-43 or the wild-type TDP-43 using fluorescent dyes or genetically encoded nanosensors. Astrocytes with TDP-43 inclusions exhibited a 3-fold increase in the accumulation of lipid droplets versus astrocytes expressing wild-type TDP-43, indicating altered lipid droplet metabolism. In these cells the noradrenaline-triggered increases in intracellular cAMP and Ca2+ levels were reduced by 35% and 31%, respectively, likely due to the downregulation of β2-adrenergic receptors. Although noradrenaline triggered a similar increase in intracellular lactate levels in astrocytes with and without TDP-43 inclusions, the probability of activating aerobic glycolysis was facilitated by 1.6-fold in astrocytes with TDP-43 inclusions and lactate MCT1 transporters were downregulated. Thus, while in astrocytes with TDP-43 inclusions noradrenergic signaling is reduced, aerobic glycolysis and lipid droplet accumulation are facilitated, suggesting dysregulated astroglial metabolism and metabolic support of neurons in TDP-43-associated ALS and FTD.Most cases of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) have cytoplasmic inclusions of TAR DNA-binding protein 43 (TDP-43) in neurons and non-neuronal cells, including astrocytes, which metabolically support neurons with nutrients. Neuronal metabolism largely depends on the activation of the noradrenergic system releasing noradrenaline. Activation of astroglial adrenergic receptors with noradrenaline triggers cAMP and Ca The pathologic hallmark of ALS are cytoplasmic inclusions in motor neurons. In most cases (~95%) of sporadic and familial ALS, TAR DNA-binding protein 43 (TDP-43), encoded by the TARDBP gene, has been identified as the key component of these inclusions9. Moreover, TDP-43 has also been identified as the major protein in inclusions in frontotemporal dementia with ubiquitin-positive inclusions (FTD-U)6.Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the loss of both upper and lower motor neurons in the brain and spinal cord, and by the progressive paralysis of voluntary muscles and death13. TDP-43, an RNA-binding protein, is implicated in multiple aspects of RNA processing, including regulation of transcription, splicing, transport, and stabilization of mRNAs. It also regulates microRNA biogenesis and interacts with DNA. Therefore, its perturbance may lead to significant changes in the transcriptome and proteome17. It consists of an N-terminal domain, two RNA recognition motifs and a C-terminal prion-like glycine-rich domain that mediates protein-protein interactions with other heterogeneous ribonucleoprotein (hnRNP) family members11.TDP-43 is a highly conserved protein (414 amino acids), ubiquitously expressed in all tissues and under physiological conditions, primarily localized to the nucleus; however, low levels are also present in the cytoplasm18. Although ubiquitination targets TDP-43 aggregates for degradation, TDP-43 begins to accumulate in the cytoplasm, suggesting that additional perturbance in either the ubiquitin-proteasome system or the autophagy pathway can facilitate the accumulation of TDP-43 in ALS and FTD-U19. 25-kDa C-terminal fragments of TDP-43 (TDP-43208–414) are commonly detected in ALS and FTD-U pathologic specimens, especially in the cerebral cortex, and generation of these fragments is sufficient to initiate a number of events that mirror TDP-43 proteinopathies20.In most pathologic cases, TDP-43 is hyperphosphorylated and ubiquitinated21. Astrocytes with TDP-43 inclusions are sufficient to cause motor neuron death in animal models23 and exhibit autocytotoxicity7. Thus, astrocytes were recently proposed to play an active role in controlling ALS disease progression and may even be the primary driver of TDP-43 proteinopathies7.TDP-43-containing cytoplasmic inclusions are not restricted to motor neurons but are also found in non-neuronal cells, in particular in astrocytes24, regulating CNS metabolism25. With their numerous processes, they are in tight contact with neurons, including motor neurons, and blood vessels. They transport nutrients from the blood stream to neurons and store blood-derived glucose in the form of glycogen as the CNS fuel reserve26 and perhaps also as free glucose in endoplasmic reticulum27. Astrocytes are considered an important cellular target of noradrenaline (NA), released from the locus coeruleus (LC) noradrenergic neurons, which regulates CNS energy metabolism32. NA binds to G-protein-coupled adrenergic receptors  on the surface of brain cells, including astrocytes34, where ARs are abundantly expressed35, changing the intracellular concentration of cyclic adenosine monophosphate ([cAMP]i) and Ca2+ ([Ca2+]i)39. This activates astroglial metabolism, which is mainly controlled by β-AR/cAMP signaling, enhancing glucose uptake, glycogenolysis, aerobic glycolysis, and lactate production40. Lactate is considered to be then shuttled to neurons where it is used as fuel by being transformed to pyruvate and entering oxidative phosphorylation42.Astrocytes are an abundant and heterogeneous subtype of neuroglia in the central nervous system (CNS)In vitro and in vivo studies using ALS model systems of superoxide dismutase 1 (SOD1)-related familial ALS have provided first evidence of metabolic dysfunction in ALS astrocytes, particularly in the transporter responsible for the efflux of lactate43, dysfunctional astrocytic mitochondria44, and lower levels of intracellular lactate upon exposure of astrocytes to glutamate45. Besides glucose metabolism, lipid metabolism might also be impaired in neurons and astrocytes during neurodegeneration as observed in animal models48. If reactive oxygen species (ROS) are elevated in the brain like during neurodegeneration46, the glia-neuron lactate shuttle is believed to promote lipid synthesis in neurons. Peroxidized and potentially cytotoxic lipids are then shuttled via apolipoproteins (APO) E and D and fatty acid transport proteins, from neurons to glial cells, causing accumulation of lipid droplets (LDs) in glial cells47, dynamic organelles, undergoing regulated, stress-induced biogenesis and/or degradation50, that play an important role in intracellular lipid metabolism and storage51. Consistent with the observations in animal models, a reduced glucose uptake and increased glycogen storage have been observed in the CNS of patients with ALS52; both processes highly depend on astrocytes, suggesting alterations in astroglial metabolism. Whether TDP-43 inclusions, a hallmark of ALS and FTD-U pathologies, can in astrocytes per se alter astroglial glucose and lipid droplet metabolisms is not known, and was the subject of this study.2+, cAMP signaling and lactate production in astrocytes, we used real-time fluorescence microscopy, Ca2+ indicator Fluo-4 AM dye, and genetically encoded FRET (fluorescence resonance energy transfer)-based cAMP (Epac1-camps53) and lactate (Laconic54) nanosensors. NA induced increases in [cAMP]i, [Ca2+]i, and intracellular concentration of lactate ([lactate]i) in astrocytes expressing wild-type (WT) TDP-43 and in astrocytes with cytoplasmic TDP-43 inclusions . The amplitude and the time constant of the increase in [cAMP]i were, however, reduced by 35% and 50%, respectively, and the peak amplitude and the cumulative [Ca2+]i increase were reduced by 20% and 30%, respectively, in astrocytes with cytoplasmic TDP-43 inclusions  versus astrocytes transfected with WT TDP-43. The amplitude and the rate of increase in [lactate]i upon NA treatment were similar in WT TDP-43 expressing astrocytes and in astrocytes with TDP-43 inclusions, however the responsiveness of astrocytes containing cytoplasmic TDP-43 inclusions to NA with production of lactate was increased by 1.6-fold versus WT TDP-43 expressing cells. Astrocytes with TDP-43 inclusions exhibited a reduced expression of β2-ARs and MCT1 transporters, as determined with immunocytochemistry, consistent with reduced cAMP and Ca2+ signaling and suggesting decreased astroglial lactate release capacity. Thus, astroglial aerobic glycolysis is altered in astrocytes with cytoplasmic TDP-43 inclusions, which may affect astroglial metabolic support of neurons in ALS and FTD-U. Moreover, these cells accumulated LDs.The results revealed that overexpression of the C-terminal fragment of TDP-43 (amino acids 208–414) results in cytoplasmic TDP-43 inclusions in cultured rat cortical astrocytes, typical for ALS and FTD-U pathologies. These cells accumulated LDs. To measure the effect of TDP-43 inclusions in single cells on the dynamics of AR-induced Ca208–414), known to generate cytoplasmic inclusions in other cell types20, or with the pDNA encoding RFP-tagged WT TDP-43 (RFP-TDP-43wt). Using confocal microscopy, we observed that in astrocytes expressing the RFP-TDP-43wt construct, the RFP signal is visible mainly in the cell nucleus  with anti-TDP-43 was 71.5 ± 5.5% (n = 20) and 95.5 ± 2.1% (n = 23) in RFP-TDP-43wt- and RFP-TDP-43208–414-expressing astrocytes, respectively  affects the nuclear levels of endogenous TDP-43 in cultured rat astrocytes. To evaluate this, we performed colocalization analysis between Alexa488-labelled TDP-43 antibody and nuclear marker DAPI in control, non-transfected astrocytes, and in astrocytes expressing the RFP-tagged TDP-43 pDNA constructs. The degree of colocalization (%) with DAPI was 99.1 ± 0.4% (n = 28), 92.2 ± 2.9% (n = 20), and 33.9 ± 6.0% (n = 23) in control, RFP-TDP-43wt- and RFP-TDP-43208–414-expressing astrocytes, respectively , suggests that the C-terminal fragment of TDP-43 causes partial depletion of endogenous nuclear TDP-43. When we analyzed the extent of DAPI and TDP-43-colabeled nuclei of non-transfected astrocytes , positioned around astrocytes expressing the RFP-TDP-43 constructs , we observed a small, statistically insignificant reduction trend in percentage of DAPI and TDP-43-colabeled nuclei in the RFP-TDP-43208–414 experimental group compared to RFP-TDP-43wt and control experimental group  in astrocytes increased the amount of BODIPY493/503-positive cell cross-sectional area, indicating LD content increase, by ~4- and ~3-fold compared with control non-transfected (n = 58) and RFP-TDP-43wt-expressing astrocytes, respectively . Moreover, the average number of LDs per cell (32.0 ± 4.1 (RFP-TDP-43wt) versus 74.7 ± 6.7 (RFP-TDP-43208–414), P < 0.001), LD perimeter (1.6 ± 0.1 μm (RFP-TDP-43wt) versus 2.3 ± 0.1 μm (RFP-TDP-43208–414), P < 0.001), and LD diameter (0.50 ± 0.02 μm (RFP-TDP-43wt) versus 0.74 ± 0.03 μm (RFP-TDP-43208–414), P < 0.001), increased in astrocytes with TDP-43 inclusions  enabling the nuclear and cytosolic fluorescence signals to overlap, it is more likely, that the TDP-43 inclusions seen at the DAPI-labeled nuclear area are in fact located in the cytosol.40. Through binding to ARs on the surface of astrocytes, NA activates cAMP and Ca2+ signaling56, which may be dysregulated in various neurologic disorders60. To study whether ALS- and FTD-U-linked TDP-43 inclusions affect cAMP and Ca2+ signaling, we monitored NA-induced changes in [cAMP]i and [Ca2+]i using real-time confocal microscopy and genetically encoded FRET-based cAMP nanosensor Epac1-camps53 or Fluo-4 AM dye, respectively, in astrocytes expressing WT (RFP-TDP-43wt) or mutant TDP-43 pDNA construct  or 44.7% (n = 42/94), respectively  and RFP-TDP-43208–414-expressing astrocytes 37, indicating that the overexpression of RFP-TDP-43wt and RFP-tagging of the TDP-43wt does not affect NA-mediated cAMP signals in astrocytes. However, the NA-mediated increase in [cAMP]i was significantly reduced in RFP-TDP-43208–414- compared with RFP-TDP-43wt-expressing astrocytes, as reflected by the reduced amplitude in the FRET signal . Thus, noradrenergic stimulation leading to the β-AR-mediated increase in [cAMP]i is reduced in astrocytes expressing the ALS- and FTD-U-related C-terminal fragment of TDP-43 (amino acids 208–414) compared with astrocytes expressing WT TDP-43.The percentage of astrocytes co-expressing both Epac1-camps and RFP-TDP-43ely Fig. , not sigwt or RFP-TDP-43208–414 were labeled with Ca2+ indicator, Fluo-4 AM dye, and treated with NA  and the initial rates of the Laconic FRET signal increase (ΔFRET/Δtime [%/min]) in NA-responsive cells were similar in the RFP-TDP-43208–414 compared with astrocytes expressing RFP-TDP-43wt .However, when we looked at the whole population of astrocytes, we observed that the responsiveness of astrocytes to NA-induced lactate production increased by 1.6-fold (from 42.8% to 70.0%) in cells expressing RFP-TDP-433wt Fig. ; Table 2wt) versus 60.0% (RFP-TDP-43208–414); Table 208–414 than in the RFP-TDP-43wt-expressing astrocytes  and 12.0% ± 1.4% [n = 48] versus 10.0% ± 0.8% , respectively; Fig. 2-ARs was significantly lower in astrocytes expressing RFP-TDP-43208–414 , a major component of ALS- and FTD-U-associated pathologic cytoplasmic inclusions, affects astroglial cell metabolism. TDP-43 inclusions may compromise astroglial metabolic support of neurons in ALS and FTD-U and contribute to CNS hypometabolism observed in patients with neurodegeneration.Based on the new disease models, a number of recent studies have highlighted the involvement of non-neuronal cells in the pathogenesis of ALS and FTD-U, including astrocytes20, consistent with studies in other cell types64. Astrocytes with cytoplasmic TDP-43 inclusions had a 3-fold lower amount of endogenous nuclear TDP-43  compared with astrocytes expressing WT TDP-43, where most of the TDP-43 was present in the cell nucleus. This indicates that expression of the C-terminal fragment of TDP-43 in astrocytes affects trafficking of endogenous TDP-43 between the nucleus and the cytoplasm, consistent with reports on neurons5, glial cells from human tissue samples55, various cell lines (e.g. QBI-293 and tsBN2 cells10), and muscle cells65. The results suggest that aggregated TDP-43 prevents newly synthesized endogenous TDP-43 from being imported into the nucleus and/or inhibits the re-entry of existing cytoplasmic TDP-43 into the nucleus13. Interestingly, the transfection with inclusion-forming TDP-43, caused in neighboring non-transfected astrocytes  a small, albeit not significant, trend in reduced percentage of DAPI and TDP-43-colabeled nuclei, which suggests that the transfection with the C-terminal fragment of TDP-43 might affect the physiology of non-transfected cells. In pathological conditions, mimicked here by the expression of inclusion-forming TDP-43, astrocytes bear a significant functional plasticity, known as reactive astrogliosis66. These astrocytes secrete or distribute through gap junctions various cytotoxic factors, such as Lcn268, interleukin-6, ciliary neurotrophic factor, etc.69, which can affect neighboring cells. Even though we did not specifically test whether astrocytes expressing inclusion-forming TDP-43 transform into a reactive phenotype, this outcome is possible. If this is the case, the observed reduction in the nuclear TDP-43 staining in some non-transfected astrocytes adjacent to inclusion-forming TDP-43 astrocytes may be a consequence of altered physiological state of these cells affecting the TDP-43 gene expression level or the synthesis/degradation/distribution of TDP-43.Expression of C-terminal fragment of TDP-43 in isolated cortical rat astrocytes has led to the formation of cytoplasmic TDP-43 inclusions, which can mimic key biochemical features of TDP-43 proteinopathies3, including on cell metabolism. LD accumulation has been observed in glial cells, including astrocytes, in early stages of neurodegeneration46. Recently, alterations in lipid metabolism  in spinal cords from SOD1G93A transgenic mouse model have been reported that might be linked to astrogliosis and LD formation in astrocytes48, since a population of astrocytes isolated from the degenerating spinal cords of the same animal model exhibited significant abundance of LDs as well as autophagic and secretory vesicles, all characteristic features of cellular stress and inflammatory activation70. However, the mechanisms leading to increased LD accumulation in astrocytes are poorly understood and may, among others, rely on altered neuronal mitochondrial function and ROS as well as on astrocyte-neuron lactate shuttle47, in particular on astroglial lactate-derived lipid production in neurons and transfer of excess lipids in lipoprotein-like particles (ApoE) from neurons to astrocytes71. We show here that in astrocytes with TDP-43 inclusions the LD presence is enhanced (the size and the number of LDs) in the absence of neighboring neurons, suggesting the existence of an alternative astroglial-mediated mechanism triggering accumulation of LDs through altering the balance between biogenesis and degradation of LDs. Accumulation of LDs in astrocytes with cytoplasmic TDP-43 inclusions may be a response to cellular inflammation, which is typically found in the pathology of various neurologic disorders, including ALS72. Here, LDs are hypothesized to be an important source of energy for proliferation and may serve a protective role by gathering free fatty acids to protect cells against lipotoxicity73.Because TDP-43 is involved in multiple aspects of RNA processing, any changes in TDP-43 nuclear level may have detrimental effects on astroglial physiology2-ARs and a 35% reduction in NA-mediated increase in [cAMP]i. Consistent with our results, dysregulation of astrocytic β2-AR/cAMP signalling has been suspected to contribute to the pathology of a number of other neurologic disorders, including multiple sclerosis, Alzheimer’s disease, human immunodeficiency virus encephalitis, and others74. Reduction of β2-AR expression was reported in human white matter astrocytes obtained from post mortem brain tissue of patients with multiple sclerosis75. Moreover, it was demonstrated both in vitro and in vivo that the presence of Alzheimer’s disease associated amyloid beta peptide (Aβ) in prefrontal cortical neurons leads to internalization and degradation of β2-ARs, which leads to subsequent attenuation of cAMP signalling59. Since aerobic glycolysis in astrocytes is upregulated with β-AR/cAMP signaling, one would expect that astrocytes with TDP-43 inclusions and a reduced expression of β2-ARs will exhibit reduced β-adrenergic mediated aerobic glycolysis and lactate production. When we stimulated astrocytes with Iso, a selective β-AR agonist, although the responsiveness of cells to Iso was unchanged, there was a trend in reduction of [lactate]i increase, since the amplitude in [lactate]i increase was ~2-fold lower (P = 0.08) in astrocytes with TDP-43 inclusions, consistent with downregulation of β2-AR and cAMP signaling in these cells. In contrast to Iso stimulation, the amplitude and the rate of [lactate]i increase upon NA stimulation was unaltered in astrocytes with TDP-43 inclusions, but the probability of activating aerobic glycolysis, measured as increased responsiveness to NA, was increased by 1.6-fold in astrocytes with TDP-43 inclusions. When viewing the astroglial population as a whole, this means that glycolytic lactate production upon NA stimulation is facilitated in astrocytes with TDP-43 inclusions.Besides alterations in LD metabolism, changes in noradrenergic regulation of glucose metabolism were observed in astrocytes with cytoplasmic TDP-43 inclusions. Astrocytes with TDP-43 inclusions exhibited downregulation of β2+ signals through activation of α1-AR/Gq-protein signaling pathway have important role in regulation of NA-mediated glucose metabolism in astrocytes33. Abnormal Ca2+ homeostasis has been observed in astrocytes isolated from SOD1G93A animals. In particular, excess Ca2+ release from ER stores upon purinergic/Gq-protein signaling pathway activation has been reported due to abnormal ER Ca2+ accumulation76. If such a mechanism exists in astrocytes with TDP-43 inclusions, binding of NA to α1-AR may lead to excess Ca2+-release from ER and enhanced aerobic glycolysis despite downregulation of β2-AR/cAMP signaling pathway. However, contrary to the results obtained on SOD1G93A animal model, we observed a 31% reduction in Ca2+ signaling in astrocytes with TDP-43 inclusions, even though the level of α1-AR expression was unchanged. It has been reported that β-AR/cAMP and α1-AR/Ca2+ signaling pathways interact in astrocytes, enhancing each other39, therefore downregulation of β2-ARs may reduce the noradrenergic Ca2+ response via α1-ARs, explaining the reduction of both noradrenergic cAMP and Ca2+ signals despite an unaltered expression of α1-ARs.Besides cAMP, Cai37. If aerobic glycolysis in astrocytes displays a similar bell-shaped dependency on [cAMP]i, astrocytes with reduced β2-AR/cAMP signalling may actually attain enhanced metabolic responsiveness to noradrenergic stimulation. Moreover, perturbances in TDP-43 may change the transcriptome and proteome of a cell affecting the expression level of enzymes involved in LD metabolism and aerobic glycolysis, as was observed in other cell types with a TDP-43 knock down16. Whether expression level of metabolic enzymes is altered in astrocytes with TDP-43 inclusions and whether this contributes to the observed enhanced astroglial metabolism needs to be investigated in the future.It has been reported that morphologic changes in astrocytes exhibit a bell-shaped dependency on [cAMP]77. This is, however, contradictory to the CNS hypometabolism observed in patients with neurodegenerative diseases, including ALS45. Recently, decreased expression of lactate MCT1 and MCT4 transporters has been reported post mortem in the motor cortex of ALS patients compared to non-ALS patients78. Moreover, downregulation of MCT1 mRNA in the spinal cords of early symptomatic and endstage SOD1G93A transgenic mice model of ALS has been observed, presumably in glia (oligodendrocytes and astrocytes)78. Down-regulation of the lactate MCT4 transporter has been also demonstrated in spinal cord astrocytes from patients with ALS with SOD1 mutations and in pre-symptomatic SOD1G93A transgenic mice43. We show here that expression of C-terminal fragment of TDP-43 (TDP-43208–414) per se in astrocytes causes a reduction in the expression of astroglial lactate MCT1 transporters. Lower expression of astroglial MCTs may decrease the lactate release capacity in astrocytes. Thus, despite facilitated NA-mediated aerobic glycolysis in astrocytes with TDP-43 inclusions, lactate may accumulate inside cells, and the metabolic support of neurons in patients may be decreased significantly contributing to neuron degeneration in ALS and FTD-U. Consistent with this hypothesis, cAMP signaling and lactate homeostasis in the brain were noted to be reduced in patients with some forms of ALS45.Increased lactate production upon NA stimulation in astrocytes with TDP-43 inclusions may lead to a better metabolic support of neurons with lactate due to lactate flux generated between astrocytes and neurons2+ signaling, whereas both aerobic glycolysis and LD presence appear facilitated. Although NA-mediated L-lactate production was increased in astrocytes with TDP-43 inclusions, the expression of lactate MCT1 transporters was reduced, suggesting decreased astroglial lactate release capacity .79, and grown in high-glucose Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, 2 mM l-glutamine and 25 µg/ml penicillin-streptomycin in a vaporized atmosphere containing 95% air and 5% CO2 at 37 °C until reaching 70%–80% confluence. Confluent cultures were shaken overnight at 225 rpm and the medium was changed the next morning; this was repeated three times. After the third overnight shaking, the cells were trypsinized and put in flat tissue culture tubes with 10 cm2 growth area. After reaching confluence again, the cells were subcultured onto 22-mm diameter poly-l-lysine-coated glass coverslips. This procedure yielded astrocytes with > 95% purity, determined by anti-glial fibrillary acidic protein (GFAP) antibody staining80.Primary astrocyte cultures were prepared from the cerebral cortices of 2- to 3-day-old Wistar rats, as described previously53 or lactate nanosensor Laconic54 and RFP-tagged WT TDP-43 (pTagRFP-C::TDP-43wt [RFP-TDP-43wt]) or RFP-tagged C-terminal fragment of TDP-43 (pTagRFP-C::TDP-43208–414 [RFP-TDP-43208–414]) using FuGENE 6 Transfection Reagent . Transfection medium contained no serum or antibiotics.After 1–3 days, astrocytes were co-transfected with the genetically encoded FRET-based cAMP nanosensor Epac1-campsThe experimental animals were cared for in accordance with the International Guiding Principles for Biomedical Research Involving Animals developed by the Council for International Organizations of Medical Sciences and the Animal Protection Act . The experimental protocol was approved by The Administration of the Republic of Slovenia for Food Safety, Veterinary and Plant Protection , Document No. U34401-47/2014/7, signed by Barbara Tomše, DVM. Every set of data was acquired from at least 2 independent experiments.wt or RFP-TDP-43208–414 were fixed with 4% formaldehyde in phosphate buffered saline (PBS) for 15 min and then permeabilized with 0.1% Triton X-100 for 10 min at room temperature (except for MCT1 and MCT4) before being treated with 10% goat serum for 1 h at 37 °C. Astrocytes were then stained with primary antibodies against endogenous TDP-43 , α1-AR , β1-AR , β2-AR , MCT1  and MCT4  overnight at 4 °C. After washing with PBS, cells were incubated for 1 h at 37 °C with Alexa Fluor488-conjugated secondary goat anti-rabbit IgG . Excess antibodies were washed off and the coverslips were mounted onto glass slides using SlowFade antifade reagent with DAPI  and carefully sealed. Immunolabelled cells were imaged with an inverted Zeiss LSM780 confocal microscope with a Plan apochromatic 40×/1.4 oil immersion objective  using 488-nm Ar-ion, 543-nm He-Ne and 405-nm diode laser excitation. Emission spectra were acquired sequentially with 505- to 530-nm bandpass (Alexa Fluor488), 560-nm long pass (TagRFP584) and 445- to 450-nm bandpass (DAPI) emission filters.Control (non-transfected) astrocytes and astrocytes expressing RFP-TDP-43488 (immunolabelled TDP-43), TagRFP584 (RFP-TDP-43 pDNA constructs), and DAPI fluorescence signals was performed on exported TIFF files using ColocAna software 81 that counts all red, green, blue and colocalized (green and red or green and blue) pixels within the image. The threshold for the colocalized pixel count was set at 20% of the maximal green, red or blue fluorescence intensity, respectively. Colocalization was expressed as the ratio of colocalized to green or blue pixels (as percentages). Kruskal-Wallis one-way analysis of variance (ANOVA) on ranks (followed by Dunn’s test) or Mann-Whitney U test were performed to determine statistical significance between the experimental groups. P < 0.05 was considered significant.Colocalization analysis of Alexa Fluor1-AR-, β1-AR-, β2-AR-, MCT1- and MCT4-positive green fluorescence pixels with fluorescence intensity above the threshold of 10% (α1-/β1-/β2-AR-positive cell area) or 20% (MCT1 and MCT4-positive cell area) of maximal fluorescence and the number of all pixels  per cell cross-sectional area were determined for each cell separately in RFP-TDP-43wt- and RFP-TDP-43208–414-expressing astrocytes using ZEN software . The percentage of the AR- and MCT-positive cell area relative to the total cell area was calculated for RFP-TDP-43wt- and RFP-TDP-43208–414-expressing astrocytes for individual receptor and transporter type. The Mann-Whitney U test was performed to determine statistical significance between the experimental groups. P < 0.05 was considered significant.To determine the expression level of different ARs and MCTs, the number of αwt- and RFP-TDP-43208–414-expressing astrocytes  we measured the percentage of DAPI and TDP-43-colabeled nuclei per all DAPI nuclei in neighboring non-transfected cells  in the RFP-TDP-43wt and RFP-TDP-43208–414 experimental groups. In the control non-transfected group, we analyzed the nuclei of all cells. Some partially visible DAPI-stained nuclei at the edges of the confocal images were also taken into account in the analysis, however, only if no RFP signal in or around the visible part of the nuclei was observed. Kruskal-Wallis one-way ANOVA on ranks, followed by Dunn’s test was performed to determine statistical significance between the experimental groups. P < 0.05 was considered significant.To determine the extent of DAPI and TDP-43-colabeled nuclei in non-transfected astrocytes  adjacent to the RFP-TDP-43wt or RFP-TDP-43208–414 plasmid were incubated in fresh growth medium for 24 h. Cells were then fixed in 4% formaldehyde in PBS for 5 min and stained with 1 µg/ml BODIPY493/503 , a fluorescent LD marker, for 6 min at room temperature. Excess dye was washed off and the coverslips were mounted onto glass slides using SlowFade antifade reagent with DAPI  and carefully sealed. Stained cells were imaged with an inverted Zeiss LSM780 confocal microscope with a Plan apochromatic 40×/1.4 oil immersion objective  using 488-nm Ar-ion, 543-nm He-Ne and 405-nm diode laser excitation. Emission spectra were acquired sequentially with 505- to 530-nm bandpass (BODIPY493/503), 560-nm long pass (TagRFP584) and 445- to 450-nm bandpass (DAPI) emission filters.Control (non-transfected) astrocytes and astrocytes transfected with RFP-TDP-43493/503-positive green fluorescence pixels with fluorescence intensity above the threshold of 20% of maximal fluorescence (Lipid droplet S) and the number of all pixels (Cell S) were determined for each cell separately in control, RFP-TDP-43wt- and RFP-TDP-43208–414-expressing astrocytes in ZEN software . The values obtained were normalized to the average LD content in control non-transfected cells. The mean number of BODIPY493/503-labeled LDs per cell and the mean LD perimeter were determined in cross-sections of individual cells using ImageJ, Analyze Particles function after applying 20% threshold and signal intensity (watershed) segmentation on individual images. Then, assuming that all LDs are spherical, the mean LD diameter was estimated from the perimeter values with the equation d = C/π, where d is the diameter and C is the LD perimeter. Kruskal-Wallis one-way ANOVA on ranks, followed by Dunn’s test was performed to determine statistical significance between the groups. P < 0.05 was considered significant.To determine the LD content in individual astrocytes, the number of BODIPYwt or RFP-TDP-43208–414) were examined 24–30 h after transfection with a fluorescence microscope Zeiss Axio Obsever.A1 , with a CCD camera and monochromator Polychrome V  as a monochromatic source of light with a wavelength 436 nm/10 nm. Dual emission intensity ratios were recorded using an image splitter  and two emission filters . Images were acquired every 3.5 s for Epac1-camps and 10 s for Laconic. Exposure time was 100 ms.Cells co-expressing the FRET-based nanosensor Epac1-camps or Laconic and RFP-tagged TDP-43 pDNA construct ) with a Axiocam 702 camera and Colibri.2 Lamp Module  as a source of light with a wavelength 433 nm. Dual emission intensity ratios were recorded using an image splitter  and two emission filters (469-491 nm for ECFP and 530-4095 nm for EYFP). Images were acquired at intervals of 10 s with exposure time of 100 ms.In some experiments astrocytes co-expressing Laconic and RFP-TDP-43i or [lactate]i.Coverslips with transfected astrocytes were mounted in a superfusion recording chamber on the microscope stage. Imaging was performed at room temperature (22–24 °C). One cell per experiment was recorded. The FRET signals (CFP/YFP (Epac1-camps) and mTFP/Venus (Laconic) signals) were obtained from the integration of the ratio signal over the entire cell using Life Acquisition software  or ZEN . In the graphs, the FRET signal was reported as the ratio of the CFP/YFP (Epac1-camps) and mTFP/Venus (Laconic) fluorescence signals after subtracting the background fluorescence from the individual fluorescence signals using Excel . The values of the FRET ratio signals were normalized to 1.0. An increase in the FRET ratio signal reflects an increase in [cAMP]d-glucose, 135.3 mM NaCl, 1.8 mM CaCl2, 2 mM MgCl2, and 5 mM KCl, 0.5 mM NaH2PO4·H2O, 5 mM NaHCO3), and then treated with 100 µM NA (non-selective AR agonist) or 100 µM Iso (selective β-AR agonist) following a 100- to 200-s baseline. Experiments were conducted with the addition of a bolus solution; 200 µl of extracellular solution containing NA was added by pipette to 200 µl of bath solution in the recording chamber. The application of a control bolus solution without reagents had no significant effect on the FRET signal, as reported previously83. Extracellular solution osmolality was 295–305 mOsm, measured with the Osmomat 030 freezing point osmometer .Before the experiments, astrocytes were kept in extracellular solution containing sodium bicarbonate for 10 min )) were fitted to the FRET ratio signals using SigmaPlot. The time constant (τ) and amplitude changes in the FRET ratio signal (ΔFRET [%]) were determined from the fitted curves. F is the FRET ratio signal at time t, F0 is the baseline FRET ratio signal, c is the FRET ratio signal amplitude of F − F0, and τ is the time constant of the individual exponential component. In experiments with Laconic, the maximal  rates of the FRET ratio signal increase (ΔFRET/Δt) were calculated as the slope of the linear regression function (ΔFRET [%] = slope [%/min] × Δt [min]) fitting the initial FRET ratio signal increase. In these experiments, changes in the FRET ratio signal (ΔFRET [%]) were calculated by subtracting the mean maximal FRET ratio signals from the mean baseline FRET ratio signals.In experiments with Epac1-camps, single-exponential increases to maximum functions  and then transferred to dye-free medium for at least 30 min before the experiments to allow for cleavage of the AM ester group. The cells were excited with an Argon-ion laser at 488 nm, and time-lapse images were obtained every 3 s with an inverted Zeiss LSM780 confocal microscope and an 20x objective. Emission light was acquired with a 505–530-nm band-pass emission filter. Fluo-4 AM–labeled astrocytes were after 100 s baseline stimulated with 100 μM NA. Total recording time was ~400 s (135 frames). In individual cells, Fluo-4 AM intensity was quantified within a region of interest and expressed as the relative change in fluorescence: ΔF/F0 = (F − F0)/F0, where F0 denotes the baseline fluorescence level after subtraction of background fluorescence. The peak and cumulative increase in Fluo-4 AM ΔF/F0 were determined using Microsoft Excel.Astrocytes expressing RFP-TDP-43P < 0.05 was considered significant.Mann-Whitney U test was performed to determine statistical significance between the experimental groups. Supplementary material.


Acute exacerbation (AE) is a common cause of rapid deterioration and high mortality in idiopathic pulmonary fibrosis (IPF) patients. Osteopontin (OPN) plays an important role in IPF, but the studies about serum OPN in AE-IPF are unclear. We aimed to investigate whether OPN had a potential prognostic value in acute exacerbation and mortality in IPF.  Thirty-two patients with AE-IPF, 39 with S-IPF, and 20 healthy controls were included. Serum OPN and KL-6 levels were compared between AE-IPF and S-IPF. Logistic regression analysis was applied to identify the predicted value of OPN for AE. Kaplan–Meier curves were used to display survival, and Cox proportional hazards regression was used to identify risk for mortality. p < 0.001) or healthy controls (p < 0.001) or healthy controls (p < 0.001) or healthy controls (p < 0.001) or healthy controls (p < 0.001) or healthy controls (p < 0.001) or healthy controls (p < 0.001) or healthy controls (p < 0.001) or healthy controls (p < 0.001) or healthy controls (p < 0.001) or healthy controls ( In AE-IPF patients, serum OPN levels were significantly higher than in S-IPF subjects ( Elevated OPN could be a potential serum predictor for AE status and survival in IPF patients. Idiopathic pulmonary fibrosis (IPF) presents as a progressive deterioration of dyspnea and ultimately respiratory failure , charactSo far, the etiology and mechanisms of AE-IPF occurrence have been poorly known. More and more evidence demonstrated the importance of diffuse alveolar damage and enhanced epithelial injury and proliferation during AE in IPF patients . OPN as In this study, we aimed to determine whether OPN could be a biomarker of AE-IPF occurrence and mortality by comparing serum OPN levels in AE-IPF and S-IPF and by analyzing correlation between serum OPN and other variables in IPF patients.The study population includes 71 subjects (32 AE-IPF patients and 39 S-IPF patients), who were evaluated at Nanjing Drum Tower Hospital between January 2015 and April 2018. 20 healthy subjects from the Center of Physical Examination were included as a control group. The criteria used for enrollment of patients in this study were in accordance with the criteria of IPF and AE-IPF already published , 1.001 , 1.039, 1.035 , 0.950 , and 0.929 , respectively  were significantly associated with a higher risk of AE in IPF, with odds ratios of 1.305 , 1.012 , and 1.009 . Subsequently, multivariate Cox regression was performed. Several covariates were excluded for statistical limitations. We observed that OPN was also associated with the survival of IPF in multivariate Cox models, with a hazard ratio of 1.010 . The same results were also identified for serum KL-6 .To further examine the prognostic values of OPN with regard to the survival in all IPF patients, univariate Cox regression was performed . ElevateThis was the first study to assess the significance of serum OPN levels in occurrence of AE and mortality in IPF patients. IPF patients had elevated serum OPN levels, especially in AE-IPF, compared with healthy controls. In addition, the enhanced expression of OPN was also found in alveolar epithelial cells and alveolar macrophages of the lung section from IPF patients. Compared with S-IPF, serum OPN levels in AE-IPF were significantly increased, which were associated with poor outcome. We found that the survival period was obviously shorter for patients with OPN levels above, compared with those below 3.24 ng/ml; moreover, OPN was a predictor of survival. Collectively, our results suggested that OPN could predict prognosis of patients with IPF.Acute exacerbation state of IPF is an important factor contributing to IPF mortality. Unrecognized infection, diffuse alveolar damage, mechanical procedures, and secondary pulmonary hypertension have been suggested to explain acute exacerbation of IPF , 13, butOPN is a secreted phosphoprotein produced by a variety of cells such as activated macrophages, vascular smooth muscle cells, and epithelial cells, mediating cell chemotaxis, adhesion, proliferation, and migration, and plays a vital role in the pathogenesis of liver and kidney fibrosis , 19. CarIn current study, AE-IPF patients showed higher mortality than S-IPF, consistent with the reported studies , 26. ElePrevious studies demonstrate physiologic parameters such as age, FVC%, and DLCO% have been shown to be important survival factors for IPF , 28. In In this study, LDH and CRP were significantly associated with acute exacerbation status and mortality. LDH is one of the earliest for the serum marker of disease activity in different forms of pulmonary fibrosis . LDH, asMore and more studies demonstrate that acute alveolar epithelial damage and unrecognized infection play an important role in AE of lung fibrosis. This could explain why the levels of LDH and CRP increased obviously in AE-IPF. Our results indicated that elevated levels of LDH and CRP were notably associated with survival, which was consistent with previous studies, but due to their low specificity, they were difficult to be a reasonable biomarker for mortality .There are several limitations in this study. Our ability to perform serial analysis of OPN was limited due to the small study population, which was understandable in consideration of the rarity of IPF. Furthermore, there were no data about OPN in the same patient experiencing the different stages of disease , which was necessary to be a good biomarker for OPN. A comprehensive study in a larger IPF population would be helpful for further research.In summary, to the best of our knowledge, this was the first study to demonstrate elevated serum OPN levels in AE-IPF patients, which was associated with acute exacerbation state and increased mortality risk. The same results were found for KL-6. More studies are needed to verify and extend the role of OPN, to determine whether or how OPN plays a role in the progress of AE, and to confirm the clinical usefulness of OPN as a biomarker for AE-IPF occurrence and predictor of survival in IPF patients.


The distribution, Raman and photoluminescence (PL) spectra of the above prepared heterostructure samples can be respectively observed and tested by optical microscopy and Raman spectrometry, which can be used to study their growth mechanisms and optical properties. Meanwhile, the uniformity and composition distribution of heterostructure films can also be analyzed by the Raman and PL spectra. The internal mechanism of Raman and PL spectral changes can be explained by comparing and analyzing the PL and Raman spectra of the junction and non-junction regions between 2D-2D vdWs heterostructure materials, and the effect of laser power on the optical properties of heterostructure materials can also be analyzed. These heterostructure materials exhibit novel and unique optical characteristics at the stacking or junction, which can provide a reliable experimental basis for the preparation of suitable TMDs heterostructure materials with excellent performance.Due to the attractive optical and electrical properties, van der Waals (vdWs) heterostructures constructed from the different two-dimensional materials have received widespread attention. Here, MoS Nanoscale PN heterostructure research is particularly important as the device size decreases. However, due to the dangling bonds and lattice mismatch, it is difficult for the traditional materials to achieve atomically flat heterostructure interfaces, which limits the development of nanoscale PN heterostructures ,2,3. TwoGraphene has the novel physical properties, strong mechanical stability, high mobility and the unique electronic properties of zero-band gap Dirac semimetals, which can make it a basic component of vdWs heterostructures ,15. The To solve the difficulties and challenges in the application of 2D-2D vdWs heterostructure materials, vdWs heterostructure materials were prepared by the direct CVD and wet transfer methods in this paper. The resulting vdWs heterostructures have ultra-clean and atomically clear interfaces, and novel physical properties appear when 2D materials with different properties are stacked together, which can provide a multifunctional research platform for the study of interface-related characteristics . In addi2 thin film is formed on a h-BN/SiO2/Si substrate by the Frank Van der Merwe mechanism. This is because the surface of h-BN is clean and smooth, which is beneficial to the sulfidation and diffusion of MoS2 molecules. The large-sized triangular MoS2 materials are evenly distributed on graphene when the CVD growth of MoS2 material is completed, and the MoS2 and graphene materials on SiO2/Si substrate can be well distinguished with the help of contrast as shown in 2/h-BN heterostructure at different positions on the SiO2/Si substrate. The average size of the WS2 material on h-BN/SiO2/Si substrate is about 30 µm, the thickness is uniform and the surface is smooth. The edge of the h-BN sheet has many dangling bonds, which can provide nucleation sites for the growth of WS2 material. 2/graphene heterostructure, where the triangle-shaped WS2 sheet has an average size of 60 μm. In addition, there is also a stack of small triangles on the large triangle of WS2 material, which indicates the existence of multilayer triangular WS2 sheets.The morphology of 2D-2D vdWs heterostructure materials can be observed by optical microscopy. In −1 and 2631.3 cm−1, and the intensity of the 2D peak is stronger than that of the G peak, so the monolayer graphene material has good quality [2D/IG decreases, which is due to the interaction change between graphene material and the underlying SiO2/Si substrate.In  quality . Figure 2 at different positions, the lattice vibration of MoS2 has the E12g and A1g Raman active characteristic phonon peaks, where the E12g and A1g characteristic peaks were respectively derived from intra-layer lattice and inter-layer lattice vibrations. The two lattice vibration modes change accordingly with the layer number of MoS2 material decreases, and the E12g and A1g characteristic peaks are respectively red-shifted and blue-shifted, so the number of MoS2 material layers can be determined by the peak position difference. In the Raman spectrum of MoS2, the characteristic peaks of E12g mode and A1g mode are respectively located at 382.1 cm−1 and 401.4 cm−1, and the characteristic peak position difference is 19.3 cm−1, which indicates that the MoS2 sample is a single layer. 2, the peak intensities of E12g and A1g characteristic peaks accordingly increase with the laser power increases and the peak position of A1g characteristic peaks is blue-shifted to some extent.2, the E12g and A1g mode characteristic peaks are respectively located at 351.5 cm−1 and 416.4 cm−1, the peak position difference is 64.9 cm−1, and there is no peak near 310 cm−1, which indicates the existence of monolayer WS2. It can be found by observing 12g vibration characteristic peak and the peak position of A1g vibration mode characteristic peak remains unchanged. This is because the electron concentration changes as the laser power increases, and the increase of electron concentration could cause a reforming of the band gap.2 material, the resonance Raman scattering of MoS2/h-BN heterostructure material can be studied by analyzing the band structure and exciton transition. −1 and 1358 cm−1.In order to match the excitation energy to the PL spectrum exciton peak energy of MoS12g and A1g mode characteristic peaks of MoS2 on a h-BN substrate. The in-plane vibration E12g mode characteristic peak and out-plane vibration A1g mode characteristic peak are respectively located at 384.6 cm−1 and 404.2 cm−1, and the frequency difference is 19.6 cm−1. Compared with the Raman spectrum of MoS2 on SiO2/Si substrate, the characteristic peak frequencies of E12g mode and A1g mode are respectively blue-shifted by 2.5 cm−1 and 2.8 cm−1 on h-BN/SiO2/Si substrate, and the substrate effect can explain the blue shift of the Raman peak on h-BN. Therefore, Raman spectroscopy at room temperature showed that the h-BN substrate can reduce the local strain and injected impurities into MoS2. Besides, the PL spectrum can be fitted by the Lorentz function, as shown in 2 is at 681 nm, and the corresponding energy electron volt band gap is 1.82 eV, which is consistent with the band gap of monolayer MoS2. The PL exciton emission of MoS2/h-BN heterostructure material is two times larger than that of MoS2 material, and the FWHM is 26.39 nm, which is narrower than that of MoS2 material. Therefore, MoS2/h-BN heterostructure material has a tighter interlayer contact and lower charged impurities levels.At the same time, the full width at half maxima (FWHM) of the IPM mode characteristic peak is much larger than that of the ISM mode. 2 material is grown directly on graphene to form a MoS2/graphene heterostructure, their lattice vibrations would inevitably affect each other. In a MoS2/graphene heterostructure, the work function of graphene is smaller than that of MoS2 material, which is beneficial to the formation of contacts.When monolayer MoS12g and A1g modes characteristic peaks of MoS2 material and the G and 2D peaks of graphene appear in the Raman spectrum, which indicates the presence of MoS2/graphene heterostructures. Among them, the E12g and A1g mode characteristic peak positions of MoS2 material are respectively located at 382.6 cm−1 and 400.3 cm−1, and the G and 2D peak positions of graphene are located at 1602.5 cm−1 and 2689.7 cm−1, respectively. The G and 2D peak positions of the MoS2/graphene heterostructure shift to larges wavenumbers compared with the spectrum of intrinsic graphene, the the G and 2D peaks respectively change from 1585.9 cm−1 and 2631.3 cm−1 to 1602.5 cm−1 and 2689.7 cm−1. The reason is that the hole concentration becomes smaller and the electrons from MoS2 material can be transferred to the graphene. Besides, the E12g and A1g mode characteristic peaks of MoS2/graphene heter-structure are respectively red-shifted and blue-shifted by 0.5 cm−1 and 1.1 cm−1 compared with the spectrum of monolayer MoS2, which is due to the Van der Waals force interactions between MoS2 and graphene. 2/graphene heterostructure material at different positions. The strongest PL spectrum peak position of MoS2/graphene heterostructure is at 674 nm, and the corresponding electron volt energy is 1.84 eV. The peak position of strongest PL spectrum shows the blue shift, graphene has the influence on the Raman spectrum of MoS2 material. The energy band of MoS2 material bends downward, and the Schottky barrier is lower when the Fermi level of graphene and MoS2 materials are aligned. It can be found by observing 2/graphene heterostructure becomes higher, it will cause the spectrum to change. The rise of 2D and G peaks position is related to the effective interlayer coupling of MoS2 nanosheets, and the strain effect, which is caused by the high temperature heating. Besides, due to the PL phenomenon of MoS2, the background Raman signal increases with the wavenumber increase. As shown in 2/graphene heterostructure is superimposed on B exciton, and the peak position has the blue shift, which can be explained by the stacking effect [2 sample directly grown on a graphene and MoS2 sample transferred on a graphene substrate. The E12g peak position of monolayer MoS2 material is blue-shifted by 1.3 cm−1 after the transfer, and the A1g peak position only changes slightly. There is a certain tension effect when monolayer MoS2 material is grown on the SiO2/Si substrate by CVD. After the transfer, the A1 peak position of PL spectrum in MoS2 sample has a blue shift about 40 meV, as shown in 2 sample transferred on a graphene is higher than that of MoS2 sample directly grown on a graphene. The reason is that the tension of the sample is effectively released while using PMMA to transfer MoS2 material to the new SiO2/Si substrate.In g effect . Figure 2 on h-BN and graphene substrates. As we all know, the MoS2 material has a suitable and adjustable band gap, strong exciton interaction, unique nonlinear effects and a novel spin valley effect, which has the great application potential in the fields of integrated electronic chips, photoelectric conversion and the energy environment. In 12g and A1g mode characteristic peaks of MoS2/graphene heterostructure are blue-shifted relative to the MoS2/h-BN heterostructure material. The reason is that the graphene substrate has the effect on the Raman spectrum of MoS2. Vertical heterostructures can be formed by the van der Waals force between the upper and lower layers, which would lead to a strong quantum confinement effect. This can not only change the electronic band and optical properties of MoS2 material, but also give it more excellent chemical activity. It can be found from 2/graphene heterostructure material is red-shifted compared with MoS2/h-BN heterostructure, and the peak intensity is weaker than that of MoS2/h-BN heterostructure. This is because graphene has the weakening effect on the fluorescence of MoS2. Besides, the exciton absorption peak of PL spectrum in MoS2/graphene heterostructure is located at 621 nm. The change of thickness and the abundance of heterostructures can further produce and broaden the new characteristics and application research of MoS2 material.A 633 nm red laser was used to compare and analyze the spectral characteristics of MoS2/h-BN heterostructure materials where the corresponding peak positions are located at 52 cm−1 and 1346 cm−1, respectively.2/h-BN heterostructure, the peak position of ISM and IPM mode characteristic peaks are blue-shifted. The reason is that WS2 material has an influence on the spectrum of h-BN. It can be clearly observed from 12g and A1g Raman activation mode peak positions of WS2 are respectively located at 354.8 cm−1 and 419.4 cm−1, and the peak spacing is 64.6 cm−1, which corresponds to the peak spacing of monolayer WS2 on SiO2/Si substrate. The peak position of WS2/h-BN heterostructure is different from that of WS2 on SiO2/Si substrate. The reason is that the van der Waals interaction between layers, and the effective transfer between dielectric screen of long-distance Coulomb interactions. In 2 material. The FWHM of PL emission peak is related to the exciton lifetime and interface quality, which can be attributed to the extension of high crystallinity WS2 material, and the clean heterojunction interface.Compared to the spectrum of MoS2 and WS2/graphene heterostructures is shown in 1g mode characteristic peak of WS2/graphene heterostructure shifts to the short wavelength compared with the Raman spectrum of WS2, and the peak position of A1g characteristic peak has the blue shift. And the E12g and A1g mode characteristic peak intensity of WS2/graphene heterostructure is higher than that of WS2.Raman spectroscopy is used to evaluate the crystal quality and film thickness of 2D materials. The Raman spectrum contrast analysis of WS2/graphene heterostructure film has excellent crystallinity. In addition, monolayer WS2 and multilayer WS2 materials are respectively the direct bandgap semiconductor and indirect semiconductor, so the PL spectroscopy can also be used to identify the number of layers. In 2 and WS2/graphene heterostructure both show the strongest PL emission peak is located at 634 nm, and the band gap is about 1.94 eV, which indicates that it is consistent with a mechanically peeled monolayer WS2. The PL intensity of WS2/graphene heterostructure is weaker than that of monolayer WS2. This is because the work function between graphene and WS2 material does not match, thus forming the internal field, which would cause the photoelectrons from WS2 can transfer to graphene, and leave holes in WS2.There is the sharp Raman characteristic peak, which indicates that WS−1 and 2667.5 cm−1, and the intensity of 2D peak is almost twice that of D peak, which indicates the presence of monolayer graphene. The D peak appears in the Raman spectrum of graphene material when the growth of WS2 material is completed. Meanwhile, the peak position of 2D peak shifts downward, which is due to the effective interlayer coupling and strain effect. As shown in 2. At the same time, the internal field can be formed, which is due to the work function mismatch between graphene and WS2.In 2/h-BN and WS2/graphene heterostructure is shown in 2/h-BN heterostructure, the E12g and A1g mode characteristic peaks of WS2/graphene heterostructure are respectively red-shifted and blue-shifted. The reason is that graphene material has a influence on the spectrum of WS2 material. In 2/graphene heterostructure is blue shifted. The difference of peak position shift can be attributed to the influence of different substrates and the change of interaction.A Raman spectrum characteristics comparison of WSFirst, the copper foil substrate was used in the growth process of graphene and h-BN materials by the CVD growth process. Before the growth of graphene and h-BN films on copper foil substrate, it is necessary to immerse the copper substrate in hydrochloric acid for 15 min to remove the local surface oxides. Finally, the copper foil substrate can be rinsed with the acetone and isopropanol solution.2 was also punched. Next, the tube furnace was charged with 35 sccm CH4, while the flow rate of H2 was adjusted to 10 sccm, and the growth time of graphene was 30 min, as shown in 4 gas from flowing into the tube furnace.2 gas), aminoborane polymer (aminoborane) and borazine under the action of heating belt [2. Next, the borazine can dehydrogenated again due to the catalytic effect of the copper foil metal, and the B and N atoms combined to form h-BN on SiO2/Si substrate. Finally, the power supply of heating belt and furnace is quickly cut off when the h-BN growth reaction is completed, and the tube furnace enters the cooling stage. The specific temperature change curve is shown in ing belt . At the 2/Si substrate, which can be achieved by the PMMA transfer method. First, the 500 nm PMMA layer was spin-coated on the graphene or h-BN/copper foil substrate, and the PMMA film was cured at 120 °C for 3 min when the spin coating was completed. Then, the copper foil substrate was etched by 1M FeCl3 solution. Next, the PMMA/graphene or PMMA/h-BN layers were rinsed several times in deionized water, which can be transferred to the SiO2/Si substrate. Subsequently, the sample was dried in the N2 gas environment for 12 h to make the graphene or h-BN and SiO2/Si substrate adhere more closely. Immediately after, the PMMA film was washed away in the acetone solution.The graphene or h-BN material grown on Cu foil substrate by CVD method was transferred to SiO2/Si substrate hydrophilic, and the treatment is 2 min, which can help the transfer of graphene or h-BN film and the growth of MoS2 or WS2 materials [2/Si substrate was annealed at 400 °C for 8 h when graphene or h-BN thin film was transferred to SiO2/Si substrate. Meanwhile, the Ar gas with 5% H2 was continuously provided to remove residues during the transfer process.The purpose of oxygen plasma treatment is to make the SiOaterials . The SiO2 or MoS2 materials were grown on the graphene/SiO2/Si or h-BN/SiO2/Si substrate in the tube furnace, and the SiO2/Si samples coated with graphene or h-BN materials were used as the substrates. The quartz boat containing 200 mg sulfur powder was placed upstream of the tube furnace, which is the low-temperature heating zone. At the same time, the graphene/SiO2/Si or h-BN/SiO2/Si substrate were placed upside down on the quartz boat containing WO3 or MoO3 powder, which is located in the high temperature heating area of tube furnace, as shown in 3 powder (or WO3 powder) respectively rises to 50 °C, 100 °C, 100 °C within 5 min, and maintains at this temperature for 10 min. Next, the temperature of sulfur powder and MoO3 powder (or WO3 powder) can respectively reach the sublimation temperature of 150 °C, 800 °C and 950 °C within 50 min, and the growth temperature is maintained for 10 min. Finally, the tube furnace was naturally cooled to room temperature.The WSRaman spectrometry can confirm the molecular structure by the Raman shift. The incident light is scattered when a certain wavelength of laser is irradiated on the sample surface, and the frequency of most scattered light does not change, which is the Rayleigh scattering. The small part scattering of scattered light frequency is referred to as Raman scattering, and the frequency difference between scattered light and incident light is called Raman shift. Raman spectroscopy is used to monitor the crystal quality, polymorphism, doping, defects, strain, disorder, chemical modification and relative orientation of 2D materials. Besides, Raman analysis has the high spatial resolution, the fast and easy spectral imaging, which is suitable for identifying the characteristics and structure of various materials . The phoThe distribution of heterostructure samples on the substrate surface can be observed by optical microscopy, which can test and characterize the aboveprepared heterostructure materials. The spatial resolution depends on the choice of excitation laser to a certain extent, and the excitation wavelength can be optimized based on the characteristics of the sample. Meanwhile, a Raman spectrometer  with a 633 nm laser red light was used to measure and obtain the single-point Raman and PL spectrum of the heterostructure materials, and the surface uniformity and composition distribution of heterostructure film were analyzed by Raman and fluorescence surface scanning. The surface morphology of heterostructure materials can be observed by optical microscopy. For the Raman and PL spectrum tests, the corresponding laser power is respectively fixed at 10 mW and 20 mW, the laser spot diameter is 1 μm, and the objective magnification of microscope is 10. The Raman data of characteristic peaks were analyzed by the classical least squares (CLS) fitting.2/h-BN, MoS2/graphene, WS2/h-BN and WS2/graphene van der Waals heterostructures were successfully prepared in this paper. The growth mechanism and optical characteristics of heterostructure materials can be studied by optical contrast microscopy and Raman PL spectroscopy, which can obtain the spectral characteristics of heterostructure materials. These heterostructures exhibit the novel and unique optical properties at the stacking or junction of several materials. The Raman and PL spectroscopy models are complementary and can describe the temperature-dependent relationships between interlayer coupling and physical origin. Meanwhile, the electron-phonon coupling and vdW interaction can be enhanced by reducing the local strain and charged impurities. Compared with the Raman spectrum of MoS2 on SiO2/Si substrate, the characteristic peak frequencies of E12g mode and A1g mode are blue-shifted by 2.5 cm−1 and 2.8 cm−1 respectively on a h-BN/SiO2/Si substrate, and the E12g and A1g mode characteristic peaks of MoS2/graphene heterostructure are respectively red-shifted and blue-shifted by 0.5 cm−1 and 1.1 cm−1. The G and 2D peak positions of MoS2/graphene heterostructure shift to the large wavenumber compared with the spectrum of intrinsic graphene. Besides, E12g and A1g mode characteristic peaks of MoS2/graphene heterostructure are blue-shifted compared with MoS2/h-BN heterostructure, and the strongest peak position of PL spectrum is red-shifted. The crystal orientation between WS2 or MoS2 growth and hBN or a graphene substrate is significantly different, which is due to the interlayer Coulomb interactions. Our research can effectively improve the interlayer coupling basic understanding of heterostructures and temperature-dependent Raman and PL spectra were used to explore the optical properties of vdWs heterostructures. Therefore, nanoscale vdW heterostructures have the great application prospects in the fields of nanoelectronics and optoelectronics.MoS


KL determined for the Langmuir isotherm is high: KL = 1.27 × 108 M−1, enabling highly sensitive control of the gated-RET by PD-L1. Thus, with the gated-RET technique, one can determine PD-L1 within the dynamic range, extending from 1.2 to 50 nM. Moreover, we have found that the Gibbs free energy for PD-L1 binding to AuNP@Cit is −46.26 kJ/mol , indicating a strong adsorption with supramolecular interactions. The proposed gated-RET system, with the fluorescence intensity of the fluorophore probe molecule modulated by plasmonic quenching with AuNP and shielding of energy transfer by the adsorbed PD-L1 can be further developed for determination of PD-L1 in pharmaceutical formulations for immune checkpoint control in cancer therapy.The resonance energy transfer (RET) between an excited fluorescent probe molecule and a plasmonic nanoparticle (AuNP) has been investigated to evaluate the effect of protein molecules on the RET efficiency. We have found that the energy transfer to a functionalized AuNP can be modulated by a sub-monolayer film of programmed death-ligand 1 (PD-L1) protein. The interactions of PD-L1 with AuNP@Cit involve incorporation of the protein in AuNP shell and formation of a submonolayer adsorption film with voids enabling gated surface plasmon resonance energy transfer (SPRET). A model of the gated-RET system based on the protein size, estimated using Fisher–Polikarpov–Craievich density approximation, has been developed and can be utilized for other proteins, with minimum data requirement, as well. The value of the equilibrium constant  Significant progress, achieved in the last two decades, in understanding processes that enable cancer to grow and metastasize freely, while evading the immune response of the host, has largely been associated with exploring pathways controlling the apoptosis  and the immune checkpoints . Still, many other proteins may influence the expression of cancer controlling genes and they may also need to be regulated to win the battle with cancer. The use of nanomaterials in cancer research, diagnostics, and therapy is rapidly growing and spans from the targeted drug delivery using nanocarriers ,3,4, to The programmed death-ligand 1  is a transmembrane protein of 290 amino acids, consisting of immunoglobulin V- and C-like domains, a hydrophobic transmembrane domain, and a cytoplasmic tail of 30 amino acids. The elevated expression of PD-L1 is observed on the surface of tumor cells and associated with worse prognosis for many tumors ,22,23,24The structure of human PD-L1 (hPD-L1) was determined by Garboczi’s group  and ChenIn our previous paper, we have demonstrated that the resonance energy transfer (RET) from a fluorescein isothiocyanate (FITC) fluorescent dye to citrate-capped gold nanoparticles (AuNP@Cit) can be modulated by a gating sub-monolayer film of cytochrome c (Cyt c) , bovine KL for the Langmuir adsorption isotherm and the Gibbs free energy ΔG°ads for PD-L1 binding to a citrate-capped gold nanoparticle surface. Furthermore, we have been able to utilize the principle of gating of a plasmon-enhanced RET for the detection of PD-L1 at low concentrations. A generalized model of the gated-RET for any protein that binds to a functionalized AuNP has been developed based on the average protein density calculated using Fisher–Polikarpov–Craievich equation  and d is the average density [g/cm3]. Thus, for M = 52 kDa, one obtains d = 1.4136 g/cm3. Assuming a spherical shape of PD-L1, one obtains for 5 nm dia. AuNPs, the maximum monolayer surface coverage γmax of PD-L1 on AuNPs as 8.9 pmol/cm2. It corresponds to 9.3 molecules of the protein per single AuNP@Cit at close packing density. To estimate the protein surface coverage for this model, for any protein, we can assume a spherical shape for the protein molecules and calculate their maximum surface coverage based on the protein molar mass. The average protein density, needed for these calculations, can be determined using Fisher–Polikarpov–Craievich equation :d = 1.41The thickness of the PD-L1 layer was estimated from the volumetric density of the protein and its molecular mass, assuming the spherical shape and a compacted cube shape, as follows.M is the molar mass of a protein and d is the protein density.The molar volume of a protein is given by:N0 is the Avogadro number (N0 = 6.023 × 1023 mol−1). Assuming spherical shape of the protein molecule, we can estimate the diameter 2r of the protein and thus, the monolayer film thickness h:Thus, the volume of a single molecule of a protein can be estimated using the equation:a:Similarly, we can estimate the film thickness for protein molecules compacted to cubes with sides r and a, is:The ratio of the two values, 2h for any protein shape from an ideal sphere to a compacted cube by taking the mean value between 2r and a:Emax in the estimated height h of the film will be less than ±10.7%:Hence, if we characterize the film thickness M = 52 kDa and d = 1.4136 g/cm3, one obtains: 2r = 4.89 nm, a = 3.94 nm, and h = 4.41 ± 0.48 nm. A slight modification of this number could be due to the non-regular protein shape, orientation on the surface, and denaturation (if it would occur). For KL as follows:θ is the fraction of the AuNP@Cit surface covered by PD-L1 and CPD-L1 is the concentration of PD-L1 in solution. For such a gated-RET system, the fluorescence intensity of FITC modulated by plasmonic quenching by AuNP and shielding of energy transfer by the adsorbed PD-L1, according to our recent studies [Another model parameter for gated-RET system is the equilibrium constant for the adsorption of PD-L1 on AuNP@Cit. Assuming Langmuirian adsorption with no self-interactions between the adsorbed PD-L1 molecules, we can express the equilibrium constant  studies , is giveI0 in the absence of PD-L1 and the saturated intensity Isat observed for a full monolayer coverage of PD-L1. It is clearly seen from this equation that KL is the key parameter controlling the RET behaviour of the system. To determine the value of the Langmuir constant KL, we note that for:This equation describes the modulation of fluorescence intensity of a FITC probe between the intensity Equation (4) reduces to:KL can be determined graphically from the graph in C1/2 for the fluorescence intensity equal to I1/2. The value of KL is given by:Therefore, C1/2 = 7.9 × 10−9 M and the value of KL is 1.27 × 108 M−1. Based on the value of KL, the Gibbs free energy ΔG° for PD-L1 binding to AuNP@Cit can then be calculated using the formula: R is the gas constant (R = 8.3144 J/(mol K)) and T is the absolute temperature. For T = 298.1 K, the Gibbs free energy of PD-L1 binding to AuNP@Cit is −46.26 kJ/mol . This value of ΔG° indicates on a strong adsorption with supramolecular binding.Hence, from the graph in IPD-L1 = 6.76. The FITC molecules in this solution are deprotonated (pKFITC = 6.4) and most of the citrate ions are also deprotonated as well  [The surface of PD-L1 protein is predominantly negatively charged at pH = 7.4 of the measurement buffer since the solution pH > p = 6.39) . Therefo = 6.39) , which iDue to the small size of the citrate ligands and their surface mobility in AuNP’s shell, the adsorption of PD-L1 on the surface of AuNP@Cit nanoparticles is random. Since PD-L1 protein contains six cysteine residues in its structure, some of them exposed on its surface, a direct local covalent binding to a gold nanoparticle is also possible after replacement of citrate ions from AuNP surface. However, due to the large PD-L1 size and complex nature of the protein surface, only some of the citrates can be displaced by these cysteine residues. E of gRET is evaluated from the equation:IFL,0 and IFL are the fluorescence intensities for a FITC probe in the absence of AuNP quenchers and in the presence of AuNP@Cit/PD-L1sub-mono quenchers with different sub-monolayer films of PD-L1, respectively.The quenching efficiency of gated resonance energy transfer (gRET) for different concentrations of PD-L1 protein is shown in E = 0.55. The protein forming an external shell around AuNP partially blocks the energy transfer process and creates the gates (voids) for controllable FITC access to AuNP surface. At the maximum PD-L1 surface coverage by PD-L1, the gates are minimized, resulting in the residual RET with E = 0.31 (E greater than 0 is due to the remaining voids between PD-L1 molecules at saturated coverage on AuNP and direct quenching of the probe through the protein if any. This value would be lowered virtually to zero if a protein would undergo a denaturation while binding to a plasmonic host. The non-zero residual RET efficiency at surface saturation with PD-L1 indicates that no denaturation of the protein occurs. The linear part of the graph of E vs. CPD-L1 for low analyte concentration has been expanded in σ-method.The FITC quenching efficiency calculated for AuNP@Cit solution in the absence of PD-L1 is: E = 0.31 A. Note tKL = 1.27 × 108 M−1. A model mechanism of the gated-RET controlled by PD-L1 has been developed. This work demonstrates the interactions of immune checkpoint protein PD-L1 with AuNP@Cit and the high sensitivity of gRET transduction, thus developing the base model for future applications of gRET biosensors with biorecognition capabilities for the detection of proteins in complex real samples in studies of immune checkpoint cancer therapy.The experiments performed in this work indicate that the immune checkpoint protein ligand PD-L1, in the form of a sub-monolayer adsorption film on a functionalized AuNP, is able to control the gated-RET between a fluorophore probe molecule and a plasmonic core of AuNP@Cit. We have demonstrated a substantial de-quenching of the FITC probe fluorescence by partial coating of AuNP@Cit with PD-L1. The strong adsorption of PD-L1 on citrate-capped AuNPs occurs despite of electrostatic repulsions, as both the PD-L1 and AuNP@Cit are negatively charged at neutral pH. Therefore, the dominant binding forces must be due to thiolate binding and supramolecular interactions. In fact, several hydrogen bonds may be formed between high-oxygen content citrate shell on AuNPs and numerous functional groups on the protein surface. Also, the counterions in electrolyte ensure effective screening of excessive negative charges and cations provide bridging options to negatively charged surface functional groups. The strong supramolecular binding of PD-L1 to AuNP@Cit is corroborated by the high value of the equilibrium constant characterizing the Langmuir adsorption isotherm for PD-L1 on AuNP@Cit:


Blumeria graminis f. sp. hordei. However, the mechanism is not well understood. In this study, the morphological and cytochemical analyses revealed that substances containing proteins leaked from the tip of the appressorial germ tube of conidia without the formation of appressorium under a high humidity condition. In addition, exposure to high humidity prior to the formation of appressorium caused the aberrant formation of the appressorial germ tube without appressorium formation, resulting in failure to penetrate the host cell. These findings suggest that the formation and maturation of the appressorium requires a low humidity condition, and will be clues to improve the disease management by humidity control.High humidity decreases the penetration rate of barley powdery mildew  Blumeria graminis f. sp. hordei (Bgh). An obligate biotrophic pathogen Bgh can make a success of the asexual life cycle only on the living host cells, resulting in the spread of airborne conidia through the formation of conidiophores. After the arrival to the leaf epidermis, the primary germ tube (PGT) emerges within 2 h. Although PGT does not penetrate the host cell wall, it is hypothesized that PGT has roles not only in the transduction of host-derived signals to initiate the coming morphogenesis and the suppression of host resistance, but also in the absorption of water from host cells under dry condition [Bgh forms the second lobe from the lateral part of AGT as another attempt to penetrate a neighbor cell. Successfully penetrating Bgh forms a haustorium surrounded by a host-derived extrahaustorial membrane and establishes infection. Subsequently, the fungus develops secondary hyphae by absorbing nutrients with the haustorium and forms conidiophores to produce new conidia. Because the entire life cycle is accomplished on the surface of host leaves, it is assumed that the infection processes are largely affected by conditions surrounding the leaves  [Bgh. For example, as with other fungi, high humidity is required for the sporulation and the germination of conidia [Bgh more likely to fail the normal development of the germ tubes [Bgh.Powdery mildew is a major fungal disease of barley, caused by ondition ,2,3. A sondition . In casendition) ,6. Of pa conidia ,8. Howevrm tubes ,10,11. BBgh were exposed to the high humidity condition (more than 95% relative humidity (RH)), the infection was considerably suppressed  or more than 95% RH (high humidity) after inoculation with Bgh conidia. The penetration rates were measured 24 h after inoculation. As a result, the penetration rate was significantly reduced under the high humidity condition  staining was carried out after incubation under the high humidity condition. The barley coleoptiles were inoculated with Bgh conidia and kept at more than 95% RH for 4 h. The inoculated coleoptiles were transferred to the incubator kept at about 70% RH and then stained with CBB solution 20 h later. As a result, the leaked substances were observed at the tip of AGT and stained blue  were grown in vermiculite supplemented with 300-fold diluted HYPONeX  in growth chambers  at 20 °C under continuous light condition (ca. 23.6 Wm−2). Powdery mildew fungus Bgh RACE1 isolate was cultured on the barley leaves at 70% relative humidity (RH) in culture chambers  as described by Wahara et al. [Seedlings of barley .Conidia were collected from barley leaves around 7 days after inoculation and were inoculated onto coleoptiles or cellulose membranes. The coleoptiles were inoculated with fresh conidia with a brush and were floated on 1 mM CaCleviously . In vitrescribed  with sliBgh on the coleoptiles or the cellulose membranes were soaked in CBB solution  for 5 min, rinsed with 30% ethanol and gently washed with distilled water three times. Leaf segments inoculated with Bgh were stained with trypan blue solution 4 d after inoculation as described by Yaeno et al. [o et al. . To quanBgh were fixed and dehydrated with a series of ethanol 75%, 80%, 95%, twice 100%, isoamyl acetate–ethanol (1:1) for 30 min and twice isoamyl acetate for 1 h. Samples were dried with a critical point dryer , coated with platinum, and observed by a scanning electron microscope  with a secondary electron detector at an accelerating voltage of 5 kV. To observe the penetration pore, Bgh conidia with the appressorium or the second lobe were shifted with a micromanipulator  before fixation.Coleoptiles inoculated with The cellulose dialysis membranes on agar plates were inoculated with conidia and were transferred from 70% RH to more than 95% RH. The germinated conidia were photographed 18 h after inoculation and the conidia with the leakage at the tip of AGT were counted in randomly selected photographs. The length of AGT was measured using software .


Based upon the approximate Crank–Nicolson (CN) finite-difference time-domain method implementation, the unconditionally stable algorithm is proposed to investigate the wave propagation and transmission through extremely thin graphene layers. More precisely, by incorporating the CN Douglas–Gunn algorithm, the piecewise linear recursive convolution method and the auxiliary differential equation method, the analytical model is proposed for Drude-like graphene model. To obtain the solution of the governing equations, the perfectly matched layer and the periodic boundary condition are applied to the graphene structure with two dimensional nano-materials. Numerical examples are carried out for further investigation. During the simulation, the influences of the parameters such as the grating slit and its thickness on the wave transmission are investigated and discussed. The result shows that not only the graphene grating has high transmission performance but also the proposed methods have considerable performance and accuracy. Graphene has attracted much attention during the last decades due to its very interesting properties, such as mechanical, physical, electrical and optical properties4. For the remarkable physical property, GSs are being studied in the field of communication and optoelectronic nanodevices6. Graphene is an ideal plasmonic material to explore tunable devices, including the tunable nanoantennas and the tunable reflectors in the range of terahertz and mid-infrared frequencies7. In the recent year, the absorption enhancement of graphene in near-infrared light has attracted considerable interest due to its unique electronic and optical properties9. Graphene is a kind of lossy and multi-scale material, and thus it is difficult to accurately simulate the wave propagation characteristics of GSs10.Graphene sheet (GS), a two dimensional one-atomic-thick monolayer of graphite, is the strongest ever measured and the thinnest known material11. Shen et. al. studied the wave propagation in thin GSs by using the FDTD method12. Compared with a theoretical solution, they verified the proposed FDTD method through two numerical examples, and proved its accuracy and efficiency. Liang et. al. investigated the extraordinary optical transmission of periodic metallic gratings based on the locally one-dimensional FDTD13. In14, the analysis of electromagnetic phenomena in graphene structures was carried out by an efficient FDTD formulation. Numerical verification, involving comparison with closed-form solutions and the results of other schemes, exhibited the high accuracy of the algorithm.The finite-difference time-domain (FDTD) method can be used as an effective numerical analysis technique to model the wave propagation in GSs because of its direct iterative solution in the time domain15. However, when calculating the large amount of time steps problems, the simulation duration will be unacceptable resulting in the limitation application of the FDTD algorithm. In order to improve the computational efficiency and maintain the accuracy, the unconditionally stable algorithms are proposed. Among existed algorithms, the Crank–Nicolson (CN) algorithm with higher computational accuracy is firstly proposed in one dimensional cases16. By applying the original CN algorithm directly to 2D problems, large sparse matrices must be calculated at each step resulting in the computational more expensive. To avoid the calculation of the sparse matrices, approximate CN algorithms are introduced including the CN approximate-decoupling (CNAD) and CN Douglas–Gunn (CNDG) algorithms18. It has been testified that the CNDG algorithm shows higher accuracy compared with CNAD algorithm19. In addition, from the previously works, CNDG algorithm has been extensively employed merely in TM cases21. This limits the development of the CNDG algorithm.However, analytical models and mesh sizes are key for obtaining correct results in numerical analysis technique. In order to ensure the stability of the numerical scheme, an adequate time step must be taken to assure the Courant-Friedrichs-Lewy (CFL) condition. The CFL limit is determined by the smallest cell size in the space domain22. Among modified PML schemes, unsplit-field SC-PML is developed to simplify the corners and edges of the PML regions23. The CFS-PML with advantages in attenuating low-frequency propagation waves and reducing late-time reflections is carried out24. It has been testified that the low-frequency propagation waves can not be absorbed efficiently by employing both SC-PML and CFS-PML. Thus, the higher order PML is proposed not only to alleviate such problem but also further enhanced the absorbing performance27.In order to simulate the infinite computational domain in the limited space, an efficient absorbing boundary condition is necessary to truncate the computational domain, such as the perfectly matched layer (PML)15. Among existed CN-PML algorithms, the employment of PBC have not been investigated21. Due to the existence of PBC, the Thomas algorithms can not be employed directly to the unconditionally stable algorithm. Thus, an alternative method should be investigated.Moreover, the periodic boundary condition (PBC) is also applied due to the existence of the periodic structureHere, based on the CNDG algorithm, higher order PML and Drude model, unconditionally stable implementation is proposed for evaluating the wave propagation characteristics through GSs in TE case. Combining with the piecewise linear recursive convolution (PLRC) and the auxiliary differential equation (ADE) methods, the extraordinary optical transmission through extremely thin periodic graphene can be simulated and calculated. Two numerical examples with different situations in one period are discussed. The results reveal the characteristics of extremely thin graphene and efficiency of the proposed algorithm.22. According to the Drude model, the relative dielectric function n is the density of electrons, e is the electron charge, and 24. The source-free and normalized frequency-domain modified Maxwell’s curl equations in two dimensional Drude media can be written asThe Drude dispersion model can be applied to GSsIntroducing the auxiliary variables, they can be given as the following formsBy substituting (11)-(14) into (15)-(17), the equations can be rewritten as26Based on the CN scheme and PLRC methods, we obtain the FDTD difference equations as followsAnd the operator The updated equation of auxiliary field obtained by PLRC method can be calculated asBy substituting (21) and (22) into (23), the updated equation of magnetic component in the y-direction can be given asTo obtain the solution of Maxwell’s equations for the periodic structure, the PBC is introduced. Figure For the top boundary condition, we obtainThus, by employing the PBC, the matrices in (29)-(30) can be given as27.It can be observed that the matrices are no longer tri-diagonal matrices which can not be solve by employing the Thomas algorithm. Thus, an alternative method, the RCM method is employed to decrease the dimension of the matrices and improve the computational efficiency,28. The observation point is located at the left bottom corner of the domain to observe the waveform.Before the investigation of GSs, the effectiveness and efficiency of the proposed CNDG-HO-PML algorithm is validated through a full-filled domain with graphene with the parameters of entclass1pt{minimaTo ensure the accuracy of the calculation and validate the effectiveness of the algorithm, cell per wavelength (CPW) is chosen as 200. The CFL number (CFLN) is defined as The waveform observed at the observation point obtained by different CFLNs are shown in Fig. The accuracy of the calculation also affects by the absorbing performance of the PML regions. The absorbing performance of the PML regions can be reflected by the relative reflection error in the time domain which can be defined as29. The computational efficiency, memory consumption and time reduction are shown in Table Figure The wave transmission through a graphene sheet is studied with regard to transverse electric The sketch picture of the computational domain for investigation is shown in Fig. It should be noticed that the mesh size and the time step is no longer limited by the CFL condition. Thus, the physical size can be employed during the simulation. To ensure the accuracy of calculation, the thickness of single-layer GS is divided into 10 cells as Firstly, the wave transmission through GSs can be investigated through the maximum value of the waveform obtained from the observation point. Figure In addition, it can be observed that the normalized transmitted energy decreases with the increment of the thickness. This indicates that the thicker sheet transmits less energy. This phenomenon can be reflected by the normalized reflection energy, shown in Fig. 30. As defined in the microwave theory, the transmission can be reflected by S21. Thus, the port 1 is defined at the left side of sheet with the incidence plane wave. Port 2 is defined at the right side of the sheet. In this simulation, the S21 is obtained by CNDG-HO-PML when CFLN = 20, as shown in Fig. 21 decreases with the increment of the thickness. At the frequency of 6.2 PHz, the graphene sheets show the least S parameters. At the low-frequency, the graphene sheets have higher S parameters indicating the less loss during the wave transmission. Figure 21 parameters obtained by different thickness. It can be observed that the S21 becomes lower in small wavelength.To demonstrate the wave transmission in the frequency domain, the S parameters in the microwave theory is introduced during the simulationTo investigate the extraordinary optical transmission phenomenon through the graphene sheet, a periodic graphene structure with slit is proposed. Figure Compared with the graphene sheet without slit in the previously numerical example, due to the existence of slit, the interference of wave transmission results in the generation of the crest and trough in the observation plane. Thus, it should be noticed that the energy from each mesh grid in the observation plane should be employed during the calculation. Figures 21 parameters can be employed in evaluating the performance and investigating the properties of graphene. Figures 21 parameters versus frequency and wavelength with different slit width under 8-layer-sheet, respectively. It can be observed that the S parameters increases obviously with the increment of slit width. This indicates that the wave is much easier to transmit with the increment of slit width before the 6 PHz frequency.Following the same step as the numerical example above, the SAs is demonstrate in the previously that the wave is no longer uniform at the observation plane. The reason is that the interference of electric current occurs when wave transmitting through the slit. The crest and the trough are formed at the observation point. Thus, it is important to investigate the crest and the trough of the waveform.34.In this simulation, the parameter 21 parameters.Figures Based on the CNDG algorithm, the Drude-like model and higher order concept, an unconditionally stable CNDG-HO-PML algorithm is presented to investigate the wave propagation through graphene gratings. The Maxwell’s equations are applied to the model to study the transmission characteristics. The ADE- and PLRC-FDTD methods with faster calculation and less memory is used to solve the partial differential equations for time stepping of the electromagnetic fields. The effectiveness and efficiency are testified through a full-filled structure, a single sheet and the sheet with slit. Based on numerical analysis, the obtained result shows that the characteristics of graphene with high transmission performance. The grating graphene layer structures can be used in the field of optoelectronic detection technology and systems.