We investigated the potential protective role of MOMAST(®) HY100 and MOMAST(®) HP30 on isolated rat colon, liver, heart, and prefrontal cortex specimens treated with Escherichia coli lipopolysaccharide (LPS), a validated ex vivo model of inflammation, by measuring the production of prostaglandin (PG)E2, 8-iso-PGF2α, lactate dehydrogenase (LDH), as well as cyclooxygenase (COX)-2, tumor necrosis factor α (TNFα), and inducible nitric oxide synthase (iNOS) mRNA levels. MOMAST(®) HY100 decreased LPS-stimulated PGE2 and LDH levels in all tested tissues. Following treatment with MOMAST(®) HY100, we found a significant reduction in iNOS levels in prefrontal cortex and heart specimens, COX-2 and TNFα mRNA levels in heart specimens, and 8-iso-PGF2α levels in liver specimens. On the other hand, MOMAST(®) HP30 was found to blunt COX-2, TNFα, and iNOS mRNA levels, as well as 8-iso-PGF2α in cortex, liver, and colon specimens. MOMAST(®) HP30 was also found to decrease PGE2 levels in liver specimens, while it decreased iNOS mRNA, LDH, and 8-iso-PGF2α levels in heart specimens. Both MOMAST(®) HY100 and MOMAST(®) HP30 exhibited protective effects on multiple inflammatory and oxidative stress pathways.MOMAST Olea europaea) polyphenols have healthy beneficial effects, including the prevention of several chronic diseases, such as cancer and aging-associated degenerative diseases [It has been well established that olive tree  HY100 and MOMAST(®) HP30  are polyphenolic liquid complexes from olive  pressing juice with a total polyphenolic content of 100 g/kg (at least 50% as HT) and 36 g/kg (at least 30% as HT), respectively ((®) HY100 and MOMAST(®) HP30 are also characterized by the presence of tyrosol and oleuropein.MOMASTectively . In addi(®) HY100 and MOMAST(®) HP30, both including HT and oleuropein, on the burden of oxidative stress/inflammation occurring on various isolated rat tissue  specimens exposed to Escherichia coli lipopolysaccharide (LPS), a well-established inflammatory stimulus. Specifically, we studied the effects of MOMAST(®) HY100 and MOMAST(®) HP30 on multiple inflammatory and oxidative stress pathways, by measuring the production of prostaglandin (PG)E2, 8-iso-PGF2α, lactate dehydrogenase (LDH), as well as cyclooxygenase (COX)-2, tumor necrosis factor α (TNFα), and inducible nitric oxide synthase (iNOS) mRNA levels. The results support a rational use of these polyphenolic complexes in the prevention of tissue damage occurring during inflammation.Considering the antioxidant effects displayed by both HT and oleuropein, the aim of the present study was to investigate the putative protective effects of MOMAST(®) HY100  and MOMAST(®) HP30  were tested in vitro to evaluate their effects on cell viability. We observed that both polyphenolic liquid complexes were well tolerated by Hypo-E22 and C2C12 cell lines (MOMASTll lines . Particu(®) HY100  and MOMAST(®) HP30  supplementation on oxidative stress and multiple inflammatory pathways in colon, liver, heart, and prefrontal cortex specimens challenged with LPS. As previously reported [2α, an isomer of prostaglandins produced by free radical-catalyzed peroxidation of membrane arachidonic acid, is a stable marker of lipid peroxidation and oxidative stress [(®) HP30 (110 and 220 µg/mL) was able to decrease 8-iso-PGF2α levels on rat prefrontal cortex, colon, liver, and heart tissues, challenged with LPS inflammatory stimulus  HY100  was effective in inhibiting LPS-induced 8-iso-PGF2α in rat liver specimens ((®) HY100 and MOMAST(®) HP30 . HT was also shown to decrease low density lipoproteins oxidation [(®) HY100 and MOMAST(®) HP30 on LDH level in inflamed tissues. LDH is a cytosolic enzyme, which can be considered a marker of tissue destruction [(®) HY100  treatment, we found a significant inhibition of LPS-induced LDH level in all tested tissues ((®) HP30  was also able to decrease LDH level induced by LPS in heart specimens . Actually, the reduction of LDH level could be related to the presence of HT [(®) HY100 and MOMAST(®) HP30. Finally, we evaluated the modulatory effects of MOMAST(®) HY100 and MOMAST(®) HP30 on pro-inflammatory markers, including PGE2, COX-2, TNFα, and iNOS. LPS was found to induce macrophage production of inflammatory cytokines such as TNFα, interleukin-1β (IL-1β), and IL-6, along with inflammatory mediators including nitric oxide (NO) and PGE2 [2 in both neoplastic and inflamed tissues [(®) HP30 was able to reduce COX-2, TNFα, and iNOS mRNA levels in prefrontal cortex, colon, and liver specimens  HY100 in heart tissue specimens ((®) HY100 in prefrontal cortex specimens ((®) HP30 in heart tissue specimens  HY100 was effective in reducing LPS-induced PGE2 levels in all tested tissues ((®) HP30 was able to decrease PGE2 levels only in liver tissue ((®) HY100 and MOMAST(®) HP30 on PGE2 levels, along with COX-2, TNFα, and iNOS mRNAs, support the protective effects of both polyphenolic liquid complexes in prefrontal cortex, colon, liver, and heart specimens. Accordingly, HT was shown to exert anti-inflammatory effects in LPS-stimulated RAW264.7 mouse macrophages by suppressing nuclear factor-kB (NF-κB) signaling and downregulating gene expression of iNOS, COX-2, TNFα, and IL-1β, and production of NO and PGE2 [(®) HY100 and MOMAST(®) HP30 could also be related to the presence of other phenolic compounds, including tyrosol and OE. In this context, tyrosol was found to significantly inhibit COX-2 gene and protein expression, as well as PGE2 secretion in human glioblastoma cells [®) HY100 and MOMAST(®) HP30 could display different effects in tissues. On one hand, Takeda et al. [As regards PGE tissues , MOMAST HY100 and MOMAST(®) HP30 exhibited protective effects as indicated by the blunting effect on the tested pro-inflammatory mediators. On the basis of these results, HT seems to be the main extract component involved in the pharmacological effects. Nevertheless, considering the inherent limitations of the ex vivo experimental model, further investigations including oxidative stress and inflammation biomarkers in in vivo studies are needed for a more accurate evaluation of MOMAST(®) HY100 and MOMAST(®) HP30 efficacy.In conclusion, both MOMAST® CRL-1772™) were cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal bovine serum and 1.2% (v/v) penicillin G/streptomycin in 75 cm2 tissue culture flask . The cultured cells were maintained in a humidified incubator with 5% CO2 at 37 °C. For cell differentiation, Hypo-E22 and C2C12 cell suspensions at a density of 1 × 106 cells/mL were treated with various concentrations  of phorbol myristate acetate  for 24 h or 48 h (induction phase). Thereafter, the PMA-treated cells were washed twice with ice-cold pH 7.4 phosphate buffer solution (PBS) to remove PMA and non-adherent cells, whereas the adherent cells were further maintained for 48 h (recovery phase). The morphology of the cells was examined under an inverted phase-contrast microscope. To assess the basal cytotoxicity of MOMAST(®) HY100 and MOMAST(®) HP30, a viability test was performed on 96-microwell plates, using a 3--2,5-diphenyltetrazolium bromide (MTT) test. Cells were incubated with MOMAST(®) HY100  and MOMAST(®) HP30 , corresponding to HT , for 24 h. Ten microliters of MTT (5 mg/mL) were added to each well and incubated for 3 h. The formazan dye formed was extracted with dimethyl sulfoxide and the absorbance was recorded as previously described [Rat Hypo-E22 cells (Cedarlane Cellution Biosystem) and mouse myoblast C2C12 cell lines  were housed in Plexiglass cages (40 cm × 25 cm × 15 cm), two rats per cage, in climatized colony rooms , on a 12 h/12 h light/dark cycle (light phase: 07:00–19:00 h), with free access to tap water and food, 24 h/day throughout the study, with no fasting periods. Rats were fed a standard laboratory diet .Housing conditions and experimentation procedures were strictly in accordance with the European Union ethical regulations on the care of animals for scientific research.2 inhalation (100% CO2 at a flow rate of 20% of the chamber volume per min) and colon, liver, heart, and prefrontal cortex specimens were immediately collected and maintained in a humidified incubator with 5% CO2 at 37 °C for 4 h, in DMEM buffer with added bacterial LPS (10 μg/mL) (incubation period).According to the recognized ethical principles of “Replacement, Refinement, and Reduction of Animals in Research”, colon, liver, heart, and prefrontal cortex specimens were obtained as residual material from vehicle-treated rats randomized in our previous experiments approved by the Local Ethical Committee of University “G. d’Annunzio” and the Italian Health Ministry . Rats were sacrificed by CO®) HY100  and MOMAST(®) HP30 . Tissue supernatants were collected, and the PGE2 and 8-iso-PGF2α levels (ng/mg wet tissue) were measured by radioimmunoassay (RIA), as previously reported [2α and anti-PGE2 were developed in the rabbit; the cross-reactivity against other prostanoids is <0.3%. One hundred microliters of prostaglandin standard or sample were incubated overnight at 4 °C with the 3H-prostaglandin  and antibody , in a volume of 1.5 mL of 0.025 M phosphate buffer. Free and antibody-bound prostaglandins were separated by the addition of 100 μL 5% bovine serum albumin and 100 μL 3% charcoal suspension, followed by centrifuging for 10 min at 4,000× g at 5 °C and decanting off the supernatants into scintillation fluid  for β emission counting. The detection limit of the assay method was 0.6 pg/mL. Additionally, tissue supernatants were assayed for lactate dehydrogenase (LDH) activity [−ΔΔCt method was used to quantify the relative abundance of mRNA and then determine the relative changes in individual gene expression (relative quantification) [During the incubation period, tissues were treated with scalar concentrations of MOMAST .p < 0.05. The number of animals randomized for each experimental group was calculated on the basis of the “Resource Equation” N = (E + T)/T (10 ≤ E ≤ 20) [https://www.nc3rs.org.uk/experimental-designstatistics.Statistical analysis was performed using GraphPad Prism version 5.01 for Windows . Means ± S.E.M. were determined for each experimental group and analyzed by one-way analysis of variance (ANOVA), followed by Newman–Keuls comparison multiple test. As for gene expression analysis, 1.00  was considered the theoretical mean for the comparison . Statist E ≤ 20) ,59,60, a


Graphene, having a perfect two-dimensional crystal structure, has many excellent features such as a high specific surface area, and extraordinary electrical, thermal and mechanical properties. However, during the production process, lattice defects will inevitably be produced. Therefore, the performance of graphene with various defects is much lower than its theoretical value. We summarize the major advances of research into graphene defects in engineering in this paper. Firstly, the main types and causes of defects in graphene are introduced. Secondly, the influence of different defects in graphene on the chemical, electronic, magnetic and mechanical properties is discussed. Also, the control methods of graphene defects are reviewed. Finally, we propose the future challenges and prospects for the study of the defects of graphene and other nano-carbon materials. According to the Mermin-Wagner theory, the long-range order of two-dimensional crystal structure could be destroyed by long wavelength fluctuation ,2. In ad2 hybrid orbital carbon atoms. Its thickness is about 0.335 nm, corresponding to the thickness of one carbon atom. Three-dimensional graphite, one dimensional nanotubes 95], Pt ,97, Ir , Cu 92]92], Ni  and so oID (the peak intensity of the defective structure) to IG (the peak intensity of the regular graphene structure) does not change or even increase. A recent study solves this problem by placing the graphene oxide into plasma methane [Although the above methods can reduce the defects of oxygen extrinsic atoms, the removal of external defects is often accompanied by the loss of carbon atoms. This will result in intrinsic defects. In Raman spectroscopy, the ratio of  methane . This isDue to its excellent physicochemical properties, graphene plays a significant role in today’s nanoscience. A variety of defects, including vacancies, interstitials, topological defects and line defects are unavoidable in experiments. The defects of graphene have a direct impact on the properties of the graphene. Although a large number of experiments or theoretical simulations have been carried out for the defects of graphene, based on the wide application prospect of graphene materials, the study of its defect characteristics needs further enrichment.


A new temperature-controllable humidifier for X-ray diffraction has been developed. It is shown that the humidifier can successfully maintain protein crystal growth at a temperature lower than room temperature. The room-temperature experiment has been revisited for macromolecular crystallography. Despite being limited by radiation damage, such experiments reveal structural differences depending on temperature, and it is expected that they will be able to probe structures that are physiologically alive. For such experiments, the humid-air and glue-coating (HAG) method for humidity-controlled experiments is proposed. The HAG method improves the stability of most crystals in capillary-free experiments and is applicable at both cryogenic and ambient temperatures. To expand the thermal versatility of the HAG method, a new humidifier and a protein-crystal-handling workbench have been developed. The devices provide temperatures down to 4°C and successfully maintain growth at that temperature of bovine cytochrome c oxidase crystals, which are highly sensitive to temperature variation. Hence, the humidifier and protein-crystal-handling workbench have proved useful for temperature-sensitive samples and will help reveal temperature-dependent variations in protein structures. Here, protein crystals are coated with a polymer hydrogel , which are grown at 4°C and are immediately damaged by rising temperature. These devices have already been applied to TR structural analysis of CcO with the SPring-8 Angstrom Compact Free Electron Laser (SACLA) ].To produce humid air from ambient temperature down to 4°C, our original humidity-control device , and bubbled in a water bottle equipped with a rubber heater kept at 28°C. The humid gas is transported and then cooled to room temperature (RT) during transportation before it reaches the air-mixing area. Mixing of the dry and humid gases is regulated using type SEC-N digital mass-flow controllers . The temperature t1 (°C) and humidity h1 (%RH) of the mixed air from the humidifier are measured using a type HYT271 humidity and temperature sensor . t1 is not controlled but is about RT.The humidifier works at ambient temperature and supplies wet gas to the air cooler. The wet gas is adjusted to a vapour pressure corresponding to the relative humidity (RH) at the desired temperature by mixing dry and saturated humid gases. The saturated humid gas is produced from the dry gas, which is supplied by an NThe air cooler comprises a heat exchanger and a double-pipe transfer tube. The heat exchanger was assembled with a tube that is passed through once in the shell to even out the coolant’s cooling. The transfer tube is lagged with an insulator and connects the heat exchanger to a humid-air nozzle. The coolant for the heat exchanger is chilled using an F32HE or F50HE refrigerated–heating circulator  and is flowed continuously through an outer side passage of the air cooler. The temperature of the outlet is adjusted using the temperature setting in the circulator.b) and 1c), respectively]. The outlet temperature, t2 (°C), is measured inside the humid-air nozzle using a T-type thermocouple, which is made of copper–constantan and is used for low-temperature applications such as cryogenics in crystallography.The humid-air nozzle was fabricated from a polyacetal tube . To prevent the influence of ambient air, the measurement sample is inserted into the nozzle tube 10 mm from its edge. In addition, two holes were created in the nozzle wall at the sample position. A 5 mm-diameter hole is used as a path for incident X-rays and to monitor the sample using a co-axial CCD camera. A second, 7 mm-diameter hole at the opposite side acts as a path for diffraction up to 1.67 Å  and 1.32 Å  resolution at a wavelength of 1 Å . The airstream is supplied by an N2 gas generator (KOFLOC Corporation Ltd) and is cooled using a coolant chilled by the F50HE refrigerated–heating circulator (JULABO GmbH) with an HE-25-025T2 multi-tubular heat exchanger . The dry N2 gas is charged into the chamber of the workbench to create a positive pressure inside it. The chamber has a large inner volume of 0.36 m3  and leaks because of two holes which allow insertion of bare hands into the chamber. Therefore, the airstream was supplied at a greater flow rate of 15 l min−1, and the chamber itself was also cooled by the coolant flowing in the wall piping of the chamber’s insulation panel. However, the workbench does not control humidity.The experimental environment around protein crystals should be maintained at the desired temperature while they are being collected. Gloveboxes are commonly used to maintain the experimental environment; however, most gloveboxes are not temperature controllable and are unsuitable for delicate work with bare hands. For such experiments, we developed a temperature-controlled airflow workbench, TeC-W . The beam size was 200 µm (height) × 100 µm (width) at the sample position with a photon flux of 9.5 × 1010 photons s−1 at a wavelength of 1 Å.To evaluate CcO crystal quality during the diffraction experiment, the mosaicity and unit-cell parameters for each diffraction image were processed using the 3.c)], was successful. Meanwhile, the low-temperature TeC-W workbench worked well to maintain the temperature within 4.0 ± 0.2°C. During CcO crystal-handing with bare hands, the temperature of the TeC-W workbench increased by 0.5°C.The TeCH-1 humidifier with the F50HE refrigerated–heating circulator successfully provided humid gas at a wide range of temperatures and humidity levels: 4–20°C and 20–99.5%RH, respectively. When using the F32HE refrigerated–heating circulator, the humidity SD tended to increase with higher humidity at 4°C, and the PID control did not work stably. The results suggested that improving the heat-insulating performance and the heat-exchange efficiency of the air cooler would allow more precise temperature and humidity control. The newly designed humid-air nozzle was also important for maintaining samples. When using the previous humid-air nozzle, which was similar to those of conventional cryostreams, the CcO crystal quality was not maintained, even at low temperatures. To avoid interfusion of ambient air into outlet air, insertion of CcO crystals into the humid-air nozzle, as described before . We previously reported a similar relaxation time and long-term crystal stability in tetragonal lysozyme crystals at RT (Baba et al., 2013To determine the optimum humidity for CcO crystals, we performed three trials at 4°C. First, we investigated temporal changes of lattice constants of the CcO crystals to evaluate the equilibration time in response to environmental changes in the CcO crystals under the almost saturated humidity of >98%RH by manual flow operation without PID control. This humidity value was the maximum without dew condensation inside the humidifier. Diffraction images were collected every 10 min for 60 min. It took ∼10 min for the lattice constants to reach equilibrium, and the lattices were then stably maintained for over 1 h Fig. 2. During Next, we decreased the humidity from 98%RH to determine the optimum humidity without PID control. A new CcO crystal was mounted, and we waited for 5 min after relaxation. Then we gradually decreased the humidity in 0.5–1% steps after each time delay of 2–3 min. The CcO crystal diffraction quality evaluated by crystal mosaicity and the resolution of the observed Bragg reflections remained intact at 96%RH or higher, but at 95%RH, the CcO crystal diffraction degraded in 5 min. The damage was not recovered with either rehydration to 96%RH or exposure to >99%RH.Finally, to improve the reproducibility of the humidity-control experiments, we examined PID control by comparison with lattice constants of three CcO crystals after relaxation under an optimum humidity of 98%RH. Under these conditions Fig. 3, the lat4.The TeCH-1 humidifier and TeC-W workbench successfully controlled temperature between 4 and 20°C. Using the combined device, we were able to perform a diffraction experiment on CcO crystals which were temperature controlled for crystal-handling operations and humidity controlled under the crystallization temperature. Because the HAG method has proved adaptable for protein crystals under various crystallization conditions, we intend to attempt to improve the performance of the TeCH-1 humidifier over a wider temperature range in future. In this study, we expanded the HAG method to low-temperature experiments.10.1107/S1600576719006435/ei5041sup1.pdfTo explain crystal damage at room temperature. DOI:


In this paper, the advances in radio-frequency (RF)/microwave biosensors based on graphene nanomaterials including graphene, graphene oxide (GO), and reduced graphene oxide (rGO) are reviewed. From a few frontier studies, recently developed graphene nanomaterials-based RF/microwave biosensors are examined in-depth and discussed. Finally, the prospects and challenges of the next-generation RF/microwave biosensors for wireless biomedical applications are proposed. Graphene is a carbon allotrope consisting of an atomically thin two-dimensional 2D) hexagonal lattice . This thD hexagon2·V−1·s−1) [−1·K−1) [In terms of physical properties, a pristine graphene without defects and impurities has a number of superior qualities such as high electronic mobility  , high op[−1·K−1) . Pristin[−1·K−1) , as show[−1·K−1) . In addi[−1·K−1) , graphen[−1·K−1) , microst[−1·K−1) , and gra[−1·K−1) .Meanwhile, graphene has excellent merits for biomedical applications, e.g., drug delivery and tissue engineering, because of its large surface area , chemicaIn particular, graphene oxide (GO) and reduced graphene oxide (rGO) have received much attention as excellent nanomaterials for drug delivery due to their structural and surface properties that are well suited for biomedical applications ,22. In pThis review outlines and highlights the recent trends and advances of RF/microwave biosensors based on graphene nanomaterials with their particular focus on the following: (1) graphene nanomaterials as biosensing materials, (2) the basic concept of RF/microwave biosensors, (3) RF/microwave devices and circuits based on graphene nanomaterials, (4) RF/microwave biosensors based on graphene nanomaterials, (5) the recent advancements and trends, and finally (6) the conclusions and prospects.In biomedical applications, all graphene derivatives are useful and extensively applied as components or as ingredients for biosensors, real-time bioimaging, cancer diagnosis and treatment, catalysis, and water purification ,33,34,352 hybridized carbon atoms organized in the form of a honeycomb lattice. Preparation or synthesis methods have been well reported such as mechanical exfoliation from graphite, chemical vapor deposition (CVD), and molecular beam epitaxy (MBE). For GO, its synthesis procedures introduce a wide variety of oxygen-containing functional groups, such as carboxyl, hydroxyl, carbonyl, ethoxy, and epoxy, on both planes of the GO sheets, thereby increasing its stability in aqueous solutions. This also provides easy functionalization and derivatization of these materials through both covalent and non-covalent modifications with different biocompatible polymers such as polyethylene glycol and chitosan, and conjugation with targeting moieties, including peptides and antibodies, to develop bio-functionalized nanocomposite systems with improved biological properties [As previously mentioned, graphene is a very thin nanomaterial with a single-layer of spoperties ,37. rGO For this reason, the excellent physicochemical and biocompatible properties of these graphene nanomaterials have created a wide variety of applications in the field of biomedicine. These are mostly in cancer research, ranging from nanocarriers and photo agents for drug/gene delivery to cancer cells as well as for photothermal and photodynamic therapies of cancer, both in vitro and in vivo, to its use as bioimaging agents for developing multifunctional theranostic platforms for carrying out more efficient detection and treatment of cancers . As showGenerally, a biosensor is an analytical device that combines a biological sensing element with a transducer . As showIn the previous session, the biological effects of graphene nanomaterials using diverse synthesis methods have been exploited for biomedical applications because of a large surface area that is capable of being immobilized on its surface and has easy-functionalization. Indeed, there are many possible approaches in the biomedical applications such as bioelectronics , tissue In particular, as pristine graphene has a thin film of infinite size with no imperfections which provides a large number of chemically active sites for charge-biomolecular interactions due to the large surface area, however these also lead to an enhanced sensitivity to the target biomolecules of very low concentrations and improved selectivity . For insOver the past decade, the feasibility of graphene in RF/microwave electronics was investigated owing to its excellent physical properties. As shown in 11 (or S22) is the reflection coefficient of the input (or output) port, and S21 (or S12) is the transmission coefficient of the output port to the input port (or output port to the input port), respectively. The measured S-parameters can be converted to many other parameters such as admittance (Y), impedance (Z), hybrid (H), transfer (T), and ABCD matrix [In RF/microwave measurement system, the measured data are primarily related to the S-parameters (or S-matrix), which is defined as the ratio of the output power (voltage) to the input power (voltage) in the frequency domain. In particular, the S-parameters for the two-part network are expressed asD matrix . Owing tGO has received considerable interests as a sensitive material for biosensing because electron transfer from the biomolecular binding mainly occurs at the defects or edges of GO structure. In addition, owing to the opposite properties of a hydrophobic and hydrophilic functional group on GO, this material exhibits good biocompatibility, high affinity for specific biomolecules. Hence, these properties of GO can provide many opportunities for the various approaches of biosensing platforms, including biosensors based on fluorescence resonance energy transfer, laser desorption/ionization mass spectrometry, surface-enhanced Raman spectroscopy, and electrochemical detection . R component revealed the sensing parameter for effectively detecting the glucose level with stable linearity and small fluctuation [ctuation .One of the excellent physical properties in graphene is a transparent and conducting material with low cost and low environmental impact. This is an ideal material for the integrant of biosensing devices in a range of transduction modes, from electrochemical transduction to optical transduction .Odorrana grahami, H. pylori, E. coli, and S. aureus [L)-capacitor (C) circuit, i.e., a resonant circuit for selecting a specific frequency. Also, this study demonstrated integration onto a tooth for specific bacteria detection in saliva via wireless circuitry [. aureus . These bircuitry .Recent advances in integrated biosensing platforms associated with remote sensing via RF/microwave wireless systems have focused on design and architecture of point-of-care (POC) diagnosis, attracting considerable interest in the biomedical applications. In particular, POC has significant diagnosis possibilities for use in the continuous and real-time monitoring of human metabolites as well as cancer biomarkers . In addiS. aureus, E. coli and so on, via bifunctional peptide. Recent advances in graphene nanomaterials such as synthesis techniques, electrical, thermal and mechanical analysis, surface treatment and device design have accelerated the development and application of graphene nanomaterials-based nanoelectronics as well as bioelectronics. In this review, we have examined the emerging advances of graphene nanomaterials-integrated biosensors including structures and merits of graphene nanomaterials and their biological functionalization in RF/microwave biomedical applications. From the developed RF/microwave biosensors, these biosensing schemes could be classified with passive RF/microwave devices and RF/microwave systems with graphene nanomaterials. Firstly, it was used as a biosensing scheme utilizing simple RF/microwave devices such as resonators and capacitors, with graphene nanomaterials like GO or rGO. In the case of latter, it was used as a biosensing scheme utilizing RF/microwave systems with graphene nanomaterials, e.g., graphene. These RF/microwave biosensors could be detectable of biomolecules, e.g., glucose, DNA, as well as bacteria, e.g., However, the research and development of these materials-based biosensing systems are in their infancy in the RF/microwave biomedical applications. This is because it is not only difficult to find the optimized frequency for biosensing, but devices and circuits also are dependent on the frequency. However, since there are great merits such as real-time, non-invasive, non-contact function, as a graphene nanomaterials-based RF/microwave biosensor, the biosensing scheme still needs to develop the robust biosensing platform integrated with wireless and flexible devices and circuits. In this case, there are also remains challenges how to find effective integration methods and how to secure stability for good performance of RF/microwave devices and systems with graphene nanomaterials. Before this challenge, the optimization of material fabrication and modification techniques to obtain large area, high quality, and uniform arrays will be essential for the highly sensitive and reproducible RF/microwave biosensors. Furthermore, the integration of graphene nanomaterials-based RF/microwave device needs to be optimized to minimize the entire device volume for portable, disposable and POC diagnosis and healthcare in the future.


The ‘leaky pipeline’ in academia is a clearly described phenomenon, but has not been examined in palliative care. We analysed the gender balance of speakers at the 9th World Research Congress of the European Association of Palliative Care (EAPC) to test the null hypothesis that there is no difference in the proportion of women and men with senior academic visibility in palliative care conference programmes.The final programme of the 2016 EAPC World Congress was examined, and the gender of each speaker was recorded. Presentations were assessed using a three-tier hierarchy of senior academic visibility: Free Communication sessions, Themed sessions and invited Plenaries (low to high). As there was only one Invited Plenary at EAPC 2016, we examined the gender balance at EAPC Plenaries from 2012 to 2016.2=25.4, p<0.001).Overall, the majority of speakers at EAPC 2016  were women. The proportion of women was highest in the Free Communication sessions . In the Themed sessions, women made up just over half of speakers . In 2016, there was 1 invited Plenary speaker, a man. From 2012 to 2016, just 6 of 23 invited Plenary speakers at EAPC conferences have been women (26.1%)  and unconscious gender bias need further exploration, as well as actions to address the imbalance. The gender balance in academia is the subject of considerable debate.Visibility, for example through conference talks and plenaries, is an important dimension of gender equality. A paucity of women conference speakers has been demonstrated in several academic disciplines.In June 2016, 1200 delegates attended the 9th World Research Congress of the European Association of Palliative Care (EAPC) in Dublin, making it one of the largest research conferences in palliative care globally. The programme featured 17 Free Communication sessions where researchers were chosen on the quality of anonymous (and gender-blind) abstracts to present their work in 8 min talks. In addition, there were seven Themed sessions in which speakers were invited on the basis of their level of authority on a specific subject to give 20 min talks. The most prestigious speaking opportunity is the invited Plenary where high-profile academics are invited to give longer talks (30 min) on the main conference stage.We aimed to analyse the gender balance of speakers at the 2016 EAPC World Congress to test the null hypothesis that there is no attrition of women in senior academic positions in palliative care, using the three-tiered hierarchy of Free Communication sessions, Themed sessions and Plenaries as a proxy for seniority.2 tests were used to assess differences. We did not analyse Meet the Expert sessions or Poster Discussion sessions.The final programme of the 2016 EAPC World Congress was examined, and the gender of each speaker was recorded. Speaker gender was determined either by direct observation of their talk or by inference from their first name. In eight cases, Google was used for more information. Where there was a substitution of speaker at the last minute, the gender of the speaker originally planned was used. At the 2016 EAPC, five top scoring abstracts were presented as 15 min Plenaries on the main stage. Since the decision to feature these presentations was made on the basis of an anonymous abstract, these were analysed with the Free Communication sessions. Since there was only one invited Plenary speaker at 2016 EAPC, we examined the gender balance of invited EAPC Plenary speakers over 5 years (2012–2016) by searching through relevant scientific programmes online. χAt EAPC 2016, there were a total of 17 Free Communication sessions comprising 102 talks. An additional five top scoring abstracts were presented on the main stage. There were seven Themed sessions which included 22 speakers. There was a single invited Plenary, the Ventafridda Lecture.Overall, the majority of speakers at EAPC 2016 were women. The proportion of women was highest in the Free Communication sessions. In the Themed sessions, women made up just over half of speakers. The Ventafridda Lecture was given by a man .2=25.4, p<0.001).Since there was only one invited Plenary speaker at EAPC 2016, we examined previous EAPC conference programmes to obtain a more representative view of gender balance of invited Plenary speakers for a 5-year period . At EAPCPalliative care is a profession where women make up the majority of the workforce,Given the large number of women working in palliative care, it is of concern that the most prestigious invited Plenaries are so infrequently given by women, and it is noteworthy that two EAPC conferences over the past 5 years have included no female Plenary speakers at all (2014 and 2016). These data suggest that there is attrition of women along the ‘academic pipeline’ in palliative care. However, the relative contribution of other potential factors such as self-selection  and unconscious gender bias  is unclear. In addition, palliative care is multiprofessional, with gender unequally represented within each of the professions. The potential contribution of discipline should also be explored.Given that there was a single Plenary speaker in 2016, we examined the gender balance of Plenary speakers over 5 years. This was based on the assumption that variation from conference to conference is likely to outweigh any temporal trends. The proportion of female Plenary speakers was one-third or fewer for each of the 5 years examined. There was no indication that the proportion of female Plenary speakers improved over time. Indeed, the 2012 and 2013 conferences had the greatest proportion of female Plenary speakers (one-third).Speaking at academic conferences is important for career advancement. Such opportunities not only facilitate networking and collaboration, but are used as a marker of quality by promotions panels.Failure of promotion on merit is deleterious for academia and ultimately for patient care. Whether these data represent a leaky pipeline, gender bias, self-selection or all three, it is essential that as a specialty we take this issue seriously. We hope that the data presented here provide a starting point for more in-depth consideration of this issue and appropriate strategies to tackle it.


CA-170 is currently the only small-molecule modulator in clinical trials targeting PD-L1 and VISTA proteins – important negative checkpoint regulators of immune activation. The reported therapeutic results to some extent mimic those of FDA-approved monoclonal antibodies overcoming the limitations of the high production costs and adverse effects of the latter. However, no conclusive biophysical evidence proving the binding to hPD-L1 has ever been presented. Using well-known in vitro methods: NMR binding assay, HTRF and cell-based activation assays, we clearly show that there is no direct binding between CA-170 and PD-L1. To strengthen our reasoning, we performed control experiments on AUNP-12 – a 29-mer peptide, which is a precursor of CA-170. Positive controls consisted of the well-documented small-molecule PD-L1 inhibitors: BMS-1166 and peptide-57. Alongside chemo- and radiotherapy, surgery, and other “targeted treatments”, cancer immunotherapy  is now regarded as the fifth pillar of cancer treatment, mainly due to a rapid development of potent immune checkpoint-blocking (ICB) therapeutic inhibitors ,2,3,4. T2 and is maintained by polar and hydrophobic interactions. More polar interactions comprising, hydrogen bonds and salt bridges, are observed at the periphery of the hydrophobic core based mainly on alkyl-π and π-π interactions. Interestingly, only PD-1 undergoes significant structural rearrangement upon binding, most notably in the CC′ loop, which transits from an open conformation in the apo form to a closed one in C β strand ..10]. PD-Physiologically, the PD-1/PD-L1 complex negatively regulates the immune response and provides so called T cell homeostasis ,15. CancCA-170, a small-molecule inhibitor against PD-L1/L2 and VISTA, with low nanomolar potencies for the treatment of advanced solid tumors and lymphomas . CA-170 is also undergoing phase II clinical trials for lung cancer, head and neck/oral cavity cancer, MSI-H positive cancers and Hodgkin lymphoma in India . According to the preclinical in vitro and in vivo data reported by the company, CA-170 demonstrated dose-dependent enhancement in the proliferation of PD-L1, PD-L2, and VISTA-inhibited T lymphocytes, and exhibited antitumor effects similar to those of antibodies, including tumor shrinkages and prolonged stable disease without adverse effects [In the recent years, several attempts for the development of non-mAb PD-1/PD-L1 inhibitors, such as macrocyclic peptides, peptidomimetic molecules and nonpeptidic small molecules, were presented and disclosed in particular by Bristol-Myers Squibb (BMS) and Aurigene Discovery Technologies Limited  is routinely employed to characterize small proteins, protein-protein and ligand-protein interactions . PerformCA-170 and AUNP-12 to hPD-L1 were assessed with the 1H and 1H-15N HMQC NMR spectroscopy. In the first experiment, the CA-170 compound was titrated against the 15N-labeled hPD-1-binding single domain of hPD-L1 (amino acids 18-134) and against the entire extracellular domain of the hPD-L1 protein (residues 18-239). In both 1D and 2D NMR spectra, no changes were observed for signals of the protein of hPD-L1, even when the 10-fold excesses of compound was used  with the KD at the millimolar level indicated by slight chemical shift perturbations (AUNP-12 at the molar ratio of 1/5. The rest of the results of NMR experiments for AUNP-12 with hPD-L1 (18-239) and with hPD-1 showed no interaction of the peptide and the proteins , which is currently a standard methodology to determine potency of the inhibitors in numerous publications and patents, due to its robustness and absence of false-positive results [50 of well-known hPD-L1 inhibitors as the positive reference: BMS-1166 and peptide-57 to cross-validate the results with the literature  or hPD-1 either. Lack of the activity in the precursor peptide does not rule out the possibility that its fragments alone may be potent. Moreover, we do not exclude the possibility that this family of compounds may act downstream from the hPD-1 receptor or on any other T cell-activating pathway, since the compounds clearly present promising therapeutic results according to the data provided by Aurigene and Curis [Based on the above consideration we state that neither nd Curis .AUNP-12, is a hybrid of the peptide sequences extracted from hPD-1, that are supposed to be close to its interaction surface with hPD-L1. However, if we look at the locations of particular peptide fragments of AUNP-12  Cas: 1673534-76-3, AUNP-12 was purchased from SelleckChem  BMS-1166 was synthesized according to the protocol from described in [peptide-57 was synthesized as described in [ribed in , peptideribed in .Escherichia coli BL21 (DE3). Bacterial cells were cultured at 37 °C in LB or M9 minimal medium containing 15NH4Cl as the sole nitrogen source to achieve 15N isotope labeling. Proteins expression was induced with 1 mM Isopropyl β-d-1-thiogalactopyranosid (IPTG) at OD600 of 0.8 and the cells were cultured overnight. For hPD-1, hPD-L1 and mPD-L1 temperature was lowered to 28 °C, for hPD-L1(18-239) temperature was left at 37 °C. The inclusion bodies purification was carried out as described previously [l-Arginine hydrochloride, 2 mM EDTA, 5 mM cystamine and 0.5 mM cysteamine for hPD-1 and 0.1 M Tris pH 8.0, 1 M l-Arginine hydrochloride, 0.25 mM oxidized glutathione and 0.25 mM reduced glutathione for hPD-L1, human PD-L1(18-239) and mPD-L1. After refolding, proteins were dialyzed 3 times against solution containing 10 mM Tris pH 8.0 and 20 mM NaCl. Finally, proteins were purified by SEC (size-exclusion chromatography) on HiLoad 26/600 Superdex 75 column  in 25mM sodium phosphate pH 6.4 with 100 mM NaCl for hPD-1 or in PBS pH 7.4 for hPD-L1, hPD-L1(18-239) and mPD-L1.The gene encoding hPD-1  was cloned into pET-24d, the gene encoding hPD-L1  was cloned into pET-21b, the genes encoding hPD-L1  and mPD-L1 (amino acids 38-134) was cloned into pET-28a, respectively. Proteins were expressed in the v/v) of D2O was added to the samples to provide the lock signal. All spectra were recorded at 300 K using a Bruker Avance III 600 MHz spectrometer. Binding of the compounds was analyzed by titrating the 15N-labeled hPD-L1/hPD-1 and recording the 1H and 1H−15N HMQC spectra prior to and after the addition of the compounds.For NMR measurements, the buffer was exchanged by gel filtration to PBS pH 7.4. 10% (50) of tested compounds, measurements were performed on individual dilution series. After mixing all components according to Cis-Bio protocol, the plate was left for 2h incubation at room temperature followed by TR-FRET measurement on Tecan Spark 20M. Collected data was background subtracted on the negative control, normalized on the positive control, averaged and fitted with normalized Hill’s equation to determine the IC50 value using Mathematica 12.HTRF assay was performed using the certified Cis-Bio assay kit at 20 µL final volume using their standard protocol . To determine the half maximal inhibitory concentration   and Jurkat T cells overexpressing hPD-1 and carrying a luciferase reporter gene under the control of Nuclear Factor of Activated T-cells Response Element (NFAT-RE)  were cultured in RPMI-1640 medium  supplemented with 10% Fetal Bovine Serum  and 200 mM L- Glutamine (Biowest) in the presence of G418  and Hygromycin B Gold  as selection antibiotics. The overexpression of hPD-L1 and TCR ligand in aAPCs and PD-1 in ECs were confirmed by flow cytometry and western blot analysis, respectively. PCR tests for 50 values) were calculated from the Hill’s curve fitting to the experimental data.The activity of the inhibitors of hPD-1/hPD-L1 immune checkpoint was examined using the hPD-1/hPD-L1 Blockade Bioassay (Promega), according to the manufacturer’s instructions. hPD-L1 aAPCs were seeded on 96-well (white) plates at the density 10 000 cells/well 17h prior to the experiment. The 2.5-fold dilutions of the small molecules or peptide-57 were first prepared in DMSO. On the day of the assay the compounds were diluted 1000-fold in the assay buffer  to maintain the constant concentration of DMSO . The 2.5-fold dilutions of nivolumab, a positive control anti-hPD-1 monoclonal antibody , were prepared in the assay buffer on the day of the assay. The culture medium was discarded from the wells and serial dilutions of either the small-molecule or antibody was added. Afterwards, Jurkat hPD-1 cells were seeded at the density of 20,000 cells per well in the assay’s plates. After 6h of the incubation in standard culture conditions, assay plates were equilibrated at ambient temperature for 10min, followed by a 20min incubation with the Bio-GloTM Assay reagent (Promega). The luminescence was detected using the Infinite M200 reader. Half maximal effective concentrations  containing protease inhibitor cocktail (Sigma Aldrich). Following the electrophoresis and transfer to PVDF membranes, the membranes were blocked with 4% BSA  in TBS-N buffer and incubated with primary antibody solution at 4 °C overnight. After that, four washes with TBS-N buffer, the incubation with secondary antibody for 1h at room temperature, and additional four washes were done. Then, the detection was performed with the use of Clarity Western ECL Substrate  and ChemiDoc MP system (BioRad).The following antibodies and dilutions were used: rabbit monoclonal anti-hPD-L1 , cat. 13684), rabbit monoclonal anti-α-Tubulin , goat peroxidase-conjugated anti-rabbit .For the analysis of the expression of human PD-L1 on the surface of aAPC cells flow cytometry analysis was performed. The cells were seeded on 6-well plates and cultured till sub-confluency. The cells were detached from the plates with TrypLe Select Enzyme , placed on ice, washed 2 times with Flow Cytometry Staining Buffer  and stained with mouse anti-human PD-L1 antibody . 17-4714 antibody was used as an isotype control of the staining. The cells were analyzed with the FACSCanto II cytometer.CA-170 is the first small-molecule inhibitor targeted at PD-L1 that was introduced to phase I and II clinical trials. We, however, unambiguously determined that there is in fact no direct binding between CA-170 and its peptidic precursor AUNP-12 to PD-L1. In the NMR binding assay, neither of these molecules showed any interactions with the one- or two-domain PD-L1 in the human and mice variants as well as human PD-1. CA-170 and AUNP-12 were also not able to dissociate complex formation in the isolated system of the HTRF assay and cell-based assay mimicking in vivo conditions. All measurements were cross-validated with positive controls: peptide-57 and small-molecule BMS-1166. Therefore, proposed mechanism of the direct blocking of hPD-1/hPD-L1 interactions with CA-170 has to be changed.


Bilayer graphene consists of two stacked graphene layers bound together by van der Waals interaction. As the molecular analog of bilayer graphene, molecular bilayer graphene (MBLG) can offer useful insights into the structural and functional properties of bilayer graphene. However, synthesis of MBLG, which requires discrete assembly of two graphene fragments, has proved to be challenging. Here, we show the synthesis and characterization of two structurally well-defined MBLGs, both consisting of two π−π stacked nanographene sheets. We find they have excellent stability against variation of concentration, temperature and solvents. The MBLGs show sharp absorption and emission peaks, and further time-resolved spectroscopic studies reveal drastically different lifetimes for the bright and dark Davydov states in these MBLGs. Molecular bilayer graphene — a discrete assembly of two stacked graphene fragments — is challenging to produce in pure form. Here, the authors are able to synthesize and characterize stable molecular bilayer graphenes by the π−π stacking of two identical, well-defined nanographene sheets. SLG sheets display an inherent tendency to stack into multilayered structures due to the inter-planar π−π interaction3. As the simplest manifestation of such structures, bilayer graphene (BLG) exhibits novel characteristics absent in SLG, such as bandgap opening4, bound excitons6, etc., and therefore provides a fertile ground7 for studying two-dimensional materials. Advances in construction and characterization of BLG have also provided theoretical frameworks and instrumental tools in exploring the emerging field of van der Waals heterostructures9. However, current methods, such as chemical vapor deposition11 and layer-by-layer assembly12, often restrict BLG formation on substrate surfaces. Moreover, tendency of solution-dispersed graphene sheets to self-aggregation3 leads to a mixture of multi-layered graphene sheets in solution and merely the dispersions of enriched bilayer or trilayer graphene sheets could be achieved by exfoliation of specific graphite intercalation compounds13.Single-layer graphene (SLG) possesses a variety of unique optical and electronic properties, which stem from its mono-atomic network of sp14. Research on nanographene has provided deep insights into the structure–property relationships of graphene15 and stimulated the field of organic synthesis of graphenic materials with a well-defined architecture16. Although peripheral substitution of nanographene with sterically bulky substituents can preserve its monolayer structure19, stacking nanographenes in a discrete bilayer form is much more challenging. By using surface supramolecular assembly method, bilayer assembly of nanographene can only be found at liquid/solid interfaces in a very limited amount21. This problem is further compounded by the fact that the generated bilayer nanographene is often bound to, and thus difficult to be separated from, the substrate surface. An alternative strategy involves tethering two nanographene sheets together with a covalent linker23. However, the resultant product cannot be considered as a molecular cutout of bilayer graphene because the covalent attachment disrupts the van der Waals interactions between the two layers. On the other hand, although stable bilayer nanographene assembled by π−π interaction can qualify as molecular bilayer graphene (MBLG), to the best of our knowledge, such an MBLG has not been obtained in a pure form so far, because the propensity for multilayer stacking of nanographene often results in a dynamic mixture of columnar superstructures with indefinite numbers of layers28.Nanographene is widely regarded as the molecular model of graphene114H24R6)2, 1 and (C96H24R6)2, 2, R = mesityl], each formed by π−π stacking of two identical nanographene sheets. Mass spectrometric analysis shows that these MBLGs possess a bilayer structure and can dissociate into the corresponding monolayer when exposed to enhanced laser ablation. Nuclear magnetic resonance (NMR) spectroscopy and single-crystal X-ray diffraction (SXRD) clearly validate their bilayer structures. The interlayer H···H proximity in the bilayer structure is revealed by two-dimensional (2D) nuclear overhauser effect spectroscopy (NOESY). The bilayer structure of MBLGs is highly stable against varying temperature, concentration, and solvent. The absorption and emission of the MBLG shows clear vibronic fine structures. The time-resolved fluorescence (TRPL) and transient absorption (TA) spectroscopies indicate that the bright and dark Davydov states of MBLGs have drastically different lifetimes.Herein we report the synthesis and characterization of two stable MBLGs  , the upper level is the allowed state (bright state). On the other hand, if the transition dipoles are in in-line alignment, the upper level is the forbidden state (dark state). Owing to the parallel dipole alignment in π–π stacking configuration of 1 and 2, the optical transition from ground state to the upper and lower Davydov states are allowed and forbidden, respectively46. Taking into account this energy splitting, we tentatively attribute the bright and dark state to the upper and lower states, respectively, and the fast energy relaxation from upper to lower states is responsible for the short lifetime of bright states.Owing to the van der Waals interaction between two aggregated molecules, originally degenerate excited states from the two monomers will form excited states with different energies (Davydov splitting) after forming a dimer. The optical transition to one of these two Davydov states is allowed, while the transition to the other one is forbidden. The energetics of the allowed and forbidden states depend on the transition dipole alignmentWe have demonstrated the chemical synthesis of well-defined MBLGs and validated their structures. The MBLGs are remarkably stable, showing no sign of aggregation or dissociation. The MBLGs here offer the well-defined molecular cutout of bilayer graphene and can help in the understanding of structure and properties of bilayer graphene at the molecular level. Further investigation on rational design of MBLGs with tunable size, edge, and twist angle could be readily envisioned, and perhaps even more ambitious goals, such as hetero-structural MBLG and bilayer graphene nanoribbons, could be aimed. Our current study can also inspire the synthesis of other stable bilayer carbonic nanomolecules, such as geodesic polyarenes, carbon nanobelt, and nanorings.Supplementary InformationPeer Review File


Phenotypic changes in lung fibroblasts are believed to contribute to the development of Idiopathic Pulmonary Fibrosis (IPF), a progressive and fatal lung disease. Long intergenic non-coding RNAs (lincRNAs) have been identified as novel regulators of gene expression and protein activity. In non-stimulated cells, we observed reduced proliferation and inflammation but no difference in the fibrotic response of IPF fibroblasts. These functional changes in non-stimulated cells were associated with changes in the expression of the histone marks, H3K4me1, H3K4me3 and H3K27ac indicating a possible involvement of epigenetics. Following activation with TGF-β1 and IL-1β, we demonstrated an increased fibrotic but reduced inflammatory response in IPF fibroblasts. There was no significant difference in proliferation following PDGF exposure. The lincRNAs, LINC00960 and LINC01140 were upregulated in IPF fibroblasts. Knockdown studies showed that LINC00960 and LINC01140 were positive regulators of proliferation in both control and IPF fibroblasts but had no effect upon the fibrotic response. Knockdown of LINC01140 but not LINC00960 increased the inflammatory response, which was greater in IPF compared to control fibroblasts. Overall, these studies demonstrate for the first time that lincRNAs are important regulators of proliferation and inflammation in human lung fibroblasts and that these might mediate the reduced inflammatory response observed in IPF-derived fibroblasts. The etiology and pathogenesis of the disease are still unclear, although recent research has indicated that persistent epithelial injury and/or exposure to pathogens, leads to the secretion of fibrotic, proliferative and inflammatory mediators such as TGF-β14, PDGF5 and IL-1β6 . These are then thought to act upon surrounding fibroblasts, to induce an exaggerated wound healing response that contributes towards the development and progression of IPF1.Idiopathic pulmonary fibrosis (IPF) is a fatal progressive chronic disease characterised by scar tissue accumulation in the lungs leading to impaired gas exchange and restricted ventilation8 and diminished capacity to synthesis cyclooxygenase 2 and prostaglandin E29. Differences have also been observed in proliferation and release of fibrotic components, although these have resulted in contradictory observations13. Attempts to understand these persistent phenotypic changes at the epigenetic level have shown differences in the pattern of DNA methylation15. However, although there are reports of histone changes associated with individual genes linked to IPF16, there has been no attempt to determine if there are genome wide changes in the profile of histone modifications.Comparison of the phenotype of lung fibroblasts derived from IPF patients with those from non-fibrotic patients has shown that these exhibit multiple differences including reduced apoptosis17. In contrast, little is known about lncRNAs which are commonly divided into three groups: long intergenic non-coding RNAs (lincRNAs) that are located between protein-coding genes, antisense that are transcribed across protein coding genes on the reverse strand and pseudogenes, that are non-translated versions of protein coding genes19. In most cases, it is believed that the actions of lncRNAs are mediated through domains that interact with proteins, acting as scaffolds or to modulate their activity21. At the present time, a number of miRNAs have been implicated in the regulation of fibroblast function and in the development of IPF including let-7d22, miR-17~9223, miR-10124 and miR-15525. In contrast, although there is accumulating evidence to indicate that lncRNAs are important regulators of biological response19, little is known regarding the role of lncRNAs in lung fibroblast function or IPF.High-throughput sequencing indicates that much of the human genome is transcribed into non-coding RNAs (ncRNAs). The majority of ncRNAs (>90%) are involved in house-keeping activities such as translation , splicing (short nuclear RNAs) and post-transcriptional RNA modifications (short nucleolar RNA) whilst the others are broadly classified as either short ncRNAs (<200 nt (nucleotides)) or long ncRNAs (lncRNAs) (>200 nt). The microRNA family of short ncRNAs are the best characterised and are known to induce mRNA degradation or block messenger RNA (mRNA) translation via the RNA interference pathway27 and up-regulation of two lincRNAs, LINC00960 and LINC01140 in IPF fibroblasts. Functional analysis has shown that the both LINC00960 and LINC01140 are required for proliferation and that LINC01140 is a negative regulator of the inflammatory response. Given that LINC01140 is upregulated in both IPF fibroblasts and lung biopsies, our data suggest that LINC01140 mediates the reduced inflammatory response in IPF fibroblasts.In this report, we have demonstrated differences in the functional responses between fibroblasts derived from control and IPF lungs. These are reflected by changes in H3K4me1, a histone epigenetic marker of primed genes and enhancersOur initial aim was to determine whether there were significant differences in the phenotypic responses of lung fibroblasts derived from control lung and IPF patients and to examine the potential role of lincRNAs in these variations. We selected high-throughput approaches to measure the time- and concentration dependency of their TGF-β1-induced fibrotic response, PDGF-induced proliferation and IL-1β-stimulated inflammatory response.28. To assess potential difference in the fibrotic response in control and IPF fibroblasts we examined the time- and concentration-dependent release of PAI-1 release in response to TGF-β1.TGF-β1-induced activation of lung fibroblasts triggers the expression of PAI-1 , a protein known as an important regulator of fibrinolysis and wound healing and therefore implicated in the process of fibrosisInitial studies showed a time-dependent release of PAI-1 from non-stimulated IPF cells, which reached significance at 48 hours (h) and 72 h Fig. . However50 between the control (319 pg/ml) and IPF (139 pg/ml) individuals showed a significant difference . In relation to PAI-1 release, these studies show IPF fibroblasts are more fibrotic since they demonstrate increased sensitivity to TGF-β1 activation.To examine the concentration-dependent response, cells were incubated for 72 h with 0.01 to 10 ng/ml TGF-β1 and compared with time matched non-stimulated cells. As expected, TGF-β1 induced a concentration-dependent increase in PAI-1 release in both control Fig.  and IPF 29. To assess the proliferative response in control and IPF fibroblasts, we investigated the time- and concentration-dependent effect of PDGF-AB on the fibroblast cell number. Initial examination of the proliferation in non-stimulated fibroblasts showed increased proliferation in control versus IPF at 72 h , we observed a concentration-dependent increase in proliferation at 72 h, although we observed wide variation in the response of IPF fibroblasts Fig.  compared31. To assess potential differences in the inflammatory response, we examined the time- and concentration-dependent IL-1β induced IL-6 release from control and IPF lung fibroblasts.The pro-inflammatory cytokine IL-1β has been shown to potently induce IL-6 release from various cell types including fibroblastsInterestingly, we observed a time dependent IL-6 release in non-stimulated fibroblasts, which was significantly increased in control (67.1 ± 19.8 pg/ml) versus IPF (3.7 ± 0.9 pg/ml) fibroblasts at 24 h, 48 h and 72 h Fig. . Exposur50 values showed a significant reduction (p = 0.0234) between control (EC50 = 101 pg/ml) and IPF (EC50 = 550 pg/ml) fibroblasts. Based upon IL-6 release, this data would indicate that IPF fibroblasts are less inflammatory than control cells as they demonstrated both reduced sensitivity to IL-1β and a lower basal release of IL-6.To examine the concentration-dependency, cultured cells were incubated for 72 h with increasing concentrations of IL-1β ranging from 0.03 to 30 ng/ml . Comparison of the logEC31. Exposure to 10 ng/ml TNF-α induced a comparable time-dependent release of IL-6 from control and IPF fibroblasts, which continued to increase over the 72 h period  and IPF (EC50 = 2.2 ng/ml) fibroblasts  despite no history of smoking in either the control or IPF patients. To examine further the potential role of epigenetics changes, we have employed Western blotting to compare the levels of H3K4me1, H3K4me3 and H3K27ac in the five control and IPF patient samples . Amongst this group, PAI-1 (Serpin E1) gave one of the highest fold changes in both the control (44-fold) and IPF fibroblasts (31-fold). Comparison of the lists of genes that were changed following TGF-β1 exposure identified only 77 that were differentially expressed between control and IPF, which includes a single lincRNA (LOC100507516) whose expression was reduced in IPF . To this end, we compared the expression profile in control and IPF lung fibroblasts in non-stimulated cells and those exposed to 3 ng/ml TGF-β1 for 24 h, using the Affymetrix GeneChip™ Human Transcriptome Arrays 2.0  that were increased in IPF  and IPF (n = 20) lungs32  antisense sequences against each lincRNA that produced 50–85% knockdown following overnight transfection into fibroblasts and stimulation with TGF-β1 for 24 h Fig. . FollowiFollowing overnight transfection with LNA antisense to LINC00960, LINC01140 and the 2 negative controls, we observed no effect upon cell count, indicating that this procedure had no immediate action upon cell viability Fig. . FollowiWe once again demonstrated LNA mediated knockdown of LINC00960 and LINC01140 in both control and IPF fibroblasts following overnight transfection and 24 h exposure to IL-1β Fig. . LINC0003. These included TGF-β1-stimuated PAI-1 release as a marker of the fibrotic response34, PDGF-induced changes in cell numbers as a model of proliferation36 and IL-1β-stimulated IL-6 release as a marker of inflammation37.In this report, we have for the first time investigated the role of lincRNAs in the regulation of lung fibroblast function and whether changes in their expression might be involved in the development of IPF. In our initial studies, we employed high throughput assays to undertake detailed examination of the time and concentration responses of control and IPF fibroblasts, examining phenotypes associated with IPF13. In contrast, the concentration response curves showed a significant leftward shift in IPF fibroblasts, indicating that these had increased sensitivity to TGF-β1.As previously reported when using collagen release as a measure of fibrosis, we observed no difference in the magnitude of PAI-1 release from control and IPF fibroblast in the absence and presence of a maximally effective TGF-β1 concentrationet al.10.In the absence of PDGF, we observed a reduction in the proliferation of IPF fibroblasts. Following exposure to PDGF, the small increases and the variability of the response between individual fibroblasts samples, meant there was no significant difference in the magnitude or concentration dependency of the proliferation. This variability in the PDGF-induced proliferative response had previously been observed by Jordana 38. Unlike TGF-β1 and PDGF, the role of IL-1β in the development of IPF is yet to be established although there is a report of showing increased levels in bronchoalveolar lavage39. In addition, there are conflicting reports as to whether IL-1β elicits profibrotic or antifibrotic activities41. IL-1β was previously shown to drive IL-6 expression in orbital and synovial fibroblasts in vitro42, although nothing is known regarding its effect on lung fibroblasts. Significantly, measurement of IL-6 indicated that non-stimulated IPF fibroblasts were less inflammatory and showed a reduced sensitivity to IL-1β stimulation. In contrast, examination of the IL-6 release in response to TNF-α showed no difference between control and IPF fibroblasts indicating that reduced sensitivity might be specific to IL-1β. In support of these phenotypic observations, a recent meta-analysis of four previous microarrays studies in control and IPF fibroblasts which showed repression of inflammation and immune pathways43. Interestingly, this reduced inflammation in non-stimulated and IL-1β-stimulated IPF fibroblasts may explain the lack of efficacy observed when corticosteroids and immunosuppressants have been examined as therapeutic options for the treatment of IPF3.In general, IL-1β is considered to be a potent pro-inflammatory cytokine that can induce the release of multiple pro-inflammatory mediators, including IL-615, with none having examined histone modifications. We therefore measured the distribution of H3K4me1, a marker for priming at promoter and enhancer regions that is thought to work through the recruitment of multiple chromatin-remodelling complexes44. Significantly, we identified multiple regions demonstrating differential expression of H3K4me1, with unsupervised hierarchical clustering showing a clear separation between control and IPF fibroblasts. In addition, although the patients variation meant that we failed to reach significance, we have shown a trend towards reduced expression of H3K4me1, H3K4me3 and H3K27ac in IPF fibroblasts and speculate that this might to linked to phenotypic changes such as the reduced inflammatory response. Although there have been a number of reports showing changes is histone marks localised to specific genes implicated in IPF16, this is the first evidence to show genome-wide changes in histone modifications and that targeting the acetylation of H3K4 and other histones might provide a novel therapeutic strategy. Indeed, this latter contention is supported by studies into Brd4, a bromodomain containing protein that can bind to acetylated histones and act as a scaffold to attract components of the transcriptional machinery. Thus, inhibitors of Brd4 have been shown to attenuate migration, proliferation and IL6 release in isolated fibroblasts and inhibit fibrosis in a bleomycin-induced model of flung fibrosis45.Having demonstrated significant differences in fibrotic and inflammatory responses between control and IPF fibroblasts, we then proceeded to show that this was also reflected at the epigenetic level. To date, the only genome wide epigenetic studies in IPF have examined the changes in the profile of DNA methylation47 and pulmonary fibrosis associated lncRNA (PFAL)48 contribute to lung fibrosis by acting as sponges (and therefore inhibitors) for miRNAs. Using microarray analysis and qRT-PCR, we identified 2 lincRNAs, LINC00960 and LINC01140 that were up-regulated in IPF compared to control fibroblasts. Given that isolated and cultured fibroblasts might not reflect the situation in the whole lung, we were able to analyse historical RNA sequencing data obtained from lung biopsies32 and confirm up-regulation of LINC01140 but not LINC00960. Knockdown studies indicated that neither appeared to regulate TGF-β1-induced PAI-1, although it is not possible to eliminate the possibility that these lincRNAs might regulate another aspect of the fibrotic response. In contrast, knockdown studies indicated that both LINC00960 and LINC01140 were required for proliferation. Interestingly, these knockdown studies appeared to contradict the results in non-stimulated cells which showed increased proliferation in control fibroblasts despite reduced levels of LINC00960 and LINC01140. The reason for this discrepancy is unknown, although we speculated that other pathways, rather than LINC00960 and LINC00960, might play a dominant role in regulating the increased proliferation in non-stimulated control fibroblasts. It might also be argued that measurement of cell number is not the optimal approach and that lincRNA knockdown may have impacted upon cell viability rather than decreased proliferation. However, we believe that this is unlikely since we observed no significant effect of lincRNA knockdown upon TGF-β1-induced PAI-1 release and increased IL6 release in response to IL-1β exposure, when decreased cell viability might have been expected to also reduce these responses. Examination of the inflammatory response showed that LINC01140 was a negative regulator of IL-1β-induced IL-6 release. In the latter case, there was a greater elevation in IL-6 release from IPF fibroblasts following LINC001140 knockdown, possibly reflecting the increased expression of LINC01140 in IPF versus control fibroblasts. This increased LINC01140 levels in IPF fibroblast might also explain the absence of IL-6 release in non-stimulated IPF fibroblasts compared with controls and the shift in the concentration response curve in response to IL-1β exposure. Although the mechanism of action is yet to be determined, negative regulation of the inflammatory response is commonly observed with both lncRNAs and miRNAs including interleukin-7-antisense (IL7-AS)49 and miR-146a50. Of relevance, we have recently shown that increased inflammatory response is IPF fibroblast might also be related to reduced expression of MIR3142HG/miR-146a51.At the present time, little is known regarding the role of lincRNA either in the regulation of fibroblast function or whether changes in their expression are associated with the phenotypic changes associated with IPF. The exception is studies showing that H1953. With regards to inflammation, immune-modulatory lncRNAs have been shown to bind and regulate the activity of (i) transcription factors such as nuclear factor-κB (NF-κB), signal transducer and activator of transcription 3 (STAT3) and the glucocorticoid receptor (GR), and (ii) chromatin associated proteins including the heterogeneous nuclear ribonucleoproteins (hnRNPs) family and components of the polycomb repressor complex 2 (PRC2)55. However, the identification of these functional domains (conserved sequences) has been hindered by their poor evolutionary conservation, which in contrast to protein coding genes, does not require the maintenance of a conserved open reading frame for optimal translation. Instead, it is thought that lncRNAs conservation is geared towards the maintenance of genomic position (synteny) and short conserved domains (microdomains)53. In addition, it has been suggested that the action of lncRNAs might be mediated through acting as sponges for miRNAs, although this remains a controversial issue,57. Future studies will examine the mechanism of action of LINC00960 and LINC01140 in fibroblasts responses.Overall, this report is the first to demonstrate that phenotypic differences between control and IPF fibroblasts are associated with genome-wide changes in histone modifications and increased expression of the lincRNAs, LINC00960 and LINC01140. Significantly, we also demonstrate that these lincRNAs can regulate fibroblast proliferation and inflammation whilst changes in LINC01140 expression might mediate the reduced inflammatory in IPF fibroblasts. Currently, the mechanisms by which these lincRNAs regulate these responses is unknown. By analogy with proteins and the non-coding RNAs involved in mRNA translation (tRNAs/rRNAs) and splicing (snRNA), it is speculated that the biological actions of lncRNAs are mediated through domains containing conserved sequences, that interact with proteins and/or base pair with RNA/DNA2 humidified incubator. Upon reaching approximately 80–90% confluency cells were washed in sterile 1x PBS   followed by treatment with StemPro® Accutase® cell detachment solution . All experiments were performed using cells plated at passage 6 to 7.Control  and IPF fibroblasts  were obtained from Professor Carol Ferghali-Bostwick  and the Coriell Institute of Medical Research . Approval was obtained from the Medical Board of the Medical University of South Carolina and the Coriell Institute of Medical Research with patients proving material with informed consent. All methods were performed in accordance with the relevant guidelines and regulations. Neither the control or IPF patients had a history of smoking. Isolation of lung fibroblasts was initiated using explants of minced lung tissue. Fibroblasts were cultured in DMEM  growth media  supplemented with 10% (v/v) FBS  , 1% (v/v) Penicillin-Streptomycin  and 0.1% (v/v) Fungizone . All cultures were maintained in a 37 °C, 5% (v/v) CO4 cells were plated in 96 cell culture wells on day 1 and allowed to adhere overnight. On day 2, cells were serum-deprived by reducing FBS to 0.1% in 200 μl of starvation media. Fibroblasts were then treated with the required concentration of TGF–β1 , IL-1β , TNF-α  and PDGF-ΑΒ  and incubated for the indicated time before supernatants were collected for protein quantification assessment or cell viability assays.1 × 105 cells of control and IPF lung fibroblasts were seeded in 25 cm2 cell culture flasks  and medium was replaced until the cells reach near-confluency state. Their growth medium was then replaced with 3 ml starvation medium (0.1% FBS) and left overnight. The following day, the cells were treated with and without 3 ng/ml TGF-β1 for 24 h before all supernatants were collected and cells were lysed.5 × 10Lung fibroblast proliferation was evaluated by measuring cell viability with the Cell Counting Kit-8 . Cells (5000) were seeded in a 96-well plate and incubated overnight in 100 μl growth media (10% FBS). The following day, the cells were serum-deprived and treated with the indicated concentration of PDGF-ΑΒ and then incubated for the indicated times. Before the end of each assay, 10 μl of the CCK-8 solution was added into a final volume of 100 μl cultured media and incubated for 2 h before absorbance was measured using a microplate reader . The absorbance wavelength was measured at 450 nm and 600 nm which was then subtracted during data analysis.5 cells per well in 200 µL of growth media and incubated with the transfection mixes at 37 °C, 5% (v/v) CO2 overnight. The next day, 800 µL of media (0.1% FBS) was added to the wells to dilute out the lipid-LNA complexes and reduce the toxicity of the reaction. The cells were stimulated with either 3 ng/ml of TGF-β1 or 3 ng/ml of IL-1β and incubated for 24 h before harvesting for RNA extraction and ELISA analysis. The same transfection protocol was followed for the cell viability assays (CCK-8 kit) with or without 100 ng/ml of PDGF-AB stimulation for 72 h, however 96 well plates were used and the reaction volume and reagents were reduced accordingly. LNA Gapmer sequences: LNA1 –TCATACTATATGACAG; Negative Control LNA2 – GACGGTAAGTAGGCGA; LINC00960 LNA1 – GGCGTGAGAGTAAAGC; LINC00960 LNA2 – GTGCTTAGGCTTAGAG; LINC01140 LNA1 – TTTAATTGGGCCGTCT; LINC01140 LNA2 – TTGACACGGCTGACTT.On the day of transfection, 5 µL of HiPerFect (Qiagen) was mixed with 200 µL of growth media without antibiotics, serum or antifungals to prepare the transfection mix. LNA Gapmers were added to 200 µL of the transfection mix at a final concentration of 30 nM, placed in 12-well plates and incubated for minimum 10 mins at room temperature. Fibroblasts were then seeded at a density of 5 × 10Supernatants of cultured lung fibroblasts were collected and used to assess secretion of IL-6 and PAI-1, using the DuoSet ELISA  Development System Kits  following the manufacturer’s instructions. Samples and standard curve samples were diluted as appropriate in reagent diluent. Absorbance was measured at 450 nm with wavelength correction at 570 nm using a microplate reader .For all samples, total RNA was extracted using the RNeasy kit (Qiagen), included an on-column DNase treatment (Qiagen), according to the manufacturer’s guideline. RNA concentration was determined using the Qubit 2.0 (Life Technologies). RNA quality was measured using the Agilent Bioanalyzer and produced RIN values of >8.0.−(ΔΔCt) method was used to determine relative-quantities of individual mRNAs and lncRNAs which were normalized to 18S ribosomal RNA. qRT-PCR primer sequences: 18S – AAACGGCTACCACATCCAAG (Forward), CCTCCAATGGATCCTCGTTA (Reverse); IL-6 – ACTCACCTCTTCAGAACGAATG (Forward), CCATCTTTGGAAGGTTCAGGTTG (Reverse); LINC00960 – TCCAGGCGTCATAACCAACC (Forward), CGGTGCTTAGGCTTAGAGGG (Reverse); LINC01140 – CATCTCATCGGCATGGACCT (Forward), CAAACTGGACTGACTTTCACCA (Reverse).For quantitative PCR (qPCR), cDNA libraries were prepared from total RNA using the High capacity cDNA RT kit . Expression of mRNAs and lncRNAs were determined by qPCR using the SYBR® Green PCR mix  supplemented with a protease inhibitor cocktail . SDS-PAGE electrophoresis was used to separate proteins using the Mini-PROTEAN® TGX™ Precast Gels  with Precision Plus Protein™ Dual Xtra Prestained Protein Standards . Samples were transferred to PVDF membranes using the Trans-Blot® Turbo™ Transfer System (Bio-Rad) and the membranes were blocked for 1 h at room temperature (RT) in blocking buffer (3% BSA in PBS) before they were incubated overnight at 4 °C in a 3% BSA-TTBS  blocking buffer with the following antibodies: β-actin , H3K4me1 , H3K4me3  and H3K27ac . The membranes were washed 3 times in TTBS for 5 mins and incubated with IRDye® 800CW Goat anti-Mouse  and IRDye® 680RD Donkey anti-Rabbit  for 1 h at RT before they were washed and imaged on the LI-COR Odyssey CLx imaging system.The Affymetrix GeneChip™ Command Console Software was used to summarise probe intensity data and to generate a CEL file for each sample. The CEL files were then processed by the Affymetrix Expression Console™ using Robust Multi-chip Analysis (RMA) to generate CHP files (Probe-level summarisation) following the manufacturers manual. The CHP files were then used for Quality Control analysis which generated a full report with the array QC metrics and appropriate algorithm parameters. Using the data of the CHP files, a 3-dimensional Principle Component Analysis (PCA) graph was generated accounting for the majority of variance based on a set of variables PCA1, PCA2 and PCA3 in the original data set. CHP files generated by the Expression Console Software were used in the Transcriptome Analysis Console (TAC) Software to perform statistical analysis and obtain a list of differentially expressed genes. To run the TAC software, a library folder containing the annotation files was required (HTA-2_0.na36.hg19.probeset.csv) and installed. CHP files were imported and separated into different condition groups for analysis using the Gene Level Differential Expression Analysis tool. A master table was generated containing signal expression levels of all 67528 transcript clusters (genes) covered in the arrays, 44699 protein coding and 22829 non-protein coding. Signal intensity for each transcript cluster was presented as a Bi-weight average signal (log2) value and depending on which two conditions were compared the values of ANOVA p-value, FDR p-value (based on Benjamini-Hochberg Step-Up FDR-controlling Procedure) and gene fold change (linear) were adjusted accordingly.32 were downloaded from Sequence Read Archive (SRA) (https://www.ncbi.nlm.nih.gov/sra) using the following command in SRA tools: fastq-dump −I–split-files <file_name>. The paired end reads were aligned to the human reference genome (hg38) using Hisat2 (version 2.0.4)59 using the following command line options: hisat2 −q–dta–rna-strandness FR −x < reference-genone.gtf > −1 < forward_strand.fa > −2 < reverse-strand file.fa > −S < output.sam > . Output SAM files were then sorted and converted to BAM files (samtools sort −@ 8 −o output.bam output.sam) and indexed (samtools index −b output.bam) in Samtools60. The profile of gene expression (using the Gencode v27 database) in the BAM files for each samples were determined using Stringtie61: stringtie < sample.BAM > −G < GenCodev26.gtf > −1o < samples.gtf > −e −A < sample.txt > . Following feature counting: featureCounts -a < reference-genome.gtf > −g gene_name -o counts.txt Control_*.bam IPF_*.bam the differential gene expression was assessed using DeSeq2 and the following R script: curl −s -O http://data.biostarhandbook.com/rnaseq/code/deseq 2.r cat simple_counts.txt | Rscript deseq2.r 19 × 20 > results_deseq2.txt.Previously reported sequencing data obtained from the biopsies of 19 control and 20 IPF lungs (GSE92592)62: bowtie2 −q–very-fast < reference_genome.gtf > −U < file_name.fastq > −S < output_file.sam > . Output SAM files were then sorted and converted to BAM files (samtools sort −@ 8 −o output.bam output.sam), indexed (samtools index −b output.bam) in Samtools60 and then converted to BigWig format using BamCoverage (which is part of the deepTools suite63) using the following command line: bamCoverage −b < input_bam.bam > –normalizeUsingRPKM–binSize 30–smoothLength 300 -p 10–extendReads 200 −o < output_file.bw > . Significant ChIPseq peaks (q =< 0.1) in each sample were called with MACS264 using the broadpeak options: macs2 callpeak –t < sample > −c < backgrounf_igG > −broad − < output_files > −g hs. The differential expression of H3K4me1 peaks was determined by inputing the individual Bam files (Bowtie2) and BED files (Broadpeaks output - MACS2) for control and IPF samples into Diffbind (version 2.2.1) on Galaxy 65. Control 1 was omitted as an outlier following PCA analysis.Chromatin immunoprecipitation (ChIP) using a H3K4me1 antibody  and the iDeal ChIP-seq kit for Histones kit  was performed on the 5 control and 5 IPF fibroblast samples. In addition, we performed ChIP on a single control and IPF using an IgG antibody to provide a background control. Paired-end 75 bp sequencing data was obtained using the Illumina HiSeq. 4000 at the Oxford Genomics Centre at the Wellcome Centre for Human Genetics (funded by Wellcome Trust grant reference 203141/Z/16/Z). FASTQ sequencing data from control (n = 5) and IPF (n = 5) fibroblasts was aligned to hg38 using Bowtie 266. Data was log2 transformed following the addition of 1 FPKM. The threshold for reporting gene expression at FPKM > 1 is based upon the ability to validate sequencing data using qRT-PCR.The abundance of Gencode v27 defined genes in individual samples was defined as the fragments per kilobase exon per million reads mapped (FPKM) and determined using Stringtie (RNA). PCA and hierarchical clustering on Gencode v27 genes demonstrating an expression > 1 FPKM was performed using Genesis (v1.7.7)All statistical analysis and graphs were generated using GraphPad Prism 7 software.Dataset 1 Table showing ChIPseq H3K4me1 binding in control and IPF fibroblasts (MOESM1)Dataset 2 Images of original Western blot gels showing expression of H3K4me, H3K4me 3 and H3K27ac in control and IPF fibroblasts (MOESM2)Dataset 3 Overall microarray data showing gene expression in non-stimulated and TGFß-stimulated control and IPF fibroblasts (MOESM3)Dataset 4 Differential gene expression in TGFß-stimulated control fibroblasts (MOESM4)Dataset 5 Differential gene expression in TGFß-stimulated IPF fibroblasts (MOESM5)Dataset 6 Differential gene expression between TGFß-stimulated control and IPF fibroblasts (MOESM6)Dataset 7 Differential gene expression between non-stimulated control and IPF fibroblasts (MOESM7)Dataset 8 Differential gene expression between control and IPF biopsies (MOESM8)


New architectures of transparent conductive electrodes (TCEs) incorporating graphene monolayers in different configurations have been explored with the aim to improve the performance of silicon-heterojunction (SHJ) cell front transparent contacts. In SHJ technology, front electrodes play an important additional role as anti-reflectance (AR) coatings. In this work, different transparent-conductive-oxide (TCO) thin films have been combined with graphene monolayers in different configurations, yielding advanced transparent electrodes specifically designed to minimize surface reflection over a wide range of wavelengths and angles of incidence and to improve electrical performance. A preliminary analysis reveals a strong dependence of the optoelectronic properties of the TCEs on (i) the order in which the different thin films are deposited or the graphene is transferred and (ii) the specific TCO material used. The results shows a clear electrical improvement when three graphene monolayers are placed on top on 80-nm-thick ITO thin film. This optimum TCE presents sheet resistances as low as 55 Ω/sq and an average conductance as high as 13.12 mS. In addition, the spectral reflectance of this TCE also shows an important reduction in its weighted reflectance value of 2–3%. Hence, the work undergone so far clearly suggests the possibility to noticeably improve transparent electrodes with this approach and therefore to further enhance silicon-heterojunction cell performance. These results achieved so far clearly open the possibility to noticeably improve TCEs and therefore to further enhance SHJ contact-technology performance. Graphene has been regarded as a promising candidate for the new emerging generation of transparent electrodes in several applications such as displays, touch screens and/or solar cells ,2. Its uSignificant effort has been devoted to improve the overall performance of PV devices using graphene. Thus, this material has been reported to play diverse positive roles acting as electrode, active layer, interfacial layer or electron acceptor in photovoltaic cells ,10,11,12PV market is dominated by crystalline-silicon technology that requires exhaustive technological solutions to achieve thinner and cheaper wafers. In this sense, silicon-heterojunction (SHJ) technology is emerging as a reliable low-temperature and high-efficiency solution, where new architectures of transparent conductive electrodes (TCEs) to generate and extract the current in a more efficient way are being required . In thatRegarding the prominent and unique properties of graphene, several approaches incorporating it have proven to be especially interesting. In particular, hybrid concepts combining graphene with metal grids  or nanowIn the present work, new architectures of TCOs and graphene electrodes incorporating one, two and three atomic graphene layers in different configurations have been fabricated in order to evaluate the optoelectronic properties of the resulting structures. The key parameters for choosing the most appropriated and reliable combination of layers for the electrode have been determined.A commercial copper foil was used as a catalyst for the chemical vapor deposition (CVD) of the graphene . This foil was cut into 5 cm × 6 cm rectangles and subsequently dipped in 55 wt.% phosphoric acid and then electro-polished for 10 min. After that, a hot peroxide treatment at 80 °C for 10 min was carried out.2), and methane (CH4)) connected to the CVD chamber were flushed before the growth. After that, the temperature was raised to the synthesis temperature (1050 °C) under Ar atmosphere. Once the temperature was stabilized, a mixture of 0.7% CH4/31% H2/68.3% Ar (in volume) was introduced for 15 min at 50 mbar pressure. A cold-wall CVD reactor  for 4-inch-diameter samples was used for the synthesis of graphene monolayer. The system has two heaters, a bottom one under the sample and one at the top heating the gases from the showerhead and also capable of radiatively heating non-flat or thick samples . All the4)2S2O8) 0.3 M), which is gradually changed into deionized water (DIW) for the final steps. The synthesized graphene on the Cu foil was transferred to the different substrates used throughout the study using a home-built system designed to automatically transfer graphene to arbitrary substrates . This sy−5 Pa. The purity of Ar gas used for the sputtering processes is 99.999%, and its flux is controlled with a mass-flow controller. In this work, aluminum-doped zinc oxide (AZO) and indium tin oxide (ITO) were chosen as TCO materials. The 4-inch commercial ceramic targets of ZnO:Al2O3  and In2O3:SnO2  for AZO and ITO deposition respectively were sputtered by radio-frequency (RF) and direct-current (DC) power for AZO and ITO, respectively.A commercial UNIVEX 450B sputtering system from Leybold, Cologne, Germany equipped with four magnetron sources was used for the fabrication of TCO thin films. This system has a confocal geometry where the guns are placed at around 0.15 m from the substrate centre. The base pressure of the process chamber is 1 × 104 Ω·cm). The native silicon oxide (SiOx) was removed from the silicon-wafer surface by chemically etching with a dilute (2%) hydrofluoric acid. After that, the wafer was rinsed in DIW and finally, dried by blowing nitrogen over it. On the other hand, glass substrates were also used to determine the TCE optical transparency. The glass substrates were ultrasonically cleaned using an special detergent that is a phosphate free surface active cleaning agent radioactive decontaminant from DECON laboratories , rinsed in DIW, and finally, immersed for 2 min in isopropyl alcohol and dried by blowing nitrogen over it.The TCE structures were deposited on 4-inch polished resistive float zone <100> silicon wafers (resistivity > 10λ/4) in order to lead to the destructive interference of the beams reflected from the silicon and TCO surfaces, thus acting as an anti-reflectance (AR) coating. The number of GML was chosen in order to obtain a TCE with a good conductivity and low optical losses.Two different configurations were studied for the graphene-based TCE. In In the particular case of the configuration 1, an Ar plasma bias etching on the substrate surface was carried out at 120 W under 0.5 Pa for 5 min without intentional heating prior to the sputtering deposition. This process was used to improve TCO thin-film adherence. After that, the substrate was rotated at 20 rpm, and heated at 190 °C in the holder system. Regardless the TCO material deposited, the gas flow rate and working pressure were maintained constant at 5 sccm and 0.18 Pa, respectively. The samples were sputtered at the constant values of 75 W DC power and 250 W RF power applied to ITO and AZO targets, respectively. In the case of configuration 2, no bias was applied and softer sputtering conditions were used to avoid the possible damage to GML structure, that is, at room temperature and 25 W DC power and 150 W RF applied to ITO and AZO targets, respectively.The structural quality of the graphene in the TCE was evaluated from Raman measurements. A WITec Alpha 300AR, Ulm, Germany, confocal microscope was used for the Raman spectroscopy of the samples. Raman spectra were obtained in backscattering geometry using a ×100 objective lens  in ambient conditions. A 532 nm wavelength laser set to a power of 3 mW was used as excitation source. 2 quartz substrates. Four metal coplanar parallel electrodes unequally spaced, pictured in TCER) and to compare them to the theoretical values expected corresponding to an electric circuit with two parallel electric resistances, as described by the following Equation: TCOR is the sheet resistance of the TCO film and GGR, the corresponding to GML.The metal combination tested to have a low contact resistance was Ti (50 nm)/Ag (500 nm). Transmission-line-model (TLM) measurements were done by testing 4-point electrical resistance between all the possible electrode pairs with two contacts on each electrode. Contacts were made by means of 4 micro-positioners, a power supply was used to bias the samples, an electrometer measured currents and a voltmeter provided voltages. These TLM measurements were used to determine the actual sheet resistance of the hybrid TCEs developed  is the global spectral AM1.5G irradiance [The total optical reflectance spectra were measured in the wavelength range from 300 to 1500 nm by using a UV/Visible/NIR Perkin-Elmer Lambda 1050 spectrophotometer equipped with a 6 mm integrating sphere accessory. From these measurements the weighted reflectance radiance .T) of the fabricated TCEs deposited on glass were measured by using a three-detector module in the Perkin Elmer spectrophotometer. This measurement serves to determine the fraction of light that reaches the absorber of the solar device. Finally, optical transmission maps were obtained from the TCEs fabricated on glass by means of a home-made system combining a focused white-light lamp, an X-Y linear-positioner set, a pair of current preamplifiers, a reference photodiode and a pair of digital voltmeters. Specular transmittance spectra (BT), and the other one is directly related to the combined transmittance of the sample plus the background (SAT + BT). SAT comprises the transmittance of both the substrate and the different TCEs (TCO and/or GML). Thus, knowing the transmittance of the substrate (SUBT), it is possible to calculate the amount of light transmitted by the different layers (GGT for the GML and TCOT for the TCO) as follows:The system is controlled by specific software that returns an image where each point registered is associated with a specific transmittance value. Once the data is collected, it is represented in the form of a histogram where two peaks appear: one of them corresponds to the background transmittance  , where tTCO of 120 ± 10 Ω/sq, and GGR of 390 ± 10, 230 ± 30 and 120 ± 5 Ω/sq for one, two and three GML, respectively, were used. The evaluation of the compatibility between the TCO and the GML was carried out in terms of the electrical and the optical properties of the whole structure. 2. As it can be appreciated, the measured TCER values differed considerably from the theoretical ones, especially in the case of configuration 2 where the TCO is sputtered on top of the transferred graphene. This suggests that GML might get damaged by the bombardment of the high-energy sputtered atoms during the sputtering process of the TCO thin film. This process could induce significant disorder, affecting the electrical performance of the GML [The contact resistivity of the Ti/Ag metallization was in the range of 3 to 10 mΩ·cm the GML ,22. TCER in configuration 1 (graphene transferred onto sputtered TCO), summarized also on On the other hand, the results of the TCOR is 85 ± 5 Ω/sq was used. Therefore, another TCO material free of aluminum in its composition, such as ITO, was tested. This material was preferred because of its good conductivity and its optical transparency. The electrical performance of the ITO-based TCEs are summarized in The results from With regard to optical performance of hybrid TCEs developed, the optical characteristics were also addressed in both total reflection and transmission geometries. Thus, Finally, the white-light optical-transmission maps of the samples on glass substrates can reveal the number of GML transferred and the homogeneity of the transfer process. This technique is very useful because of its high sensitivity to small variations in thickness (either produced during the graphene growth or due to folds and wrinkles arising from the transfer process). Optical-transmission maps yielded white-light transmissions of 95% that corresponds to On the other hand, regardless the TCE configuration, the white-light transmission values for the AZO-based TCEs are slightly lower than the bare AZO, as it was expected. In addition, no re-cover effect during thermal evaporation process was observed in 12 cm−2.The structural quality of the optimum hybrid ITO-based TCE, with configuration 1, was assessed. 2/min. The laboratory relative humidity was 50% and its temperature was 23 °C. All the electrical values were extracted using mathematical models detailed in [Finally, THz-TDS conductance and resistance maps acquired in reflection-mode for optimum hybrid ITO-based TCE deposited on Si substrate are pictures in ailed in . From Fiailed in . In compRegarding the resistance b, a centIn this work, the compatibility of the graphene and the TCO deposition processes was analyzed with the aim of developing hybrid TCEs for silicon-based solar cell technology. The evaluation of TCEs made by coating the GMLs with a sputtered TCO layer indicated that this process severely affected the structure of graphene, leading to a significant detriment of sheet-resistance . In contrast, the GML placed on top of a TCO led to TCEs with relatively good sheet resistances, particularly when the TCO material was ITO. This hybrid TCE showed sheet resistance values as low as 55 Ω/sq. These results suggest the convenience of avoiding aluminum in the TCO material, and hence, the choice of ITO is considered as suitable to achieve an optimum TCE structure. The in-depth electrical analysis using the THz-TDS technique led to average conductance values as high as 13.12 mS and a sheet resistance of 76.2 Ω/sq, improving the values of a common TCO. Optically, it was also verified that graphene transferred on TCO caused a total reflectance decrease, reaching values of weighted reflectance 2–3% lower than those obtained without graphene. All these results suggest the possibility of a noticeable improvement for TCEs by developing structures combining graphene with TCOs. Such hybrid TCEs would have optical and electrical properties favoring the absorption of more photons, hence leading to positive effects on the short circuit current, as well as lowering the cell series resistances.