Rhus chinensis Mill.) ointment has been shown as an effective treatment for keloids. However, the protective mechanisms of Wubeizi ointment are not fully understood. The mammalian target of rapamycin (mTOR) has been demonstrated to be associated with keloid pathogenesis. In the present study, we investigated if Wubeizi ointment suppressed keloid formation through the modulation of key molecules of the rapamycin (mTOR) pathway including phosphatase and tensin homolog (PTEN), phosphatidylinositol 3-kinase (PI3K), and protein kinase B (Akt).  Wubeizi  followed by pairwise q-tests.  A keloid mouse model and human keloid-derived fibroblasts were developed and treated with Galla chinensis. Immunohistochemistry, western blot, and reverse transcription-PCR were used to detect PI3K, PTEN, Akt, and mTOR in keloid tissues and keloid fibroblasts. The apoptosis and proliferation rate of keloid fibroblasts was, respectively, analyzed by flow cytometry according to the MTT assay. Statistical analysis was done using SPSS version 20.0. For two variable comparisons, a two independent samples  Our in vivo and in vitro studies showed that Wubeizi ointment suppressed keloid formation through inhibition of fibroblast proliferation and promotion of fibroblast apoptosis. The underlying basis involves downregulation of p-Akt and p-mTOR as well as upregulation of PTEN.  These findings may contribute to a better understanding of the mechanisms of Wubeizi ointment for treating keloids. Keloids are formed because of abnormal overgrowth of scar tissue during the healing of skin wounds and are characterized by the proliferation of dermal fibroblast and the aberrant accumulation of extracellular matrix (type I and III) collagens, mucins, and glycosaminoglycans . Wubeiziβ (TGF-β1), PI3K, Akt, and mTOR was significantly increased in pathological scar fibroblasts than in normal skin tissue or fibroblasts [mTOR is a serine/theronine kinase which plays an important role in the regulation of metabolic processes and translation rates. mTOR as a regulator of collagen expression and inhibition of mTOR reduced extracellular matrix deposition. Tissue extracts obtained from keloid scar showed an increase in the expression of mTOR, p70KDa S6 kinase, and their activated forms, suggesting an activation of mTOR in keloid scars . The exproblasts . In addiroblasts . TherefoIn the present study, we investigated the effect of Wubeizi ointment on key molecules of the mTOR pathway including PTEN, PI3K, and Akt. The results may contribute to a better understanding of the protective effect of Wubeizi ointment against keloid pathogenesis.Salvia miltiorrhiza, Clematis, black vinegar, Galla Chinensis, Cortex Lycii, and alum. It was prepared as described previously [The formula of Wubeizi ointment includes Chinese herbs eviously . BrieflyThe keloid scar tissues were obtained from the Department of Dermatology of Xuzhou Hospital affiliated to Nanjing University of Traditional Chinese Medicine . The tissues were taken from the anterior part of chest after surgical resection for treatment, from 18 Chinese patients . The normal skin tissue was resected near the keloid scar tissue.n = 6 for each group). For the Wubeizi ointment-treated group, the corresponding concentration of Wubeizi ointment was applied to the skin . For the control group, only the matrix of Wubeizi ointment was applied to the skin .Eighteen nude mice were purchased from the animal facility of Xuzhou Medical University. They were housed under conditions of constant temperature and humidity and with a 12 h light–12 h dark cycle. Keloid models were generated as described previously . BrieflyAfter 30 days of Wubeizi ointment treatment, mice were anesthetized by intraperitoneal injection of 0.1% pentobarbital sodium at 0.1 ml/10 g of body weight and then killed by cervical dislocation, and the keloid tissues were disassociated. The size of the keloid tissues was measured by a vernier caliper as described previously .3) and placed into a flask. Subsequently, 5 ml of 0.25% trypsin solution  was added into the flask. After 14 hours of digestion at 4°C, protease was removed, and 5 ml of complete media containing 10% fetal bovine serum  was added into the flask. Single cell suspension was prepared. Cells were then subcultured in a 25 ml flask and incubated in an incubator  at 37°C with 5% CO2. The generation was passaged every 2–3 days. The 3rd generation cells at logarithmic growth phase were analyzed.The primary cells of the human keloid-derived fibroblasts were disassociated and cultured according to our published procedure . Brieflyμl DMEM containing 5% FBS was added to the culture medium. For the IGF-1-treated group, 20 μl IGF-1  was added. IGF-1 is an agonist of the mTOR signaling pathway. For the IGF-1+Wubeizi ointment-treated group, 20 μl mixture of IGF-1 and Wubeizi ointment was added. For the Wubeizi ointment-treated group, 20 μl of the Wubeizi ointment solution was added. The Wubeizi ointment solution used for in vitro experiments was prepared by dissolving the extract of Wubeizi ointment in water at a concentration of 0.5 mg/ml.Keloid fibroblasts were divided into four groups; i.e., control group, IGF-1-treated group, IGF-1+Wubeizi ointment-treated group, and Wubeizi ointment-treated group. For the control group, 20 μm thickness by Rotary Paraffin Microtome Slicer paraffin embedding machine , and stained with hematoxylin and eosin for routine examination. Subsequently, the keloid sections were incubated with antibodies against p-PI3K , PTEN  monoclonal antibody, Santa Cruz, CA, USA), p-Akt , or p-mTOR . Bound antibodies were visualized using 3,3′-diaminobenzidine (DAB) as a chromogen , and the slides were counterstained with hematoxylin. The positive expression of p-PI3K, PTEN, p-Akt, and p-mTOR was evaluated in four randomly selected fields under a light microscope . The mean optical densities (MOD) of positive expression were quantified with the Image Pro-Plus image analysis system .Immunocytochemistry was conducted as described previously . BrieflyΔΔCt−. Each RT-qPCR experiment was repeated three times.The mRNA levels of PI3K, PTEN, Akt, and mTOR in the keloid tissues and cultured keloid fibroblasts were analyzed as described previously . Brieflyμg) was separated on 15% SDS-polyacrylamide gel, transferred to nitrocellulose membrane, and blocked with 5% skim milk powder for 2 hours at room temperature. Membranes were incubated with primary antibody against PI3K , p-PI3K , PTEN , Akt , p-Akt , mTOR , p-mTOR , and actin  overnight at 4°C, washed with PBS, and then incubated with secondary antibody  and conjugated to horseradish peroxidase for 1 h at room temperature. The membranes were washed with PBS, and immunoreactivity was visualized on Odyssey infrared fluorescence imager . Images were analyzed by using the Image Pro-Plus image analysis system.Western blot was performed as described previously . Brieflyμl MTT was added to each well. After incubating for 4 h at 37°C, the liquid was removed, and dimethyl sulfoxide (150 μl/well) was added. After dissolution of the blue-purple formazan, optical density (OD) was measured 1 h later in a microplate reader  at a wavelength of 490 nm.The proliferation rate of keloid fibroblasts at specific time points  was measured by the MTT assay. The MTT assay was performed as described previously [μl of Annexin V-FITC was added to 2 × 105 cells for 15 min followed by 5 μl PI and incubated in dark at 4°C for 5 min. The cells were subjected to flow cytometry . Dot intensities in both Q2 and Q3 regions indicated the number of apoptotic cells.The apoptosis of keloid fibroblasts was analyzed by flow cytometry according as described previously . Brieflyt-test was used. For multiple variable comparisons, data were analyzed by the χ2 test or one-way analysis of variance (ANOVA) followed by pairwise q-tests.Statistical analysis was done using SPSS version 20.0 . Data were presented as mean ± S.D. For two variable comparisons, a two independent samples 3 (n = 6), which were significantly smaller than the sizes of keloid tissues disassociated from 3% Wubeizi ointment-treated nude mice  and control nude mice  (P < 0.05).As show in P < 0.05). In contrast, compared to the control group, the expression of PTEN significantly increased in keloid tissues from the 3% and 5% Wubeizi ointment-treated group, and the effect was dose-dependent (P < 0.05). However, there was no significant difference between the Wubeizi ointment-treated and control group in the expression of p-PI3K in keloid tissues (P = 0.735).The results of immunohistochemistry showed that most of the p-PI3K, p-Akt, and p-mTOR were expressed in cytoplasm and a small amount of p-PI3K, p-Akt, and p-mTOR was observed in nucleus . In contP < 0.05). In contrast, compared with the control group, mRNA and protein levels of PTEN were significantly increased in keloid tissues from the Wubeizi ointment-treated group, and the effect is dose-dependent . However, there were no significant differences found between the Wubeizi ointment-treated and control group in the mRNA of PIK3CA and phosphorylated protein levels of PI3K in keloid tissues .The results of qPCR and western blot showed that, compared to the control group, mRNA of Akt1 , and phoThese data suggested that Wubeizi ointment could downregulate the mRNA expression levels of Akt1 and mTOR and upregulate PTEN mRNA expression levels in keloid tissues. It also could reduce phosphorylated protein levels of Akt and mTOR in keloid tissues.P < 0.05). In addition, the inhibitory effect of Wubeizi ointment on fibroblast proliferation could be attenuated by IGF-1  (n = 24) (Results of the MTT assay showed that IGF-1 promoted fibroblast proliferation whilst Wubeizi ointment inhibited fibroblast proliferation (by IGF-1 . Results < 0.05) . The inh(n = 24) .P < 0.05).The results of qPCR showed that, compared to the control group, mRNA levels of PIK3CA, Akt1, and mTOR were significantly increased in keloid fibroblasts from the IGF-1-treated group (P < 0.05). However, there was no significant difference between the Wubeizi ointment-treated and control group in PI3K mRNA levels in keloid fibroblasts (P = 0.418). Compared to the control and IGF-1-treated group, mRNA levels of PTEN were significantly increased in keloid fibroblasts from the IGF-1+Wubeizi ointment-treated group and Wubeizi ointment-treated group (P < 0.01). However, there was no significant difference between the IGF-1-treated and control group in the mRNA levels of PTEN in keloid fibroblasts (P > 0.05). In contrast, compared to the control group, mRNA levels of Akt1 and mTOR were significantly decreased in keloid fibroblasts from the IGF-1+Wubeizi ointment-treated group and Wubeizi ointment-treated group (P < 0.05). The mRNA levels of Akt1 and mTOR in keloid fibroblasts from the IGF-1+Wubeizi ointment-treated group was higher than those in keloid fibroblasts from the Wubeizi ointment-treated group (P < 0.05).Compared to the control and Wubeizi ointment treated-group, mRNA levels of PIK3CA were significantly increased in keloid fibroblasts from the IGF-1+Wubeizi ointment-treated group and IGF-1-treated group (P < 0.05).The results of western blot showed that, compared to the control group, phosphorylated protein levels of p-PI3K, p-Akt, and p-mTOR were significantly increased in keloid fibroblasts from the IGF-1-treated group (P < 0.05). However, no significant difference was found between the control and Wubeizi ointment-treated-treated group in the protein levels of p-PI3K in keloid fibroblasts (P > 0.05). Compared to the control and IGF-1-treated group, protein levels of PTEN were significantly increased in keloid fibroblasts from the IGF-1+Wubeizi ointment-treated group and Wubeizi ointment-treated group (P < 0.05). No significant difference was found between the control and IGF-1 group in the protein levels of PTEN in keloid fibroblasts (P = 0.364). In contrast, compared to the control group, protein levels of p-Akt and p-mTOR were significantly decreased in keloid fibroblasts from the IGF-1 + Wubeizi ointment-treated group and Wubeizi ointment-treated group (P < 0.05). The protein levels of p-Akt and p-mTOR in keloid fibroblasts from the IGF-1+Wubeizi ointment-treated group was higher than those in keloid fibroblasts from the Wubeizi ointment-treated group (P < 0.05).Compared to the control and Wubeizi ointment-treated-treated group, protein levels of p-PI3K were significantly increased in keloid fibroblasts from the IGF-1+Wubeizi ointment-treated group and IGF-1-treated group . Aberrant accumulation of extracellular matrix collagens, mucins, and glycosaminoglycans because of an imbalance between collagen production and degradation of the extracellular matrix along can lead to the elevated production of cytokines and proinflammatory responses. Faulty fibroblast regulation resulting in proliferation of fibroblasts and apoptosis inhibition plays an important role in the keloid development and scar formation .Our previous study demonstrated that Wubeizi ointment increased fibroblasts at the S phase thereby greatly reduced dividing fibroblasts in addition to inhibiting the proliferation of keloid-derived fibroblasts in a time- and dose-dependent manner . Moreoveβ1 [β1 through upregulation of the miR-21 and PTEN/AKT signaling pathway [β. Scarring can be reduced in rodents by inhibition of TGF-β signaling proteins [β1 signal transduction [It has been demonstrated that the proliferation of keloid fibroblasts could be regulated by various cytokines, such as TGF-β1 . Keloid  pathway . Keloid proteins . The mTOsduction –12. The sduction . The mTOsduction –19. Pathsduction . These fIt was found that inhibition of mTOR can reduce extracellular matrix deposition , 21. In In conclusion, the present study investigated if Wubeizi ointment inhibited the keloid formation through modulation of key molecules of the mTOR pathway including PTEN, PI3K, and Akt. The results of the in vivo and in vitro studies suggested that Wubeizi ointment inhibited the proliferation of keloid fibroblasts and promoted the apoptosis of keloid fibroblasts, probably through downregulation of Akt and mTOR and upregulation of PTEN. These findings may contribute to a better understanding of the mechanism of Wubeizi ointment for treating keloid and provide a theoretical basis for its clinical application.


The superlattice potential generates a series of Dirac points (DPs) and van Hove singularities (vHSs) within an experimentally accessible low energy state, providing a platform to study orbital moments with respect to band structure. In this work, theoretical calculations and magnetothermoelectric measurements are combined to reveal the emergence of an orbital magnetic moment at vHSs in graphene/h-BN moiré superlattices. The thermoelectric signal for the vHS at the low energy side of the hole-side secondary DP exhibited significant magnetic field-induced valley splitting with an effective g-factor of approximately 130; splitting for other vHSs was negligible. This was attributed to the emergence of an orbital magnetic moment at the second vHS at the hole-side.Bloch electrons lacking inversion symmetry exhibit orbital magnetic moments owing to the rotation around their center of mass; this moment induces a valley splitting in a magnetic field. For the graphene/ h-BN moiré superlattice. Here, Moriya et al. report thermoelectric evidence of magnetic field induced valley splitting for a van Hove singularity in this superlattice, suggesting the emergence of an orbital magnetic moment.An orbital magnetic moment emerges as a result of inversion symmetry broken at the graphene/ The presence of the Berry curvature induces electrons to have anomalous velocity perpendicular to an applied electric field, thus exhibiting the valley Hall effect. The valley Hall effect has been demonstrated at the gapped Dirac band such as gapped bilayer graphene and monolayer graphene/h-BN moiré superlattice6. In contrast to this, an orbital magnetic moment will induce an energy shift under the application of the perpendicular magnetic field; this is known as valley Zeeman splitting and has been demonstrated in gapped bilayer graphene9 as well as transition metal dichalcogenides10. Both Berry curvature and orbital magnetic moment, in principle, strongly depends on the local structure of the band. Here we choose graphene/hexagonal-boron nitride (h-BN) as a model system to investigate the orbital magnetic moment in relation to the band structure.Berry curvature and orbital magnetic moment are two pseudovectors that are observed in the presence of spatial inversion asymmetry. These enable us to control valley contrasting phenomena in two-dimensional materialsh-BN insulator through van der Waals force, two significant changes occur in the band structure. First, the interaction between graphene and h-BN breaks the spatial inversion symmetry of graphene. Second, the lattice mismatch between two crystals generates a moiré pattern  derived from Fig. 19. These result in local minima in the DOS  and both electron- and hole-side SDPs  at this vHS. This is an analog of a spin angular moment of the electron. However, m(k) is orbital in nature, and it is responsible for the splitting between the K and K′ valley instead of the up- and down-spin. A general form of the m(k) is expressed as the following equation9:H(k) is the Bloch Hamiltonian, and ε(k) is the dispersion of band. We calculated m(k) for the band structure in Fig. m(k) with respect to the wavevector of the first, second, and third bands in the hole-side of the main DP, as depicted in Fig. m(k) is large for the gapped DPs, including both the main DP and SDPs3. Moreover, the calculation shows that there is a noticeably large m(k) presented at the second vHS. The second vHS(h) yields an m(k) value of 66 μB, corresponding to a valley g-factor of ~130. This value is noticeably large as an orbital magnetic moment of vHS, considering that m(k) is negligibly small at other vHSs such as Y-point in the first band , first vHS(h), and second vHS(h). The first vHSs on both the electron- and hole-side had previously been investigated25; here, we investigated the second vHS(h) which was observed27 but not discussed before. In addition to these, it is noteworthy that the Vind signal largely increases around nL = −4.65 × 1012 cm−2 in Fig. 2 dielectric.The two-terminal resistance Vind versus nL are measured in a magnetic field from B = + 0 to 2.23 T and results are shown in Fig. B , whereas the Vind signals from other vHSs, i.e., the first vHS(h) and the first vHS(e) , we present a comparison between the experimental values and the calculated DOS in aligned graphene/h-BN obtained from the continuum model12. In Fig. Vind as a function of nL and B plotted for +B and −B. The signals arising from three vHSs—the first vHS(e), first vHS(h), and second vHS(h)—are extracted from the data, and plotted with green, blue, and magenta solid circles, respectively in Fig. Vind data in high magnetic field region are presented in Supplementary Note n/n0 and B in Fig. E is also provided in Supplementary Note B-induced splitting of vHSs as well as the Landau fan diagram of the DPs were completely reproduced by the calculation. By plotting the K- and K′-valley contributions to the DOS separately in Fig. n/n0 value with the magnetic field. Furthermore, we plotted individual traces of Fig. Vind shown in Fig. To investigate the origin of the magnetic field-induced splitting in m(k) at the second vHS(h). To obtain a finite m(k) from Eq. 28  carbonate (PPC)-based dry release transfer method38, and the thickness of h-BN was chosen to be ~30–40 nm. Using e-beam (EB) lithography and reactive ion etching employing a mixture of CF4 and Ar gases, a trench was fabricated in the h-BN located between the two graphene flakes. Thus, the connection through h-BN between the two graphene flakes was eliminated. Finally, a contact pattern was created using an EB lithography with PMMA resist, and a 90-nm-thick Au/5-nm-thick Cr stack was deposited using EB evaporation. It should be noted that during device fabrication, the device was annealed at 350 °C under an Ar/3% H2 atmosphere after each flake transfer. This annealing caused the transferred graphene flakes to rotate within the plane toward the preferred orientation with respect to the adjacent h-BN40. In the device presented in Fig. h-BN, to generate moiré superlattice potential; however, the graphene on the right was not aligned.A device was fabricated using sequential dry release transfer of an individual flake. Two different flakes of monolayer graphene were successively transferred on top of the Iac = 10 nA, with a frequency of 18 Hz; the AC voltage was measured using a lock-in amplifier. A source measure unit (Keithley 2400) was used to apply constant power to the heater graphene.A liquid He-cooled variable temperature cryostat equipped with a superconducting magnet was used for transport measurement. The differential resistance of graphene was measured by applying an AC current Supplementary InformationPeer Review File


A novel graphene H-waveguide is proposed for active terahertz components. A graphene film illuminated by strong pumping light shorts the parallel conductor plates. The terahertz modes propagating along this film are amplified at certain conditions. A rigorous electromagnetic (EM) quasi-linear method of analytical calculations of  Today much attention is paid to two-dimensional materials and electronic components. Graphene is the first one consisting of carbon atoms and composing a flexible and stretchable honeycomb crystal lattice discovered by Geim and Novoselov . The there known ,5,6,7,8.The graphene sheets or strips can be placed over a dielectric substrate surface and combined with conductors forming the waveguides . The EM A part of graphene’s unique properties is its controllability by electric, magnetic, light fields, and chemical doping ,13. The Interesting techniques are when the real part of graphene complex conductivity njection ,19,20. Tic sheet , metal/gic sheet , conductic sheet , a dieleic sheet , and hybic sheet .The effectiveness of optically induced or charge injection lasing can be enhanced using the multilayered graphene ,25. For The nearest competitors to the graphene-based devices are the resonant tunneling diodes showing the amplification and generation of terahertz irradiation ,32. A prIn this contribution, a novel low-loss graphene-based waveguide  is propoThe terahertz range of frequencies is the place of complicated wave and material effects, and the development of new components is a challenging process . A part In microwaves, an H-waveguide consisting of two parallel conductor plates and a dielectric insert forming a figure of the letter H is known. The main mode of this waveguide is the quasi-TEM (transverse-EM) one  with Ey aveguide . This wain Refs. ,37,38,39In this paper, the central dielectric sheet of a nonradiative dielectric waveguide is substituted by a single or multilayered graphene film , the boundary conditions (4) and (5) at This expression gives a pair of forward and backward modes  whose prThe found longitudinal modal constant The Using (4), (5), (9) and (10), an analytical formula is derived for the modal propagation constants Similarly to the A graphene conductivity formula used here is from Refs. ,18(12)σThe zero position of quations  for charIn practice, according to the data of different authors, it is supposed that for the charge densities in order of –150 meV ,22,27. T devices . The gaiThe photo-induced carriers pump energy to traveling plasmons of the terahertz range, and the EM coupled-wave is amplified. This effect is approximately described by a graphene conductivity Formula (12) obtained using Kubo’s approach. In Ref. , it is sAnalyzing Formulas (8) and (11), it is seen that a critical frequency In The simulation of the imaginary parts of propagation constants shows that the negative conductivity effect makes these modes propagate with amplification occurring below and over their critical frequencies m)/k0)>0 . RemarkaAnother variant is when ed range . These wfilm see .As mentioned in y states . In grapuctivity . Below fsolution ,46,49,50Another mode marked as graphene . In thisFor comparison, consider the backward (0) mode  and calcThe imaginary part of the longitudinal propagation constant  is negatIn this way, other Concluding the research with the modes of different polarizations, it is seen that their interactions with graphene films highly depend on the modal type, as it was shown earlier in  in the rComing back to the An important question is on the controllability of amplification parameters. The modal parameters can be adjusted in part by dielectric filling. The increase in permittivity leads to some decrease in the phase z(1)/k0)  and lossonstants  as it fohe field  and the The proposed waveguide’s guiding properties’ overall review shows that all its complex It is worthwhile to notice that these results are obtained using the quasi-linear approach. Localization of the field close to graphene leads to increased field values, and if this field is strong enough, it influences the graphene conductivity. It limits the quasi-linear modeling’s applicability to the cases where amplification and signals are not so strong. In general, the problem should be formulated in its self-consisted nonlinear form and solved using, for instance, the iteration methods ,15. AnotAlthough the active graphene-based devices demonstrate an extremely high level of gain in order of 000 cm−1 , the queUsually, in terahertz frequencies, the conductor loss prevails the dielectric one. The loss level around 10–15 dB/mm is found below 10 THz, even in a parallel-plate waveguide for ing mode ,51.The conductor loss of complex modes below their cut-off frequencies is exceptionally high, and this worst-case scenario is studied below.The conductor loss constant tance Rs ,52,53,54tance Rs .An analytical formula used here to calculate the conductor loss constant The found propagation constant corrected by loss in conductor plates allows calculating the field components’ longitudinal dependence along the Overall review of the obtained results shows that the proposed structure can provide a very high gain of terahertz signals, similarly to the studied earlier planar structures. Still, the impact of many other factors should be additionally investigated in the future. Among them is the influence of nonlinearity ut loads ,32.In this paper, a novel guiding structure has been proposed for terahertz lasing applications. The waveguide consists of two parallel-plates of conductors shorted by a graphene film and composing an H-waveguide. The negative conductivity of graphene is induced by pumping light, and the THz modes are amplified propagating along with this structure. At the difference to known designs, the proposed waveguide can have decreased loss due to the complete exclusion of the sharp-edged conductors and graphene edged strips from its design and using the This waveguide has been analyzed in a quasi-linear regime using a rigorous EM method of field matching. The algorithm is formulated for the hybrid, The waveguide’s design can be enhanced further using the multilayered graphene conductors and heterostructures to increase pumping light absorption ,56. It hThe obtained results highlight the EM interaction mechanisms with graphene films showing the modal-type and mode-dependent amplification and irradiation in the studied open graphene-based structure. Based on our theory, a set of waveguides and components can be designed for the loss-compensated interconnections and active antennas. Self-oscillating devices can be developed by creating the active resonators in which the instability condition is provided by choosing the proper geometry of a proposed H-waveguide and loads .


Idiopathic pulmonary fibrosis (IPF) is a chronic disease characterized by fibroblasts activation, ECM accumulation, and diffused alveolar inflammation. The role of inflammation in IPF is still controversial and its involvement may follow nontraditional mechanisms. It is seen that a pathological microenvironment may affect cells, in particular mesenchymal stem cells (MSCs) that may be able to sustain the inflamed microenvironment and influence the surrounding cells. Here MSCs have been isolated from fibrotic (IPF-MSCs) and control (C-MSCs) lung tissue; first cells were characterized and compared by the expression of molecules related to ECM, inflammation, and other interdependent pathways such as hypoxia and oxidative stress. Subsequently, MSCs were co-cultured between them and with NHLF to test the effects of the cellular crosstalk. Results showed that pathological microenvironment modified the features of MSCs: IPF-MSCs, compared to C-MSCs, express higher level of molecules related to ECM, inflammation, oxidative stress, and hypoxia; notably, when co-cultured with C-MSCs and NHLF, IPF-MSCs are able to induce a pathological phenotype on the surrounding cell types. In conclusion, in IPF the pathological microenvironment affects MSCs that in turn can modulate the behavior of other cell types favoring the progression of IPF. Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and fatal interstitial lung disease (ILD) , charactUp to five different hypotheses for inflammation involvement in IPF have been proposed   secrete a wide pattern of pro- and anti-inflammatory cytokines ; in addiSOX2, OCT4, NANOG gene expression, while the expression of KLF4 was lower in IPF-MSCs than in C-MSCs (Lung tissues from three patients affected by IPF and from three patients undergoing lobectomy for adenocarcinoma (intended as controls limited to the IPF pathology) were used to establish cell cultures. Cells derived from IPF samples were named IPF-MSCs while the others served as controls (C-MSCs). Cells appeared homogeneous, with a fibroblastoid morphology A. All sun C-MSCs F.Finally, cells were strongly positive for CD73, CD90, and CD105 and negative for HLA-DR, CD14, CD19, CD34, CD45, and CD9 .https://imagej.net/Fiji) [Since it has been demonstrated that MSCs from pathological tissues acquire specific features , the expet/Fiji) , in IPF-Cell proliferation was evaluated after 72 h of culture. In mock samples, the proliferation of IPF-MSCs was significantly slower than C-MSCs A. After IL1A, IL1B, IL4, IL6, IL8, IL12, TNF-α and G-CSF) and to other connected pathways such as hypoxia , oxidative stress  and ECM pathway  was evaluated by qRT-PCR. First, the analysis was referred only to C- and IPF-MSCs to assess potential differences caused by the pathological microenvironment. The results showed a general increase of the expression of all the selected genes in IPF-MSCs with respect to C-MSCs except for NRF2 which is significantly downregulated ) showed significantly higher levels of expression of all the analyzed genes, and exception of nificant A, than Cnificant B.An ELISA assay was performed to evaluate the secretion of cytokines directly related to inflammation  in C- and IPF-MSCs. The results showed as the secretion of all the analyzed cytokines was significantly greater in IPF-MSC than in C-MSC  confirmiAfter co-cultures between MSCs, as observed for the gene expression, IPF-MSCs induced a significant increase of secretion by C-MSCs of all the analyzed cytokines, except for IL1B A. Again,Despite recent advances in the comprehension of IPF pathogenesis, the involvement of MSCs on IPF onset and development is still completely unknown. The aim of this study was to understand the effects of pathological microenvironment on MSCs and their consequent role in the onset of this pathology. First, cells isolated from control (C-MSCs) and fibrotic lung (IPF-MSCs) were characterized , confirmThe evaluation of stemness was integrated by the analysis of the expression of genes referred to as self-renew like SOX2, OCT4, NANOG, and KLF4. OCT4, SOX2 and NANOG form a reciprocal regulatory circuit whereby the equilibrium between stem cell self-renewal, proliferation, and differentiation is in perpetual balance . No signOxidative stress can play a key role in the development of IPF by the increasing ROS and consequent imbalance in the production of antioxidant enzymes . To evalNRF2 is known to protect against the development of pulmonary fibrosis by regulating the cellular redox level and lung Th1/Th2 balance . In addiAlthough some aspects are yet to be clarified, and further studies on animal model are necessary to evaluate the underlying mechanisms, in conclusion we can say that the pathological microenvironment affects the MSCs behavior: IPF-MSCs present a dysregulated expression/secretion of molecules related to ECM, inflammation, and interdependent pathways. In addition, when co-cultured with control cells, IPF-MSCs are able to drive an exceeding response by the other cell types moving them toward a pathological phenotype.The study was conducted in collaboration with the Pulmonology and Thoracic Unit of Azienda Ospedaliero-Universitaria “Ospedali Riuniti” ; all subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Institutional Ethics Committee of Università Politecnica delle Marche . Three patients with a final multidisciplinary diagnosis of IPF undergoing transbronchial lung cryobiopsy (TBLC) for diagnostic purpose according to current guidelines  were enrOCT4, SOX2, NANOG, KFL4) was analyzed by qRT-PCR. Cell pellet was collected from 3 × 105 cells after 72 h of culture and total RNA was extracted with Total RNA Purification Plus Kit . cDNA synthesis was performed using 5× All-In-One RT MasterMix . After amplification qRT-PCR was carried out with SsoFast EvaGreen Supermix  and fluorescence and melting curves were acquired. The parameter threshold cycle (Ct) was defined as the cycle number at which the first detectable increase above the threshold in fluorescence was observed. All samples were tested in duplicate with the housekeeping genes GAPDH for data normalization. mRNA expression was calculated by the 2−ΔΔCt method [Tissue fragments were cultured with MSCGM medium  . MSCs ist method . The pri4 cells were incubated with anti-collagen type I (COL1A1), anti-cellular fibronectin, and anti-α-SMA followed by goat anti-mouse FITC-conjugated antibody. Nuclei were visualized using Hoechst 33342 . For immuno-cytochemistry (ICC), 1.5 × 104 cells were incubated with anti-TGF-β1 (Sigma-Aldrich) and then immune-stained using the streptavidin–biotin–peroxidase technique  and incubated with 3,3-diaminobenzidine. Slides were counterstained with Mayer’s hematoxylin. To quantify the expression of the proteins, the percentage of area occupied by the protein inside the cells was calculated using Fiji-ImageJ software (available online at https://imagej.net/Fiji) [For indirect immuno-fluorescence (IIF), 1.5 × 10et/Fiji) .2.The cell line NHLF  was cultured in DMEM (Dulbecco’s modified Eagle’s medium)/Hams F-12 50/50 Mix , 10% FBS (Corning), 1% penicillin/streptomycin  at 37 °C and 5% CO5 IPF-MSCs were seeded at the lower surface and then 3 × 105 of C-MSC were added at the upper surface of a polycarbonate transmembrane filter  for 72 h [5 of NHLF were seeded at the lower of surface and then 3 × 105 of C- or IPF-MSCs were individually added at the upper surface of a polycarbonate transmembrane filter. After 72 h mock and co-cultured cells were counted with Countess Automated Cell Counter , collected and stored to −80 °C. The conditioned medium after co-cultures was collected, filtered with 0.2 µm filters and stored to −80 °C.Co-cultures were performed between C-MSC and IPF-MSC to evaluate the paracrine effects that could have one above the other. Total of 3 × 10for 72 h . In addiIL1A, IL1B, IL4, IL6, IL8, IL12, TNF-α, and G-CSF) and to other related pathways such as oxidative stress , hypoxia , and ECM . The primer sequences are summarized in −ΔΔCt method [Pellets collected from mock and co-cultured cells were used to analyze the expression of genes referred specifically to inflammation  were investigated by ELISA test with Multi-Analyte ELISArray kit  following manufactured instructions as previously described . ELISA tt-Student’s test. While, for three-experimental group comparisons significance was calculated by one-way or two-way ANOVA. The significance was set at p < 0.05.All experiments were performed in triplicate. For statistical analysis it was used GraphPad Prism 6 Software. All data are mean ± SD. For two-sample comparisons, significance was calculated by unpaired


Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease caused by the progressive death of both upper and lower motor neurons. The disease presents a poor prognosis, and patients usually die 2–5 years after the onset of symptoms. The hallmark of this disease is the presence of phosphorylated and ubiquitinated aggregates containing trans-active response DNA-binding protein-43 (TDP-43) in the cytoplasm of motor neurons. TDP-43 pathology has been associated with multiple pathways in ALS, such as metabolic dysfunction found in patients and in in vivo models. Recently, it has been described as a “prion-like” protein, as studies have shown its propagation in cell culture from ALS brain extract or overexpressed TDP-43 in co-culture and conditioned medium, resulting in cytotoxicity. However, the cellular alterations that are associated with this cytotoxicity require further investigation. Here, we investigated the effects of conditioned medium from HEK293T (Human Embryonic Kidney 293T) cells overexpressing TDP-43 on cellular morphology, proliferation, death, and metabolism. Although we did not find evidence of TDP-43 propagation, we observed a toxicity of TDP-43-conditioned medium and altered metabolism. These results, therefore, suggest (1) that cells overexpressing TDP-43 produce an extracellular environment that can perturb other cells and (2) that TDP-43 propagation alone may not be the only potentially cytotoxic cell-to-cell mechanism. Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease caused by the rapid deterioration of the superior and inferior motor neurons, leading to death of the patient 2–5 years following symptom onset . To thisThe C-terminus of TDP-43 has been associated with its propensity to aggregate. This region also exhibits a prion-like structure; it is a low-complexity domain, rich in glycine, asparagine, and glutamine residues, and has an undefined tertiary structure. Prions are classified as abnormally folded, transmissible proteins that seed the misfolding of otherwise normally folded copies of itself, usually causing their aggregation and cytotoxicity . In cellDrosophila overexpressing TDP-43 [Increasing evidence supports the hypothesis that metabolism is heavily modified in ALS, with reports on hypermetabolism, altered lipid/glucose metabolism, tricarboxylic acid (TCA) cycle, and mitochondrial function  in ALS pg TDP-43 , as wellg TDP-43 . AccordiTo investigate whether TDP-43 “prion-like” behavior was involved with metabolic disturbances, we performed metabolomics on HEK-293T cells cultured in conditioned medium from other HEK-293T cells having overexpressed TDP-43. Briefly, we overexpressed wild-type (WT) TDP-43 and added the corresponding conditioned medium to naïve recipient HEK-293T cells . AlthougThe full-length wild-type human TDP-43 sequence was cloned into the mammalian expression vector pcDNA3.3  using the following primers: forward_5′- TCTGAATATATTCGGGTAACCGAAG-3′ and reverse_5′- CTAGTGGTGATGGTGATGATGAGAACCCCCCATTCCCCAGCCAGAAGACTTAG-3′ . We were interested in the wild-type form of TDP-43 because this is the predominant form found in ALS patients, as mutated TDP-43 accounts for 5% of cases . A histiv/v) fetal bovine serum (FBS) at 37 °C  and in an incubator maintaining an atmosphere of 5% CO2 . In all, 700,000 cells were seeded in T-25 flasks . After 24 h, the seeded cells were either transfected with 8 µg of pcDNA3.3-wtTDP43-6×His vector and 16 µL of jetPEI transfection agent  or treated only with an equal quantity of jetPEI. After 4 h, the growth media were replaced with 8 mL of fresh media, and overexpression was allowed for 72 h. Conditioned medium from TDP-43-transfected cells was referred to as TDP-43 CM and that of cells only treated with jetPEI referred to as NT CM. After 72 h of overexpression, the conditioned media were collected and centrifuged at 1500× g for 5 min at room temperature to remove floating dead cells and debris. The media were then added to Amicon Ultra-15 centrifuge tubes  and concentrated by centrifugation at 10,000× g at room temperature until the volume was reduced 10-fold , representing a 10-fold concentrated conditioned medium that was then applied to downstream analyses.HEK-293T cells  were the cell line of choice due to its robust transfection efficiency and common application in studies on TDP-43 proteinopathy ,13,16,18w/v) powdered milk  in PBS (MPBS) for 2 h at room temperature, rinsed with PBST and PBS in the same manner, and incubated with each of the 10-fold concentrated NT CM and TDP-43 CM, and 10 µg/mL of purified GFP-wtTDP-43-6×His (positive control) for 1 h at room temperature. Wells were then rinsed five times with PBST, one time with PBS, and incubated in MPBST with anti-6×His antibody  for 1 h at room temperature. After rinsing in the same manner, each well was incubated with 100 µL of room-temperature TMB-ELISA solution  for 5–10 min. Finally, 50 µL of 2 M H2SO4 were added to stop the reaction, and the absorbance was measured at 450 nm.Nunc MaxiSorp 96-well plates  were coated overnight at 4 °C with 10 µg/mL of anti-TDP-43 antibody targeting the N-terminus . After 24 h, the coated wells were rinsed three times with phosphate-buffered saline supplemented with 0.1% Tween-20 (PBST) and one time with PBS. Wells were saturated with 4% (Recipient cells were either non-transfected or transfected with pcDNA3.3-wtTDP43-6xHis. Each cell type received either NT CM or TDP-43 CM. Non-transfected cells in NT CM (NT CM/NT cells) represented the healthy control. Transfected cells in NT CM (NT CM/TDP-43 cells) represented cells exhibiting TDP-43 proteinopathy in control medium. Non-transfected cells in TDP-43 CM (TDP-43 CM/NT cells) represented cells without TDP-43 proteinopathy in the conditioned medium of cells having endured TDP-43 proteinopathy. Finally, transfected cells in TDP-43 CM (TDP-43 CM/TDP-43 cells) represented cells displaying both TDP-43 proteinopathy and conditioned medium from the same type of cells.®, Lyon, France) was added to samples of 250,000 cells at a final concentration of 10 µg/mL in PBS/1% FBS. This molecule is taken up by dead cells because their plasma membrane becomes less intact. After 30 min of incubation at 37 °C with 5% CO2, cells were placed on ice before being analyzed by a Becton Dickinson Accuri™ C6 Plus flow cytometer . Recipient HEK-293T cells were seeded in 6-well plates  at 250,000 cells/well in 2 mL of DMEM with 5% FBS. After 24 h, cells were either treated only with 6 µL of jetPEI/well (NT cells) or transfected with 3 µg of pcDNA3.3-wtTDP-6×His vector and 6 µL of jetPEI/well (TDP-43 cells). After 24 h, 200 µL of medium were removed from each well, and 200 µL of 10-fold concentrated NT CM or TDP-43 CM were added to the corresponding wells to dilute the conditioned media to 1×. The rationale was to introduce recipient cells to a 1× concentration of conditioned medium without depriving them of the basic nutrients provided by fresh culture medium. This would control for non-specific effects related to old, nutrient-deprived medium. Following a 24 h incubation at 37 °C, cells were trypsinized in 0.25% trypsin , washed in PBS, and resuspended in PBS/5% FBS. For cell death, propidium iodide  and forward scatter (FSC: size). The distribution of cells from the SSC/FSC ratio of NT CM/NT cells was considered the healthy, morphological control. Densities that deviated from this ratio were considered to have aberrant morphologies.®, Lyon, France) in 200 µL of DMEM with 5% FBS. Replicates of three were seeded per condition. After 24 h, cells were either treated only with 240 nL of jetPEI/well (NT cells) or transfected with 120 ng of pcDNA3.3-wtTDP-6×His vector and 240 nL of jetPEI/well (TDP-43 cells). After a 24 h incubation at 37 °C with 5% CO2, 20 µL of medium were removed from each well, and 20 µL of 10-fold concentrated NT CM or TDP-43 CM were added to the corresponding wells to dilute the conditioned media to 1×. Following the 24 h incubation at 37 °C, the medium was removed and replaced with 0.5 mg/mL of MTT diluted in Hank’s Balanced Salt Solution . Following 30 min of incubation at 37 °C, the medium was withdrawn, the precipitated formazan was solubilized with 100 μL of dimethyl sulfoxide , and proliferation was quantified by spectrophotometry at a wavelength of 570 nm.Proliferation was measured by MTT -2,5-diphenyltetrazolium bromide) reduction assay . Recipient cells were seeded in 96-well plates at 10,000 cells/well  coated with 100 µg/mL poly-D-Lysine  and universal nuclease  with agitation for 15 min at 4 °C. Protein content of whole-cell lysates was measured by the Lowry method . Then, 30 µg of protein from each cell lysate were separated in 4–20% SDS-PAGE gel apparatus  and transferred to PVDF (polyvinylidine fluoride) membranes. After blocking with 5% (w/v) Régilait in Tris-buffered saline supplemented with 0.2% Tween-20 (TBS-T), membranes were incubated for 1 h at room temperature with the following primary antibodies: mouse HRP-conjugated anti-β-actin  for an internal control, mouse anti-GFP  for GFP detection of GFP-6xHis, and mouse HRP-conjugated anti-6×His  for detection of GFP-6xHis and wtTDP-43-6×His, respectively. For GFP-specific detection, the primary incubation was followed by 1 h incubation with a secondary antibody coupled to horseradish peroxidase . Chemiluminescence was observed by adding Clarity Western ECL substrate to the membrane followed by visualization using Chemidoc apparatus  after incubation with ECL. Band intensity was measured with Image Lab software v6.1 .One million recipient cells in each condition were harvested in ice-cold PBS and centrifuged at 900× 2, 20 µL of medium were removed from each well and 20 µL of 10-fold concentrated NT CM or TDP-43 CM were added to the corresponding wells to dilute the conditioned media to 1×. Following a 24 h incubation, the cells were analyzed by a Seahorse XF analyzer  to measure the following metabolic parameters: basal respiration, proton leak, maximal respiration, spare respiratory capacity, non-mitochondrial respiration, ATP production, non-glycolytic acidification, glycolysis, glycolytic capacity, and glycolytic reserve.Recipient HEK293T cells were seeded in a Seahorse XF96 microplate  at 10,000 cells/well in 200 µL of DMEM with 5% FBS. Replicates of three were seeded per condition. After 24 h, cells were either transfected with 120 ng pcDNA3.3-wtTDP43-6×His vector and 240 nL jetPEI (TDP-43 cells) or only supplemented with an equal amount of jetPEI (NT cells). After a 24 h incubation at 37 °C with 5% COg for 5 min at 4 °C. Approximately one million cells were resuspended in 1 mL of ice-cold PBS and centrifuged using the same parameters as above. Cells were dried by removing all of the PBS and then stored at −80 °C. On the day of analysis, cells were thawed and resuspended in ice-cold ethanol/1× PBS at a ratio of 85:15, then subjected to 3 freeze/thaw cycles in liquid nitrogen. Samples were centrifuged at 18,000× g at 2 °C for 5 min. Samples were loaded onto a filter paper and dried in a stream of nitrogen for derivation with a solution of 5% phenyl isothiocyanate. Afterward, dried residues were extracted with methanol containing 5 mM ammonium acetate. The MetIDQ® software  was used to calculate the concentrations of individual metabolites. Quality controls (QCs) were analyzed regularly on the plate (every 8 samples) to ensure the stability of the mass spectrometer over time.A targeted, quantitative approach was employed based on the AbsolutIDQ™ p180 kit  using a flow injection analysis and high-performance liquid chromatography (HPLC) tandem mass spectrometry (MS/MS) protocol . This assay permits the quantification of 188 metabolites . Recipiehttp://www.metaboanalyst.ca). The classification method was based on partial least squares discriminant analysis (PLS-DA). Values of variable importance in projection [p < 0.1) after a non-parametric Wilcoxon test (volcano plot), while the multivariate analysis of metabolites depended on the accuracy of the variable importance in the projection (VIP). Therefore, the metabolites that had VIP values > 1 of the multivariate analyses and those that were determined by an FC > 2 (for each comparison) with p < 0.1 of the univariate analyses were retained as the most relevant for further discussion [For metabolomics analysis, two separate analytical methods were employed to identify discriminant metabolites—multivariate and univariate analyses. These analyses were performed using MetaboAnalyst v4.0 . Results were considered significant with p < 0.05.Results are shown as mean ± standard error of the mean (SEM). Cytometry results were analyzed using FlowJo VX software v.10.0 . When relevant, the Mann–Whitney non-parametric p = 0.0159) within 72 h post-transfection (p = 0.0357) as a positive control , we observed that overexpressed TDP-43 had been released into the culture medium , compared to control (NT CM/NT cells). Conversely, non-transfected cells incubated for 24 h in TDP-43-condtioned medium (TDP-43 CM/NT cells) exhibited a slightly, but significantly, higher proliferation rate (p = 0.0286) in comparison to the control (p = 0.0079) when compared to the control group. A similar alteration was observed in TDP-43 CM/NT cells (p = 0.1270). Regarding cell death by propidium iodide uptake, we did not find significant differences among the conditions but observed the following two notable trends: NT CM/TDP-43 cells tended to reveal a higher amount of uptake (p = 0.1), as well as TDP-43 CM/NT cells (p = 0.1), compared to NT CM/NT cells (The proliferation rate of HEK293T cells overexpressing TDP-43 and incubated for 24 h in NT-conditioned medium (NT CM/TDP-43 cells) was significantly reduced . Therefore, these data suggest that neither TDP-43 overexpression nor TDP-43-conditioned medium caused severe alterations in energy metabolism.To assess an effect of TDP-43-conditioned medium on the energy metabolism of cells, the rates of oxidative phosphorylation (oxygen consumption rate (OCR)) and glycolysis (extracellular acidification rate (ECAR)) were measured. Across all metabolic parameters, TDP-43-conditioned medium did not appear to perturb energy metabolism . NeitherFollowing a multivariate analysis comparing the metabolomes of recipient cells in each condition, we found clusters corresponding to the particular metabolomes of the different conditions A. We canTo further identify the most significant metabolites that would explain the separation of the different clusters, we performed 2-by-2 multivariate models for these conditions. First, we investigated the metabolome profile of cells overexpressing TDP-43 in control conditioned medium (NT CM/TDP-43 cells vs. NT CM/NT cells). The top 15 significant metabolites revealed by multivariate analysis (100% accuracy) are displayed in Finally, the 15 metabolites of each metabolome profile were compared E. Six meTo determine whether the disturbed metabolites associated with TDP-43 overexpression was a phenomenon associated with the overexpression of whichever protein, we also evaluated the metabolome profiles of TDP-43 and GFP-overexpressing cells without conditioned medium . The metTo our knowledge, two other studies have investigated the effect of TDP-43 in conditioned medium from overexpressing HEK-293T cells, which present contradictory findings pertaining to TDP-43 propagation within conditioned medium. One study indicated the transmission of TDP-43; overexpressed TDP-43 was detected in the medium encapsulated in microvesicles/exosomes and transmitted to recipient HEK-293T and primary neurons, in which cytotoxicity followed . The othThe recovery of a fraction of the overexpressed TDP-43 in the cell-free medium  corroborWe were unable to observe by immunoblot the propagation of overexpressed TDP-43 in recipient cells incubated in conditioned medium . This coIn addition to the factor of assay sensitivity, the state of TDP-43 oligomerization could have influenced the discrepancy between our findings and those of Feiler et al. . BecauseHEK-293T cells overexpressing TDP-43 demonstrated compromised morphology, decreased proliferation, and exacerbated cell death, which have been reported in various cellular models of TDP-43 overexpression ,18. HoweRemarkably, the conditioned medium seemed to increase proliferation . This coG93A NSC-34 cells [Given the different clusters of metabolomes A, we ded34 cells . Interes34 cells . TherefoDrosophila TDP-43 overexpression model [Furthermore, the eight altered metabolites that were only identified in the model comparing NT CM/NT cells and naïve cells cultured in TDP-43-conditioned medium suggested separate ALS-associated defects not highlighted in TDP-43-overexpressing cells. These metabolites included biogenic amines , glycerophospholipids, hydroxysphingomyelins, and acylcarnitines E. Carnoson model . As a reon model . These fInterestingly, the effects in biogenic amines, lipid distribution, and acylcarnitines not found in TDP-43-overexpressing cells were then observed in these cells once in contact with TDP-43-conditioned medium E. This sAltogether, we observed a metabolism alteration associated with TDP-43 overexpression and TDP-43 conditioned medium with common and distinct discriminant metabolites, suggesting that the putative mechanisms of toxicity are not completely the same. As we showed that these metabolites were not found in the other tested conditions, it will be necessary to show the relationship between these metabolism alterations and cell toxicity and to focus on these alterations, regardless of the specificity to TDP-43.The findings of this study suggest that the conditioned medium resulting from cells overexpressing TDP-43 provokes deleterious effects in other cells. These effects seem related to the changes in the metabolome profile of these cells, which could represent the underlying toxic mechanisms. On the one hand, the common metabolome alterations between TDP-43-overexpression and TDP-43-conditioned medium could represent mechanisms that would occur in both cells overexpressing TDP-43 and naïve cells in contact with the associated conditioned medium. On the other hand, the modified metabolites only found in naïve cells with TDP-43-conditioned medium probably indicate distinct and deleterious mechanisms. However, the scope of this study did not allow for the determination of the toxic outcome of such metabolic changes. Therefore, it will be important in future studies to investigate more closely these metabolites to elucidate whether specifically altering them will lead to cytotoxicity. Moreover, future endeavors should include neuronal cell types, such as iPSC-derived motor neurons, to validate these findings.This study also raises the question as to which species in the conditioned medium acted to bring about such changes. The results shed light on the hypothesis that TDP-43 propagation could not be the only threatening factor in the medium surrounding cells. Since many avenues of the metabolome appeared to be altered in recipient cells, it is unlikely that TDP-43 propagation would be responsible for all outcomes. Furthermore, the release of TDP-43 does not exclude the possibility that other yet-to-be identified species were released in parallel. At this stage, it remains unclear whether the additional factors are specifically linked to overexpressed TDP-43 or non-specifically triggered from the resulting cellular stress. In order to better understand the role of TDP-43 propagation in the context of ALS, these other factors should not be overlooked.


FES1A leads to a defect in thermotolerance of Arabidopsis; however, independent fes1a mutants exhibit a range in the extent of thermosensitivity. Here, we found that BRF2, a gene adjacent to FES1A and encoding a component of transcription factor IIIB, affects the thermosensitivity of fes1a mutants. Knockout of BRF2 suppressed fes1a thermosensitivity, while overexpression of BRF2 increased thermosensitivity of fes1a. BRF2 in fes1a mutants regulates the transcriptional strength of RNA Polymerase II and accumulation of heat shock proteins and eventually affects the thermotolerance of fes1a. There is a cross-talking between RNA Pol III and Pol II. The cross-talking is initiated by BRF, magnified by the mutation of FES1A, and finally has an effect on thermotolerance.FES1A is a heat shock protein 70 binding protein. Mutation of  Plants are sessile organisms. To cope with high-temperature stress, plants have evolved various adaptive mechanisms to optimize their growth and development to ultimately achieve successful reproduction. Thermotolerance in plants is associated with a number of regulatory and functional genes which constitute complex signaling pathways. It has been found that abscisic acid, salicylic acid, ethylene, reactive oxygen species, membrane fatty acid composition, ubiquitin, heat shock proteins (HSPs), programmed cell death, and photooxidation are all involved in thermotolerance.Arabidopsis mutant shows a defect in thermotolerance  and a substrate-binding domain (SBD). ATPase activity is contained in the NBD. In the ATP-bound state, the SBD of HSP70 loosely associates with its substrates . Once ATtibility .Eukaryotic genes and RNAs are transcribed by three RNA polymerases. RNA polymerase (Pol) I and Pol II are responsible for transcribing rRNAs and mRNAs, respectively, while RNA Pol III transcribes tRNAs, 5S ribosomal RNAs, 7SL RNAs, RNase P, and U6 spliceosomal small nuclear RNAs . Genes tfes1a exhibited defects in thermotolerance but to different degrees . T-DNA insertions were confirmed using PCR and sequencing (quencing .ScBRF1-ΔN10 cDNA fragment (lacking the coding sequence for the first 10 amino acids) was cloned into pYX242WS ×2-pMY72. The PCR-amplified fusion fragment of BRF2-(HA)×2 .The cDNA of ® 480 (LC480) using the SYBR Green system. The gene specific primers used in the qRT-PCR analysis are listed in ACTIN8 gene was used as an internal control.Total RNA was isolated from 15-day-old seedlings using TRIzol reagent (Invitrogen). First-strand cDNA was synthesized using a PrimeScript™ RT Reagent Kit with a gDNA Eraser (Takara). RT-PCR was performed using the primers shown in Western blot was performed as described previously  using thP < 0.05).All experiments in this study were performed at least three times, and the values are expressed as the mean ± SD. Statistical significance was determined by Duncan's multiple range test  plants, finally perished, indicating that all fes1a mutants possessed thermotolerance defects, but with different sensitivities. In addition, the survival rates of the seedlings undergoing an acquired thermotolerance challenge indicated that fes1a-3 had the best thermotolerance among the three fes1a mutants, with the survival rate reaching 92.5%, while fes1a-2 and fes1a-1 had 41.7% and 0.8% survival, respectively , BRF2 (At3g09360), and BRF3 (At2g01280). We further bred the double mutants of BRFs. The homogenous double mutants of brf1 brf3 and brf2 brf3 were generated, while the double mutation of BRF1 and BRF2 led to sterility  of RNA Polymerase II largest subunit 1 (RPB1), using anti-S2P-CTD and anti-S5P-CTD antibodies. The CTD of RPB1 is composed of tandem heptad repeats that are critical for the activity of RNA Pol II  and the Research Fundamental Capacity Improvement Project for Middle Age and Youth Teachers of Guangxi Universities (2019KY0517). Research in the lab of DZ was supported by the National Science Foundation grant IOS-1322796 and the Research Growth Initiative (RGI). The funder had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cytoplasmic inclusion of TAR DNA-binding protein 43 (TDP-43) is a pathological hallmark of amyotrophic lateral sclerosis (ALS) and a subtype of frontotemporal lobar degeneration (FTLD). Recent studies have suggested that the formation of cytoplasmic TDP-43 aggregates is dependent on a liquid–liquid phase separation (LLPS) mechanism. However, it is unclear whether TDP-43 pathology is induced through a single intracellular mechanism such as LLPS. To identify intracellular mechanisms responsible for TDP-43 aggregation, we established a TDP-43 aggregation screening system using a cultured neuronal cell line stably expressing EGFP-fused TDP-43 and a mammalian expression library of the inherited ALS/FTLD causative genes, and performed a screening. We found that microtubule-related proteins (MRPs) and RNA-binding proteins (RBPs) co-aggregated with TDP-43. MRPs and RBPs sequestered TDP-43 into the cytoplasmic aggregates through distinct mechanisms, such as microtubules and LLPS, respectively. The MRPs-induced TDP-43 aggregates were co-localized with aggresomal markers and dependent on histone deacetylase 6 (HDAC6), suggesting that aggresome formation induced the co-aggregation. However, the MRPs-induced aggregates were not affected by 1,6-hexanediol, an LLPS inhibitor. On the other hand, the RBPs-induced TDP-43 aggregates were sensitive to 1,6-hexanediol, but not dependent on microtubules or HDAC6. In sporadic ALS patients, approximately half of skein-like TDP-43 inclusions were co-localized with HDAC6, but round and granular type inclusion were not. Moreover, HDAC6-positive and HDAC6-negative inclusions were found in the same ALS patient, suggesting that the two distinct pathways are both involved in TDP-43 pathology. Our findings suggest that at least two distinct pathways  are involved in inducing the TDP-43 pathologies. TDP-43 is a DNA/RNA-binding protein that is predominantly localized in nuclei and plays multifunctional roles in RNA metabolism, including pre-mRNA splicing, translational control, and mRNA stability3. However, in most of the ALS and FTLD-TDP cases, TDP-43 is mislocalized into the cytoplasm and forms inclusion bodies5. In addition to this, more than 50 mutations in the TARDBP gene, encoding TDP-43, have been identified as a cause of inherited ALS6. These observations suggest that dysfunction of TDP-43 is a significant component of ALS pathogenesis. Thus, understanding the mechanism of TDP-43 aggregation will uncover the mechanistic basis for TDP-43 pathology and neurodegeneration in ALS/FTLD.Abnormal accumulation of TAR DNA-binding protein-43 (TDP-43) is a pathological hallmark of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease characterized by a selective loss of motor neurons, and a subtype of frontotemporal lobar degeneration (FTLD-TDP)8. LLPS is a process in which proteins and nucleotides abruptly segregate into two distinct phases, enabling the formation of intracellular membrane-less organelles9, such as p-bodies10 and stress granules12. Under physiological conditions, LLPS enables to achieve high local concentrations for molecular interactions and rapid chemical reactions, and allow fast changes of molecules upon signaling for facilitating various intracellular biological processes, e.g., transcriptional regulation and signal transduction13. However, once excess amounts of proteins are accumulated together with dysregulation of LLPS, the complexes quickly transform into pathological inclusions that are often found in neurodegenerative diseases15. ALS causative gene products, including FUS, TIA-1, and, of course, TDP-43, are proposed to form aggregates via LLPS. Consistent with this hypothesis, recent studies have discovered that optical multimerization of cytoplasmic TDP-43 induces the aggregation and sequestration of endogenous nuclear TDP-43 into the cytoplasmic aggregates that are dependent on LLPS17.Recently, a number of studies revealed that many kinds of RNA-binding proteins (RBPs), including TDP-43, spontaneously develop granule-like structures via a liquid–liquid phase separation (LLPS) mechanism19. For instance, SQSTM1 and TBK1 encode proteins involved in autophagy, while PFN1 and TUBA4A encode cytoskeletal proteins. Considering that TDP-43 pathology is also observed in the most of inherited ALS/FTLD patients, there may be the intracellular mechanisms other than LLPS stimulating the formation of TDP-43 aggregates in ALS/FTLD lesions.However, whether LLPS is solely responsible for the cytoplasmic aggregation of TDP-43 remains unclear. Among the over 25 inherited ALS/FTLD causative genes, there is a considerable number of the genes encoding the proteins that have the functions different from RNA regulationIn this study, we aimed to determine the intracellular pathways involved in the formation of TDP-43 aggregates. Using a screening system of cytoplasmic TDP-43 aggregations induced by the expression of inherited ALS/FTLD causative genes, we established that, in addition to the RBPs, microtubule-related proteins (MRPs) also co-aggregated with TDP-43 in the cytoplasm. The MRPs induced co-aggregation of TDP-43 via the formation of aggresomes, cytoplasmic aggregations that are dependent on microtubules. In contrast, the RBPs-induced aggregations were dependent on LLPS. Therefore, both the TDP-43 co-aggregation pathways were independent from each other. Moreover, we also showed that a subset of TDP-43 inclusions in spinal motor neurons of sporadic ALS cases were co-localized with histone deacetylase 6 (HDAC6), an aggresome marker. Our findings suggest that at least two distinct pathways  are involved in inducing the TDP-43 pathologies.Antibodies used in this study are as follows; anti-HDAC6 , anti-γ-tubulin , anti-vimentin , anti-TDP-43 , anti-β-actin , anti-RFP  Co LTD, Nagoya, Japan).20. To establish the cell line stably expressing EGFP-fused human TDP-43 wild-type or nuclear localization signal (NLS)-deficient mutant21, we transfected linearized pcDNA3.1(+)  inserted each TDP-43 complementary DNA (cDNA) with Lipofectamine 2000 (Thermo Fisher). The cells were cultured for two weeks in a growth medium containing 1.0 g/L G-418 . The colonies were isolated and confirmed the expression of EGFP-TDP-43 by fluorescent microscopy and immunoblotting. The established cell lines were maintained in a growth medium containing 1.0 g/L G-418. HeLa (CCL-2) and HEK293 (CRL-1573) cells, both obtained from ATCC, were maintained in Dulbecco’s Modified Eagle’s’ Medium (DMEM) containing 4.5 g/L glucose supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin  at 37 °C in a humidified chamber containing 5% CO2. Total RNAs of HeLa and HEK293 cells were isolated using the RNeasy-micro kit .Mouse neuroblastoma Neuro2a (RRID: CVCL_0470) (N2a) cells were maintained and differentiated, as described elsewhereHdac6 gene was obtained from Thermo Fisher. siRNA (20 nM) was transfected into N2a cells using Lipofectamine RNAi Max (Thermo Fisher) as described by the manufacturer.Stealth siRNA against the murine 22. ALS/FTLD-causing mutations were introduced according to the guidance of the Primestar Mutagenesis Basal Kit .Full-length wild-type cDNA of each familial ALS causative gene was amplified using the gene-specific primers from cDNA synthesized from total RNA of HeLa or HEK293 cells. The amplified cDNA was inserted into pmCherry-N1/C1 vectors  using SLiCE reaction4 /well on a 4 well cover-slide chamber  coated with poly-D-lysine. On the next day, 0.25 µg/well plasmids to express inherited ALS/FTLD causative genes were transfected with Lipofectamine 2000 (Thermo Fisher) according to the manufacturer’s guide. After 6 h of the transfection, the medium was changed into a differentiation medium (DMEM + 2%(v/v) fetal bovine serum and 2 mM N6,2′-O-dibutyryladenosine-3′,5′-cyclic monophosphate ), and cultured for 48 h. Then, the non-fixed cells were observed using laser scanning confocal microscopy  and the equipped software .N2a cells stably expressing EGFP-fused TDP-43, which is described above, were seeded at 5.0 × 10For the treatment with 1,6-hexanediol  (Sigma), the medium was replaced with a fresh one with/without 4%(v/v) 1,6-HD and incubated for 10 min at room temperature. Then, the non-fixed cells were observed using laser scanning confocal microscopy.23. For TDP-43 insolubility assay, the cells were first lysed in TBS supplemented with 1 mM EDTA, 1%(v/v) Triton X-100 and protease inhibitor cocktail by brief sonication on ice. After centrifugation at 15,000 × g, at 4 °C for 5 min, the supernatants were collected as soluble fraction. The pellets were resuspended in TBS supplemented with 1 mM EDTA and 2%(w/v) sodium-dodecyl sulfate (SDS) by brief sonication, then they were subjected to centrifugation at 15,000 × g, room temperature for 5 min. The supernatants were collected as insoluble fractions. Aliquots containing 15 µg/lane of soluble fractions and the same volume of corresponding insoluble fractions were analyzed by immunoblotting. For immunofluorescence analyses of γ-tubulin and vimentin, cultured N2a cells were transfected and cultured in a differentiation medium for 48 h. Then, the cells were fixed with ice-cold methanol at −30 °C for 20 min. After three times wash with TBS (50 mM Tris-HCl (pH 7.4), 150 mM NaCl), the cells were immunostained as described previously23. Briefly, the cells were incubated with primary antibodies at 4 °C for over-night, following incubation with secondary antibodies at room temperature for 2 h. Immunofluorescence images were obtained by a confocal laser scanning microscopy  and the equipped software .Immunoblotting analyses were performed as described elsewhereSpecimens of spinal cords from six patients with sporadic ALS and two control patients with other neurological diseases were obtained by autopsy with informed consent .All the data from immunofluorescence and semi-quantitative immunoblotting were analyzed by an unpaired WT or TDP-43∆NLS, respectively) as reporter cell lines to visualize TDP-43 aggregates  and RBP (FUSP525L) in N2a reporter cells stably expressing EGFP-TDP-43∆NLS revealed that the aggregates induced by MRPs and RBPs were distinctly partitioned in cytoplasm , an inhibitor of LLPS, to assess the involvement of LLPS in the co-aggregation of RBPs and TDP-43. As shown in Fig. FUS and TIA1. Consistent with the decreased number of RBPs-induced aggregates, sequestration of TDP-43 into the aggregates was also significantly prevented  or co-aggregates with TDP-43  and TDP-43  were not affected by nocodazole  recognizes misfolded proteins and transports them to aggresomes through microtubuleslls Fig. . As showThe results of TDP-43 co-aggregation obtained by our screening system are based on the overexpression of the inherited ALS/FTLD causative genes. However, it is unclear whether the mechanisms for the formation of TDP-43 aggregation observed in our study are involved in the TDP-43 pathology of sporadic ALS. To examine the involvement of the aggresome pathway in TDP-43 pathology, we examined the co-localization of TDP-43 and HDAC6, an aggresomal marker, in spinal motor neurons of sporadic ALS patients. We found that approximately half of TDP-43 skein-like inclusions were co-localized with HDAC6  Fig. . It shouIn this study, we demonstrated that: (i) at least two distinct intracellular pathways were involved in TDP-43 cytoplasmic aggregation, including aggresome formation and LLPS; (ii) the two pathways were independent of each other; and (iii) in spinal motor neurons of sporadic ALS, both HDAC6-positive and HDAC6-negative aggregates were found in the same ALS patient. Therefore, to the best of our knowledge, we are the first to confirm that HDAC6-dependent aggresome formation is partially involved in skein-like TDP-43 inclusion of sporadic ALS.Our study revealed that over 70% of the examined inherited ALS/FTLD causative 19 genes induced TDP-43 co-aggregation in cultured neuronal cells and the fluorescent-based observation of TDP-43 co-aggregates was well consistent with the TDP-43 insolubility examined by immunoblotting. Our screening system enabled us to assess whether any genes of interest induce cytoplasmic TDP-43 aggregation by a simple transfection-based screening. Moreover, our screening system has been proved useful for investigating the mechanism of TDP-43 aggregation. Indeed, as described above, we identified two distinct mechanisms involved in TDP-43 aggregation by using this system . Our screening system will also be useful for identifying compounds that prevent cytoplasmic TDP-43 aggregation, which may contribute to a novel therapeutic strategy for ALS/FTLD.19. Notably, we did not find any evidence of TDP-43 aggregation in the neuronal cells expressing the inherited ALS/FTLD causative genes related to protein degradation , although the previous studies reported TDP-43 pathology in inherited ALS patients with these mutations18. Moreover, the co-localization of TDP-43 and the inherited ALS/FTLD causative gene products are still controversial even though TDP-43 pathology is widely observed in the patients with ALS. For example, some studies have suggested the co-localization of TDP-43 with TIA-131 or FUS32 but other studies do not34. One possible interpretation of this inconsistency is an experimental limitation of our screening system based on the overexpression of the inherited ALS/FTLD causative genes with a short incubation time before evaluation. Therefore, a careful interpretation is required to compare our results with the inherited ALS/FTLD neuropathology data.Although our screening system generally well recapitulated cytoplasmic TDP-43 aggregation in ALS/FTLD, our results were partially inconsistent with some previous reports on the pathology of inherited ALS36. Indeed, the previous study demonstrated that an artificially designed RNA, which specifically binds to TDP-43, prevents intracellular aggregation of TDP-43 triggered by light-driven forced multimerization of TDP-43 in the cytoplasm17. This mechanism could explain our observations that RBPs only aggregated with TDP-43 when they leaked from nuclei into the cytoplasm. On the other hand, the MRPs-induced co-aggregation of TDP-43 was dependent on aggresome formation. The role of aggresomes in the accumulation of misfolded proteins has been pointed out in various neurodegenerative diseases37. ALS-linked PFN1 mutant proteins that aggregated in the cytoplasm were dependent on microtubules, which was similar to our observations38. Intriguingly, these two pathways of TDP-43 aggregation are not complementary and are independent of each other. Inhibiting LLPS prevented RBPs-mediated TDP-43 aggregation, but did not affect MRPs-induced TDP-43 aggregation. Conversely, HDAC6 suppression and microtubular destabilization only affected MRPs-induced TDP-43 aggregation. All these observations indicate that targeting the intracellular mechanism specific to each pathway is key to preventing TDP-43 accumulation. Nonetheless, there is still a possibility that other independent pathways are also involved in cytoplasmic TDP-43 aggregation. Further investigation into the potential mechanisms of TDP-43 accumulation is required.The co-aggregation of TDP-43 and RBPs was sensitive to 1,6-Hd, suggesting that LLPS drives cytoplasmic RBP-induced aggregation. Nucleotides, enriched in nuclei, prevent aggregation driven by LLPS37. The involvement of HDAC6 has been reported in various neurodegenerative diseases such as Huntington’s disease29, Parkinson’s disease28, and Alzheimer’s disease30. In ALS, it was reported that deletion of Hdac6 ameliorates the disease progression in SOD1-ALS mice39 and TDP-43 deficiency drastically reduces HDAC6 expression in cultured cells40. Moreover, HDAC6 inhibition reversed axonal transports, which are defective in iPS-derived motor neurons from ALS patients with FUS mutation41. These observations suggest the possibility that HDAC6 is also involved in the ALS pathology similar to other neurodegenerative diseases and that it may be a therapeutic target for ALS through restoring axonal transport. In addition to this, our results strengthen the possibility that inhibition of HDAC6 is a potential therapeutic target for ALS by providing a novel mechanism for preventing microtubule-dependent TDP-43 pathology. HDAC6 inhibition is probably effective for MRPs-linked inherited ALS and partially to sporadic ALS.HDAC6 is a key molecule in aggresome formation. HDAC6 facilitates the transport of misfolded proteins by connecting microtubules and ubiquitinated proteins42. Consistent with the cited study, we found that TDP-43 was sequestered into aggregates of MRPs in N2a cells and approximately half of TDP-43 skein-like inclusions in sporadic ALS patients were co-localized with HDAC6, an aggresomal marker. Our results suggest that the HDAC6-dependent aggresome pathway may be involved in the formation of skein-like inclusion in ALS/FTLD. However, we also found that both HDAC6-positive and HDAC6-negative skein-like inclusions were observed in the same sporadic ALS patient, as shown in Fig. Since most of the ALS cases are sporadic, it is important to identify the mechanism responsible for TDP-43 aggregation in the sporadic ALS. Recent reports showed that cytoplasmic TDP-43 was sequestered into aggresome dependent on microtubules in fibroblasts derived from sporadic ALS patientsSupplementary InformationSupplementary Figure S1Supplementary Figure S2Supplementary Figure S3Supplementary Table S1


Structural colorful cholesterics show impressive susceptibility to external stimulation, leading to applications in electro/mechano-chromic devices. However, out-of-plane actuation of structural colorful actuators based on cholesterics and the integration with other stimulation remains underdeveloped. Herein, colorful actuators and motile humidity sensors are developed using humidity-responsive cholesteric liquid crystal networks (CLCNs) and magnetic composites. The developed colorful actuator can exhibit synergistic out-of-plane shape morphing and color change in response to humidity, with CLCNs as colorful artificial muscles. Through the integration with magnetic control, the motile sensor can be navigated to open and confined spaces with the aid of friction to detect local relative humidity. The integration of multi-stimulation actuation of cholesteric magnetic actuators will expand the research frontier of structural colorful actuators and motile sensors for confined spaces. In contrast, structure color remains vivid under daylight and is free of photobleaching problems.Colorful outlooks have several benefits for living creatures, for example chameleons and octopi, in the purpose of camouflage, body protection, signaling, and physiology.–24 As a typical kind of photonic crystals, cholesteric liquid crystals consist of rod-like liquid crystalline mesogens and periodically align in a helical manner. Cholesterics reflect the light of certain wavelength following Bragg's Law: λ = npsinθ, where λ is the central wavelength of reflected light, n is the average refractive index of liquid crystals, p represents the pitch of cholesteric liquid crystals and θ is the incidence angle. Impressive progress has been made in the development of dynamic colorful cholesteric liquid crystal devices.–31 However, most of the previously reported devices were in the form of static coatings/liquid crystal glass cells or polymer film sheets with 1D in-plane shrinkage/elongation. Despite impressive reports on the dynamic color change of the humidity-responsive CLCNs, attention was mainly focused on the chromatic response of CLCN coatings adhered to substrates.–34 The mechanical deformation parameters of hygroscopic CLCNs that are critical for robotics remain unexplored, and 3D actuation remains rarely demonstrated. While for 3D actuators based on liquid crystal polymers,,35–43 the sample cutting direction couples with the deformation modes  since LC actuators usually adopted spatially non-uniform molecular configurations, e.g., radial/splay/twist-nematic alignment.–43 This coupling complicates the fabrication of the actuator with complex shapes and desired actuation modes. Such molecular configurations are also colorless and could not be directly used to develop colorful actuators.Most of the structural colors result from ordered microstructures, including scattering, thin-film interference, gratings, metasurfaces, total internal reflection, and photonic crystals.In the present study, we show the application of the humidity-responsive CLCN as a platform to develop colorful actuators with synergistic color change and shape morphing when the CLCNs act as colorful artificial muscles, and the control of actuators by humidity and magnetic field; we also showcase CLCNs' applications in robotic and motile sensing fields while CLCN coatings act as humidity sensors with the integration of magnetic actuation and aid of friction, since currently most of previously sensors were usually static. The aforementioned coupling between the actuation mode and actuator cutting direction is overcome by using cholesteric alignment where mesogens rotate along the helical axis/film thickness direction, which is accompanied by the benefit of endowing the actuator with structural color. We first utilize humidity-sensitive contractile CLCNs as colorful artificial muscles to develop structural colorful actuators with synergistic out-of-plane shape morphing and color change in response to external stimulus, i.e., humidity, and demonstrate the multi-stimuli robotic control using the magnetic field in addition to humidity. Specifically, we develop colorful actuators mimicking blooming flowers and butterfly flapping wings to show their synergistic shape morphing/color change performance under humidity and magnetic stimulation. We then show the application of magnetic actuation that could navigate motile sensors to different places and sense local humidity where commercial tethered sensors are not facile to implement.22.1p = 1/(HTP·c), where HTP and c are helical twisting power and concentration of the chiral dopant, respectively. Photoinitiator 7 (IRGACURE 369) was used to initiate photopolymerization while inhibitor 8  was used to prevent premature thermal-induced polymerization during sample processing. The compositions of monomers were optimized to afford a broad nematic phase and enable LCN to show wide color spectra change upon base treatment. To prepare LC films, the LC monomer mixture in isotropic phase at 80 °C was filled into the LC cell composed of two glass substrates with a gap of 20 μm that were coated with antiparallel-rubbed PVA. In the monomeric state, the cholesteric monomer mixture showed temperature-dependent optical reflection. The reflection band was red-shifted upon cooling  monomers . Humidity-responsive carboxylic acid groups-containing monomers .Here we employ cholesterics to break the coupling between actuation modes and cutting direction as the LC molecular long axis rotates along the helical axis in the thickness direction. The introduction of out-of-plane 3D actuation into structural colorful actuators provides opportunities for developing photonic devices with more degree-of-freedom. In detail, two CLCN films and magnetic Ecoflex  with NdFeB microparticles  were processed to form a triple-layer structure with the magnetic Ecoflex layer in-between . Firm inMaratus robinsoni. To empower humidity-responsive actuation, asymmetry was introduced into the actuator via base treatment of only one side CLCN of the CLCN/Ecoflex/CLCN composite. During actuation, the hygroscopic LCN layer expanded when exposed to moisture and resulted in shape change. Experimentally, the colorful stripe (dimension of 2 mm × 20 mm) curled in response to moisture and the color also synergistically changed; after stopping the moisture flow, the strip recovered its initial curling state. Simulation results also match well with the experimental phenomena  into a circular shape with the aid of a fixture propoxy)benzoic acid), 2 (4-((6-(acryloyloxy)hexyl)oxy)benzoic acid), 3 (4-methoxyphenyl 4-((6-(acryloyloxy)hexyl)oxy)benzoate, synonym RM105) and 5 -hexahydrofurofuran-3,6-diyl bis(4-((4-(((4-(acryloyloxy)butoxy)carbonyl)oxy)benzoyl)oxy)benzoate), synonym LC756) were obtained from Synthon Chemicals. LC monomer 4 propoxy)benzoate), synonym RM257) was purchased from Wilshire Technologies company. Chemical 6 , photoinitiator 7 (IRG369), inhibitor 8 (butylated hydroxytoluene), and poly (Mw 85–124 kDa) were purchased from Sigma-Aldrich. NdFeB microparticles  were purchased from Magnequench.Glass substrates were cleaned in acetone and n-isopropanol with ultrasonication for 10 min, and then treated with UV ozone for 20 min. PVA solution (3% PVA in water) was spun coated onto substrates at a speed of 1500 rpm for 45 s and then dried on a 120 °C hotplate for 30 min. The PVA-coated glass substrates were then rubbed with a cloth and assembled into liquid crystal cells with 20 μm spacers to determine the cell gap. The LC mixture consists of compounds 1–8 with the mass ratio of compound 1 : 2 : 3 : 4 :5 : 6 : 7 : 8 = 25 : 25 : 15 : 12 : 7 :15 : 1 : 0.5. The ratio of monomers was adjusted carefully to have a proper temperature range of cholesteric phase from 22 to 71 °C  and then treated with vinyltrimethoxysilane (2% in methanol) via spin-coating at a speed of 1500 rpm for 45 s. Then magnetic Ecoflex0030 composite (mass ratio of Ecoflex part A : part B : NdFeB microparticles : vinyltrimethoxysilane = 100 : 100 : 200 : 2) was sandwiched between two LCN films. The gap (thickness of magnetic Ecoflex) between LCN films was 300 μm determined using suitable spacers. The magnetic Ecoflex was cured at 95°C for 30 min. The triple-layered composite was carefully harvested from the glass substrate after soaking in water to dissolve the sacrificial PVA layer. LCN/Ecoflex/LCN sheets with predesigned shapes were fabricated with a laser cutter (LPKF ProtoLaser U3 Cutter).The walking robot robotic sensor was composed of three pairs of magnetic Ecoflex legs with a CLCN-coated PDMS body. Ecoflex (Part A + Part B) was thoroughly mixed with magnetic NdFeB microparticles, degassed in a vacuum chamber for 10 min, poured onto a plastic substrate and blade-coated into a film with a thickness of ≈300 μm, which was cured at 45 °C for 4 h. Magnetic legs were laser cut from the magnetic Ecoflex film and magnetized in the designed direction using MicroSense EZ7 vibrating sample magnetometer. To prepare the PDMS body, CLCN was plasma treated and coated with silane to form firm bonding with PDMS. A proper amount of PDMS (mass ratio of base: curing agent = 10:1) mixed with black dye  was poured onto the CLCN film sticking on a glass substrate. After curing in a 90 °C oven for 3 h, the composite was immersed in water to release the composite from the glass substrate by dissolving the PVA layer from the CLCN preparation step. To assemble the magnetic robot, the magnetic legs were glued onto the PDMS body with a small amount of Ecoflex as the glue.To fabricate the cylindrical magnetic robotic humidity sensor, uncured magnetic Ecoflex (Ecoflex: magnetic microparticles = 1:1 in mass) was injected into a plastic tube (inner diameter: 2 mm) and cured in a 60 °C oven for 4 h. After curing, the cylindrical magnetic Ecoflex was carefully pulled out from the plastic tubular mold. The cylindrical magnetic Ecoflex composite was cut into 3.5 mm long pieces and magnetized with a sinusoidal magnetization profile by confining the cylindrical Ecoflex rod in a circular fixture  with a temperature ramp rate of 10 °C minFinite element analysis (FEA) was performed using commercial software  to verify the deformation of the LCN/magnetic Ecoflex/LCN sandwich structure. Eight node hexahedral elements with reduced integration (C3D8R) were used to model the structures and included at least 8 elements along the width to accurately capture the bending deformation. The swelling of the LCN was predicted by using the analogy to thermal expansion. Orthotropic thermal expansion properties were defined for the LCN material and a temperature differential was applied to induce the swelling. The thermal expansion constants were found by experimentally measuring the shape change of LCN samples. Both LCN and magnetic Ecoflex were modeled as linear elastic materials and the elastic constants were found experimentally. The change in elastic modulus of LCN material due to NaOH treatment was incorporated by defining a temperature-dependent Young's modulus.A commercial humidifier was used to blow moisture to the humidity-responsive actuator. To investigate the responsive rate, moisture was applied to the colorful stripe in Supplemental MaterialsSupplemental Movie 1Supplemental Movie 2Supplemental Movie 3Supplemental Movie 4Supplemental Movie 5