The fracture properties of pre-cracked monocrystalline/polycrystalline graphene were investigated via a finite element method based on molecular structure mechanics, and the mode I critical stress intensity factor (SIF) was calculated by the Griffith criterion in classical fracture mechanics. For monocrystalline graphene, the size effects of mode I fracture toughness and the influence of crack width on the mode I fracture toughness were investigated. Moreover, it was found that the ratio of crack length to graphene width has a significant influence on the mode I fracture toughness. For polycrystalline graphene, the strain energy per unit area at different positions was calculated, and the initial fracture site (near grain boundary) was deduced from the variation tendency of the strain energy per unit area. In addition, the effects of misorientation angle of the grain boundary (GB) and the distance between the crack tip and GB on mode I fracture toughness were also analyzed. It was found that the mode I fracture toughness increases with increasing misorientation angle. As the distance between the crack tip and GB increases, the mode I fracture toughness first decreases and then tends to stabilize. Pristine graphene, a two-dimensional material, consists of carbon atoms and possesses excellent electronic, thermal, and mechanical properties ; it has The fracture properties of monocrystalline/polycrystalline graphene have been investigated by experimental and theoretical methods. The GBs (repeating pentagon–heptagon ring) of graphene were observed via transmission electron microscopy ,11, and The fracture toughness is very important for the stability of materials. For practical engineering, the available strength of graphene relies not on the failure strength which governs the fracture of the carbon bonds, but on the fracture toughness . Hence, In this paper, the molecular structure mechanics  and the The interatomic potential energy of graphene can be described through the steric potential energy :(1)U=∑Url is the length of the beam element; A is the area of the cross section; I and J represent the moments of inertia and polar moment of inertia, respectively; and E and G are the elastic modulus and shear modulus, respectively. However, in this method, the different parameters can be derived by different force constants, and the in-plane flexural rigidity of the beam element is equal to the out-of-plane flexural rigidity.Based on molecular structure mechanics , the equb and h are the width and height of the beam element, respectively; In order to eliminate the above problems, the sectional properties of the beam element are derived from modified molecular structural mechanics , so the Further, the nonlinear behavior of materials is very important for the result of simulations. In this research, the nonlinear mechanics behavior of graphene is described by the modified Morse potential ,28: (8)UThe variations in force with increasing strain of the C–C bond are exhibited in on point . The equstrainU is the total strain energy, and m and n are the width and length of the graphene nanoribbon, respectively.From the perspective of the strain energy per unit area, the initial fracture site in polycrystalline graphene is now explored. The following equation is proposed for calculating the strain energy per unit area:D is the correction factor, 2a is the crack length, W is the width of the graphene, and f is the proportion of the crack.The mode I critical stress intensity factor is calculated by the Griffith criterion in classical fracture mechanics ,30,31: were created to study the influence of GB on the mode I fracture toughness as shown in oughness . In this2, respectively. The results of this paper are in good agreement with those in the literature [In this section, the initial fracture site of graphene is explored. The strain energy per unit area of pristine zigzag graphene was obtained via Equation (12). terature .The strain energy per unit area of different graphene nanoribbons was investigated through the model in How is the fracture toughness affected by the GB? To answer this question, the influence of the misorientation angle and the distance between the crack tip and GB on the mode I fracture toughness will be discussed in this paper. It is well known that graphene is a nanoscale material. Its fracture process is different from that of macroscopic material. The fracture toughness of graphene could be affected by many factors such as the crack width, the crack proportion, etc. Thus, the influence of crack width and crack proportion on the mode I fracture toughness and the size effects of mode I fracture toughness were investigated in pre-cracked monocrystalline graphene. Further, the mode I fracture toughness can be expressed by the mode I critical stress intensity factor, and the parameters of the polycrystalline graphene model can be determined through the analysis of the mode I critical stress intensity factor in monocrystalline graphene.f = 0.3. It can be seen from this figure that the mode I critical SIFs of the two different graphene models have similar variation tendencies. When the graphene length is smaller than the width, the mode I critical SIF decreases sharply as the graphene length increases. However, the mode I critical SIF hardly changes with increasing graphene length when the graphene length is larger than the width. Moreover, it is interesting that the turning point in the variation trend of mode I critical SIF can be obtained when the graphene length is equal to the width. Thus, it can be inferred that there is a size effect of mode I fracture toughness, and the graphene length has little influence on the mode I critical SIF when the graphene length is larger than the width.The size-dependent mode I critical stress intensity factor (SIF) was studied using the model in f on the mode I critical SIF, as shown in 2 and those of the square models are 400 nm2 and 900 nm2. The variation in the mode I critical SIF with increasing crack proportion f is shown in f increases, the mode I critical SIFs of the three graphene models first increase and then tend to stabilize. The sizes of the three mode I critical SIFs are basically the same. In addition, the curvilinear variation tendencies show that the mode I critical SIF of graphene almost becomes a constant value when the crack proportion f is between 0.2 and 0.6. The range of the SIF then is 3.4–3.5 MPam1/2. These results are consistent with those in the literature [f has a reasonable range (f = 0.2–0.6).Two types of graphene models were created to study the effect of crack proportion terature ,35. Fromf was a constant value. L = 30 nm and different crack proportions f. It can be seen that as the graphene width increases, two types of mode I critical SIF are almost a constant value. For a graphene model with a fixed width, the changes in the mode I critical SIFs can be neglected. The SIF in The effect of graphene width on the mode I critical SIF is also discussed in the graphene model (as shown in 2), as shown in f = 0.3 has a more reasonable range when the crack width is B ≥ 1.6 nm.In the process of graphene fracture, there is a crack-tip blunting effect. It is difficult to observe the crack-tip blunting effect in experiments, and numerical simulations are thus one of the best methods to investigate the crack-tip blunting effect of graphene. From previous numerical simulations ,24, it i2, 10 nm, and 1.6 nm, respectively, as shown in Based on all the above analysis, the other influence factors can be eliminated when the effect of GB on the mode I fracture toughness is investigated. Therefore, in this research, the size of polycrystalline graphene model and the crack length and width are 30 × 30 nmThe model in In this paper, the mode I fracture toughness of pre-cracked monocrystalline/polycrystalline graphene was investigated via molecular structure mechanics and the Griffith criterion. The initial fracture site (near grain boundary) of polycrystalline graphene was revealed by the strain energy per unit area.For monocrystalline graphene, it was found that the crack proportion and crack width have a significant influence on the mode I fracture toughness. The mode I fracture toughness has size effects. The mode I critical stress intensity factor (SIF) of graphene decreases sharply with increasing graphene length when the graphene length is less than the width (the loaded edge). However, the mode I critical SIF almost becomes a constant value when the graphene length is larger than the width. Moreover, the mode I critical SIF of graphene hardly changes when the crack proportion is between 0.2 and 0.6.For polycrystalline graphene, the initial fracture site was discussed in terms of the strain energy per unit area. It was inferred that the initial fracture site appears in the vicinity of the GB. In addition, the conclusion was obtained that the GB has an influence on the mode I fracture toughness, but the affected region is limited. The mode I fracture toughness of pre-cracked polycrystalline graphene is less than that of pre-cracked pristine graphene due to the existence of the GB. The mode I critical SIF of pre-cracked polycrystalline graphene increases with increasing misorientation angle. However, as the distance between the crack tip and GB increases, the mode I critical SIF first decreases and then gradually stabilizes.


Giardia lamblia provides an excellent opportunity to explore this question.It is widely accepted that the last eukaryotic common ancestor and early eukaryotes were intron-rich and intron loss dominated subsequent evolution, thus the presence of only very few introns in some modern eukaryotes must be the consequence of massive loss. But it is striking that few eukaryotes were found to have completely lost introns. Despite extensive research, the causes of massive intron losses remain elusive. Actually the reverse question -- how the few introns can be retained under the evolutionary selection pressure of intron loss -- is equally significant but was rarely studied, except that it was conjectured that the essential functions of some introns prevent their loss. The situation that extremely few (eight) spliceosome-mediated cis-spliced introns present in the relatively simple genome of Giardia. Without finding any special features or functional importance of these introns responsible for their retention, we noticed and experimentally verified that some intron-containing genes form sense-antisense gene pairs with transcribable genes on their complementary strands, and that the introns just reside in the overlapping regions.Our investigation found three types of distribution patterns of the few introns in the intron-containing genes: ancient intron in ancient gene, later-evolved intron in ancient gene, and later-evolved intron in later-evolved gene, which can reflect to some extent the dynamic evolution of introns in Giardia’s evolution, despite constant evolutionary selection pressure of intron loss, intron gain can still occur in both ancient and later-evolved genes, but only a few introns are retained; at least the evolutionary retention of some of the introns might not be due to the functional constraint of the introns themselves but the causes outside of introns, such as the constraints imposed by other genomic functional elements overlapping with the introns. These findings can not only provide some clues to find new genomic functional elements -- in the areas overlapping with introns, but suggest that “functional constraint” of introns may not be necessarily directly associated with intron loss and gain, and that the real functions are probably still outside of our current knowledge.In This article was reviewed by Mikhail Gelfand, Michael Gray, and Igor Rogozin.The online version of this article (10.1186/s13062-018-0226-5) contains supplementary material, which is available to authorized users. Encephalitozoon species  ferredoxin (GL50803–27266) intron, has a noncanonical splice signal CT-AG [To find the reason why these few introns can be retained in al CT-AG . Most ofal CT-AG , 23.Interestingly, on the complementary strands, we found that two intron-containing genes, GL50803–17244  and GL50803–37070 , each have an antisense gene, GL50803–20429 and GL50803–28204, which are just annotated as “hypothetical protein” and “unspecified product” in the genome database, respectively. That is, the two intron-containing genes and their antisense gene form SAS gene pairs. We thought this phenomenon might be related to the intron retention. Nevertheless, the two anti-sense genes need to be further verified, and the details of the overlaps with their sense genes also need to be analyzed in detail, both of which are carried out at below.The strand-specific RT-PCR of the two antisense genes, GL50803–28204 and GL50803–20429, generated two products with the expected lengths of 172 and 288 bp, respectively. The sequencing further confirmed that the two products just seem to be transcribed from the opposite direction of the two sense (intron-containing) genes, GL50803–37070 and GL50803–17244, respectively, and the two introns are just located within the two overlapping regions of the two SAS gene pairs, respectively  Polymerase to add a poly(A) tail at the 3′ends before we performed rapid amplification of their cDNA 3’ends, thus from the experiment we still do not know whether the transcripts of GL50803–28204 are polyadenylated or not, namely, it is unclear whether the transcripts of GL50803–28204 are mRNA or not. Therefore, we can only conjecture that GL50803–28204 is either a non-protein-coding or Giardia-specific protein-coding gene. Considering that many antisense genes overlapped with protein-coding genes are non-coding RNA genes in Giardia [Interestingly, we noticed that on the complementary strands of two of the eight intron-containing genes, GL50803–37070 and GL50803–17244, there exist correspondingly two anti-sense genes, GL50803–28204 and GL50803–20429. By strand-specific RT-PCR, RACE and sequencing, we got the transcripts and sequences of the two genes, and found they both have no introns. Thus the two anti-sense genes have been verified to be really genes that are actively transcribed. And actually the anti-sense gene GL50803–20429 has been reported to be a mRNA gene being expressed during excystation and encystation, and in trophozoites but not cysts [ Giardia , the antGiardia isolates with genome sequenced, and found that the two anti-sense genes (exactly corresponding to GL50803–28204 and GL50803–20429 in isolate WB) also exist in isolate DH, but not in the other three isolates. However, interestingly, other ORFs (not exactly corresponding to GL50803–28204) can be found on the complementary strands to overlap with the intron corresponding to the intron in the gene GL50803–37070 in P15, GS, GS-B . But considering that some genes overlap with the UTRs of the adjacent genes on the opposite strand  are transcribed, no transcripts were found  and thaTheoretically, overlapping with any genomic functional elements on either the same strand or the complementary one  can result in intron retention, as long as the introns are just in the overlapping areas. Therefore, since such overlapping structures are widely distributed in eukaryotes , it can Giardia, we have revealed some interesting findings about the dynamic evolution of introns in the intron-poor eukaryotes: the evolutionary selection pressure of intron loss may constantly exist in these eukaryotes, but new introns can still arise either in ancient genes or later-evolved genes, but only a few introns can be retained in the genome; the retention of the few introns may not be necessarily caused by functional constraint of the introns themselves but due to the reasons outside of the introns, and “overlap constraint” imposed by other genomic functional elements overlapping with the introns is at least one of such causes. Our finding may conversely provide a clue to find new genomic functional elements  in such kinds of overlap regions. Most importantly, our work implicates that “functional constraint” of introns is not necessarily directly associated with intron loss and gain, and even that the real functions or the way of functioning of introns are probably still outside of our current knowledge. Therefore this work may be able to shed some new lights on the research of evolution and function of introns and genomes.In conclusion, by investigating the extremely intron-poor eukaryote Giardia genes were downloaded from GiardiaDB (1 december 2017 release) [Giardia’s corresponding sequences as queries. Protein alignments were generated with ClustalX 2 applying default alignment parameters. The introns in the genes were determined by comparing cDNA and gene sequences. The other four intron-containing genes with annotated as hypothetical protein also identified by sequence comparative analysis to determine whether they are Giardia-specific or not.The template sequences for designing primers of release) . The sofThe cell line of WB isolate (assemblage A), namely WB clone C6 (ATCC 50803), was used in the study. Its cultures were routinely grown in filter-sterilized TYI-S-33 medium supplemented with bovine bile in glass screw cap tubes at 37 °C and were sub-cultured every 3 to 4 days.Giardia total RNA was extracted and treated to remove any contaminated genomic DNA by RNAprep Pure Cell/Bacteria Kit (TIANGEN) using about 5 × 106Giardia trophozoites that were harvested by ice-slush incubation and centrifuged at 6000 g for 5 min according to the manufacturer’s instructions. The quality and quantity of the RNA preparation were assessed with agarose gel electrophoresis and the absorption at 260 and 280 nm.E. coli. Colony PCR with vector-specific primers provided in the kit was adopted to select colonies. These selected colonies were sequenced using vector-specific forward and reverse primers by Sangon Biology Company .First-strand cDNAs of the two antisense genes, GL50803–28204 and GL50803–20429, were synthesized from 500 ng DNase-I treated total RNA per reaction at 54 °C 30 min, 99 °C 5 min, and 5 °C 5 min with the specific antisense primer 4C and 9C respectively instead of the reverse primers in the kit, and then amplified with specific primers pairs of 4A/4S and 9A/9S by using RNA PCR Kit (AMV) Ver.3.0. The PCR conditions were as follows: 94 °C for 30 s, followed by 30 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 45 s. The PCR products were purified using the Wizard SV Gel and PCR Clean-Up System kit , and cloned into pMD-19 T vectors from 35 ng purified PCR products using TaKaRa pMD-19 T Vector Cloning Kit  according to the manufacturer’s instructions. Then, the ligation products were transformed into DH5α Chemically Competent The total RNA were processed using Poly(A) Polymerase to add a poly(A) tail at the 3′ends of the RNA before performing rapid amplification of their cDNA 3’ends. We experimentally determined the 3′ends by using nested PCR primer (3R4O/3R4I and 3R9O/3R9I) according to the RNA PCR Kit (AMV) Ver.3.0 . 5’-RACE was performed by using a SMARTer RACE 5’/3’Kit  with 500 ng total RNA as the template and the gene-specific 5’-RACE primers 5R4 and 5R9 for the two antisense genes, GL50803–28204 and GL50803–20429, according to the manufacturer’s instructions. Both the 3’-RACE and 5’-RACE primers Additional file 2:Giardia isolates. (DOCX 51 kb)The Analysis results of the two SAS gene pairs in Additional file 3:G. lamblia. (DOC 517 kb)Results of strand-specific RT-PCR of the complementary areas of the other six introns of Additional file 4:G. lamblia. (DOC 79 kb)The primers designed for the strand-specific RT-PCR and RACE of the complementary areas of the eight introns of Additional file 5:Reviewers’ comments. (DOCX 20 kb)


To fabricate a tunable optical filter with a fast response in the near infrared region, a tunable guided-mode resonance (GMR) filter using graphene was proposed and its performance was optimized. In this study, a rigorous coupled wave analysis method was employed to systematically investigate the effects of geometrical configuration of graphene-integrated GMR filters and the optical properties of constituent materials including graphene on their spectral response in terms of tunability and extinction ratio. It was found that as the graphene is located close to the waveguide and the evanescent-field strength at the interface increases, the GMR filter exhibits better tunability. The bandwidth of the filter could be drastically reduced by adopting a low-index contrast grating layer, so that the extinction ratio of an optical signal could be greatly improved from 0.91 dB to 27.99 dB as the index contrast decreased from 0.99 to 0.47, respectively. Furthermore, new practical device designs, that is easy to fabricate and effectively implement the electric-field doping of graphene at low gate voltage, were also suggested and theoretically validated. These results demonstrate not only the excellent potential of a graphene-based tunable GMR filter but also provide practical design guidelines for optimizing the device performance. In a GMR filter, only the 0th-order diffraction component of a specific wavelength that satisfies a phase-matching condition can produce a resonance band in transmission or reflection spectra. Higher-order components are coupled into the waveguide modes. The guided modes are coupled out into the radiation mode interacting with the grating again, which results in a high efficiency at resonance. The GMR filter has excellent out-of-band rejection and has a very narrow bandwidth with a high efficiency of almost 100%. In addition, the resonance wavelength highly depends on the geometrical parameters, such as the period of the diffraction grating, the duty cycle, the optical thickness of the waveguide layer, and the refractive index of a surrounding material4. Due to these characteristics, GMR filters have attracted much attention in many practical applications such as narrow band-stop filters6, optical polarizer7, and biosensors9.A guided-mode resonance (GMR) filter is an optical component that filters light through an optical interaction between a zero-order grating with a period smaller than the incident wavelength and an adjacent waveguide11, thermo-optic12 or electro-optic materials14. However, since the refractive index of these materials are not significantly modulated, operation based on the bulk response is necessary to obtain an adequate tuning range. This limits the response time or prevents cost-effective fabrication of the device. Fang et al.15, suggested a low-cost gradient-period-grating-based tunable GMR filter whose spectral resonance wavelength is tuned mechanically by changing the illumination position on the device. This approach is still far from solving the fundamental issues.Various attempts have been also made to give active tunability to GMR filters. Most of them replace bulk components of GMR filter with photo-isomerizable polymers17. Therefore, its integration into the GMR filter could produce an active device with high sensitivity and fast switching of resonance wavelength. Recently, a graphene-based active tunable GMR filter has been proposed18.Therefore, there is a need to apply a material which exhibits a large change in the refractive index and the fast response to external stimuli without relying on the bulk effect. Graphene could be a promising candidate. Graphene is a two-dimensional, planar material consisting of a single layer of carbon atoms, which has excellent electrical conductivity and low optical loss. In addition, its optical constants can be altered drastically when exposed to external stimuli such as light, electric-field, heat, and chemical doping19:The dielectric constant of graphene can be derived from the Kubo formula representing the surface conductivity given by the following Eq. 19:1\docuE is the energy, τ is the scattering time of electron related to quality of graphene (1/2τ = Γ is the electron relaxation energy), T is the temperature, μc is the chemical potential of graphene (or Fermi level shift), ω is the angular frequency, ħ is the reduced Planck’s constant, e is the electron charge, and kB is Boltzmann’s constant). It should be noted that if the values of T and τ−1 are fixed in Eq.  is the thickness of the monolayer graphene.In Eq. , ε0 is tT = 300 K and Γ = 0.66 meV. Figure ε1 and the imaginary part ε2 of the dielectric constant on the chemical potential of graphene for the wavelength λ = 1.5 μm. The ε1 has positive values at μc smaller than 0.5 eV and peaks at 0.4 eV. Then it crosses zero and turns to a negative value when μc exceeds 0.5 eV, exhibiting a dielectric-to-metal transition. Graphene reveals an absorptive characteristic in the dielectric region, as evidenced by the large ε2 related with optical loss. On the other hand, the ε2 becomes negligible in the metallic region indicating low-loss nature of graphene. Figure μc value and is gradually blue-shifted with increasing μc. When μc is larger than 0.6 eV, graphene behaves as a low-loss metal in the near-infrared communication band of 1.3 to 1.55 μm. Since the dielectric constant of graphene undergoes a tremendous change depending on the μc value, it is expected that a tunable optical filter with a fast response and large tunability can be realized by incorporating graphene as an active component of GMR filter.Figure In this study, we simulated the effects of geometrical parameters of GMR filter and optical properties of constituent materials including graphene layer on the spectral response of the tunable filter using a rigorous coupled wave analysis (RCWA) method. We systematically and theoretically compared the device performance of different configurations in terms of the tunability and extinction ratio of signal channel. On the basis of the results obtained, the key factors that determine the performance of the tunable GMR filter and the design rules for optimizing the performance were suggested.2. In a single layer GMR, the grating layer also serves as a waveguide layer. Because of the open structure of the waveguide core region, the single layer GMR structure is sensitive to a change in the bulk environment, but the degree of freedom of designing properties is limited. On the other hand, for the multilayer GMR, the waveguide and grating layers are separately provided. The multilayer GMR structure has high degree of freedom of designing, which is advantageous in optimizing the filter spectrum. For example, the filter can have very low sideband reflectance by designing the grating to be an antireflection layer. The filter linewidth can also be very easily adjusted by controlling the index contrast of grating.The GMR filter can be classified into two types, namely, a single layer GMR and a multilayer GMR depending on whether a grating and a waveguide layer are separately providedIn this study, dual-layer GMR structure, which is the simplest type of multi-layer GMR, was adopted, as shown in Fig. 2 cladding layer, a Si3N4 waveguide layer, and an upper periodic linear grating. In the initial calculation, the upper grating layer was assumed to be a monolithic grating composed of Si3N4. The number of the graphene layers was assumed to be four layers and their dielectric constant was set using Eq.  and thickness of the grating layer were determined by considering it as an effective medium satisfying the antireflection condition.The basic structure consisted of a semi-infinite lower SiOsing Eq.  for in-p21:nH is the refractive index of the diffraction grating, nL is the refractive index of the surrounding material, and d is the duty cycle. For the reference wavelength of 1.5 μm, the optimum condition for antireflection was calculated based on the Fresnel equations and found to be met when the refractive index and thickness of the effective medium were 1.374 and 0.250 μm, respectively. From this, the duty cycle of the Si3N4 grating was determined to be 0.3.According to the effective medium theory (EMT), the effective refractive index of a linear diffraction grating for the TE polarized light at normal incidence was calculated using the relation given asμc < 0.5 eV), the resonance spectrum is damped due to the optical absorption loss caused by high ε2, resulting in lower and broader reflection peak. In addition, as μc increases to 0.4 eV, the resonant wavelength is shifted towards the longer wavelength, which is believed to be ascribed to the increase in ε1 , the reflection spectrum of the GMR filter doesn’t seem to suffer from optical damping and the resonance wavelength was continuously blue-shifted with increasing μc. This is due to the fact that in this region, ε2 has a negligible value close to 0, while ε1 decreases continuously with increasing μc of graphene.Figure  ε1 Fig. . On the 3N4 waveguide layer, show much better tunability with the chemical potential. On the other hand, the tunability becomes smaller when the graphene is located away from the optical waveguide.The changes in resonance wavelength as a function of the chemical potential of graphene for four different configurations are compared and summarized in Fig. i.e. by making the waveguide leakier. In our model structure, the refractive index of the lower SiO2 cladding (neff = 1.374) and the surrounding air. Thus, the refractive index difference is the smallest at the interface between the waveguide and the lower SiO2 cladding and the evanescent field is the largest at point D in Fig. When the tunability of graphene-GMR filters is defined as the extent of resonance wavelength shift with respect to the unit change in chemical potential of graphene, the tunability of each configuration in the metallic region can be quantitatively compared, as shown in Fig. However, as shown in Fig. z axis direction, the grating located from z = 0 to 0.25 μm, and the waveguide and cladding layers sequentially placed thereon. It is observed that the E-field intensity is confined in the waveguide core representing the guided-mode and the position of maximum intensity is repeated every 0.5p. In addition, it is noted that the maximum intensity and the portion of E-field confined in the waveguide core increases with increasing its thickness. For more quantitative comparison, the distribution of E-field intensity normalized in the z-axis direction at the center (x = 0) of the GMR filter was extracted from Fig. First, the effect of waveguide thickness on the performance of tunable GMR filter was analyzed. In the D configuration, only the thicknesses of the waveguides were varied, namely, 200, 377, and 800 nm, while the other factors remain unchanged. The calculated reflection band spectra are shown in Fig. 2 instead of Si3N4. Since the refractive index of SiO2, nSiO2 = 1.47 at a wavelength of 1.5 μm, the duty cycle was estimated to be 0.765 by Eq. (neff = 1.374) as in the case of Si3N4 grating. The reflection band spectra of the GMR filter were calculated respectively for Si3N4 and SiO2 gratings in the configuration D, where all parameters were the same except for the duty cycle, as shown in Fig. 3N4 grating with the same waveguide material (Si3N4)  Fig. , use of N4) Fig. . This ca3N4 grating gives a FWHM of the reflection band of about 22 nm. Their FHWMs are remarkably reduced to 6.5 and 1.5 nm, respectively  is used. On the other hand, the extinction ratio, representing a degree of output signal change with reference to a specific resonance channel (at μc = 0.6 eV), shows a quite different characteristic (Fig. μc = 0.6 eV. When the chemical potential increases from 0.6 to 1.0 eV, the way of increasing the waveguide thickness in the GMR filter with a high-index contrast grating such as Si3N4 suffers from the lower tunability caused by reduced leaky-field at the waveguide/cladding interface where the graphene is located, consequently resulting in the absence of improvement in the extinction ratio. However, for the GMR filter with the low-index contrast grating, the extinction ratio increases up to 27.99 dB when the baseline corrected. Therefore, these results demonstrate that use of a low-index contrast grating is an essential design rule for achieving narrower FWHM and improving the extinction ratio.Figure ely Fig.  when thetic Fig. . Here, t24.So far, we have explored the optimal design of optical geometry that improve the tunability and signal extinction ratio of graphene-GMR filters. On the other hand, a method of charge doping to graphene has to be provided for the practical device implementation. Graphene doping can be accomplished by optical, chemical, or electrical means. Among them, charge doping via gate voltage control has been regarded as the most convenient and effective method. Our design based on the multilayer GMR can be easily incorporated into a typical back-gated graphene field effect transistor device layout as shown in Fig. As a solution to this problem, we propose two new practical device configurations for experimental implementation which drastically reduce the level of gate voltage while following the design rules developed in this work, as shown in Fig. 2 is semi-infinite and a 30 nm thick Al2O3 layer is inserted as the gate oxide. The optical constants of TCO were obtained from the Drude model25 calculation by setting several typical parameters, which include the electron effective mass, m* = 0.3me, the relaxation time, τ = 1.7 × 10−14 s, and the background dielectric constant, ε∞ = 3.6. The free electron density, ne was adjusted to 1 × 1019 cm−3 to ensure good optical transmittance in the 1.5 μm wavelength region and a conductivity suitable for use as a back-gate electrode, simultaneously. The calculated refractive index of TCO was about 1.86 at 1.5 μm and its thickness was set to 250 nm. For the calculation of reflective type device, it is assumed that Si3N4 is used as the waveguide and the Au electrode is used as a metal cladding. In both transmissive and reflective type configurations, diffraction gratings made of SiO2 were used with their period adjusted to 950 and 960 nm, respectively. The duty cycles of the gratings were determined to be 80 and 65%, respectively, considering optimum anti-reflection effect.To validate the proposed device configurations, RCWA simulations were performed for each model structure. For the calculation of the transmissive tunable filter with TCO waveguide, it is assumed that the cladding layer of SiOThe reflection band spectra of the newly proposed back-gated tunable graphene-GMR filter configurations were calculated with varying chemical potentials of graphene and shown in Fig. 2. The first method increased the ratio of the E-field confined in the waveguide core, thereby reducing the spectrum linewidth. However, this caused the deterioration in the tunability due to the decreased intensity of the evanescent field to be perturbed by an index modulation of graphene. Second, use of the SiO2 grating resulted in a high extinction ratio as a result of the narrowed linewidth of the filter spectrum without loss of tunability. Furthermore, we suggested new practical device designs that is easy to fabricate and effectively implement the graphene doping with low gate voltage, which is achievable by applying the TCO waveguide with a thin gate oxide inserted between the graphene and TCO, or by replacing the dielectric cladding with a metal one. Theoretical calculations confirm that these approaches are very effective in providing excellent tunability while significantly lowering the level of gate voltage for electric-field doping of graphene. Consequently, our results provide practical guidelines to design and implement the tunable GMR filters based on the refractive index modulation of the graphene with large and fast tunability.In this study, a tunable GMR filter based on a large change in refractive index of graphene caused by the external stimuli was proposed. The effects of the geometrical configuration of the GMR filter and the optical properties of constituent materials including graphene on the spectral response of the filter were analyzed systematically in terms of the extinction ratio of the signal channel and the tunability. It was found that the filter displayed better tunability when the graphene was located adjacent to the waveguide and the evanescent field strength at the interface increased. However, the increase of the leaky-field, observed in the GMR filter with a thin waveguide, accompanies a line-broadening and causes the overlap between the tuned spectra to increase. Consequently, it has an adverse effect on the extinction ratio of signal. In order to reduce the linewidth of reflectance band, two approaches were proposed, i.e. thickening waveguide and employing a low-index contrast grating such as SiO


Down-regulating mitochondrial protease LonP1 increased mitochondrial TDP-43 levels and exacerbated TDP-43-induced mitochondrial damage as well as neurodegeneration. Together, our results demonstrate that TDP-43 induced mitochondrial impairment is a critical aspect in TDP-43 proteinopathy. Our work has not only uncovered a previously unknown role of LonP1 in regulating mitochondrial TDP-43 levels, but also advanced our understanding of the pathogenic mechanisms for TDP-43 proteinopathy. Our study suggests that blocking or reversing mitochondrial damage may provide a potential therapeutic approach to these devastating diseases.Mutations in or dys-regulation of the TDP-43 gene have been associated with TDP-43 proteinopathy, a spectrum of neurodegenerative diseases including Frontotemporal Lobar Degeneration (FTLD) and Amyotrophic Lateral Sclerosis (ALS). The underlying molecular and cellular defects, however, remain unclear. Here, we report a systematic study combining analyses of patient brain samples with cellular and animal models for TDP-43 proteinopathy. Electron microscopy (EM) analyses of patient samples revealed prominent mitochondrial impairment, including abnormal cristae and a loss of cristae; these ultrastructural changes were consistently observed in both cellular and animal models of TDP-43 proteinopathy. In these models, increased TDP-43 expression induced mitochondrial dysfunction, including decreased mitochondrial membrane potential and elevated production of reactive oxygen species (ROS). TDP-43 expression suppressed mitochondrial complex I activity and reduced mitochondrial ATP synthesis. Importantly, TDP-43 activated the mitochondrial unfolded protein response (UPR mt in both cellular and animal models of TDP-43 proteinopathy. LonP1, one of the key mitochondrial proteases in UPRmt, protects against TDP-43 induced cytotoxicity and neurodegeneration. Our study uncovers LonP1 as a modifier gene for TDP-43 proteinopathy and suggests protecting against or reversing mitochondrial damage as a potential therapeutic approach to these neurodegenerative disorders.TDP-43 proteinopathy is a group of fatal neurological diseases. Here, we report a systematic examination of the role of mitochondrial damage in TDP-43 proteinopathy using patient brain tissues, as well as cellular and animal models. Our data show that TDP-43 induces severe mitochondrial damage, accompanied by activation of UPR TDP-43 proteinopathy is characterized by the presence of TDP-43 immunoreactive inclusion bodies in the affected tissues. Clinically, TDP-43 proteinopathy manifests as a spectrum of different neurodegenerative diseases, ranging from dementia  and motor neuron disease (MND) to traumatic brain injuries –4. FTLD Mitochondrial damage is associated with a range of neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD) and MNDs –19. Mitomt), which includes induction of mitochondrial chaperones assisting in proper protein folding, and of proteases promoting clearance of misfolded proteins  with different concentrations of purified LonP1 protein for 90 min at 37°C. The reaction products were analyzed by Western blotting using the corresponding specific antibodies to detect TDP-43 and LonP1 proteins.A cDNA encoding the human LonP1 protein (amino acid residues 115–959) was cloned into vector pET32M3C [a modified version of the pET32a vector ], expressed as an N-terminal thioredoxin and 6XHis-tagged protein and purified from E. coli  following the published protocol . PurifieTotal RNA was isolated from HEK293 cells or fly heads using TRizol reagent (Invitrogen) as described previously . cDNA syThe adult fly locomotor assay was carried out as described previously with minor modifications . BrieflyThe protein solubility was examined as described previously with minor modifications . Brieflyt-test. Multiple group comparisons were performed using a one-way or two-way analysis of variance (ANOVA) followed by post-hoc tests. The bar graphs with error bars represent mean ± standard error of the mean (SEM). Significance is indicated by asterisks: *, P < 0.05; **, P< 0.01; ***, P< 0.001.Data were collected in Excel (Microsoft) and analyzed using GraphPad Prism 6 unless specified otherwise. Differences between two groups were analyzed using a Student’s S1 TableAll samples used were sequenced and confirmed that there were no mutations in known genes associated with ALS or FTLD, including TDP-43, FUS, C9orf72, GRN, SOD1 and MAPT, as reported previously . Age, ge(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 FigA) Western blotting experiments revealed that the Myc-tagged TDP-43 (Myc) was detected in the purified mitochondria 24 hr following induction of TDP-43 expression, and that the endogenous (Endo) TDP-43 was detected in the purified mitochondria from both control and TDP-43 expressing cells. Western Blotting experiments were performed as described for B, C). Quantification of mitochondrial size (B) or percentage of damaged mitochondria in EM analyses of the corresponding HEK293 cells, vector control (Ctr) or cells expressing Wt or A315T-mutant TDP-43. Approximately 200 mitochondria  were quantified for each group. Data represents 3 independent experiments [one-way ANOVA with Bonferroni post hoc test (***: P<0.001)].((TIF)Click here for additional data file.S2 Fig(A) Induction of either Wt or A315T-mutant TDP-43 expression does not affect the total cellular ATP level. The total cellular ATP level was measured in cells expressing the control, or Wt or A315T-mutant TDP-43 at different time points after induction of TDP-43 expression using tetracycline (1ug/ml Tet). (B) Induction of either Wt or A315T-mutant TDP-43 expression does not lead to a general reduction in the mRNA levels of respiratory complex I genes. Quantitative RT-PCR experiment was performed at 24 hr post-induction using specific primers to examine the expression of a number of components of the complex I, including ND3, ND6, NDUFAB1, NDUFS8, NDUFAF4 and NDUFA13. Data represent 3 independent experiments and are analyzed using a one-way ANOVA with Bonferroni post hoc test .(TIF)Click here for additional data file.S3 Fig(A) A diagram illustrating the heat-shock induction strategy. Adult flies were collected after eclosion and subjected to heat shock daily at 28°C for 4 hours followed by culturing at 25°C for 20 hours every day.  TEM analyses revealed that the expression of A315T-mutant TDP-43 under the GMR-Gal4/Tubulin-Gal80ts driver leads to age-dependent progressive retinal degeneration and mitochondrial damage, whereas the retinae and mitochondria of control flies retained their normal morphology even at day 30 post-induction (Day30 post-Ind).TDP-43 expression was induced by heat shock in the retinae (GMR) or all neurons (Elav) under the Gal4 driver containing a Tubulin-Gal80ts regulatory element. (TIF)Click here for additional data file.S4 Fig(A) Confocal ratiometric imaging of VNC motor neurons of larvae expressing control RFP (Ctr), Wt or A315T-mutant TDP-43 revealed increased mitochondrial ROS production (excitation for reduced/oxidized mito-roGFP2-Grx1: 405-nm/488-nm). Scale bars: 25μm. (B) Quantification of the mitochondrial redox index (405-nm/488-nm) in MNs of fly larvae expressing Ctr, Wt or A315T-mutant TDP-43. Thirty-two to forty images were taken from more than 15 flies in each group . Data represent 3 independent experiments, analyzed using a one-way ANOVA with Bonferroni post hoc test (***:P<0.001). Fly genotypes: Ctr: OK371-Gal4/UAS-mito-roGFP2-Grx1/UAS-RFP; Wt: OK371-Gal4/UAS-mito-roGFP2-Grx1/UAS-Wt-TDP-43-RFP;A315T: OK371-Gal4/UAS-mito-roGFP2-Grx1/UAS-A315T-TDP-43-RFP.(TIF)Click here for additional data file.S5 Fig TDP-43-induced cytotoxicity is enhanced by the LonP1 inhibitor, but not by proteasome or autophagy inhibitors. Control (Ctr) cells or cells expressing Wt or A315T-mutant TDP-43 were induced with Tet (1μg/mL) for 24 hours. Following PBS washes to remove Tet, cells were cultured for an additional 24hours in media containing LonP1 inhibitor CDDO , proteasome inhibitor MG132  or autophagy inhibitor 3MA . Cell viability or cytotoxicity was determined using a CytoTox-ONE Homogeneous Membrane Integrity kit (Promega) in cells treated with CD, MG or MA. Data represent 4 independent experiments and are analyzed using StatPlus (one-way ANOVA with Bonferroni post hoc test). (C) Increased LonP1 expression suppresses TDP-43 cytotoxicty see  without  Western blotting analyses show increased LonP1 expression following LonP1 transfection (+) in cells expressing control vector (Ctr), Wt or A315T-TDP-43. The total TDP-43 levels in cell lysates were not changed by LonP1 overexpression (OE). (D) TDP-43 degradation by LonP1 is ATP-dependent. The in vitro degradation assay was carried out as described for (TIF)Click here for additional data file.S6 Fig(A) There was no detectable interaction between ClpP and TDP-43 in the co-Immunoprecipitation assay. (B) Down-regulating ClpP did not affect mitochondrial TDP-43 protein level. ClpP was down-regulated in HEK293 stable inducible cells expressing Wt or A315T-mutant TDP-43. ClpP down-regulation did not alter mitochondrial TDP-43 levels. Data represent 3 independent experiments.(TIF)Click here for additional data file.S7 FigA) qPCR experiment was carried out to determine mRNA levels of Drosophila Lon (dLon) in the corresponding fly lines expressing vector control (Ctr) or siLon (#1 and #2) or overexpressing Lon (dLonOE). Because siLon#1 line showed consistently more robust down-regulation (reducing dLon expression to ~30% of the control level), this line was used in subsequent experiments. Data represent 3 independent experiments. Overexpression of dLon in control flies led to retinal degeneration, preventing us from testing effect of dLonOE in TDP-43 flies. (B) Western blotting experiments using anti-TDP-43 antibody showed that the total TDP-43 was expressed in eyes at equivalent levels in TDP-43 and TDP-43/siLon flies at day 20 following heat shock. (C) Western blotting experiments using anti-TDP-43 antibody showed that the total TDP-43 was expressed at equivalent levels in heads of TDP-43 and TDP-43/siLon flies at day 4 following heat shock. The pan-neuronal marker Elav was used as a loading control in panels B and C.((TIF)Click here for additional data file.S8 Fig(A). Mitochondria were purified from heads of flies expressing control or Wt or A315T-mutant TDP-43 under the GMR-Gal4 driver. Purified mitochondria were sequentially extracted in RIPA buffers containing 0.5% NP-40 or 2%SDS and finally 8M Urea (see B). Quantification of WB band intensity shown in panel A. Data from 3 experiments were analyzed by a Student’s t-test. Data represent 3 independent experiments.Urea see . Corresp(TIF)Click here for additional data file.S9 Fig(A) Down-regulation of Lon in control (Ctr) flies did not affect retinal morphology or mitochondrial ultra-structure. (B) Down-regulating Lon in control flies did not affect the number of rhabdomeres per ommatidium, with 81 and 89 ommatidia analyzed in Ctr and siLon groups respectively. (C) Quantification of EM data shows that down-regulating Lon in control flies did not alter the percentage of damaged mitochondria, with 431 mitochondria in Ctr group and 521 mitochondria in siLon group examined. Data represent 3 independent experiments, analyzed using a one-way ANOVA with Bonferroni post hoc test .(TIF)Click here for additional data file.S10 FigElectron-dense aggregates (marked by black arrows) were detected inside mitochondria in flies expressing A315T-mutant TDP-43 when Lon was down-regulated by the specific siRNA. These structures were not detected in other groups, including the control flies or flies expressing Wt (with or without Lon knockdown) or A315T-mutant TDP-43 alone.(TIF)Click here for additional data file.


Idiopathic pulmonary fibrosis (IPF) is a chronic fatal lung disease without a cure and new drug strategies are urgently needed. Differences in behavior between diseased and healthy cells are well known and drug response can be different between cells isolated from IPF patients and controls. The macrolide Azithromycin (AZT) has anti-inflammatory and immunomodulatory properties. Recently anti-fibrotic effects have been described. However, the anti-fibrotic effects on primary IPF-fibroblasts (FB) directly compared to control-FB are unknown. We hypothesized that IPF-FB react differently to AZT in terms of anti-fibrotic effects.Primary normal human lung and IPF-FB were exposed to TGF-β (5 ng/ml), Azithromycin (50 μM) alone or in combination prior to gene expression analysis. Pro-collagen Iα1 secretion was assessed by ELISA and protein expression by western blot , p62, Bcl-xL). Microarray analysis was performed to screen involved genes and pathways after Azithromycin treatment in control-FB. Apoptosis and intraluminal lysosomal pH were analyzed by flow cytometry.AZT significantly reduced collagen secretion in TGF-β treated IPF-FB compared to TGF-β treatment alone, but not in control-FB. Pro-fibrotic gene expression was similarly reduced after AZT treatment in IPF and control-FB. P62 and LC3II/I western blot revealed impaired autophagic flux after AZT in both control and IPF-FB with significant increase of LC3II/I after AZT in control and IPF-FB, indicating enhanced autophagy inhibition. Early apoptosis was significantly higher in TGF-β treated IPF-FB compared to controls after AZT. Microarray analysis of control-FB treated with AZT revealed impaired lysosomal pathways. The ATPase and lysosomal pH regulator ATP6V0D2 was significantly less increased after additional AZT in IPF-FB compared to controls. Lysosomal function was impaired in both IPF and control FB, but pH was significantly more increased in TGF-β treated IPF-FB.We report different treatment responses after AZT with enhanced anti-fibrotic and pro-apoptotic effects in IPF compared to control-FB. Possibly impaired lysosomal function contributes towards these effects. In summary, different baseline cell phenotype and behavior of IPF and control cells contribute to enhanced anti-fibrotic and pro-apoptotic effects in IPF-FB after AZT treatment and strengthen its role as a new potential anti-fibrotic compound, that should further be evaluated in clinical studies. Idiopathic pulmonary fibrosis (IPF) is a devastating progressive lung disease causing dyspnea and cough, which ultimately leads to respiratory failure . IPF affTo develop or apply existing drugs for new use, the underlying pathomechanisms need to be fully understood. The pathogenesis of IPF remains still unclear. Presently it is believed that injury of the alveolar epithelial lining followed by aberrant wound healing is the initiating event for IPF development . AlthougFibroblasts isolated from IPF patients show particular properties such as insufficient autophagy , 10. ImpIn our study, we tested the anti-fibrotic properties of AZT. We hypothesized that IPF fibroblasts react differently to AZT treatment than normal fibroblasts. We thus investigated whether AZT has enhanced anti-fibrotic effects on IPF fibroblasts compared to control fibroblasts. We further investigated the underlying mechanisms of divergent responses from control and IPF fibroblasts specifically for cell death, autophagy as well as lysosomal function.We obtained control fibroblasts from healthy lung tissue from patients undergoing tumor resection at the University Hospital of Bern, Switzerland. All study participants provided their informed written consent, as approved by the local Ethics Committee, Bern, Switzerland (KEK-BE:042/2015). We received additional lung fibroblasts from control and IPF patients from diagnostic biopsies and/or lung explants isolated and cultured as previously described and kindly provided by Prof. Crestani, Paris, France .We used Ham’s F-12 K  media supplemented with 10% Fetal Bovine Serum (FBS)  and 1% Penicillin Streptomycin  for cell culture. We treated cells for 24 h as follows: 1. Starvation media control including the respective vehicle control (Media without FBS plus 50 μM Ethanol as the dissolvent for AZT), 2. Azithromycin   3. TGF-β (5 ng/ml plus 50 μM Ethanol)  or 4. Azithromycin  with simultaneous TGF-β stimulation. Each condition was tested and repeated in three independently performed experiments. The following antibodies were used for immunofluorescent staining: primary monoclonal antibody mouse anti-αSMA , monoclonal anti-vinculin-FITC  and secondary antibody Alexa Fluor 488 goat anti-rabbit . Lysotracker Green DND-26  dye for life lysosomal staining. Following western blot antibodies were used: mouse anti-αSMA , rabbit anti-Fibronectin (FN) , rabbit anti-ATP6V1B2, rabbit anti-LC3 AB (II/I), monoclonal Smad3 and pSmad3 rabbit antibodies , rabbit anti-p62  and rabbit anti-Bcl-xL antibodies . Infrared IRDye (680 or 800 CW) secondary antibodies .We isolated fibroblasts from human lung tissue, as described previously . Cell cuWe used Nucleospin RNA kit  to isolate RNA according to the manufacturer’s instructions. We used the ΔΔCt method to calculate mRNA expression levels. RNA copy numbers were normalized to β2microglobulin (β2m) expression as previously described . Human fWe seeded control and IPF fibroblasts on IBIDI® chamber slides . The cells were grown until 80% confluency was reached and kept in resting media for 24 h. Afterwards, the cells were fixated and incubated with the respective primary and secondary antibodies. To identify lysosomes, we used Lysotracker® Green dye  life staining. Pictures were taken with Leica DMI4000 B fluorescence microscope using the 20x and 63x magnification (Plan-Apochromat 63x/0.7 Oil).We treated control and IPF fibroblasts with TGF-β (5 ng/ml) and/or AZT (50 μM) for 48 h for the analysis of αSMA, Fibronectin, ATP6V1B2, LC3 AB (II/I) and p62. Incubation time for Bcl-xL analysis was 24 h and 1 h to evaluate SMAD3 phosphorylation. Cells were harvested using Pierce IP Lysis Buffer®  according to the manufacturer’s instructions. We normalized infrared fluorescent signals to internal control monoclonal β-actin mouse antibody  and quantified their intensities as previously described .Fibroblasts were grown to 80% confluency on 6-well plates preceding treatment. We used Annexin V (A) and Propidium Iodide (PI)  dyes according to manufacturer’s instructions for flow cytometry analysis. To evaluate cytotoxicity of Azithromycin in control and IPF fibroblasts, we measured the release of lactate dehydrogenase (LDH) in cell supernatants after 24 h of treatment by a colorimetric assay according to the manufacturer’s instructions. We measured the optical densitiy at 540 nm using an Infinite M1000 PRO microplate reader  as previously described . Bcl-xL We determined lysosomal pH using FITC-Dextran loaded fibroblasts following a previously published protocol . We seedWe used a monoclonal ELISA kit  according to the manufacturer’s instructions to determine concentrations for pro-collagen Iα1 (pro-Col1A1).AZT-induced vacuolar structures of control and IPF fibroblasts were analyzed in Hannover, Germany at the Institute for Anatomy by transmission electron microscopy. The preparation for electron microscopic evaluation was performed according to established methods . In briewww.kfb-regensburg.de). RNA microarray data from control lung fibroblasts treated with Azithromycin versus non-treated can be downloaded from the ArrayExpress database at EMBL-EBI (www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-8488. Summarized probe set signals in log2 scale were calculated by using the GCCN-SST-RMA algorithm with the Affymetrix GeneChip Expression Console v1.4 Software. After exporting into Microsoft Excel, average signal values, comparison fold changes and significance P values were calculated. Probe sets with a fold change above 2.0 fold and a student’s T-test P value < 0.05 were considered statistically significant. Differentially expressed genes were further analyzed and evaluated in the Department of Biostatistics, Bern, Switzerland (Cedric Simillion). Microarray data were analyzed using custom CDFs and data was normalized and log-transformed using the RMA method [We performed microarray analysis to screen involved pathways after AZT treatment in control lung fibroblasts. Microarray analysis was performed on isolated total RNA samples. RNA isolation is described above and concentration was determined spectrophotometrically using Nanodrop (NanoDrop 2000). Sample preparation for microarray hybridization was carried out as described in the Affymetrix GeneChip WT PLUS Reagent Kit User Manual . In brief, 100 ng (50 ng of sample no. 09) of total RNA was used to generate double-stranded cDNA. 15 μg of subsequently synthesized cRNA was purified and reverse transcribed into sense-strand (ss) cDNA, whereat unnatural dUTP residues were incorporated. Purified ss cDNA was fragmented using a combination of uracil DNA glycosylase (UDG) and apurinic/apyrimidinic endonuclease 1 (APE 1) followed by a terminal labeling with biotin. 3,8 μg fragmented and labeled ss cDNA were hybridized to Affymetrix Clariom S human arrays for 16 h at 45 °C in a GeneChip hybridization oven 640. Hybridized arrays were washed and stained in an Affymetrix Fluidics Station FS450, and the fluorescent signals were measured with an Affymetrix GeneChip Scanner 3000 7G. Fluidics and scan functions were controlled by Affymetrix GeneChip Command Console v4.1.3 software. Sample processing was performed at an Affymetrix Service Provider and Core Facility, “KFB - Center of Excellence for Fluorescent Bioanalytics” . Student’s T-test was used to compare the mean (± SE) between only two treatment conditions. We investigated whether Azithromycin (AZT) differently affects the fibrotic response in lung fibroblasts from normal human lung fibroblasts and IPF fibroblasts (referred to as controls and IPF-FB). We found that gene expression of the pro-fibrotic markers collagen Iα1 (Col1A1) and fibronectin (FN) were expectedly increased after TGF-β stimulation over 24 h in both cell types   Fig. a indicat05) Fig. b. The ly05) Fig. c. We fur05) Fig. d. Cathep05) Fig. e. Last, 05) Fig. f.Fig. 4AZT-induced vacuolar structures of control and IPF fibroblasts were analyzed in Hannover, Germany at the Institute for Anatomy by electron microscopy (EM). The vacuolar structures were identified in control Fig. a1–3 and Microarray analysis Fig. a revealeOur study shows that AZT has anti-fibrotic and pro-apoptotic effects on primary fibroblasts and that these effects are enhanced in IPF fibroblasts compared to control fibroblasts. We confirmed that AZT reduces gene expression levels of pro-fibrotic genes after stimulation with TGF-β in vitro in control as well as in IPF fibroblasts as recently reported . In our Studies with antimicrobial therapies have been previously proposed for IPF treatment as increased bacterial burden in the BAL of IPF predicted lung functional decline . PreventAutophagy inhibition is observed after AZT treatment and prevented myofibroblast differentiation through pro-fibrotic NOX4 suppression , howeverLysosomes are organelles with a low internal pH of 4.5–5 which is known to be regulated by vacuolar ATPases . The maiIn addition to enhanced anti-fibrotic effects of AZT in IPF-FB, we have observed increased apoptosis of fibroblasts after AZT treatment compared to control-FB. We showed that AZT induces a stronger increase of the lysosomal pH in IPF-FB, which has been proposed to lead to lysosomal membrane permeabilization and trigger the so-called lysosomal apoptotic pathway , 64. FurToday, two drugs approved for IPF treatment (Nintedanib and Pirfenidone) are able to slow down disease progression, but adverse effects are common and no improvement can be achieved in most patients. New therapeutic treatments and treatment combinations are thus urgently needed to not only stop, but also to reverse fibrosis. AZT is a well tolerated macrolide antibiotic with tolerable side effects and could be a potential new additional therapeutic drug for IPF, but additional in vivo studies and prospective clinical trials are needed to confirm its anti-fibrotic effects.Our study shows that AZT has a greater anti-fibrotic effect regarding collagen and fibronectin secretion as well as myofibroblast differentiation in IPF compared to controls. Moreover, AZT has enhanced pro-apoptotic effects on IPF fibroblasts. The impaired lysosomal function in IPF fibroblasts after additional AZT treatment could explain cell damage with higher apoptosis levels in IPF fibroblasts. Our findings may stimulate new treatment strategies for IPF patients, but need to be tested in controlled clinical trials.Additional file 1.Figure S1. Azithromycin reduces gene expression of fibronectin and αSMA in IPF and control fibroblasts. Figure S2. SMAD phosporylation is not influenced by Azithromycin in IPF and control fibroblasts. Figure S3. Azithromycin does not alter protein expression of ATP6V1B2 in IPF and control fibroblasts.


Since 2009, IPF patients across Europe are recruited into the eurIPFreg, providing epidemiological data and biomaterials for translational research.The registry data are based on patient and physician baseline and follow-up questionnaires, comprising 1700 parameters. The mid- to long-term objectives of the registry are to provide clues for a better understanding of IPF phenotype sub-clusters, triggering factors and aggravating conditions, regional and environmental characteristics, and of disease behavior and management.p = 0.001).This paper describes baseline data of 525 IPF subjects recruited from 11/2009 until 10/2016. IPF patients had a mean age of 68.1 years, and seeked medical advice due to insidious dyspnea (90.1%), fatigue (69.2%), and dry coughing (53.2%). A surgical lung biopsy was performed in 32% in 2009, but in only 8% of the cases in 2016, possibly due to increased numbers of cryobiopsy. At the time of inclusion in the eurIPFreg, FVC was 68.4% ± 22.6% of predicted value, DLco ranged at 42.1% ± 17.8% of predicted value . Signs of pulmonary hypertension were found in 16.8%. Steroids, immunosuppressants and N-Acetylcysteine declined since 2009, and were replaced by antifibrotics, under which patients showed improved survival .The eurIPFreg and eurIPFbank are listed in ClinicalTrials.gov( Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and usually fatal fibrotic disease of the lung that typically affects elder patients beyond 60 years of age. The overwhelming fibrotic processes and the distortion of the lung’s ultrastructure result in a progressive loss of pulmonary compliance and a decline in gas exchange properties . The preChanges in the knowledge on etiology and pathogenesis of IPF, technical improvements in radiological diagnostics, as well as new treatment modalities resulted in a modification of the existing ATS/ERS/JRS/ALAT guidelines in 2011 . Still, As the natural course of IPF is quite heterogeneous, and as the response to the novel anti-fibrotic drugs has been reported to show great variability, it is essential to identify reliable predictive factors indicating the risk of deterioration and the response to medical treatment, as well as side effects in a broad non-selected patient cohort , 13. In www.pulmonary-fibrosis.net). The eurIPFreg and eurIPFbank were launched in November 2009 to collect a broad range of clinical data and of biomaterials from European IPF patients in a longitudinal fashion [In order to meet these demands, to better explore the pathogenesis and natural course of IPF, and also in order to facilitate translational research in biomaterials from IPF subjects, the European IPF Registry (eurIPFreg) and the European IPF Biobank (eurIPFbank) were set up in the frame of the European IPF Network, a research consortium funded by the European Commission under the FP7 program from 2008 until 2011  [, Methods, and Infrastructure for Networked Medical Research e.V.) and authorities . Both, eurIPFreg and eurIPFbank have also been reviewed and received positive votes from institutional review boards in Germany , France, Italy, Austria, Spain, Czech Republic, Hungary and the UK. The research was conducted strictly according to the principles of the Declaration of Helsinki. Patients were included into the registry starting November 2009. The eurIPFreg and eurIPFbank are listed in ClinicalTrials.gov (NCT02951416).The eurIPFreg has been designed as an Internet-based, multicentre registry interlinked with the European IPF Biobank  . The datIn the period between 2009 and 2016, the following sites recruited patients: Universities of Giessen and Marburg Lung Center, Germany and the nearby AGAPLESION Lung Clinic Waldhof-Elgershausen; Competence Center for Rare Pulmonary Diseases of Hopital Bichat in Paris, France; Interstitial Lung Disease Unit of Royal Brompton Hospital in London, United Kingdom; Reference Center for Rare Pulmonary Diseases, Centre Hospitalier Universitaire Dijon-Bourgogne, France; Dept. of Clinical and Molecular Biomedicine of Università degli Studi di Catania, Italy; Vienna University Hospital, Austria; Hospital Clinico San Carlos in Madrid, Spain; Department of Pulmonology Semmelweis University in Budapest, Hungary; Thomayer Hospital in Prague, Czech Republic; Ospedale San Gerardo in Monza, Italy; Università degli Studi di Napoli Federico II, Italy, and Ruhrlandklinik, University Hospital of Essen, Germany.secuTrial®) was developed by the German Parkinson Network and is certified by the Food and Drug Administration (FDA) as well as the European Medicines Agency (EMEA). The patients’ data are transferred via a secure internet-based data collection form and all data entries are based on the unique patient’s encrypted ID number (“pseudonym”).The software solution underlying the registry  or other ILDs (as comparator group) as diagnosed by the expert site, and had provided written informed consent prior to the inclusion. In order to facilitate research, inclusion of other lung diseases such as lung cancer, COPD as well as healthy subjects or family members as additional comparator groups was made possible in the frame of an amendment in March 2013.On a local level, each patient’s IPF diagnosis was evaluated in a multidisciplinary discussion including at least chest physicians, pathologists and radiologists on the basis of the respective ATS/ERS/JRS/ALAT guidelines. The registry had no explicit exclusion criteria, thereby reducing selection bias. The clinical data were collected at the time of enrolment (baseline) and in intervals 3 to 12 month thereafter .After entry into the registry, each case was checked by a documentation officer (BP) for data quality and by FD and AG for internal plausibility of medical data and the diagnosis of IPF (e.g. hints for collagen/vascular diseases or hypersensitivity pneumonia in patient questionnaires). Also, in 2015 and 2016, all previous IPF cases diagnosed as having IPF according to the previous guideline from 2000, were checked if they would also fulfil the guideline criteria from 2011 [www.pulmonary-fibrosis.net). The patient questionnaire included patient’s demographics, a detailed medical history making use of the WHO classification, complaints as well as report of co-morbidities [The clinical data acquisition took place primarily via patient and physician baseline questionnaires, which can be retrieved upon logging in to our website (bidities . Alongsibidities –19. Theswww.whocc.no/atc_ddd_index).The physician questionnaire contained data of physical examination and laboratory tests, pulmonary function, radiology, echocardiography, 6 MWD as well as other information concerning relevant patient’s diagnosis and therapy; the form had to be filled out online in English language. Import of historical medical data was also possible. Medication was assessed making use of the official WHO list of drugs 2018 from WHO Collaborating Centre for Drug Statistics and Methodology, allowing categorial (group-wise) analysis of co-medication . In addition to the data as provided by the questionnaires, high resolution computed tomography (HRCT) and any other DICOM formatted images could be uploaded; during this procedure, the patient’s personal data in the DICOM header were replaced by the pseudonym.Biological materials such as blood, bronchoalveolar lavage fluid (BALF) and tissue samples as well as exhaled breath condensates and electronic Nose (eNose) profiles were centrally recorded and managed through generation of patient-, time-, and specimen-specific Lab IDs and they were stored both, locally as well as in the centralized European IPF Biobank (eurIPFbank) located in Giessen.Quality of data was improved by introduction of internal plausibility checks, in which different items were put into a logical context, causing the generation of queries in case inconsistent entries were noted (e.g. if physician’s and patient’s report were not consistent with regard to signs of underlying collagen/vascular disease). These queries were addressed to the respective site investigator, asking for clarification of the issue. In addition, site investigators were asked to conduct on-site data verification.All statistical procedures were performed using SPSS 24 . For baseline data, the summary descriptive statistic was generated with categorical data displayed as absolute numbers and relative frequencies. Continuous data are shown as mean (SD) for normally distributed data or as median (interquartile range) for nonparametric data. Comparisons between groups were performed using a t-test or Mann–Whitney U test, as appropriate. For assessment of overall survival Kaplan-Meier analyses were applied.The period of recruitment covered in this paper was chosen as November 2009–October 2016. By October 2016, a total of 2090 patients were included in eurIPFreg. One thousand eighty-six of these patients had suspected or proven ILD, among them 525 patients with IPF. The data of another 50 IPF patients, who had been recruited into the registry, could not be taken into consideration, as they were still under final assessment.In the period between 2009 and 2016, a total of 1086 patients with ILDs were recruited into the eurIPFreg. The distribution of these ILDs is shown in Fig. With regard to the 525 IPF patients included in the eurIPFreg, the baseline characteristics are outlined in Table The most common clinical symptoms reported by the patients are shown in Fig. To classify dyspnea in a patient questionnaire, we graded severity of dyspnea and impairment of physical activity in analogy to well-known NYHA classification . Our cohDiagnoses of IPF were made based on the respective ATS/ERS/JPS/ALAT guidelines 2000 and 2011 , 7, 16. Two different scales were used for the grading of HRCT. Prior to the release of the 2011 guidelines, the scale used in the eurIPFreg consisted of four grades and all the 151 patients diagnosed according to the 2000 guidelines were, independent of the existence of an open lung biopsy, classified into highly probable UIP , somewhat probable , weakly probable  and not probable UIP pattern .After the publication of the 2011 guidelines, the new HRCT classification  was applied. In this regard, a definite UIP pattern was encountered in 63.7% of all IPF cases, a possible UIP pattern in 27.7%. In 8.6% of the cases an “inconsistent with UIP” pattern was found. In this cohort covering the time span between 2003 and 2014, 79% of the patients received open lung surgery incl. VATS; 3.5% did show features of UIP in coincidentally larger transbronchial biopsies, for which reason VATS was not recommended anymore. In all these cases, histological findings were consistent with a pathologic UIP pattern. In 17.5% of patients a surgical procedure was not recommended because of the general condition of the patients, but longitudinal follow up and discussion in a MDD rounds was very suggestive of UIP/IPF, for which reason this diagnosis was the preferred differential diagnosis.Data regarding bronchoscopy were available for 455 IPF patients (86.7%). According to these, bronchoscopy was performed as part of the diagnostic procedure in 309 out of these 455 patients (67.9%) and biopsies (incl. cryobiopsies) were taken in 128 cases, corresponding to 24% of all IPF patients.BALF was performed in 263 cases (85.1%). The BAL differential revealed elevated neutrophil (14 ± 15.7%) and eosinophil (5.4 ± 8.5%) counts in face of normal lymphocyte (9.8 ± 10.7%) and reduced macrophage counts .20–30% of all patients diagnosed as having IPF in 2010 or 2011 underwent open lung surgery or video-assisted thoracoscopy (VATS), but these numbers went down in following years, possibly due to the increasing use of cryobiopsy, as shown in Fig. n = 12; 10 of which revealed fibroblast foci, spatial and temporal heterogeneity and absence of another pattern, hence only missing information as to the reference to the pleural surface), NSIP (n = 5), HP (n = 1), and unclassifiable IIP (n = 1). In addition, while we did not observe any exacerbation with a temporal association to the conductance of cryobiopsy, we have seen two exacerbations related to VATS.When evaluating the whole DPLD cohort for the entire observation period (1086 patients from 2009 until 2016), we found that a pattern “inconsistent with UIP” was documented in 177 cases. Next to VATS, cryobiopsy was undertaken 19 (10.7%) of these cases and forwarded criteria of an UIP pattern  values exceeding 50 mmHg . Tricuspid annular plane systolic excursion (TAPSE) of less than 1.5 cm was found in 14 patients (3.8%). The n-pro brain natriuretic peptide (nBNP)-value was assessed in 273 patients; the mean n-pro BNP was 301.24 pg/ml (range < 1–6716 pg/ml). Forty-one IPF patients (15%) showed an n-pro-BNP value exceeding 150 pg/ml.A six-minute walk test was performed and was available at baseline for 420 patients (80%). The mean distance walked was 388 ± 122 m.Co-morbidities were common in patients with IPF and were assessed via both, physician- and patient questionnaires. Because co-morbidities are often exclusion criteria for clinical trials, this real-world data represents a broad IPF population without selection bias . As showAt the enrollment time, various therapeutic regimes were used throughout Europe. Prior to the commercial release of anti-fibrotic drugs pirfenidone and nintedanib, a significant number of the patients were treated with diverse immunosuppressants and/or N-acetylcysteine. In our study, the most used IPF medications were classified into following groups: antifibrotics , N- acetylcysteine, prednisolone, azathioprine, as well as mycophenolic acid. Figure There is accumulating evidence that rehabilitation improves quality of life and symptoms in patients with IPF. In our cohort, 81 patients participated in an in-patient and 26 patients in an out-patient rehabilitation program.Of the 525 IPF patients included in the registry from November 2009 until baseline cut-off in October 2016, definite outcome data (date of death or last known visit) were available for 210 cases.Of those, eight patients underwent lung transplantation (corresponding to 3.9%) and 78 patients (corresponding to 38%) had died until data cut-off in 2016. These 78 patients were enrolled in the registry at a mean age of 63.5 years, with a range of 42–88 years. The mean age of these subjects at the time of death was 71 years (range 44.5–90 years). The most common reasons for death were bronchopulmonary infections, i.e. pneumonia, leading to sepsis and multiorgan failure, followed by right heart failure due to progressive pulmonary hypertension.p = 0.001).When assessing survival via Kaplan-Meier analysis in correlation to the date of first IPF diagnosis, our results indicate that the median survival on antifibrotics was 123.1 months , as compared to a median survival of 68.3 months in patients treated with any other medication . Figure The data reflect the inhomogeneity of the natural course of IPF in a “real world” clinical setting, ranging from slow progression with long periods of stable disease to a rapid progressive fibrosis, with successive lung function impairment and death within first 2 years upon diagnosis. In this study we did not explicitly compare outcomes of IPF vs. non-IPF cohort, leaving this topic to our further research.In the frame of the European IPF Network “Natural course, Pathomechanisms and Novel Treatment Options in Idiopathic Pulmonary Fibrosis” (eurIPFnet), the multicenter, European-wide IPF registry (eurIPFreg) and biobank (eurIPFbank) were launched in 2009. In this report we summarize the clinical characteristics of our large European IPF cohort. Our study results include survival outcomes extend beyond baseline findings.The primary purpose of eurIPFreg was to characterize a natural course of IPF, as well as to capture diagnostic and treatment strategies. This is in line with a previous note, according to which improved survival of IPF depends on better understanding of the epidemiology of the disease, its diagnostic spectrum and outcomes from emerging therapies , 22. FurWith regard to demographics, the data obtained in our IPF cohort were similar to those reported in large randomized clinical trials and other registries , 23–29. Bronchoscopy was performed in a significant number of patients. Forceps biopsies, undertaken primarily in the first yeas of the patient recruitment, were reported to be somewhat indicative of interstitial lung disease in only about 25% of all the patients. As a result, VATS was a common procedure in the beginning of the registry, but significantly declined in numbers with the time due to knowledge of causing IPF exacerbations, but also with increasing use of less invasive cryobiopsy , 33. BALThe prevalence and impact of comorbidities on the clinical course of IPF remains unclear and is the subject of our further follow-up studies. From all reported co-morbidities, cardiovascular were most common, followed by GERD, sleep disorders, hyperlipidemia, diabetes and pulmonary hypertension. This appears to be also consistent with other registries , 34.As the latest IPF guidelines were released in 2011, the eurIPFreg comprises patients initially diagnosed and treated according to the ATS/ERS Consensus Statement of 2000, as well as those diagnosed and treated according to the recent ATS/ERS/JRS/ALAT IPF guidelines of 2011 , 16. HenIn this regard, the diversity of this European IPF cohort may reflect not only the variety in natural course of the disease, but also changes in clinical management of the patients, especially when comparing our results to historic IPF cohorts . Thus, tp = 0.001), in accordance with randomized controlled trials, such as CAPACITY, ASCEND as well as open-label extension study (RECAP) [Our registry also reflects the steady increase of usage of antifibrotic drugs. In October 2016 for example 27 patients received nintedanib and 224 patients received pirfenidone. Our results showed an improved survival in patients on anti-fibrotic medication ( (RECAP) . PrognosHowever, our data indicate that IPF still has a high mortality rate and that survival times are quite heterogenous. As mentioned above, these data may reflect both, the heterogeneous natural course of IPF in a clinical setting, ranging from stable disease to a rapid progressive fibrosis, and the change in the pharmacological approach to IPF subjects, with absence of antifibrotic drugs in the beginning, but widespread use at the end of the period.The interpretation of the real-world data has several limitations. First, as a databank, the information available for each patient is based on clinical practice instead on a trial protocol. Despite our expectation to have the questionnaire for each visit and every patient fully completed, some categorical data were missing. Additionally, the co-medication, as well as co-morbidities, are partly self-reported as a part of the structured patient questionnaire and thus might lack accuracy.We report a clinical characteristic of a large European IPF cohort with outcome data extending up to 7 years. Our patients are diverse in age, impairment of lung function, therapeutic regimes and co-morbidities. The data reflect changes in the diagnostic and therapeutic approach in IPF in the last 10 years, supporting the important role of large real-world data registries to document and scrutinize changes in IPF management.


Graphene provides a unique way of sensing the local pH level of substances on the micrometric scale, with important implications for the monitoring of cellular metabolic activities where proton excretion could occur. Accordingly, an innovative biosensing approach for the quantification of the pH value of biological fluids, to be used also with small amounts of fluids, was realized and tested. It is based on the use of micro-Raman spectroscopy to detect the modifications of the graphene doping level induced by the contact of the graphene with the selected fluids. The approach was preliminarily tested on aqueous solutions of known pH values. It was then used to quantify the pH values of cell culture media directly exposed to different doses of X-ray radiation and to media exposed to X-ray-irradiated cells. The Raman response of cells placed on graphene layers was also examined. Thus, an estimation of the electron carrier area density n could be obtained from the spectral position p-doping. In this regime, the carrier density n was negative (hole-carriers), and its dependence on n- (p-) doping changes in graphene were investigated.The measured position of the G mode , the graphene doping level was low and the carrier density n changes strongly depended on the We used this relation for evaluating tion see a. The acsolution b, which n is the carrier density evaluated for the sample and for the bare graphene reference and The Samples of DMEM-A exposed to ionizing radiation, for doses of 2, 4, 6, 8 and 10 Gy, were considered along with unexposed sample (dose of 0 Gy). The Raman response of graphene covered by a drop of unexposed DMEM is reported in n was estimated also in the case of irradiated DMEM-A samples. The data reported in The value of In The radiation-induced bystander effect is the phenomenon for which radiobiological effects are also present in un-irradiated cells in the vicinity of cells that have been irradiated. This is in contrast to the central dogma of radiation biology, affirming that ionizing radiation must interact with the DNA of the cell in order to produce radiobiological effects. Experimental evidence of this effect has been widely observed using different ionizing radiation ,30. The Measurements were performed on MDA-MB-231 cells placed on graphene film, both on the cells and the nearby areas, as schematically shown in the microscope image of n concentration by using the relation of A single cell, placed on the graphene substrate, was selected (Position A in Based on the unique properties of graphene, the dependence of the electronic doping of this material induced by the proximity of alkaline or acid liquids was probed by micro-Raman spectroscopy and exploited for a new pH-sensing approach. The method allows the determination of the pH level of solutions even when very small volumes are considered (smaller than 1 adiation , which m


Over the past few years, there has been an increasing demand for stretchable electrodes for flexible and soft electronic devices. An electrode in such devices requires special functionalities to be twisted, bent, stretched, and deformed into variable shapes and also will need to have the capacity to be restored to the original state. In this study, we report uni- or bi-axially wrinkled graphene–silver nanowire hybrid electrodes comprised of chemical vapor deposition (CVD)-grown graphene and silver nanowires. A CVD-grown graphene on a Cu-foil was transferred onto a bi-axially pre-strained elastomer substrate and silver nanowires were sprayed on the transferred graphene surface. The pre-strained film was relaxed uni-(or bi-)axially to produce a wrinkled structure. The bi-axially wrinkled graphene and silver nanowires hybrid electrodes were very suitable for high actuating performance of electro-active dielectric elastomers compared with the wrinkle-free case. Present results show that the optical transparency of the highly stretchable electrode can be successfully tuned by modulating input voltages. Smart windows, which can control the level of light transmission, have recently been attracting great interest, because they can be applied to a variety of applications, including vehicle windows, exterior wall windows and skylight windows. Until now, most smart window technology has been based on the redox reaction of a molecular element and a chromic material in response to external stimuli such as light, electricity, or temperature ,2,3,4. HAmong various approaches to smart window technology, there has been recent interest in techniques for modulating transmittance by changing the surface morphology of an elastomer actuator. In order to generate such a morphological change of soft materials, a flexible and stretchable conducting material should be used as compliant electrodes, which can be stretched or shrunk according to the movement of the soft material, such as nanowires, graphene, and hybrid materials ,10,11,12However, since most devices were operated with flat electrodes, the changes in transmittance were not so large, resulting in a limited application. In order to resolve this performance problem, research on smart windows using graphene and silver nanowire hybrid electrodes has been carried out, but a wrinkled structure implemented with stretchable hybrid materials has not been reported yet.In this study, we developed a wrinkled hybrid electrode, comprised of CVD-grown graphene and silver nanowires, and reported a tunable transmittance by applying electrical fields to the dielectric elastomer actuators sandwiched with the wrinkled graphene–silver nanowire hybrid electrodes. A monolayer of graphene grown by CVD on a Cu-foil was transferred onto a bi-axially pre-strained elastomer substrate and silver nanowires were sprayed on the transferred graphene surface. The pre-strained film was released bi-(or uni-)axially to develop a wrinkled structure. The morphological and electrical characteristics of the wrinkled structures with bi-(or uni-)axially compressive strains were investigated using scanning electron microscopy (SEM). Also, the bi-axially wrinkled graphene and silver nanowire hybrid nanostructures were successfully applied to produce high actuating performance compared with the wrinkle-free case. Using the fabricated electrode, we demonstrated a device whose optical transparency was tunable with input voltages.3. The dielectric constant of the VHB 4905 film is 3.21 at 1 kHz. The dissipation factor and dielectric breakdown strength are 0.0214 and 630 V/mm, respectively [VHB 4905 film  is an appropriate material for dielectric elastomer actuators because the film is easy to handle, and both sides of the film are sticky enough for coating with electrode materials such as graphene and silver nanowires. In addition, the VHB 4905 is almost transparent, with a thickness of around 5 mm and a density of 960 kg/mectively ,19.Silver nanowires (AgNWs) are nanostructures, generally with a diameter of ten to several hundred nanometers and lengths of tens of microns. Silver nanowire material is a kind of grayish powder and is often dispersed in solvents, such as water, ethanol, and isopropanol . The silThe fabrication process of the graphene and silver nanowire hybrid electrode with a wrinkled structure is illustrated in The first step is the transfer of the CVD-grown graphene onto the surface of the pre-stretched elastomer film. In this step, the copper layer is removed using a wet-etching process to allow the graphene transfer. In order to maintain electrical conductivity between the graphene islands on the elastomer surface, silver nanowires were sprayed on the wrinkled graphene surface. Then, the pre-stretched elastomer film was released, to prepare a hybrid electrode with a wrinkled structure. In order to fabricate a dielectric elastomer actuator with a wrinkled structure, the electrode formation method described above was also applied to the opposite side of the elastomer. Using this method, a wrinkled elastomer actuator whose light transmittance could be controlled was manufactured.To induce the electro-mechanical deformation of the dielectric elastomer actuator, a high input voltage over 1 kV needs to be applied to both electrodes. For this purpose, a high voltage power supply  was connected to both the top and bottom electrodes of the actuator. To connect each compliant electrode to a cable from the power supply, conductive copper tape was used instead of the conventional cable-wires. Actuation testing was performed on an experiment table, and video data was acquired using a complementary metal-oxide-semiconductor (CMOS) image sensor to show the areal expansion of the actuator. To observe the variation in optical transmittance under actuating conditions, we placed the fabricated actuator on the sample holder of a UV-VIS spectroscopy. The optical transmittance was then examined, as input voltages were applied to the actuator.In The working principle of the elastomer actuator is based on an attraction force which is generated between the two electrodes. This force causes an expansion of the electrode area in the in-plane direction. In order to examine the change of the surface in the multilayered elastomer-electrodes according to the pulling forces in the in-plane direction, the surface morphology was examined under tensile conditions, as shown in R/R0) remains stable in the initial section, and as the strain value is increased, the magnitude of the relative resistance value is changed sharply. These results can be explained based on the wrinkled structure of the electrode. As shown in The relative resistance of the electrodes was examined in order to investigate the influence of the wrinkling morphology. Three types of specimens with different compressive strain values were prepared for this experiment. The compressive strain values applied to the specimens were 20%, 33% and 42%, respectively. As shown in The dielectric elastomer actuator has a sandwich structure consisting of an elastomer film with two electrodes on both sides. For application, the elastomer film was fixed in a rigid frame made of polyacetal. A/A0) of 33%. Within 4 kV, the areal change of the actuator is almost similar in both cases, but there is a remarkable maximum difference of about 6% at 6 kV.In order to verify the operating performance of the actuator, we connected the electrode of the actuator to a DC high voltage supply capable of generating 0–10 kV. Then, the applied voltage was gradually increased from 0 to 6 kV, and the change in the area of the actuator was examined. For more precise performance analysis, it is essential to determine the area of the increased actuator under electrical stimuli. For this purpose, the moving images of the actuator were captured frame-by-frame, and analyzed using an image processing program. The actuation of conventional dielectric elastomer actuators using a high input-voltage caused the electrodes to expand with an isotropic areal strain as shown in As the applied voltage increases, the strength of the interaction force in the thickness direction increases in the elastomer actuator. This force has the effect of reducing the thickness of the film and increasing the area of the electrode. At this time, the transmittance of the active area is increased due to the reduced thickness of the film and the change in surface morphology on the surface of the elastomer film. Using the same method as that used for the wrinkle-free sample, the change in active area of the dielectric elastomer actuator with wrinkled graphene–AgNWs hybrid electrodes was measured with respect to the applied voltages. Then, the transmittance change according to area changes was also examined. The wrinkled electrode structure exhibited about a 33% change in area ratio, and a transmittance change of about 15%. That is, although the wrinkled structure showed the same area change rate, it can be confirmed that the transmittance was about twice as large as that in the case of the wrinkle-free sample. When compared with the structure without wrinkles, the cause of this phenomenon is understood to be the difference in transmittance due to the scattering of light.A novel dielectric elastomer actuator with wrinkled graphene and silver nanowire hybrid electrodes was developed in this study. Graphene and silver nanowires were used to realize the highly conductive and compliant electrodes. The developed dielectric elastomer actuator with the wrinkled surfaces exhibited a large in-plane deformation of up to 33%. As compared with wrinkle-free electrodes, the proposed actuator with wrinkled electrodes exhibited a 10% higher areal change rate because of the synergistic effect of the ultrahigh conductivity of the silver nanowires and the remaining capacitance of the graphene under large deformation. This novel elastomer actuator with wrinkled hybrid electrodes may be a candidate to tune the transmittance of a smart window system.