Humidity was an unfavorable factor for patients with rheumatoid arthritis (RA). RA disease activity was severe in high humidity conditions. However, there is no evidence to demonstrate the effects of humidity on arthritis in the animal experiments and explore its relevant mechanism.Using the DBA/1 mice, this study addressed the effects of a high humidity (80 ± 5%) on arthritis in collagen-induced arthritis (CIA) mice. Then, this study used the gas chromatography-mass spectrometer (GC-MS) to explore alterations in serum metabolome caused by the high humidity. Furthermore, xylitol and L-pyroglutamic acid, which were both significantly upregulated by the high humidity, were selected to further study their effects on arthritis in the CIA mice.The high humidity (80 ± 5%) could aggravate arthritis variables including increasing arthritis score and swelling, serum autoantibodies (anti-COII and anti-CCP), and proinflammatory cytokines . In addition, the high humidity could cause significant alterations in serum metabolome in the CIA mice. Xylitol and L-pyroglutamic acid were the representative serum metabolites that were significantly upregulated by the high humidity. Further experiments demonstrated that the supplementation of 0.4 mg/mL xylitol in drinking water after inducing the CIA model and 2.0 mg/mL in drinking water before inducing the CIA model could both aggravate arthritis in the CIA mice.These data demonstrated that high humidity was not beneficial for arthritis development and its mechanism might be associated with xylitol and L-pyroglutamic acid.The online version contains supplementary material available at 10.1186/s13075-021-02681-x. Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease that arises more frequently in females than males, being predominantly observed in the elderly . The gloRA is a multifactorial disease, where various genetic and environmental factors influence the prevalence of the disease . AlthougIn the present study, to clarify the influence of humidity on the onset and development of arthritis, DBA/1 mice  were kept in a humid environment with 80 ± 5% humidity and were injected with bovine type collagen type II to induce arthritis after 3 weeks. Gas chromatography/mass spectrometry (GC-MS) was employed to analyze the influences of the high humidity on serum metabolites of DBA/1 mice. Next, this study revealed the effects of xylitol and L-pyroglutamate, which are both upregulated by the high humidity, on arthritis in collagen-induced arthritis (CIA) mice. Our results may provide novel insights into the pathogenesis of RA, for which the ambient humidity could be considered a promising factor to manage RA.Specific pathogen-free (SPF) grade DBA/1 mice  were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. All the mice were allowed to be acclimated to our animal facility for one week and then randomly divided into different groups in the SPF environment of Zhejiang Chinese Medical University laboratory animal research center. Mice were housed under a 12 h/12 h light/dark cycle and constant temperature (25 ± 1 °C) and humidity (50 ± 5%) with food and water available ad libitum. All animal experiments were performed according to the requirements of the Institutional Animal Care and Use Committee of China.N=7): (1) control group (CT): kept in 50 ± 5% humidity environment and injected with 0.9 % NaCl solution on days 21 and 42; (2) CIA group (MT): kept in 50 ± 5% humidity environment and injected with 200 μg bovine type collagen type II  in 200 μL complete Freund’s adjuvant on day 21 and injected with 200 μg bovine type collagen type II in 200 μL incomplete Freund's adjuvant on day 42; (3) humidity CIA group (HT): kept in 80 ± 5% humidity environment and the injection method with bovine was the same as the CIA group. The reasons of choosing 80% humidity were as follows: (1) the 80% humidity was regarded as the boundary of high humidity in the previous literatures [The DBA/1 mice were grouped into three groups  The blood was obtained from the eye socket vein on days 21, 42, and 56 and then centrifuged at 3000 rpm for 15 min at 4 °C for serum. The arthritis scores and ankle joint swelling were measured every 3 days after day 42. The DBA/1 mice with 15 weeks old were euthanized to obtain the hind limbs of mice including the ankle.N=7) and the detailed group information was shown in Fig. After clarifying the effect of the high humidity (80 ± 5%) on the CIA model, this study selected xylitol and L-pyroglutamate, which were up-regulated by the high humidity according to serum metabolome analysis, for further study their effects on the CIA model. The DBA/1 mice were grouped into ten groups  to evaluate the damage of ankle tissue.Serum type II collagen antibody (Col II) and anti-cyclic citrullinated peptide antibody (anti-CCP) were evaluated using CUSABIO ELISA Kits  that was based on the double antigen sandwich ELISA method. In Experiment I, serum levels of pro-inflammatory cytokines were measured by a commercial multiplex mouse cytokine magnetic bead-based immunoassay  according to the manufacturer’s instructions. The cytokine screen included IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p40, IL-12p70, IL-13, IL-17A, eotaxin, G-CSF, GM-CSF, IFN-γ, KC, MCP-1, MIP-1α, MIP-1β, RANTES, and TNF-α. The mean fluorescence intensity from all the bead combinations tested was analyzed using the Bio-Plex system equipped with Bio-Plex Manager Software v6.0 (Bio-Rad Laboratories). In Experiment II, serum levels of IL-6, IL-17A, G-CSF, and eotaxin were evaluated using the Multisciences ELISA Kits  according to the manufacturer’s instructions.Serum samples were prepared according to the previous method . After tMetabolite analysis was carried out by SHIMADZU GCMS-QP2010 GC/MS . Separation was performed by loading a 30-m × 0.25-mm × 0.25-μm HP-5MS fused silica capillary column (Agilent J&W Scientific). The inlet and ion source temperatures were 300 °C amd 230 °C, respectively. The flow rate of the carrier gas, high-purity helium (>99.999%), was 1.2 mL/min. The sample injection volume was 1 μL with a split ratio of 10:1. The GC oven temperature program consisted of 70 °C for 3 min, after which the temperature ramped to 300 °C at 5 °C/min, and held steady for 5 min. The mass spectrum scan range was set at 50−550 m/z, and the acquisition frequency was 2 Hz after a solvent delay of 4.8 min. A dispersion analysis was performed for different groups of samples, with one quality control (QC) sample inserted for every five samples to measure system drift.Raw GC-MS mass spectra were converted to CDF format files and subsequently was performed feature extraction and preprocessed with XCMS in R software as previously described . The datR2X and Q2 (goodness of prediction) values, representing the total explained variance and the model predictability, respectively, were extracted. The variable importance in projection (VIP) values of all the metabolites from the PLS-DA model were taken as criteria to find the variable importance of differential metabolites. Those variables with a VIP > 1.0 and a p value < 0.05 were considered relevant for group discrimination.For multivariate statistical analysis, the XCMS output was further processed using Microsoft Excel . The normalized data were transformed using SIMCA-P 11.0 software  for partial least Squares-discriminant analysis (PLS-DA). PLS-DA was applied to the data after mean-centering and unit variance scaling . These analyses employed a default sevenfold internal cross-validation from which the t test ). Following statistical analyses with multiple comparisons, the p value was adjusted using the Benjamini-Hochberg method to control the false discovery rate (FDR). An adjusted p value of 0.05 was used as a statistically significant cutoff.The statistical significance between the two groups was evaluated by a univariate Student’s To directly assess the effects of humidity on inflammatory arthritis in vivo, a CIA model was established in DBA/1 mice living in normal (50 ± 5%) humidity or high (80 ± 5%) humidity. Compared to the normal mice, CIA mice under both two humidities showed joint swelling, inflammation and destruction, and increased autoantibodies after the second immunization Fig. . The folvs. MT, CT vs. HT, and MT vs. HT. The proinflammatory cytokines that exhibited no statistical difference in all three comparisons included IL-1a, IL-1b, IL-3, MCP-1, and TNF-a  and VIP values, four serum metabolites significantly altered between CT and HT group before inducing CIA (day 21). The high humidity could cause significant increases of xylitol, dodecanoic acid, and L-pyroglutamic acid, a decrease of sucrose in DBA/1 mice before inducing CIA (day 21)  could aggravate arthritis and its mechanism might be associated with xylitol and L-pyroglutamic acid. However, the limitations of this study were as follows: (1) this study did not validate the results through human samples and further the mechanism of the influence mechanism of xylitol and L-pyroglutamic acid; (2) this study did not calculate the inter-rater reliability or intra-rater of arthritis scores between authors; (3) this study did not record the cage temperature vales throughout the experiment. Even so, this study could facilitate the deep understanding of the relationship between humidity environment on RA.Additional file 1: Figure S1.Additional file 2: Figure S2.Additional file 3: Figure S3.Additional file 4: Table S1 List of serum metabolites found in GC-MS analysis.Additional file 5: Table S2 The numeric values of CIA measurements (mean + SE) in Fig. Additional file 6: Table S3 The numeric values of CIA measurements (mean + SE) in Fig. Additional file 7: Table S4 The numeric values of CIA measurements (mean + SE) in Fig. Additional file 8: Table S5 The numeric values of CIA measurements (mean + SE) in Fig.


One is the electron whispering-gallery mode in graphene quantum dots arising from the Klein tunneling of the massless Dirac fermions. The other is the atomic collapse state, which has never been observed in experiment with real atoms due to the difficulty of producing heavy nuclei with charge Z > 170; however, they can be realized near a Coulomb impurity in graphene with a charge Z ≥ 1 because of the “small” velocity of the Dirac excitations. Here we demonstrate that both the electron whispering-gallery modes and atomic collapse states coexist in graphene/WSe2 heterostructure quantum dots due to the Coulomb-like potential near their edges. By applying a perpendicular magnetic field, we explore the evolution from the atomic collapse states to unusual Landau levels in the collapse regime.The relativistic massless charge carriers with a Fermi velocity of about  2 heterostructure quantum dots.The whispering-gallery mode and the atomic collapse state are two distinct resonances in condensed matter associated with relativistic massless charge carriers. Now, it is shown that these states can coexist in graphene/WSe For example, the massless Dirac fermions nature of the charge carriers in graphene enables us to demonstrate several oddball predictions by quantum electrodynamics (QED), among which the Klein tunneling5 and atomic collapse8 are the two most famous effects that have attracted much attention. Very recently, it was demonstrated that the two effects lead to the formation of two types of quasibound states in graphene18. The Klein tunneling, i.e., the anisotropic transmission of the massless Dirac fermions across the potential barrier, in graphene leads to the formation of quasibound states in circular p-n junctions, i.e., graphene quantum dots (GQDs), via whispering-gallery modes (WGMs)15. Because of the “small” velocity of the Dirac fermions, a Coulomb impurity in graphene with a charge Z ≥ 1 can result in the formation of atomic collapse states (ACSs) around it17. In previous experiments, pronounced resonances of the two types of the quasibound states were clearly observed18. Due to their distinct underlying origins, the two quasibound states are expected to be observed in the two different systems.Many exotic electronic properties of graphene are rooted in its relativistic massless charge carriers2 heterostructure QDs. Because of the Coulomb-like potential near the edges of the QDs, we observe WGMs near the edge and ACSs in the center of the graphene/WSe2 heterostructure QDs. Moreover, the ACSs are further explored in the presence of high magnetic fields. The study about effect of magnetic fields on the ACSs has a long history19. However, such a longstanding prediction remains highly controversial because that the theoretical results are contradictory25 and, more importantly, an experimental verification of this fundamental prediction is still lacking up to now26. Our experiments demonstrate that the atomic collapse resonance effect still exist in magnetic fields and evolution from the ACSs to unusual Landau levels in the collapse regime are measured.Here, we demonstrate the coexistence of the electron WGMs and ACSs in graphene/WSe2 heterostructure was obtained by using a wet transfer fabrication of a monolayer graphene on mechanical-exfoliated thick WSe2 sheets (see Methods section for details of the sample preparation). In our experiment, nanoscale WSe2 QDs were surprisingly observed on surface of freshly mechanical exfoliated WSe2 sheets, as shown in atomic force microscopy (AFM) image of Fig. 2 QDs is the same as a WSe2 monolayer and the radius is <10 nm. Such WSe2 QDs also can be observed when mechanical-exfoliated thick WSe2 is covered with graphene monolayer, i.e., in the graphene/WSe2 heterostructure, as shown in scanning tunneling microscope (STM) image of Fig. 2 heterostructure). At present, the exact origin for the emergence of the nanoscale WSe2 QDs is unclear. In our experiment, nanoscale monolayer-thick WSe2 anti-dots are usually observed around the WSe2 QDs  images inside and outside the QD are identical . A schematic side view of the graphene/WSe2 heterostructure is shown in Fig. The graphene/WSeSee Fig. , which s2 QD strongly modifies electronic properties of the graphene above it. Figure 2 QD. The STS, i.e., dI/dV, measurement of the graphene off the WSe2 QD shows a typical V-shaped spectrum profile of graphene with the Dirac point at 2 QD displays a series of resonance peaks with the Dirac point estimated as 2 QD generates a circular p-n junction, i.e., a GQD, on graphene. The almost equally spaced peaks in the spectrum are the quasibound states confined in the GQD via the WGMs15. Such a result is further confirmed by carrying out STS mapping at different resonance energies  on and off the GQDs shows positive correlation to the ratio (η) of the number of edge atoms to the number of inner atoms in the WSe2 QD . Bottom panels of Fig. I/dV spectra and corresponding simulated LDOS at different positions of the GQDs, see Fig. 13. Whereas the energy levels of the quasibound states at the center of the GQD follow an exponential function m denotes the orbital states . In the absence of the GQD, the eigen-energies m and the LLs are split into a series of sublevels, which exhibit similar behavior as that observed around charged impurities26, due to the Coulomb-like electrostatic potential. However, previous experiments26 in the presence of a magnetic field were limited to a charge impurity in the subcritical regime. Further, the small cutoff radius of a charge impurity prohibits to explore the evolution from the ACSs to the LLs in experiment. Such difficulties can be naturally overcome in the studied GQDs.The Coulomb-like potential also strongly affects the electronic properties of the GQDs in the presence of magnetic fields. By applying a perpendicular magnetic field, we can observe well defined Landau levels (LLs) of massless Dirac fermions at positions away from the GQD see Fig. . When apmeV Fig. , which rB (see supplementary information for the details). The calculated radially LDOS maps in the different magnetic fields display that the orbital degeneracy is lifted, which is well consistent with our experimental results  of the −1 LL.The detailed comparison between experiment and theory can be made by numerically solving the problem for two-dimensional massless Dirac fermion of graphene monolayer in the presence of Coulomb-like electrostatic potential lts Fig. . Based olts Fig. . Thanks β = 2.4 GQD as a function of the square root of the magnetic field β = 2.4 GQD is also plotted as a function of N = 0, N = −1, N = −2) display nonlinear dependence onm = −1, m = 0). However, we did not observe the splitting of the −2 LL in the experiment, which is probably due to the large full width at half maximum (FWHM) of the LL peaks, prohibiting the observation of the splitting in the experiment. Furthermore, the ACS-R1 resonance is obvious at lowerm = −1 orbital state of −1 LL (see section 14 for the detailed discussion). Similarly, ACS-R2 resonance has the similar characteristic, connected to the m = −1 orbital state of −2 LL. However, such a feature is harder to be recognized in the experiment due to the broadening peak of the −2 LL. Our experiments, complemented by theoretical calculations, explicitly demonstrated the existence of atomic collapse resonance effect in the presence of high magnetic fields and revealed the close connection between the ACS and the lowest orbital state (m = −1) of the LLs.To better explore the evolution of the ACS in the presence of magnetic fields, we summarize the measured LLs at the center of the 2 flow of 50 sccm were maintained throughout the whole growth process. The Cu foil was heated from room temperature to 1030 °C in 30 min and annealed at 1030 °C for 6 h. Then CH4 flow of 5 sccm was introduced for 20 min to grow high-quality large area graphene monolayer. Finally, the furnace was cooled down naturally to room temperature.The large area graphene monolayer films were grown on a 2 crystal was separated into thick-layer WSe2 sheets by traditional mechanical exfoliation technology and then transferred to 2 sheets in advance. Finally, the PMMA was removed by acetone and then annealed in low pressure with Ar flow of 50 sccm and H2 flow of 50 sccm at ~300 °C for 1 h.We used conventional wet etching technique with polymethyl methacrylate (PMMA) to transfer graphene monolayer onto the substrate. PMMA was first uniformly coated on Cu foil with graphene monolayer. We transferred the Cu/graphene/PMMA film into ammonium persulfate solution, and then the underlying Cu foil was etched away. The graphene/PMMA film was cleaned in deionized water for hours. The WSe2 and graphene/WSe2 heterostructure samples. STM/STS measurements were performed in low-temperature  scanning probe microscopes [USM-1400 (77 K) and USM-1300 (4.2 K)] from UNISOKU. The tips were obtained by chemical etching from a Pt/Ir (80:20%) alloy wire. The differential conductance (dI/dV) measurements were taken by a standard lock-in technique with an ac bias modulation of 5 mV and 793 Hz signal added to the tunneling bias.The topographical images are measured by atomic force microscope  with a tapping mode. We employed the n-doped Si tip coated with Platinum-Iridium  to characterize WSeSupplementary Information


Porous polytetrafluoroethylene (PTFE) is physically flexible, thermally and chemically stable, relatively inexpensive, and commercially available. It is attractive for various flexible sensors. This paper has studied flexible capacitive humidity sensors fabricated on porous PTFE substrates. Graphene oxide (GO) was used as a sensing material, both hydrophobic and hydrophilic porous PTFE as the substrates, and interdigitated electrodes on the PTFE substrates were screen-printed. SEM and Raman spectrum were utilized to characterize GO and PTFE. An ethanol soak process is developed to increase the yield of the humidity sensors based on hydrophobic porous PTFE substrates. Static and dynamic properties of these sensors are tested and analyzed. It demonstrates that the flexible capacitive humidity sensors fabricated on the ethanol-treated hydrophobic PTFE exhibit high sensitivity, small hysteresis, and fast response/recovery time. Flexible humidity sensors have recently gained attention in various applications including respiratory devices, incubators, sterilization processes, medicine production, and storage ,2. The sFlexible materials are mainly used as sensing layers and substrates, which meets robust, inexpensive, highly sensitive, and rapid response. Porous polytetrafluoroethylene (PTFE) is a polymer-based membrane that is breathable and physically flexible. It has not only thermal stability, chemical stability, and tear resistance, but also relatively low cost and availability in the commercial market. The porous PTFE has a large surface area, which makes it attractive for various flexible gas and humidity sensors. The hydrophobic porous PTFE has recently been utilized as the flexible substrate for gas sensors ,4,5, in Graphene oxide (GO) as the derivative of two-dimensional material graphene, contains a large number of oxygen-containing groups , which eIn this paper, a capacitive flexible humidity sensor is presented in which GO is used as the sensing layers and a pair of Ag interdigitated electrodes are fabricated using a screen-printing process. In particular, the hydrophobic and hydrophilic porous PTFE as the flexible substrates are studied and compared. An ethanol treatment process is developed here to fabricate the humidity sensors on the porous hydrophobic PTFE substrates. The yield of sensors fabricated on the hydrophobic PTFE substrates is improved by this additional process without affecting the sensors performances. Compared with the traditional hydrophobic and hydrophilic PTFE substrates, the ethanol-treated porous hydrophobic PTFE substrate increases both yield and performances of the flexible capacitive humidity sensors.A flexible capacitive humidity sensor consists of a porous PTFE substrate, a pair of Ag interdigitated electrodes, and a GO sensitive film, as shown in The fabrication processes of the flexible capacitive humidity sensors are as follows. (1) Silver layers were deposited and patterned as the interdigitated electrodes on the porous PTFE substrate by screen-printing process, then the sample was heated in vacuum at 80 °C for 30 min; (2) an aqueous dispersion of GO  was dripped on the interdigitated electrodes, and then heated in vacuum at 50 °C for 1 h.Both hydrophilic and hydrophobic PTFE  are used as the substrates, respectively. PTFE is a kind of polymer material based on the skeleton of the polyethylene (PE) molecular chain, and the hydrogen atoms connected with the carbon atoms are all replaced by fluorine atoms. As each carbon atom is connected with two fluorine atoms, the highly symmetrical structure causes the low surface energy of PTFE, which results in the difficulty of poor printing of the surface of PTFE. When the surface of hydrophobic PTFE is treated by plasma or other methods, part of the carbon-fluorine bond is destroyed, and the active group replaces the fluorine atom. The surface of PTFE becomes hydrophilic, which enhances the surface adhesion with metal.The silver layers are adhered much more easily on the hydrophilic surface than on the hydrophobic surface, which has a significant impact on the quality of screen-printing . Due to The images of interdigitated electrodes and fabricated sensors on bare hydrophobic PTFE, ethanol-treated hydrophobic PTFE, and hydrophilic PTFE are shown in The surface microstructures of the PTFE membranes and the GO films coated on different PTFE substrates are investigated with a scanning electron microscope  at an accelerating voltage of 15.0 kV after the samples are coated with platinum. The SEM image of the porous hydrophobic and hydrophilic PTFE substrates are shown in The morphology of GO films on bare hydrophobic PTFE, ethanol-treated hydrophobic PTFE, and hydrophilic PTFE substrates are shown in Compositions of the different PTFE and GO films on the different substrates are characterized by laser Raman spectrometer  with the 633 nm excitation wavelength of a 45 mW laser.−1, 386 cm−1, 732 cm−1, 1215 cm−1, 1298 cm−1, and 1378 cm−1. It means that the molecular structure of two kinds of PTFE is consistent, in other words, the material composites of the hydrophobic PTFE are not changed by the ethanol treating process.The Raman spectrums of the surface of bare hydrophilic PTFE, ethanol-treated hydrophobic PTFE, and hydrophobic PTFE are shown in −1. According to [−1 assigned to stretching vibration of C-F, at 1378 cm−1 assigned to stretching vibration of C-C, while two bands at 290 cm−1 and 386 cm−1 attributed to deformation vibration of C-C in polymer chains, respectively. The significant reduction of the Raman peak intensity at 732 cm−1 means the ratio of fluorine groups of the hydrophilic PTFE is less than that of the hydrophobic PTFE, which confirms the principle that hydrophilicity of PTFE surface originates from the substitution of the active group to the fluorine group on the carbon chain.The intensity of Raman active bands of the hydrophilic PTFE decreases obviously, especially the band at 732 cmrding to ,21, the −1, which is caused by the symmetrical stretching vibration of sp2 hybrid carbon atoms in the aromatic ring. The G peak is at 1588 cm−1, which is generated by the stretching motion of the carbon cyclic ring and all the sp2 atom pairs of the long chain [D/IG) is a critical parameter to characterize the defect density of GO. The smaller the ID/IG, the higher the degree of atomic ordering and the fewer defects of GO. The software Origin 2018 was used to perform fitting using Gaussian function, and the peak ratio ID/IG is calculated. The ID/IG of GO on the substrates of bare hydrophobic PTFE, ethanol-treated hydrophobic PTFE, hydrophilic PTFE, and silicon are 1.067, 1.063, 0.990, and 1.064, respectively. It means compare with the hydrophobic substrate, the GO on the hydrophilic PTFE substrate has the fewest defects [The Raman spectrums of GO on different substrates are shown in ng chain . Since t defects ,23. To compare the surface energy, the static water contact angles of different PTFE substrates were measured, as shown in The contact angle of the bare hydrophobic PTFE is 140°, which means great hydrophobicity and lower surface energy of the surface, whereas the contact angle of the ethanol-treated hydrophobic PTFE (before heat treatment) and hydrophilic PTFE are 62° and 39°, respectively, which means good hydrophilicity and higher surface energy of these two substrates. After heat treatment, the surface of the ethanol-treated hydrophobic PTFE recovers the hydrophobicity, and the contact angle increases to 135°.Generally, the surface with low surface energy is difficult to bond, whereas it is easy to deposit silver paste on a hydrophilicity surface. In other words, the hydrophilicity of the surface leads to higher yield of the screen-printing process. As mentioned in The typical morphology of well-printed electrodes and bad-printed electrodes are shown in The yield of the screen-printing process is defined as the ratio of well-printed electrodes to total ones, and the yield on different substrates can be obtained by observing the morphology of the electrodes under a microscope. The metal layers are adhered much more easily on the hydrophilic surface than on the hydrophobic surface . AccordiThe performance test of humidity sensors includes the static test and the dynamic test. In the test of characteristics, the output capacitance values are measured by a precision LCR meter . The test frequency is 1 kHz. The static characteristics of fabricated GO humidity sensors was tested in a humidity chamber  at 25 °C in different humidity levels from 20% RH to 90% RH. The effect of temperature was also tested in the environmental chamber.2 and O2 . The dynamic characteristics of the sensors are tested by the setup shown in The capacitance of humidity sensors depends on the test frequency. As shown in The hysteresis and sensitivity of the sensors on different substrates are shown in At low RH levels (<45% RH), sensors based on bare hydrophobic PTFE, ethanol-treated hydrophobic PTFE, and hydrophilic PTFE have the sensitivity of about 2.07 pF/% RH, 1.77 pF/% RH and 0.84 pF/% RH, respectively. At high RH levels (45 to 90% RH), the high sensitivity is obtained on the sensors on the bare hydrophobic PTFE and ethanol-treated hydrophobic PTFE substrates, which is 164.26 pF/% RH and 164.98 pF/% RH, respectively, whereas the sensitivity of the sensor on the hydrophilic PTFE is only 8.02 pF/% RH. Since the ethanol on the ethanol-treated hydrophobic PTFE is completely evaporated during vacuum evaporation, the substrate recovers back to its hydrophobicity, and the properties of the sensor on ethanol-treated hydrophobic PTFE are very similar to that on bare hydrophobic PTFE. Compared with the hydrophilic substrate, which seriously affects the hysteresis and sensitivity of the sensor, hydrophobic PTFE is more suitable as a substrate for the humidity sensor.2) of sensors based on different PTFE substrates are shown in The dynamic responses . The output capacitance of the sensor was measured at 25 °C and 60% RH, as shown in According to above test results, humidity sensors based on ethanol-treated hydrophobic PTFE exhibit both high yield and good sensitivity. The electrochemical impedance spectroscopy analysis is proceeded by an impedance analyzer . The humidity sensor is measured over a frequency range from 50 Hz to 18 MHz at different humidity levels, as shown in It can be observed from The semicircle in the Nyquist diagram represents the RC parallel network, and the low frequency tail represents the Warburg impedance caused by the diffusion mechanism. The equivalent circuit diagrams are shown in 3O+), which acts as charge carriers, the transfer of protons is difficult. The Nyquist plot is shown as a semicircle of a complete RC parallel network without Warburg impedance. It is worth noting that the plot deviates slightly from the semicircle, and exhibits the characteristic of constant phase angle. This constant phase angle behavior is primarily due to the porous structure of the electrode surface [According to the model described in  surface .3O+ + H2O → H2O + H3O+) [With the rising relative humidity, more water molecules are adsorbed on the GO surface and form continuous water layers, which exhibit liquid-like behavior. Through the Grotthuss chain reaction (H + H3O+) , protons + H3O+) .Capacitive humidity sensors have been fabricated on porous substrates of bare hydrophobic PTFE, ethanol-treated hydrophobic PTFE, and hydrophilic PTFE. The sensors based on bare hydrophobic PTFE substrates exhibit excellent static and dynamic properties, but face the problem of low yield of the electrodes fabricated by a screen-printing process. Though the hydrophilic PTFE substrates with strong adhesion to the silver electrodes, it causes the long response time and large hysteresis of the sensors. The hydrophobic PTFE with ethanol soaking before screen-printing can improve the adhesion of the substrates to the electrodes, and maintain the same performance of the sensors as based on hydrophobic substrate. The results presented here suggest that ethanol-treated hydrophobic PTFE combines the advantages of both hydrophobic and hydrophilic PTFE substrates.


The increase of normal pressure and layer number enhances the interfacial friction anisotropy for graphene and h-BN but weakens that for MoSe2. Such significant deviations in the interfacial friction anisotropy of few-layer graphene, h-BN and MoSe2 can be mainly attributed to the opposite contributions of electron kinetic energies and electrostatic energies to the sliding energy barriers and different interlayer charge exchanges. Our results deepen the understanding of the influence of external loading and thickness on the friction properties of 2D vdW crystals.Friction anisotropy is one of the important friction behaviors for two-dimensional (2D) van der Waals (vdW) crystals. The effects of normal pressure and thickness on the interfacial friction anisotropy in few-layer graphene,  Zhang et al. studied the friction behavior of a graphene flake sliding on a supported graphene substrate and showed that the friction force increases exponentially with the decreasing stiffness [h-BN) heterojunctions due to load-induced suppression of out-of-plane distortions [Layered van der Waals (vdW) materials such as graphite, boron nitride, and transition metal dichalcogenides (TMDs) which layers bind with weak interlayer vdW interactions, have been widely used as solid lubricants in engineering technology to reduce friction and wear. When size goes down to nanoscale, two-dimensional 2D) vdW crystals exhibit exceptional and excellent friction properties and have attracted numerous scientific interests D vdW cry,29,30,31tiffness ,5. Guo etiffness  find thatiffness  found thtortions , and thetortions . The novDue to surface morphology, crystal lattice, or structural deformation, the friction along various directions is usually different. Friction anisotropy is one of the important friction behaviors and has been extensively studied for solid interfaces and surfaces by theoretical and experimental methods. For 2D vdW crystals, the friction anisotropy also becomes obvious and remarkable ,44,45,46h-BN, and MoSe2 under constant normal force mode have been extensively studied by using first-principles calculations. It is found that the deviations in the energy barriers between the interlayer maximum energy sliding paths and minimum energy sliding paths increase for graphene and h-BN but decrease for MoSe2 with the applied normal pressure increases. The friction anisotropy in graphene and h-BN is therefore enhanced but reduced in MoSe2 by the normal pressure. Moreover, the increase of layer number increases the friction anisotropy in graphene and h-BN and decreases that in MoSe2. The significant difference in the influence of normal pressure and layer number on the friction anisotropy among these three kinds of 2D vdW crystals can be attributed to different interlayer charge exchanges and the opposite changes in electron kinetic energies and electrostatic energies. Our results deepen the understanding of the effects of normal pressure and thickness on the friction behaviors of 2D vdW crystals.In this work, the interfacial friction properties of few-layer graphene, h-BN and 2H MoSe2 with interlayer AB stacking in the rhombus unit cells, where graphene, h-BN and MoSe2 monolayers consist of two C atoms, 1 B, and 1 N atom, and 1 Mo and 2 Se atoms, respectively. h-BN and MoSe2. In the unit cells, a vacuum region larger than 15 Å is in the direction perpendicular to the atomic plane. All computations were performed within the framework of density-functional theory (DFT) as implemented in the FHI-aims code with “tight” computational settings [k point grid of 15 × 15 × 1 was used throughout the work. First the whole systems were relaxed by PBE + MBD until the force on each atom was less than 0.01 eV/Å. The optimized lattice constants of graphene, h-BN and MoSe2 unit cells are 2.467, 2.507, and 3.28 Å, respectively.In our model, we established 2 to 8-layer graphene, settings  in whichsettings  was emplsettings ,50. A k a1–a2 planes of the unit cells were equally divided into 81 positions where the nearest translational positions were separated by 0.274, 0.279, and 0.364 Å for graphene, h-BN, and MoSe2, respectively. The top parts of few-layer graphene, h-BN and MoSe2 including 1, 2, 3, or 4 layers were transversely moved as a whole part with respect to the bottom parts, as shown in a1–a2 planes. Different interlayer stacking were achieved when the top parts shifted to different divided positions. The interlayer distances d of the few-layer 2D crystals were modified by changing the z-direction coordinates of the atoms in the top layers. For graphene and h-BN, 1 C and 1 B atom in the top layers and 1 C and 1 B atom in the bottom layers were fully fixed at each shifted position. For MoSe2, only the Se atoms at the top and bottom surfaces were fully fixed. Then those systems were relaxed again by PBE + MBD. After relaxation, the normal forces Fn at different interlayer distances and shifted positions were calculated by summing the z-direction forces of all atoms in the uppermost layers, and the total energies Pn  by a 3-order polynomial, as shown in To simulate interlayer sliding, the whole P1 and P2, see P1 is from AB to AB via an apex point AH in which path the energy barrier P2 is AB to AA to AB in which path the energy barrier P1 is defined as AH stacking. L- and 6L-layer graphene, h-BN and MoSe2. For bilayers, both 2 decreases with the normal pressure increases. The interlayer sliding energy barriers are affected by the change in layer number. On the other hand, the deviation h-BN increase with the pressure increases, indicating that normal loading enhances the interfacial friction anisotropy. Moreover, h-BN is further increased with the increase of layer number. When the thicknesses of few-layer graphene and h-BN becomes thicker, the interfacial friction anisotropy increases as well. On the contrary, the deviation 2 decreases with the pressure increases so that normal loading weakens the interfacial friction anisotropy. Meanwhile, the interfacial friction anisotropy of few-layer MoSe2 decreases with the increase of layer number. Obviously, the normal pressure and thickness increasing impose completely different influence on the interfacial friction anisotropy of few-layer graphene, h-BN, and MoSe2.As shown in X = K, H, XC, and vdW representing electron kinetic, electrostatic, exchange-correlation, and vdW energies, respectively. Then L- and 6L-layer graphene, h-BN and MoSe2. For graphene, h-BN increase but 2 decreases but h-BN and MoSe2. It is shown from In our DFT calculations, the total energy L-layer graphene, h-BN, and MoSe2 at AB, AA, and AH stacking have been calculated under the normal pressures of 0 and 2.5 GPa byh-BN the interlayer charge depletions increase with the pressure increases, and more charges move to the interfacial C atoms and N atoms. For MoSe2, the interlayer charge accumulations and depletions are relatively weak and charge exchange mainly occurs around the interfacial Se atoms. As a result, the effect of pressure on the interfacial friction anisotropy in few-layer MoSe2 is different from that of few-layer graphene and h-BN. Moreover, we also considered a 6L-layer graphene with initial ABC stacking, h-BN with initial AA’ stacking, and MoSe2 with initial AA’ stacking. The energy barriers between the interlayer maximum energy sliding paths and minimum energy sliding paths were calculated by the same method and procedure. L-layer graphene with ABC stacking and h-BN with AA’ stacking increases with the normal pressure increases, which is consistent with that of 6L-layer graphene and h-BN with initial AB stacking. On the contrary, 2 with AA’ stacking decreases with the normal pressure increases, which is also consistent with that of 6L-layer MoSe2 with initial AB stacking. Therefore, the initial stacking mode will not change the qualitative variations of friction anisotropy with normal pressure.In order to further elucidate the mechanism of interfacial friction anisotropy, the interlayer charge density differences h-BN, the friction anisotropy increases with increasing the normal pressure and layer number. On the contrary, the friction anisotropy for MoSe2 decreases with increasing the normal pressure and layer number. The opposite contributions of electron kinetic energies and electrostatic energies to the sliding energy barriers and different interlayer charge exchanges in few-layer graphene, h-BN, and MoSe2 lead to such remarkable deviation in interfacial friction anisotropy. These results deepen our understanding of friction behaviors of 2D vdW crystals and provide some new insights into the application of 2D vdW crystals as solid lubricants.In summary, we show by comprehensive DFT calculations that under constant normal force mode the interfacial friction anisotropy in few-layer 2D vdW crystals is significantly modified by normal pressure and layer number. For graphene and


Background: A deregulated immune system has been implicated in the pathogenesis of post-cardiac arrest syndrome (PCAS). A soluble form of programmed cell death-1 (PD-1) ligand (sPD-L1) has been found at increased levels in cancer and sustained inflammation, thereby deregulating immune functions. Here, we aim to study the possible involvement of sPD-L1 in PCAS. Methods: Thirty out-of-hospital cardiac arrest (OHCA) patients consecutively admitted to the ER of Mie University Hospital were prospectively enrolled. Plasma concentrations of sPD-L1 were measured by an enzyme-linked immunosorbent assay in blood samples of all 30 OHCA patients obtained during cardiopulmonary resuscitation (CPR). In 13 patients who achieved return-of-spontaneous-circulation (ROSC), sPD-L1 levels were also measured daily in the ICU. Results: The plasma concentrations of sPD-L1 in OHCA were significantly increased; in fact, to levels as high as those observed in sepsis. sPD-L1 levels during CPR correlated with reduced peripheral lymphocyte counts and increased C-reactive protein levels. Of 13 ROSC patients, 7 cases survived in the ICU for more than 4 days. A longitudinal analysis of sPD-L1 levels in the 7 ROSC cases revealed that sPD-L1 levels occurred in parallel with organ failure. Conclusions: This study suggests that ischemia- reperfusion during CPR may aberrantly activate immune and endothelial cells to release sPD-L1 into circulation, which may play a role in the pathogenesis of immune exhaustion and organ failures associated with PCAS. Post-cardiac arrest syndrome (PCAS), which occurs in resuscitated patients undergoing cardiac arrest, is characterized by four key pathological manifestations including post-cardiac arrest brain injury, post-cardiac arrest myocardial dysfunction, systemic ischemia-reperfusion, and persistent precipitating pathophysiology ,2,3. SucProgrammed cell death-1 (PD-1) is a major check-point regulator molecule expressed on T-lymphocytes and other immune cells . Upon biThis study protocol was reviewed and approved by the Institutional Review Board (IRB) of the Mie University Graduate School of Medicine (#3027). Informed consent to participate in this study was obtained in all cases from close family members of OHCA patients admitted to the emergency department and intensive care unit (ICU) at Mie University Hospital Japan.This study enrolled 30 consecutive OHCA patients from August 2020 to May 2021. Upon arrival at the hospital, all patients were immediately subjected to our institute’s standard cardiopulmonary resuscitation (CPR) protocol. Specifically, patients were intubated, manually ventilated with 100% oxygen, and treated with standard chest compressions by ICU physicians. The end-tidal carbon dioxide (EtCO2) concentrations were monitored to assure effective heart compression during CPR. Blood samples were drawn from the femoral arteries as soon as possible during CPR in all cases and from the arterial lines daily in the ICU in ROSC cases, and were used primarily for clinical purposes . Parts of the blood samples were used to measure plasma cell-free PD-L1 concentrations. Platelet-free plasma fractions were prepared from blood samples as previously described [The concentrations of sPD-L1 in the plasma samples were measured using enzyme-linked immunosorbent assay kits  according to the manufacturer’s instructions with known concentrations of human recombinant sPD-L1 as an internal standard . Each sap-value < 0.05 was considered statistically significant.Statistical analyses were performed using SPSS software v.25.0 . The results are presented as a median ± interquartile range, unless otherwise noted. Kruskal–Wallis tests were used to compare three or more groups. Mann–Whitney tests were used for two-group comparisons. To compare the correlations, Spearman’s rank correlation was calculated between each data set. A The thirty OHCA patients enrolled in this study included 12 females and 18 males who ranged from 48 to 92 years old (75.4 ± 14.2) . The cauWe have studied the plasma levels of sPD-L1 as early as possible during CPR before ROSC, in which chest compressions partially restored circulation, potentially triggering ischemia-reperfusion injury . We haveA sub-group analysis has shown that the presence of pneumonia was associated with increased sPD-L1 levels in OHCA during CPR A. NevertWhile investigating how sPD-L1 levels affected the clinical laboratory test results sampled during CPR, we found that they correlated with increased blood urea nitrogen (BUN) and creatinine (Cre) and decreased estimated glomerular filtration rate (eGFR) values . In addiTo examine the longitudinal changes of sPD-L1 levels in PCAS, we studied 7 ROSC patients who survived for more than 4 days in the ICU  and, theIn this pilot study involving 30 OHCA patients, we observed significantly elevated levels of plasma sPD-L1 during CPR. The sPD-L1 concentrations in OHCA were as high as those of sepsis , therebysPD-L1 levels during CPR were indistinguishably elevated in both ROSC (+) and ROSC (−) patients. To further study how sPD-L1 levels unfold during PCAS, we examined 7 ROSC patients treated in the ICU for more than 4 days. In all cases, sPD-L1 levels remained high, albeit changing in proportion to the SOFA scores. This preliminary finding needs to be further substantiated in future investigations to support our contention that sPD-L1 may be included as a biomarker candidate to predict the severity of organ failures in PCAS. Of note, in sepsis patients, sPD-L1 levels have been shown to correlate with SOFA scores, as well as with specific laboratory and clinical parameters of the impaired renal, coagulation, and central nervous systems .PD-L1 is expressed not only in leukocytes such as macrophages and monocytes, but also in non-leukocytic cells such as endothelial and epithelial cells ,26. AlthAs sPD-L1 retains the ability to bind PD-1 on the cell surface, thereby transmitting signals to dampen immune cell activation , increasAlternatively, increased levels of sPD-L1 may have regulatory and protective properties, as the PD-1 signaling elicited by PD-L1 alleviates ischemia-reperfusion injuries to the kidney  and liveA potential limitation of the present study is the concern that age differences could explain the cause of the increased sPD-L1 levels in the OHCA cohort, as the OHCA cohort is older than the healthy volunteer cohort . The preIn summary, this study supports our hypothesis that elevated levels of sPD-L1 in OHCA patients during CPR reflect systemic ischemia-reperfusion, which aberrantly induces immune and endothelial cells to shed the extracellular part of PD-L1 into the circulation. Our results suggest that sPD-L1 may play a role in the pathogenesis of immune exhaustion and the multi-organ failures associated with PCAS. Thus, sPD-L1 could be added to the list of reported biomarkers of PCAS ,53,54,55


Systemic sclerosis (SSc) is an autoimmune disease characterized by vasculopathy and excessive fibrosis of the skin and internal organs. To this day, no effective treatments to prevent the progression of fibrosis exist, and SSc patients have disabilities and reduced life expectancy. The need to better understand pathways that drive SSc and to find therapeutic targets is urgent. RNA sequencing data from SSc dermal fibroblasts suggested that melanin-concentrating hormone receptor 1 (MCHR1), one of the G protein-coupled receptors regulating emotion and energy metabolism, is abnormally deregulated in SSc. Platelet-derived growth factor (PDGF)-BB stimulation upregulated MCHR1 mRNA and protein levels in normal human dermal fibroblasts (NHDF), and MCHR1 silencing prevented the PDGF-BB-induced expression of the profibrotic factors transforming growth factor beta 1 (TGFβ1) and connective tissue growth factor (CTGF). PDGF-BB bound MCHR1 in membrane fractions of NHDF, and the binding was confirmed using surface plasmon resonance (SPR). MCHR1 inhibition blocked PDGF-BB modulation of intracellular cyclic adenosine monophosphate (cAMP). MCHR1 silencing in NHDF reduced PDGF-BB signaling. In summary, MCHR1 promoted the fibrotic response in NHDF through modulation of TGFβ1 and CTGF production, intracellular cAMP levels, and PDGF-BB-induced signaling pathways, suggesting that MCHR1 plays an important role in mediating the response to PDGF-BB and in the pathogenesis of SSc. Inhibition of MCHR1 should be considered as a novel therapeutic strategy in SSc-associated fibrosis. Systemic sclerosis (SSc) is an autoimmune disease characterized by vasculopathy and excessive fibrosis of the skin and internal organs . Skin fiMelanin-Concentrating Hormone Receptor 1 (MCHR1) is a G protein-coupled receptor (GPCR), identified first as a receptor for melanin-concentrating hormone (MCH) in 1999 . MCH is Primary human dermal fibroblasts were cultured from skin tissues of patients with SSc or healthy donors as previously described , under aPrimary normal human dermal fibroblasts (NHDF) from healthy donors were treated with the following reagents; PDGF-BB (40 ng/mL) , MCH (100nM) , PI3K inhibitor , MEK inhibitor U0126, 10μM), STAT3 inhibitor , TGFβ receptor inhibitor , PDGF receptor inhibitor , MCHR1 inhibitor , dimethyl sulfoxide (DMSO) or Ethanol as a vehicle control, and used for immunoblotting, PCR and ELISA analyses as appropriate. Inhibitors were used at the indicated concentrations based on previous reports –25. Cell, 10μM, STotal RNA was extracted from each dermal fibroblast cell strain in passage 3 for gene expression analysis using the CsCl-gradient purification method . RNA intA second paired-end RNAseq analysis for MCHR1 silenced NHDF and controls treated with/without PDGF-BB was performed at Novogene  with the NEBNext Ultra TM RNA library prep kit  on the Illumina NovaSeq 6000 instrument (Illumina)   and the p-value < 0.05 were considered to be significantly perturbed. Network analysis was used to identify the hub gene; genes with the largest number of incoming edges are found in the center, and those with the fewest are at the periphery.Systems level analysis was performed using iPathwayGuide , a tool that uses a systems biology approach to identify pathways that are significantly impacted in any condition from high-throughput gene expression data . The imp® gene expression assays using StepOne Plus Real-time PCR machine , using the following protocol; A. Holding stage: 1) 15 mins at 48°C 2) 10 mins at 95°C. B. Cycling Stage: 1) 1 min at 95°C 2) 1 min at 60°C for a total of 40 cycles. Gene expression levels were normalized to Beta 2 Microglobulin (B2M) and compared using the 2−ΔΔCt method. TaqMan® probes for human Actin Alpha 2 (ACTA2), Collagen Type I Alpha 1 (Col1α1), CTGF, Fibronectin 1 (FN1), TGFβ1, MCHR1, and B2M were obtained from Applied Biosystems. The assay catalog numbers are shown in Total RNA was extracted using TRIzol (Life Tchnologies), and qPCR was performed in duplicate using TaqManFibroblast lysates were harvested directly in 2× sodium dodecyl sulfate gel-loading buffer . Samples were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto nitrocellulose blotting membranes . Membranes were then blocked with 5% milk and incubated with one of the following antibodies; MCHR1, Alpha Smooth Muscle Actin (αSMA), Fibronectin (FN), CTGF, Collagen Type I Alpha 1 (Col1A1), TGFβ1, Caspase 3, and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH). Product details are shown in 6 NHDF were cultured and collected using the Subcellular Protein Fractionation Kit (Thermo Fisher Scientific), then membrane fraction samples were electrophoresed under non-reducing conditions and transferred to a nitrocellulose membrane. Similarly, recombinant human MCHR1  was electrophoresed under non-reducing conditions and transferred to a nitrocellulose membrane. The membrane was blocked with 5% nonfat milk in Tris-buffered saline/5% Tween-20 and incubated for 1 hour with biotinylated PDGF-BB (R&D). The membrane was washed and incubated for 1 hour with horseradish peroxidase-conjugated streptavidin (Invitrogen), and the signal was detected using chemiluminescence (Perkin-Elmer) on an iBright750 (Thermo Fisher Scientific).Western ligand blotting was performed as previously described  with som5 cells/well in DMEM supplemented with 10% fetal bovine serum. MCHR1-specific small-interfering RNA (ON-TARGET plus) and control RNAi were purchased from Dharmacon . For transfection, Lipofectamine®2000 (Invitrogen) was used in accordance with the manufacturer’s instructions. A mixture of 10 or 100 nM of each RNAi and Lipofectamine®2000 was added to cells, and cells were cultured for 72 h. Fibroblasts were serum-starved at least 2 h before further stimulation. MCHR1-silenced or control siRNA fibroblasts were treated with 40 ng/mL PDGF-BB (R&D) and harvested 6hrs or 24hrs after stimulation. The culture supernatants were collected by centrifugation and aliquoted. All samples were stored at - 80°C until further analysis.NHDF were seeded in a 6-well plate at a density of 1 × 10max values were calculated. The Rmax values assume 1:1 interaction mechanism. Overnight kinetics for PDGF-BB binding to MCHR1 were performed in the presence of PBS-P. The contact and dissociation times were 60 s and 300 s, respectively. The flow rates of all analytes solutions were maintained at 50 µL/min. Two 20 s pulses of 1:1000 H3PO4  were injected for surface regeneration. Injected analyte concentrations were from 100 nM to 3.125 nM (two-fold dilutions). Analytes were injected in duplicate. For analysis, sensorgrams from the overnight kinetics were evaluated using 1:1 kinetics model fitting.All experiments were done at the Biacore Molecular Interaction Shared Resource at Georgetown University using a Biacore T200 instrument  with a sensor chip CM5 (Cytiva) at 25°C. Recombinant human MCHR1 Protein (Abnova) was used as a ligand to capture onto the CM5 chip, using standard amine coupling chemistry. Recombinant human PDGF-BB (Sigma-Aldrich) was used as an analyte to flow over the ligand captured surface. Flow Cell (FC) 1 was used as the reference for FC2. Recombinant human MCHR1 was diluted  in 10 mM sodium acetate buffer at pH 4.0 and immobilized onto FC2 to a level of ~5500 RU. PBS-P  was used as the immobilization running buffer. Based on the Immobilized response value, theoretical R5 cells/well and cultured overnight. Cells were washed twice with HBSS and pre-treated with 50nM ATC0065 for 1 hour, followed by addition of PDGF-BB, MCH, or vehicle control for 30min. Fibroblasts were harvested with 0.1M HCL and centrifuged at 1000g for 10 min, and supernatants were used to measure cyclic adenosine monophosphate (cAMP) levels. The levels of cAMP in fibroblasts were measured in duplicate samples using Cyclic AMP ELISA kits  according to the manufacturer’s instructions. The absorbance at 410 nm was measured with a SYNERGY H1 microplate reader .NHDF were plated in 6 well plate at a density of 1 × 103 cells/well in 100 µl culture medium and were allowed to adhere overnight. Cells were treated with 40ng/ml PDGF-BB or 10-7M MCH or vehicle control with or without 50nM ATC0065, then incubated for 24, 48, or 72 hrs. 10μl of Cell Counting Kit-8 reagent was added to each well 2 hrs prior to measurement of absorbance. The absorbance at 450 nm was measured with a SYNERGY H1 microplate reader (Biotec).Cell proliferation was measured by using the Cell Counting Kit-8 . NHDF were seeded in a 96-well plate at a density of 5 × 10P value <0.05 was considered significant.In addition to the described differential expression analysis, statistical comparisons were performed using Mann-Whitney U test, unpaired Student’s t-test, multiple t-test, one-way analysis of variance (ANOVA) (posthoc Tukey or Dunnett), or two-way analysis of variance (post-hoc Sidak) as indicated. All tests were carried out using GraphPad Prism version 8.0 software . A MCHR1 was a hub gene significantly upregulated in our network analysis  using dermal fibroblasts from twins discordant for SSc and healthy subjects. We identified 742 DE genes in dermal SSc fibroblasts . We determined that ysis and. Thus, wMCHR1 mRNA levels in dermal fibroblasts from SSc patients with early disease compared to fibroblasts from healthy subjects using quantitative PCR (qPCR). MCHR1 expression was significantly higher in the dermal fibroblasts of SSc patients , bleomycin (BLM), and PDGF-BB. PDGF-BB and BLM significantly increased MCHR1 levels, while TGFβ1 decreased its expression, albeit not significantly (p = 0.0586) , MEK (U0126), and STAT3  signaling. Inhibition of the PI3K and MEK signaling pathways significantly reduced the PDGF-BB-induced increase in Col1α1 at 6 hrs and 24 hrs, CTGF at 4 and 6 hrs, and TGFβ1 at 6 and 24 hrs  . NHDF were then treated with PDGF--BB or vehicle (VC) for 24 hrs. We identified 9,065 DE genes regulated by PDGF-BB in siCtr NHDF [“PDGF-BB vs VC” in siCtr NHDF] (q<0.1) and 8,927 DE genes in PDGF-BB-treated siMCHR1 [“PDGF-BB vs VC” in siMCHR1 NHDF]  , integrin subunit beta (ITGB) 4 , and vitronectin (VTN)  were in the list of DE genes  analysis was performed and revealed the enrichment of biological processes related to “cell communication”, “development”, “biosynthesis and metabolism”, “DNA and RNA regulation”, “immune responses” and “cell proliferation” among the top 40 most perturbed GO terms unique to “PDGF-BB vs VC” in siCtr NHDF .The predicted upstream regulators analysis performed using iPathwayGuide is based on the gene expression data from our DE genes. iPathwayGuide predicts the activation or inhibition of each regulator based on the number of DE target genes whose fold change is consistent with the regulator predicted activity (activated or inhibited), and the sign of the interaction between the regulator and the targets (positive or negative). This analysis identified 336 upstream regulators, out of which 34 were unique to [“PDGF-BB vs. VC” in siCtr NHDF] . Col1α1, Col2α1, Col3α1, and FN1 expression was also downregulated in PDGF-treated siMCHR1 NHDF compared to PDGF-BB treated siCtr NHDF. GO analysis revealed the enrichment of biological processes related to “ECM remodeling”, “development”, “cell communication”, “immune responses” and “secretion” among the top 40 most perturbed biological processes in PDGF-BB treated siMCHR1 NHDF vs. PDGF-BB treated siCtr NHDF   PDGF-BB increased MCHR1 levels in NHDF and 2) MCH stimulation alone did not induce the expression of fibrotic genes nor MCHR1. However, previous studies suggested that MCH had profibrotic properties. MCH infusion increased profibrotic genes including se liver , and co-se liver . Human mse liver . IL4 andse liver . These sMCHR1 silencing in NHDF significantly reduced PDGF-BB-induced TGFβ1 and CTGF levels, indicating that PDGF-BB signaling could be modulated by MCHR1. GPCRs are involved in the transmission of PDGF signaling  and PDGF-receptor-α (PDGFRA) were among the list of 34 genes predicted to be upstream regulators of DE genes regulated by PDGF-BB tr NHDF] , 57, indtr NHDF] . In conttr NHDF] , 59. OurCol1α1 and TGFβ1 expression levels in a dose-dependent manner in the liver of C57BL/6 J mice with severe hepatic steatosis (In experimental colitis, MCHR1 antagonist reduced colonic inflammation, probably by blocking IL10 upregulation, suggesting that inhibition of MCH/MCHR1 signaling could be a novel anti-inflammatory therapeutic approach , 61. Antteatosis . Previouteatosis , 61, sinteatosis , 63. Theteatosis , 65, sugThe functional enrichment analysis of our RNAseq data revealed the involvement of MCHR1 in the fibrotic and inflammatory responses induced by PDGF-BB. Genes and biological processes involved in fibrosis were exclusively enriched by PDGF-BB in NHDF. For example, PDGF-BB decreased ITGB4 expression, a signature that is associated with enhanced lipopolysaccharide-induced inflammation . GRPR isvia MCHR1. Consistent with previous reports, our data indicated MCHR1 could modulate the immune system (MCHR1 expression is silenced in NHDF, indicating that silencing of MCHR1 mediated the effects of PDGF-BB in the regulation of these genes, namely PDGFRA, PDGFRB, PDGFD, and MAPK9. Interestingly, we found several biological processes related to vascular development. Vasculopathy is one of the most common features of SSc (GO term analysis revealed some biological processes are deregulated by PDGF-BB e system  and infle system . The bioe system , likely e system . The bioe system , 70. We MCHR1 silencing or chemical inhibition did not affect cell proliferation. It is reported that PDGF-BB-induced cell proliferation is suppressed by inhibition of PDGF receptor (in vivo. We showed that PDGF-BB induces MCHR1 and confirmed that blocking PDGF-BB signaling by inhibiting PDGF receptor prevented the upregulation of MCHR1, but there is a possibility that MCHR1 is further activated by PDGF-BB-induced molecules or other factors. We also showed that PDGF-BB directly binds to MCHR1 by western ligand blot and SPR, however, we must also consider the possibility that PDGFR and MCHR1 are co-localized and PDGF-BB signals via both receptors simultaneously or sequentially.In the present study, we observed that PDGF-BB induced cell proliferation of NHDF, in agreement with previous studies , whereasreceptor . Taken treceptor  and MCHRreceptor . In contreceptor . There aIn conclusion, we are the first to show that MCHR1 contributes to the PDGF-BB-induced fibrotic response and the resulting increase in the potent profibrotic factors CTGF and TGFβ1. Increased levels of MCHR1 in SSc fibroblasts can promote PDGF-BB signaling, increase TGFβ1 and CTGF levels, modulate intracellular cAMP production, and increase the fibrotic response. Increased levels of MCHR1 can also potentially mediate the vasculopathy characteristic of SSc. Together, our data show that inhibition of MCHR1 should be considered as a potential therapeutic strategy for skin fibrosis.RNAseq data used in this study have been deposited on the NCBI GEO under access number GSE180488.The studies involving human participants were reviewed and approved by The Institutional Review Board of the University of Pittsburgh, the Institutional Review Board of the Medical University of South Carolina. The patients/participants provided their written informed consent to participate in this study.Study design: NT and CF-B. Performed experiments: NT. Gene level analysis and systems level analysis: LR and WS. Writing of the manuscript, generation of figures: NT and CF-B. Reviewing the draft, comments: LR and WS. Manuscript editing: NT, LR, and CF-B. All authors contributed to the article and approved the submitted version.This project was supported by K24AR060297 to CF-B, T32 AR050958 to LR, and the SmartState and Kitty Trask Holt endowment to CF-B.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Graphene derivatization to either engineer its physical and chemical properties or overcome the problem of the facile synthesis of nanographenes is a subject of significant attention in the nanomaterials research community. In this paper, we propose a facile and scalable method for the synthesis of thiolated graphene via a two-step liquid-phase treatment of graphene oxide (GO). Employing the core-level methods, the introduction of up to 5.1 at.% of thiols is indicated with the simultaneous rise of the C/O ratio to 16.8. The crumpling of the graphene layer upon thiolation without its perforation is pointed out by microscopic and Raman studies. The conductance of thiolated graphene is revealed to be driven by the Mott hopping mechanism with the sheet resistance values of 2.15 kΩ/sq and dependable on the environment. The preliminary results on the chemiresistive effect of these films upon exposure to ethanol vapors in the mix with dry and humid air are shown. Finally, the work function value and valence band structure of thiolated graphene are analyzed. Taken together, the developed method and findings of the morphology and physics of the thiolated graphene guide the further application of this derivative in energy storage, sensing devices, and smart materials. The intense studies on graphene oxide (GO) conversion into pristine graphene layers have revealed the inevitable introduction of defects and unintended functionalization accompanying such a process, which often diminishes all the applied efforts ,2. In adAdditionally, non-covalent modification of graphene layers with polynuclear aromatic rings via π–π interactions or covalent bonding through the so-called “click” chemistry also is widely studied. Green et al. investigated the modification of graphene by pyrene derivatives by simple mixing of their aqueous suspensions with additional sonication. It was demonstrated that pyrene derivatives anchor on the surface of graphene by π–π interactions and further alter substantially the wetting properties of graphene layers due to the presence of polar functional groups that makes possible to prevent graphene aggregations in various solvents . A well-Graphene functionalization by sulfur-containing functional groups, such as thiols or sulfates, has attracted great attention as well. The introduction of thiol groups has been found to control the nucleation and kinetic stabilization of Pt nanoparticles on graphene as well as carbon nanotubes (CNTs) that advances the promising application of these nanocarbon materials as platinum-on-carbon electrodes for fuel cells ,21. Owin4, making such a derived CNTs a promising material for vacuum microelectronics  V range via Keithley 6487 picoammeter/voltage source  to check the Ohmic behavior of contacts. For the temperature-dependent sheet resistance measurements within the 10–300 K temperature range, the rGO–Th sample was mounted on the cold finger of Janis closed cycle refrigerator system CCS-450 equipped with cryogenic temperature controller . The cryostat chamber was evacuated to the high vacuum via Pfeiffer Turbo Pumping System HiCUBE 80 eco . Current-voltage measurements were performed at each temperature point for both bias directions after a temperature equilibrium was reached.eΦm = hν − (EF − ESEC), where hν = 130 eV is the photons’ energy, and EF and ESEC are the positions of Fermi level and cut-off threshold, respectively, both represented in the kinetic energy scale  and ketones/carboxyl groups at hv = 288.2 eV ) disappear or diminish, verifying the elimination of the oxygen groups indicated by the XPS studies.The restoration of the graphene electronic structure in rGO–Th is further signified by the comparative analysis of the corresponding C -domains . It shifn length ,49. Conv network ,51. At tL-edge X-ray absorption spectrum was collected as well to delve into the composition of the introduced sulfur species (p3/2 → σ*(S-H) and 2p3/2 → σ*(S-C) transitions centered at hv = 165.4 eV and hv = 166.7 eV, respectively, can be discerned [hv = 167.4 eV comprised by a mix of 2p1/2 → σ*(S-H) and 2p1/2 → σ*(S-C) resonances, the S L-edge X-ray absorption spectrum justifies the asserted presence of thiols. Furthermore, sulfur species with disulfide bonding reveal themselves by the presence of the σ*- resonances positioned at hv = 164.1 eV, hv = 171.7 eV, and hv = 178.9 eV [hv = 174.7 eV and hv = 183.7 eV is less unambiguous and attributed to the transitions either to the σ*(S-O) molecular states or empty S 3d states [S  species b. Absorpiscerned . Combine178.9 eV . The orid states ,55, thusd states ,50.Thus, given the XPS and XAS results, rGO–Th is shown to be comprised by the extensive areas of the π-conjugated network locally functionalized predominantly by thiols and disulfide moieties with a negligible content of sulfates/sulfites and retained oxygen groups. D, D3, G, D′, and C=O/C=C bands centered at v = 1350 cm−1, v = 1540 cm−1, v = 1600 cm−1, v = 1626 cm−1, and v = 1740 cm−1, respectively. The G band comes from doubly degenerated vibrational E2g mode along the C–C bonds, whereas the D and D′ bands correspond to the full-symmetry vibrational mode of the sp2-hybridized carbon of graphene network. Both D and D′ bands indicate the defectiveness of the graphene layer since the corresponding scattering of the phonons with non-zero wave vector requires a defect according to a fundamental selection rule of Raman spectra [D3 band is commonly attributed to the presence of amorphous carbon [To assess the influence of the alterations in the chemistry upon GO conversion into rGO–Th on the morphology and structure of the material, Raman spectroscopy studies were carried out.  spectra . The oris carbon , whereass carbon ,59.D band significantly reduces from 120 cm−1 to 96 cm−1, D4 band centered at v = 1150 cm−1 appears, whereas the G band splits with the rise of the G′ band positioned at ca. v = 1626 cm−1. The observed narrowing of the D band points out the increase in the extension of the π-conjugated areas [D band intensity. Particularly, the D band evolution is non-monotonic: primarily the D band rises, but further starts to decrease due to a higher discordance degree and vice versa for the recuperation of the graphene network [D′ band is inversely proportional to the content of sp3-hybridized carbon, its appearance verifies the elimination of the predominant number of oxygen groups, although still indicating the introduction of vacancy defects, graphene edges, or folds [D4 band is less unambiguous, being attributed to either the presence of polyene chains, sp3-hybridized carbon, or functionalization by various groups, such as amines [Upon the GO reduction and functionalization by thiols, the Raman spectrum considerably changes: the full width at half maximum of the ed areas , support network . Since ts amines ,64,65. C3 and 1.3 g/cm3, respectively. Also, owing to the interlayer disulfide bonding (-S-S-) and π–π interaction between the restored areas of the π-conjugated network without sonication for more than several hours, the rGO–Th platelets tend to stack forming lamellar particles of several layers  on current (I) acquired in the range from −2 V to +2 V. The sheet resistance of the rGO–Th layer at room temperature is measured to be ~2.15 kΩ/sq, which is almost of the same magnitude as reported for conventionally synthesized pristine rGOs [R0 is a pre-factor, T0 is a characteristic temperature, and p is a characteristic exponent whose value distinguishes different types of VRH mechanism [ln R vs either T−1/3 (Mott-VRH) or T−1/2 (ES-VRH), the Mott-VRH mechanism was asserted for the rGO–Th since experimental data almost perfectly fit the T−1/3 behavior R(T)=echanism ,78,79. Dbehavior c. Howeveifficult . Therefop value in this case is obtained from the slope of ln W vs. ln T plot as drawn in p = 1/3 line with the p value estimated from the slope to be ca. 0.31. Accounting for these results, the Mott-VRH mechanism of conductivity is determined for the rGO–Th samples, which complements the spectroscopic and microscopic results on the considerably high structural quality and restoration of the π-conjugated graphene network after the functionalization.The The effect of thiol functionalization on the graphene work function and structure of the valence band has been studied as well. J centered at BE of ca. 6.2 eV attributed to the 2p-σ electron states of graphene network, which is shifted in the case of GO to ca. 6.8 eV due to the localization of π-conjugated domains by the presence of basal-plane hydroxyls and epoxides [D, E, and F and positioned at Bes of ca. 13.8 eV, 17.0 eV, and 19.8 eV, correspondingly, are distinguished in both rGO and rGO–Th samples, being related to D, converged E, and F [Z peak at BE of 24.8 eV and C peak at BE of 10.1 eV rise in the acquired spectra. The former one is related to the C 1s line excited by photons with energy of 390 eV due to incompletely damped diffraction third order, while the latter one originates from the electronic states of the σ(C=O) bonds in ketones and carboxyl groups [C′ peak centered at 11.1 eV in the GO VB spectrum is due to the mix of the aforementioned states of the σ(C=O) bonds in ketones and carboxyl groups with the states of the π(O-C-O) and σ(O-H) in carboxyls, which are abundant in highly oxidized GO. Despite this similarity, certain alterations are also to be distinguished. Particularly, a prominent A′ feature centered at BE of ~3.2 eV attributed to the contribution of the π states  transient recorded upon ethanol exposures mixed with dry (0% RH) or humid (25% RH) air, for 60 min, interrupted by an intermediate purging of air. The ethanol concentrations were varied, at a 500–16,000 ppm range, repeating the film’s exposures to vapors of each concentration twice. As one can see, in both dry and humid air, the resistance follows the ethanol concentration with some residual drift of background. The chemisresistive response towards ethanol in the humid air is almost two times lower in comparison to the case of the dry air. We assert this to be related to the concurrent adsorption of the ethanol and water molecules on the rGO–Th, which limits the interaction between the ethanol and thiolated graphene and reduces the chemiresistive response.While exposing the film to ethanol vapors, the resistance steadily increases when compared to clean air conditions similar to the chemiresistive effect frequently observed in graphene or graphene oxide layers ,31,89. Tn is equal to 0.51 ± 0.02 for the dry air and 0.67 ± 0.04 for the humid air. To compare the data obtained in the dry air with the literature ones, the sensitivity coefficient calculated as S/C was considered, yielding values in the range from 1.24 × 10−3%/ppm at low gas concentrations to 0.20 × 10−3%/ppm at concentrations of several tens thousands of ppm. The estimated sensitivity coefficient is lower than the ones observed earlier in thiol-functionalized rGO films, but taken in a N2 background that enhances the sensing performance [Notably, both in dry and humid air, the resistance drops to almost initial values after the exposure to concentrations as high as 16,000 ppm without any external effect, namely heating or UV irradiation. The chemiresistive response evaluated according to Equation (1) is plotted versus the ethanol concentration in formance . Given tSummarizing, the facile method for the scalable production of thiolated graphene is developed with a report that reveals new details and peculiarities of the effect of the thiol groups on the morphology, electronic structure, and properties of the material. The synthesized thiolated graphene combines a functionalization by up to 5.1 at.%, which is among the highest reported concentrations, negligible amounts of other functionalities, and a high degree of restoration of the π-conjugated graphene network with the corresponding C/O ratio of 16.8. Accounting for such a chemistry and corrugated structure, originating from the corrugation of graphene layer upon thiolation and granting a high porosity, the synthesized thiolated graphene is regarded as a material of choice for electrochemical and catalytic applications, particularly when forming advanced platinum-on-carbon electrodes for fuel cells or metal-free porous electrodes for the oxygen reduction and oxygen evolution reactions.A thorough analysis of the electronic structure, electrophysical, and optical properties of the thiolated graphene revealed that thiol groups have a negligible effect on the physics of the graphene layer, even at sufficiently high concentrations. This fact is reflected in an almost complete coincidence of optical absorption and conductivity characteristics as well as the work function value of the thiolated graphene when compared to the ones in a pristine rGO. Furthermore, an accurate examination of the VB of the graphene revealed the lack of appearance of localized electronic states upon the thiolation. Combined with the high reactivity of thiols, the pointed out VRH mechanism of conductivity with the finite sheet resistance values of 2–3 KΩ/sq along with the optical transparency makes thiolated graphene an attractive material for use as a sensing layer in chemiresistive and optical bio- and gas sensors. The application of rGO–Th as a promising gas-sensing material is supported by the performed preliminary tests.Taken together, the developed method and findings of the morphology and physical properties of thiolated graphene guide and outline the frames of this graphene derivative’s application in advanced sensing, optoelectronic, and energy conversion technologies as well as giving a hint on the features of the alteration of graphene physics upon derivatization.