Here, NiFe2O4 nanoparticles and nanohybrids using large-diameter graphene oxide (GO) sheets are prepared via solvothermal synthesis for low-frequency wide bandwidth shielding (L and S radar bands). The synthesized materials were characterized using XRD, SEM, FTIR and microwave magneto dielectric spectroscopy. The dimension of these solvothermally synthesized pristine particles and hybrids lies within 30–58 nm. Microwave magneto-dielectric spectroscopy was performed in the low-frequency region in the 1 MHz-3 GHz spectrum. The as-synthesized pristine nanoparticles and hybrids were found to be highly absorbing for microwaves throughout the L and S radar bands (< −10 dB from 1 MHz to 3 GHz). This excellent microwave absorbing property induced by graphene sheet coupling shows application of these materials with absorption bandwidth which is tailored such that these could be used for low frequency. Previously, these were used for high frequency absorptions  with limited selective bandwidth.The magnetic nanoparticle composite NiFe The role of multifunctional materials as absorbers is significant in both antenna engineering and electromagnetic interference shielding/radar absorption. Several strategies have been used to minimize the effective radar cross section and decrease the reflection and emissions as well ,2. SurfaMicrowave absorber materials are characterized normally based on the absorption mechanism, i.e., dielectric absorbers, magnetic absorber and hybrid materials . Dielect2O4,have been widely studied for its high microwave absorption [2O4or its composite with rare earth elements; polymers; carbon nanostructures, especially CNTs; and reduced graphene oxide (rGO) for high microwave frequencies (>4 GHz) [2O4is its limited absorption bandwidth and selective absorption frequencies [2O4nanoparticles were prepared via novel modified solvothermal route and the influence of large-diameter rGO addition is analyzed by using in situ reduction and hybrid formulation utilizing the benefits of solvothermal treatment. Their magneto-dielectric properties were tailored with an aim to get high microwave absorption in the low-frequency region (mixed L and S bands) that can cover their entire bandwidth. Here, in this research work, NiFe2O4-rGO hybrid absorbers were prepared that showed excellent microwave absorption in the low-frequency region covering the entire UHF/L/S bandwidth instead of selective frequency resonances.Soft ferrites, especially NiFesorption . Most of(>4 GHz) ,12. Addiquencies –15. Cons4 in 400ml acidic mixture (320ml:80ml H2SO4:H3PO4). The reaction mixture was stirred rapidly and continuously until the color of solution turned brown. After ceasing the oxidation, the suspension containing highly oxidized flake graphite was washed multiple times with 5M HCl and double-distilled deionized water. Nanocrystals of NiFe2O4 were synthesized via modified solvothermal approach using ethanol and DI in a definite proportion[2O4. Thesame method was carried out for hybrid preparation using a different weight percentage of GO powder [Graphene oxide was used as a source of graphene sheets through modified solvothermal reduction process. All the chemicals used as precursors at any stage were of AR grade. Graphene oxide was synthesized using a simplified Hummer method;typicallroportion. In a tyO powder .2O4 nanoparticles and the NiFe2O4-rGO hybrid to analyze the structure of the synthesized nanopowder. 2O4 were fccstructurednanoferrites. Their structure fully matches with powder diffraction file JCPDS 10–0325. Similarly, the XRD pattern of hybrid samples NFG5, NFG7 and NFG10also agreed well with the structure of NiFe2O4. Moreover, there was no peak of graphene oxide in all the three synthesized composite samples. The absence of the characteristic peak (001) associated with graphene oxide in samples shows the complete reduction under solvothermal condition mentioned in experimental synthesis.X-ray diffraction (XRD) was carried out for both the NiFe2O4and NiFe2O4-graphene composite samples NG5, NG7 and NG10, along with their chemical compositions. 2O4to graft on. The composition of graphene oxide analyzed through EDX shows that the synthesized graphene sheets have high oxygen content (around 42%) attached with individual carbon layers. 2O4and NFG5, NFG7 and NFG10. The SEM micrographs of NiFe2O4show that the synthesized nanoparticles via solvothermal medium are spherical and have grown in a leaflike pattern with a uniform size distribution, as shown in 2O4hybrid samples i.e. Scanning electron microscopy (SEM) coupled with energy dispersive x-ray spectroscopy (EDX) was used to analyze the microstructures, mainly the sizes, shapes, and distributions, and the oxidation contents of as-synthesized graphene oxide sheets, NiFe2O4 nanoparticles, graphene oxide powder and the hybrid NFG10. It is clear from the transmission spectra of NiFe2O4 nanoparticles that two majors peaks around the wave numbers 384 and 587 cm−1 correspond to the ν1 and ν2 bands, respectively, which are typical of ferrites[−1 corresponds to octahedral Ni-O stretching vibrations, while the bond lying around 587cm−1 shows the stretching vibrations due to tetrahedral Fe-O. The peak at 1110and 1383cm−1 are the result of asymmetric stretching vibrations of CO32−/NO3− adsorbed on the ferrite matrix or of the impurities from reaction precursors[−1 and at 1609cm−1 arethe result of O-H stretching and bending vibrations attached with iron ions and of moisture adsorbed on ferrite nanoparticles[−1. The peak at 1107cm−1 shows the in-plane bending vibrations of C-H bonds. The band around 1636 cm−1 shows the C = O stretching vibrations of the oxidized carbon backbones. The peak at 1386 cm−1 shows the C-OH stretching vibrations. The broad peak-like feature shown at 3446 cm−1 shows the presence of moisture in graphene oxide powder[−1. The major peak at 1571cm−1 shows the vibrations from oxygen-depleted carbon lattice showing the fair reduction process during solvothermal conditions. The peaks at 1224and 1382cm−1 appear because of CO32− in the composite sample[Fourier transform infrared spectroscopy (FTIR) was performed to analyze and confirm the bonding behavior, functionalization of graphene lattice with nanoferrite crystals and reduction of graphene oxide in composite.  ferrites. The banrecursors. The banparticles,23,24. Ide powder. In the te sample,24. Abser), respectively; μ′ and μ″ are the real and imaginary parts of complex permeability μr, respectively. 2O4 and hybrid samples NFG5, NFG7 and NFG10. 2O4, the real parts of permittivity are 4.47, 4.82, 5.57 and 8.12 at 10MHz and these decreases to 1.61, 2.19, 1.65 and 2.24 at 1GHz for NiFe2O4, NFG5, NFG7 and NFG10, respectively. Magnetic losses are smaller than the dielectric losses in all the samples because of the dielectric polarization, which shows that the higher contribution is from dielectric loss along with the magnetic loss.It is clear from the pattern of the Microwave magneto-dielectric properties usually depend on a large number of factors such asthemethod of synthesizing graphene and pristine nanoferrite particles, their attachment to carbon lattice, defects in nature and distribution, sizes and shapes of graphene sheets and nanoparticles, edge structure or chirality, the nature of dielectric relaxation and the motion of electrons,27,29–312O4, NFG5, NFG7 and NFG10 are below −10dB for the entire range of frequency from 10MHz to 3GHz. There are two absorption vortexesthat appear around 200 MHz and 900 MHz for pristine NiFe2O4 nanoparticles. With the increase in graphene concentration, the vortexes for all of samples radiate away from the center absorption frequencies of pure Ni-ferrites synthesized through modified solvothermal route, with lower vortex shifting to 100, 72 and 50 MHZ and higher vortex to 1.11, 1.36 and 1.46 GHz for NFG5, NFG7 and NFG10, respectively. The results of microwave absorption shows the nanoparticles of NiFe2O4and their composites show ultrahigh performance with absorber thickness around 2 mm in the vast frequency range of 10 MHz-3 GHz as compared to the already reported absorption with high absorber thicknesses of typically 10 mm and at high frequency (>4 GHz) [It is clear from (>4 GHz) –15,27.The dielectric permittivity shows the dipolar and electric polarization at the microwave spectrum and with the increases in reduced graphene oxide percentage in hybrid sample and the strained lattice causes change in electric and dipolar polarization. The change in dielectric properties can also be explained by hopping between Fe and Fe & spin/charge polarization . In case2O4anchored with graphene sheets. This type of hybrid multilayer stacking thereby presents a continuous path for the incident energy of microwave to be absorbed via repeated magneto-dielectric losses coupled with the nanohybrid system. The reason of absorption peaks in reflection losses are mainly due to resonance phenomenon and charge hopping in ferrites based samples i.e. domain wall resonance and defects polarization centers and its coupling. [This unusually high absorption and large absorption bandwidth can be attributed to the low dimension and uniform dispersion of nanoparticles of NiFeoupling. ,35. Thes2O4and its composites with large-diameter rGO sheets were prepared via solvothermal synthesis in mixed ethanol/DI media with an average particle size in the range of 30–58 nm. Microwave materials analysis shows that the both the NiFe2O4and the composite with rGO sheets show high absorbance (−10 dB) of microwave from 1 MHz to 3 GHz with absorption bandwidth comprising whole L and S bands instead of usual selective absorptions of microwaves. The maximum reflection loss was −68 dB at 1.11 GHz.NiFeS1 Fig2O4, NFG5, NFG7 and NFG10.Microwave magneto-dielectric properties (a) real part (b) imaginary part of complex permittivity (c) real (d) imaginary parts of complex permeability of NiFe(TIF)Click here for additional data file.


The PNA molecule, if used parallel with hydrolysis probe in QPCR analysis, greatly facilitates the detection efficiency of MSRV even if ERVWE1 is present abundantly in respect to MSRV in the analysed sample. We achieved a wide and measurable range from 1 × 10 e2 to 1 × 10 e8 copies/reaction; the linearity of the technique was maintained even at the low MSRV level of 1 % in respect to ERVWE1. Using our newly developed method we confirmed that the expression of MSRV takes place in normal human astrocytes and in human umbilical vein endothelial cells in vitro. We also found that the stimulation of human monocytes did not influence the specific expression of MSRV but it caused changes in mRNA level of distinct HERV-W templates.HERV-W is a multi-locus family of human endogenous retroviruses (HERVs) that has been found to play an important role in human physiology and pathology. Two particular members of HERV-W family are of special interests: ERVWE1  and MSRV (multiple sclerosis-associated retrovirus that is thought to play a significant role in human pathology as a result of its increased expression in the brain tissue and blood cells derived from patients with multiple sclerosis (MS)). Both ERVWE1 and MSRV mRNA share high level of similarity and hence a method that allows to exclusively quantify the MSRV expression in clinical samples would be desirable. We developed a quantitative polymerase chain reaction (QPCR) technique for the detection and quantification of the multiple sclerosis-associated retrovirus. The assay utilises fluorescently labelled oligonucleotide probe, which is complementary to the conservative fragment of MSRV  Accordigression . Brudek ividuals . Anotherividuals . Both muilarity) . On the  in 81 % , 11.Because an increased level of HERV-W RNA has been reported in brain tissue from MS patients , a preciPeptide nucleic acid (PNA) is a DNA mimicking synthetic oligonucleotide with peptide back-bones instead of sugar groups and was first reported in 1991 by Nielsen et al. from the University of Copenhagen . By compAs mentioned above, PNA-mediated PCR clamping method is especially desired if DNA of interest is present in a very low amount relative to the wild type one . This sienv mRNA copy number differed in respect of the presence or absence of the ERVWE1-specific PNA, therefore we concluded that MSRV is present predominantly over syncytin-1 in human astrocytic U-87 MG cell line. Additionally, compared to the intercalating dyes (e.g. SYBR Green I), our novel method incorporated the specific fluorescent probe that is much more suitable for virology/microbiology experiments, due to higher pathogen-specificity of the procedure primers, the utilisation of a third sequence-specific element, such as oligonucleotide probe is preferred over an intercalating dye in the virology/microbiology fields due to higher pathogen-specificity of the method. In the probe-based chemistry, both primers and probe hybridise to the common target, thus as much as three events must occur independently to give a specific fluorescent signal [In our previous report, we described a real-Time polymerase chain reaction/PNA-mediated methodology that allows the specific quantitative amplification of multiple sclerosis- associated retrovirus (MSRV). The assay is based on two sequence-specific oligonucleotides (PCR primers) that are common for both MSRV and ERVWE1 gene fragment, an ERVWE1- specific PNA probe and SYBR Green 1 intercalating dye that was essential in the quantitative assessment. Those results showed that it is possible to estimate the MSRV level in a biological sample if the ERVWE1 template is excluded from the reaction by means of PNA-mediated PCR clamping. According to our results with the PNA inhibition of ERVWE1, we showed that the overall expression of HERV-W was similar in samples treated with PNA or without PNA clamping, therefore we concluded that the expression of MSRV is predominant over syncytin-1 in human astrocytic U-87 MG cell line. Because the calculated HERV-W t signal .env sequences were derived from the GenBank database according to previously published data [env sequences denoted as MSRV with four env sequences denoted as coding for syncytin-1. Accession numbers of the investigated entries were as follows: AF331500, AF127229, AF072498 for MSRV and AY101582, AF513360, AF208161 and NM_014590 for syncytin-1. In both sequence types, a consensus sequence was built using CONS software from the EMBOSS package [HERV-W hed data , 11. We Peptide nucleic acids, 15-nucleotides in length, were synthesised and purified at the Department of Chemistry, University of Gdańsk, Poland (please see “Synthesis of peptide nucleic acids complementary to ERVWE1” caption for details). Lyophilised PNA was resolved in 5 % DMSO and left for 20 min at room temperature (RT) to obtain a stock concentration of 10 μM. PNA was then aliquoted and stored at −20 °C.env gene sequence are shown in Fig. Oligonucleotide primers for polymerase chain reaction were designed according to previously published guidelines . Three rTotal RNA was extracted from normal human astrocytes (NHA) cell line , human umbilical vein endothelial cells (HUVECs) . Peripheral blood monocytic cells (PBMCs) were obtained after centrifugation on blood in the Histopaque . Afterwards monocytes were isolated from the PBMCs by negative immunomagnetic separation using Pan-T and Pan-B Dynabeads  . Monocyt2, 2.0 μl of 10 mM dNTP mixture, 0.5 μl of RNasin Ribonuclease Inhibitor, 15 units of AMV Reverse Transcriptase and 0.5 μg of Random hexamers. Reaction mixture was incubated for 10 min at room temperature and then for 30 min at 42 °C followed by thermal inactivation of reverse transcriptase at 95 °C for 5 min. According to the manufacturer’s recommendations, reverse transcription reaction mixture was further diluted 1:4 with nuclease-free water before the use in QPCR. Five μl of diluted RT reaction mixture  was then used as a template for subsequent QPCR analysis.For NHA and HUVECs, 3 ml of TRI reagent  was used to lyse cells from one 10 mm in diameter tissue culture plate after the >80 %-confluence was achieved. Before TRI reagent was added, culture medium was removed but without washing the cells with PBS. Monocyte cultures were lysed using one ml of TRI reagent, which was added directly into wells of a 24-well culture plate after removing the culture medium. Three wells were then pooled together. After cell lysis and complete disruption of nucleoprotein complexes, total RNA was precipitated and further purified according to the one-step extraction method described by Chomczynski et al. . FinallyIn our assay, we used random hexamers as primers for the reverse transcriptase enzyme. As it is stated in Promega’s instruction for use of their product # A3500 “Choose Oligo(dT)15 when priming at the 3′poly(A) region is desired. Choose Random Primers when priming throughout the length of the RNA is desired”.15 and random primers may, in theory, improve the quality of cDNA pool. However, the use of oligo(dT)15 concomitantly with random hexamers/nonamers is problematic because both solutions require slightly different thermal conditions and finally the resulting cDNA library may not be more representative than when using random primers alone.It should be emphasised that the choice of optimal RT priming is crucial to get the most representative cDNA pool. Human endogenous retroviral-derived particles make even more complex issue because they are broadly dispersed thorough the genome and are mostly truncated; e.g. a significant part of HERV-W possesses a poly-A tail (such as syncytin-1) while other retroviral members does not. If so, a mixture of oligo(dT)For cloning purposes, DNA extract obtained from human placenta was used .Polymerase chain reaction was performed using Qiagen Taq PCR Master Mix kit . The reaction mixture in a total volume of 25 μl consisted of 12.5 μl of 2× Qiagen PCR Master Mix, 200 nM of each Syncytin_out_F and Syncytin_out_R primer and 0.5 μl of placental DNA extract as a template. After reaction, amplicons were resolved by means of agarose gel electrophoresis, excised from the gel and extracted/purified using silica-based spin columns . Four separately purified amplimers were then sequenced . BLAST analysis of obtained sequences confirmed that the specific amplification of ERVWE1 locus took place, giving a 100 % similarity to the reference sequence (GenBank: NM_014590). For cloning purposes, PCR reaction was repeated at identical conditions as described above, but less error-prone Pfu polymerase was used instead of less accurate Taq polymerase . Furthermore, Pfu polymerase generated blunt-ended amplicons that were essential for the subsequent blunt-end cloning procedure.4, 200 nM of each deoxyribonucleotide, 1 U of Pfu polymerase and of 25 pmol of each syncytin_mut_F and a mutagenic_MRSV_R primer .Both ERVWE1–derived and mutated MSRV-like PCR products were cloned into pSC-B-amp/kan plasmid using StrataClone Ultra Blunt PCR Cloning Kit according to the manufacturer’s guide . Mini preps were prepared in 3.5 ml of Luria Broth medium and were purified by NucleoSpin Plasmid kit . Obtained plasmids were given an initial examination to confirm the presence of the insert (regardless of their orientation) by PCR with a plasmid-specific universal T3/T7 primer pair. Finally, pSC-B-MSRV and pSC-B-ERVWE1 positive clones were sequenced in both directions by means of dideoxy-sequencing with the use of insert-specific primers, i.e. Syncytin_out_F and Syncytin_out_R . The mini-prep extracts were quantitatively measured using SYBR Green 1 fluorescent dye  against the known amounts of lambda DNA as a reference  in a fluorometer . The excitation wavelength of the dye was 495 nm, and emission was filtered using a 520-nm barrier filter. Plasmid DNA copy number was then calculated using dsDNA copy number calculator  and filtered with a 0.22-μm syringe filter before use. Fmoc-XAL-PEG-PS resin for the PNA synthesis was obtained from Merck KGaA . Fmoc/Bhoc-protected PNA monomers were purchased from Panagene . DNA oligonucleotides were obtained from Future Synthesis . The other reagents and solvents were obtained from Sigma-Aldrich Co. .PNA sequence: TACCAGTTTGGGTGA-amide complementary to ERVWE1 was synthesised using a Labortec AG SP-650 Peptide Synthesizer on a 3.8 µmol scale using a 2.5-fold molar excess of the Fmoc/Bhoc-protected monomers and Fmoc-XAL-PEG-PS resin (amine groups loading of 0.19 mmol/g) . MonomerThe crude oligomer obtained was lyophilised and purified by semi-preparative reverse phase, high performance liquid chromatography RP-HPLC using a Knauer system with a Kromasil C8 column  . The mobile phase consisted of 0.08 % TFA in acetonitrile (solvent A) and 0.1 % TFA in water (solvent B). The gradient profile for the mobile phase was as 0–30 % of solvent A held for 90 min. The column was maintained at ambient temperature. The flow rate was 4.5 ml/min, and the eluate was monitored with a UV detector at 254 nm. Fractions, which had purity greater than 98 % were collected and lyophilised.All analytical RP-HPLC analyses were performed on a Phenomenex Kinetex XB-C18 column   using a Beckman Gold System. The mobile phase consisted of 0.08 % TFA in acetonitrile (solvent A) and 0.1 % TFA in water (solvent B). The gradient profile for the mobile phase was as 0–30 % of solvent A held for 30 min. The column was maintained at ambient temperature. The flow rate was 1 ml/min, and the eluate was monitored with a UV detector at 254 nm.Finally, PNA was characterised by MALDI-TOF mass spectrometry , which confirmed the identity of the product synthesised.The specificity of PNA hybridisation to the complementary ERVWE1 ssDNA fragment and not complementary MRSV ssDNA fragment was analyzed and confirmed by capillary electrophoresis . Separat5 copies of pSC-B-ERVWE1 and of pSC-B-MRSV plasmids were subjected, in parallel, as the template into polymerase chain reactions. Reaction mixtures contained 1× Qiagen PCR Master Mix, 200 nM of Syncytin_out_F and 200nM of an appropriate reverse primer . A hydrolysis probe, complementary to MSRV but not to ERVWE-1 gene fragment was designed using EPRIMER3 software available from the EMBOSS package . The tar®-1, BHQ-1) at the 5′ and 3′ ends, respectively. The reporter dye’s emission is quenched when the probe remains intact. Only if the probe hybridised with its complementary target (i.e. MSRV env gene), the fluorescence of a reporter dye becomes detectable due to 5′ → 3′ exonuclease activity of Taq polymerase. In the case of MSRV, the hybridisation process of the probe is not impeded by PNA because of its non-complementarity to MSRV sequence , 2.5 mM MgCl2, 200 nM of each Syncytin_out_F forward- and sync1_REV/-1 reverse primer, 400nM of MSRV-complementary hydrolysis probe, 300nM of PNA (or an appropriate volume of 5 % DMSO).The hydrolysis probe used in the assay possessed a reporter dye  and a non-fluorescent quencher  beforehand were used for tenfold serial dilutions from 1 × 10 eIn our previous experiments, we have documented that the amplification of ERVWE1 gene fragment by means of polymerase chain reaction (PCR) can be specifically inhibited by complementary peptide nucleic acids (PNA) while no inhibition occurred when MSRV clones were used as the template. Furthermore, we also found that slight differences in PNA design or in primers localization altered drastically the efficiency of PCR blocking. A PNA probe, 14-monomers in length that hybridise at the position 1370–1383 in respect to the reference gene (GenBank: NM_014590) efficiently clamped the amplification of ERVWE1 at the concentration of at least 4 μM while we noted that its longer analogue  worked effectively even at tenfold lower concentration of 400nM or less  by the competition for a common target site between a PNA and one of the PCR primers; (2) by the interference of primer elongation and/or 3) by the product elongation arrest. According to our observations, primer elongation stoppage was the only efficient way to inhibit the reaction. Neither the competition between PNA and one of the PCR primer for a common target nor the product elongation arrest was efficient to inhibit the reaction even at the final PNA concentration of as much as 10 μM. Even in the case of an inhibition caused by primer elongation stoppage, the efficiency of the clamping was strongly influenced by the exact reciprocal primer-to-PNA localization . If PNA was not present in the reaction, the target MSRV sequence was correctly measured only at the highest concentration of the MSRV compared to ERVWE1 and an estimated molecule number was similar to that theoretically calculated only in these experimental conditions. Measurements were underestimated when the concentration of MSRV template was lower  and in human umbilical vein endothelial cells (HUVECs) cultured in vitro. We also examined whether the MSRV expression level changed after the lipopolysaccharide (LPS) - induced activation of human monocytes in vitro.3 ± 2.88 × 10 e2 and 7.47 × 10 e2 ± 3.4 × 10 e1 mRNA copies number/μg RNA, respectively. For beta actin gene expression, the results were as follow: 1.29 × 10 e5 ± 1.54 × 10 e4 and 2.52 × 10 e4 ± 1.4 × 10 e3 mRNA copies per one microgram of total RNA, respectively. As we expected, the measurement values of MSRV mRNA copy number change if ERVWE1- specific PNA was added into the QPCR reaction mixture simultaneously with MSRV-specific fluorescent probe. In the control reactions where no PNA was added but only probe was present, the obtained results were lower in comparison with that after the addition of PNA  . Interestingly, an inhibitory effect of LPS on the expression of HERV-W was observed in samples without PNA against ERVWE1 , indicating that LPS may influence the ratio between MSRV and HERV-W env expression in human monocytes, especially by the diminishing of non-MSRV-derived HERV-W copy number  did not alter the expression of MSRV because no differences in HERV-W expression were observed after addition of PNA into the reaction  patients provided by Mameli et al. showed that envelope gene intracellular domain of MSRV and ERVWE1 (syncytin-1) RNA share >89 % similarity while whole envelope gene showed >93 % similarity in both MSRV and ERVWE1.env and syncytin-1 gave rise to speculations regarding their possible physiologic and/or pathogenic effects. A major difference is that only MSRV has been detected as an extracellular virus  RNA containing terminal repeats, gag, pol and env sequences) while the syncytin-1 protein has been found only intracellularly or on the plasma membrane [The homology between MSRV membrane .env and syncytin-1 on the protein level. Thus, any results based on immunostaining or Western blot could reflect either MSRV env or syncytin-1 expression, or both.As a result of high similarity at the protein level, no specific antibody for unique HERV-W family members has been identified so far and it is not possible to discriminate between MSRV env and syncytin-1 in research assays is to utilise PCR, reverse transcriptase (RT) PCR and/or real-time PCR assays that could selectively amplify either MSRV or syncytin-1 mRNA sequences. Reliability of the assays with respect to specificity, sensitivity and inter- and intra-assay variations has been published previously [env sequences harbour a 12-nucleotide insertion in the transmembrane moiety. Based on this insertion, discriminatory real-time PCR assay was developed that can selectively amplify either MSRV env or ERVWE1. The selectivity of the method was achieved by the use of allele-specific primer pairs and appropriate probes, TaqMan-type for MSRV and ERVWE1 and locked nucleic acid (LNA) probe for the generic HERV-W env [Another approach to distinguish between the expression of MSRV eviously . With reRV-W env .env-encoding DNA and RNA exhibited statistically significant increased detection and expression in the brains of MS patients in comparison with control. Moreover, ERVWE1 env transcripts were inducible in glial cells as well. Conversely, such changes in MSRV expression were not reported in that work [A similar approach was demonstrated by Antony et al., where authors employed a real-time PCR approach using MRSV or syncytin-1-selective oligonucleotide primers and fluorescent, intercalating SYBR Green I dye which allowed the quantitative analysis of gene expression in brain tissue samples, peripheral blood leukocytes (PBLs) and monocyte-derived macrophages (MDM) from multiple sclerosis (MS) and non-MS patients as well as in an astrocytic U373 cell line. Analyses provided by Antony et al. revealed that ERVWE1 hat work .2 etc.) may result in possible primers-to-template mishybridisation and, subsequently, to the synthesis of unspecific amplimers [Due to an error-prone nature of Taq polymerase, a reverse transcriptase-quantitative polymerase chain reaction based on intercalating dyes is not recommended for the detection and quantification of mutant nucleic acids, especially when slight differences among investigated sequences are expected . Any suboptimal reaction conditions  and generates fluorescent signal, which theoretically is proportional to the number of double-stranded DNA molecules that are produced during PCR. This dye is suitable in most typical, quantitative, nucleic acid- based assays. On the contrary, if the reaction conditions are not optimal and/or if any double-stranded DNA molecules other than that from desired template arise in the reaction mixture, SYBR Green I gives inappropriate, false-positive results. Indeed, in respect to HERV-W research, Garson et al. in his Letter to the Editor of “AIDS Research and Human Retroviruses” journal ascertain some technical flaws in the real-time PCRs previously employed in the studies performed by Antony et al. , 39. ThuPeptide nucleic acid (PNA) is an artificially synthesised polymer that has properties of both nucleic acids and proteins. Importantly, PNA binds to the complementary sequence of nucleic acid with much stronger binding capacity than DNA does, because of the lack of electrostatic repulsion.Thus, in our assay, PNA oligomer is intended to bind specifically to the cDNA of the ERVWE1 locus and prevent them from being amplified by polymerase action. Our methodology allows the detection and measurement of discrete MSRV expression level due to MSRV-specific fluorescent oligonucleotide. What is more, any ERVWE1- derived templates are excluded because of their specific clamping by PNA. These factors improve greatly the analysis of MSRV quantity even if it is present in minority in respect to other similar molecules.In our previously published data, we found that SYBR Green I-based Real-Time PCR and its specific clamping by ERVWE-1-specific PNA favour the phenomenon in which virtually all “non-ERVWE1-derived” amplimers could be synthesised. Because only two HERV-W members, namely MSRV and ERVWE1 comprise the majority of HERV-W expression products at all, the rest of amplimers that arose and that are quantified in course of the Real-Time PCR are, in theory, considered as being of MSRV origin. However, any possible expression from distinct HERV-W loci cannot be excluded. The principle of an assay changes if non-specific SYBR Green-I dye is replaced by a MSRV-specific oligonucleotide probe. Thus, the fluorescent signal source for the quantification of the results is derived exclusively from the amplimers synthesised on MSRV template due to the reciprocal, independent hybridisation of both primers and the probe. To sum up, PNA molecules play a role as “scavenger inhibitors” that alleviate the impact of substantial ERVWE-1 overload that could interfere with the reaction efficiency -specific PNA oligomer and MSRV-specific fluorescent probe. This technique is intended for the discrete detection and measurement of MSRV (multiple sclerosis-associated retrovirus) expression level in biological samples regardless of syncytin-1 expression from ERVWE-1 locus.


This enables a voltage responsivity as high as 23,000 mV mW−1 with a low-frequency input signal. Taking advantage of the output channels being orthogonal to the input terminals, the noise is found to be not strongly influenced by the input. Hence, the corresponding noise-equivalent power is as low as 0.64 pW Hz−1/2. Such performance is even comparable to superconducting bolometers, which however need to operate at cryogenic temperatures. Furthermore, output oscillations are observed at low temperatures, the period of which agrees with the lateral size quantization.Although graphene has the longest mean free path of carriers of any known electronic material, very few novel devices have been reported to harness this extraordinary property. Here we demonstrate a ballistic nano-rectifier fabricated by creating an asymmetric cross-junction in single-layer graphene sandwiched between boron nitride flakes. A mobility ∼200,000 cm The high mobility of graphene has the potential to enable design of devices working in a collisionless regime of carrier transport. Here the authors fabricate a ballistic nano-rectifier based on encapsulated graphene, showing intrinsic performances comparable to those of superconducting bolometers. Despite this, even the most promising applications to commercialize graphene in the electronics industry do not take advantage of this property. Instead, they often attempt to tackle the problem that graphene has no bandgap by using, for example, vertical tunnelling transistorsλMFP. This means that the carriers would be more likely to scatter off the edges in the device than off randomly distributed phonons or defects. Hence, a simplistic billiard ball model could be used for the transport of carriers through the device. One such planar, ballistic device that can take advantage of graphene is the ballistic rectifier (BR), which is an asymmetric four-terminal cross-junction that relies on the ‘billiard ball' motion of carriers to rectify a.c. signals into d.c. outputs582, which leaves charge puddles in the graphene that scatter the carriers and lower the mobility to about 1,810 cm2 V−1 s−1, making the device not truly in the ballistic regime. This resulted in a responsivity of 110 mV mW−1 and a noise-equivalent power (NEP) of 10−9 W Hz−1/2  the neutrality point. It is useful to first check if the voltage output from these devices could be a result of the Seebeck effect. The Seebeck effect describes the process of hot carriers, travelling towards cool regions and it is one of the origins of thermo-electric effect. When the current is passed from S to D, the active region of the device will heat up due to carrier interactions with the lattice. In turn, the asymmetric nature of the device may create a temperature gradient in the vertical direction of the cross-junction and therefore a net voltage difference. This effect has been studied in graphene and other materials, and does produce a quadratic response13VLU, due to the asymmetric nature of the device. We have observed that the output is in fact negative (or positive) VLU. Therefore, any thermal rectification effect that may be present in our devices is at least less dominant than the ballistic rectifying effect at room temperature.VG=+5 V for the electrons and around VG=−8 V for the holes. The output further away from the neutrality point becomes lower, which is a direct result of increased carrier density and Fermi energy12VLU. Furthermore, the output is generally stronger in the electron transport region than for holes, which reflects the different carrier mobilities. PSD, which are in good agreement. The dependence is quite linear at low input power, as expected from the extended Büttiker–Landauer formalismJ∝Eα in the non-linear regime in graphene, where 1>α>1.5 and E is the electric field strength15VLU dependence on VG at a low-a.c. input of 1 μA at different temperatures. It can be seen that the device output in general increases as the temperature decreases, since the value of λMFP is larger at lower temperatures. When electrons are the majority carriers, the output shows more dependence on temperature than in the hole transport region, which may be a result of the different dependences in carrier mobility and mean free path with temperature as shown in VG is higher than +13 V or lower than −12 V. Both the Seebeck effect and ballistic rectifying effect are expected to present in the device, and the latter is known to weaken at high-gate voltages because of high carrier concentrations. So, the output reversal is likely due to the Seebeck effect that becomes more dominant at large gate biases14VLU/dVG against Fermi energy (EF) in E) fluctuates around a mean value of 15.1 meV. The lateral quantization fulfils k⊥WSD=πm, where k⊥ is the wave vector perpendicular to the direction of transport, WSD is the narrowest part of the injection leads and m is the number of conducting modesE=ħvFπ/WSD, where vF is the Fermi velocity (106 ms−1 in graphene). The average ΔE for this device predicts a WSD of 71 nm. The width is in the same order of magnitude as, but less than those measured with atomic-force micrograph, 122 nm for the narrowest region of the source lead and 137 nm for the drain. It is likely that the ballistically conducting region of the graphene does not extend all the way to the edge due to edge states and traps (Coulomb blockade and single-electron effect of graphene constrictions)n and hence One prominent feature that the ballistic model does not explain is the oscillations at 50 K. They may look like mesoscopic conductance fluctuations at first glance, but they are observed also at 150 K. Conductance fluctuations are typically washed out at 5 K ref. . Shubnik1920VG at room temperature by applying an a.c. to the device. The responsivity is determined by the output d.c. voltage divided by the input a.c. power (the a.c. voltage drop across S and D is measured simultaneously). The optimum intrinsic responsivity was measured to be 23,000 mV mW−1, which is to the best of our knowledge, one of the highest in diode detectors to date9252627282 had a responsivity of just 67 mV mW−1, >300 times lower2 V−1 s−1 and a responsivity ∼10,000 mV mW−1.One of the advantages of the BR is its high speed, which was measured up to 50 GHz and simulated at terahertz (THz) frequencies even when using a conventional semiconductor InGaAsf noise) and, in the case of a tunnelling diode, shot noise are both proportional to the applied current. Furthermore, the BR has an intrinsic zero threshold, and it hence does not need any biasing circuit that are not only troublesome at very high frequencies, but also may introduce significant parasitic elements. Operating at zero bias also greatly reduces the flicker noise, which often practically limits microwave detection capability of diode detectorsSV, as a function of frequency at different input currents. At zero input, the BR generated a white noise that is measured at 13.3 nV Hz−1/2. As RLU is around 15 kΩ when no gate bias was applied  can be as low as 2 kHz, which is significantly lower compared with normal detector diodesIt is useful to note that the corner frequency −1/2, which is more three orders magnitude better than the previous device made of low-mobility graphene on SiO2 without BN encapsulationThe minimum power that a microwave sensor can detect is determined by the NEP, which is given by the measured noise divided by the responsivity. In RSD≈22 kΩ, as shown in Currently, the BR still has a relatively large input resistance, To fabricate a graphene BR on an insulating substrate with a desirable carrier concentration for high-frequency applications, it is possible to use an insulating layer on top with a local gate to shift the Fermi energy. However, this will introduce some parasitic capacitance, and may hamper the high-frequency performance. Alternatively, it is possible to carry out chemical doping in various ways, such as using nitric acidet al.For certain applications, such as radio-astronomy or THz detectors, the cost of exfoliated devices might be acceptable. Devices based on chemical vapor deposition (CVD) graphene would significantly reduce the cost and therefore have a much wider range of applications. Currently, the carrier mobility in CVD graphene is generally much lower than exfoliated graphene, but it is improving constantly. The most recent paper by Banszerus −1/2 at room temperature, a value that is even comparable to superconducting bolometers, which however need to be cooled to cryogenic temperatures. The encapsulation of the device in BN layers also enables operation in atmospheric conditions and shows promise for a range of applications in high-frequency electronics.In summary, a very high responsivity has been achieved at low frequency in an asymmetric cross-junction by using graphene that has a carrier mobility close to the theoretical limit. Having an intrinsic zero threshold and being nearly immune to the noise of input current due to the orthogonal arrangement of the output and input channels, the BR exhibits very low flicker noise and the low-frequency noise is mainly limited by thermal noise. The thus obtained intrinsic noise-equivalent power is found to be as low as 0.64 pW Hz2 (ref. Graphene flakes for the device are fabricated using the exfoliation technique on 290 nm SiO2 ref.  and then2 ref.  ref. 1)2 .Supplementary Figures 1-4, Supplementary Note 1 and Supplementary References


The interaction between two different materials can present novel phenomena that are quite different from the physical properties observed when each material stands alone. Strong electronic correlations, such as magnetism and superconductivity, can be produced as the result of enhanced Coulomb interactions between electrons. Two-dimensional materials are powerful candidates to search for the novel phenomena because of the easiness of arranging them and modifying their properties accordingly. In this work, we report magnetic effects in graphene, a prototypical non-magnetic two-dimensional semi-metal, in the proximity with sulfur, a diamagnetic insulator. In contrast to the well-defined metallic behaviour of clean graphene, an energy gap develops at the Fermi energy for the graphene/sulfur compound with decreasing temperature. This is accompanied by a steep increase of the resistance, a sign change of the slope in the magneto-resistance between high and low fields, and magnetic hysteresis. A possible origin of the observed electronic and magnetic responses is discussed in terms of the onset of low-temperature magnetic ordering. These results provide intriguing insights on the search for novel quantum phases in graphene-based compounds. Due to the two-dimensional nature and Dirac fermionic behaviour of charge carriers, the electronic and magnetic properties of graphene can be easily modified, making it one of the most appealing materials for a variety of disparate applications1236789101113141516171819n-doped 6 H-SiC(0001) and undoped 4 H-SiC−6 Torr. The ampule is annealed at 230 °C in a furnace for 60 hours, while the pressure inside the ampule increased by vapourized sulfur was ~360 Torr. The presence of sulfur in the samples is confirmed by the observation of: a) sulfur 2 p core electrons in the photoemission spectra and b) Auger electrons corresponding to the sulfur LMM transition in the Auger electron spectroscopy (AES) spectraIn this work, we have combined two different but complementary probes such as angle-resolved photoemission spectroscopy (ARPES) to study the evolution of the graphene band structure upon sulfur introduction and magneto-transport to explore the electro-magnetic properties of the graphene/sulfur (G/S) system. Changes in the electronic structure are monitored by ARPES experiments. 2829ntensity , a quantntensity  that shiT is lowered, a clear depletion of states at T-dependence of G/S from G excludes the possibility of thermal smearing as the origin of the depletion of states at −1, which is far away from 0.05 Å−1 where the energy gap is observed  versus T-dependence observed at low temperatures is typical of disordered systems in the variable range hopping regime (VRH)4041T range.To gain better insight on the origin of the gap opening, we have performed magneto-transport measurements  and 4. Iwering T . Figure R versus external magnetic field H) for both samples. The most striking difference is the behaviour of the magneto-resistance at low fields, where its complete reversal is observed upon sulfur introduction. This reversal can be explained with a transition from a weak antilocalization regime to a weak localization regime as previously reported for pure graphene43R near zero field. While the magneto-resistance of the G sample (R upon increasing H) over the whole field range, the magneto-resistance of the G/S sample (R upon increasing H) at low fields, similar to the case of weak localizations. We note, however, that the crossover field is three orders of magnitude higher than the previous results for weak localizations, where the strength of localization is suppressed very quickly in the presence of magnetic fieldIn G sample  is positS sample  shows a H = 0 T reveals another surprising phenomena of G/S, magnetic hysteresis with coercive fields of 0.12 T and 0.02 T  exhibits spin-polarized states via the interaction with the buffer layer . The structural optimisation and spin-polarized calculation were performed.Calculations. How to cite this article: Hwang, C. et al. Magnetic effects in sulfur-decorated graphene. Sci. Rep.6, 21460; doi: 10.1038/srep21460 (2016).


In addition, the frequency of PD-L1 immunoreactivity was significantly higher in IL-6-positive esophageal cancer specimens. When IL-6 signaling was inhibited in vitro, the level of PD-L1 is significantly down-regulated. PD-L1 is a significant predictor for poor treatment response and shorter survival. As demonstrated through in vitro experiments, Irradiation increased PD-L1 expression in human esophageal cancer cells. The inhibition of T cell functions including proliferation and cytotoxicity against tumor cells might be the mechanisms responsible to the role of PD-L1 in radiation response. In conclusion, PD-L1 is important in determining the radiation response and could predict the prognosis of patients with esophageal SCC. Therefore, we suggest inhibition of PD-L1 as a potential strategy for the treatment of esophageal SCC.The aim of this study was to assess the significance of programmed cell death 1 ligand 1 (PD-L1) in esophageal squamous cell carcinoma (ESCC) and its association with IL-6 and radiation response. Weretrospectively enrolled 162 patients with ESCC, and examined the correlation between PD-L1 levels and clinical outcomes in esophageal cancer patients. Furthermore, the human esophageal SCC cell line CE81T and TE2 were selected for cellular experiments to investigate the role of PD-L1 in T cell functions and radiation response. Here we demonstrated that PD-L1 expression was significantly higher in esophageal cancer specimens than in non-malignant epithelium. In clinical outcome analysis, this staining of PD-L1 was positively linked to the clinical T4 stage ( Esophageal cancer is one of the most common types of human cancer and a difficult gastrointestinal tumor to treat and cure . Most paTumor-induced immune suppression in cancer patients is a major issue that not only promotes tumor progression but also inhibits the efficiency of anti-cancer treatment , 5. One Increasing evidence suggests that the inflammatory response can be rerouted into a tumor-promoting direction association with poor prognosis in various type of cancer, and proinflammatory cytokines could promote PD-L1 expression in tumor microenvironment –12. We pversus 50% (37/74) in T4, P=0.004). Furthermore, the positivity of PD-L1 was significantly associated with the risk of LN metastasis and developing loco-regional failure (Table The IHC data for TMA slides demonstrated that PD-L1 was over-expressed at the membrane or in the cytoplasm (or both) of tumor cells in the surgically resected tumor specimens compared to adjacent non-malignant epithelial tissues Figure . Figure p < 0.001). Given the positive association between IL-6 and PD-L1 expression in ESCC tumors, we examined the expression of PD-L1 in esophageal cancer cell lines whose IL-6 was regulated. Flow cytometric analysis and IF data revealed that IL-6 neutralizing antibody significantly decreased the level of PD-L1 expression at the cell surface and the cytoplasm  in non-responders, P<0.001). Furthermore, 47 among these patients received esophagectomy after neoadjuvant CCRT, PD-L1 staininig linked with lower complete pathologic response rate (pCR) (16% (3/18) in PD-L1(+) patients versus 31% (9/29) in PD-L1 (−) patients)). The role of PD-L1 in radioresistance and its underlying mechanisms were further examined in vitro. As shown in Figure For esophageal SCC, radiotherapy is a well-established therapeutic modality and provides survival benefits for responders. As shown in Table Table Malignant tumors possess mechanisms for evading host immune responses. A novel mechanism that tumor may evade host immune response through the expression of PD-L1. PD-L1 expression has been found in various human cancers, and was associated with patients' prognoses –19. In tPD-L1/PD-1 has been shown to be an important mechanism underlying T-cell dysfunction during chronic inflammation and chronic infection –22. SeveFor ESCC, treatment response is an independent prognostic factor. The identification and inhibition of key drivers of immunosuppression have the potential to improve patient outcome combined with radiotherapy. PD-L1 expression has been reported to link with the resistance to anticancer therapies , 9. Our It is known that infiltration of T cells into tumors correlates with improved prognosis in several types of GI cancers –36. FurtOur study has some limitations. First, we examined PD-L1 expression mainly in biopsy tissue specimen to evaluate its prognostic value. Biopsies only sampled a small esophageal volume, which might result in undersampling of esophageal cancer tissue. Also, to examine the prognostic value of PD-L1 in esophageal cancer patients, it is a retrospective analysis of a population with different stages from a single institution. Therefore, the issue should be best answered in context of a prospective study in a more patient population.These findings indicate that PD-L1-positive esophageal cancer provides a suitable microenvironment for the development of tumor growth and treatment resistance mediated by induction of activated IL-6 signaling. Novel immune-based therapies for the treatment of cancer are currently under development. Therefore, targeting PD-L1 could be a promising strategy for the treatment of esophageal cancer.This study was approved by the institutional review board of our hospital. Patients who did not comply with the treatment regimen and those who received surgery alone for early-stage esophageal cancer were excluded from the study. A total of 162 patients with ESCC were enrolled in the study. The curative treatment for esophageal cancer included neoadjuvant CCRT combined with surgery or definitive CCRT according to the guidelines proposed by oncology team at our hospital. On completion of neoadjuvant CCRT, patients underwent a repeat CT scan and endoscopic examination to determine the response to treatment. Specimens collected from the 162 patients at diagnosis were subjected to immunochemical analysis. The main end points were overall survival (OS), disease-free survival (DFS) and treatment response. Survival probability was analyzed statistically using the Kaplan—Meier method. Significant differences between groups were assessed using the Spearman-rank test. Multivariate analyses were performed using a Cox regression model for survival.Formalin-fixed, paraffin-embedded tissues from 162 patients with esophageal cancer were subjected to IHC staining. Dissected esophageal cancer specimens from 21 of these patients were converted into tissue microarray (TMA) blocks using an AutoTiss 1000 arrayer . The TMA block contained esophageal SCCs and the adjacent non-malignant epithelium. The quality of the TMA slides was confirmed by the pathologist using hematoxylin- and eosin-stained slides. The IHC data for the specimens were assessed using the semi-quantitative immunoreactive score (IRS), as described previously . For thePeripheral blood (PB) samples were obtained from 56 of these patients before treatment. IL-6 levels in human serum samples were analyzed using an IL-6 human Quantikine ELISA kit (R&D system). The samples were frozen and stored before assaying.The human cancer cell lines CE81T, which is derived from a well-differentiated esophageal SCC and obtained from Bioresource Collection and Research Center , and TE2+CD8+ was sorted from freshly obtained PB.To obtain single-cell suspensions, tumor tissues were digested by 1 mg/ml collagenase IV (Sigma-Aldrich) and 0.2 mg/ml DNase I (Sigma-Aldrich) for 45 minutes at 37°C. Cells were blocked and then stained with antibodies against PD-L1. For apoptosis assays, tumor cells were stained with antibodies against 7-AAD and annexin V after the removal of CD8+ cells. To isolate cytotoxic T cells from peripheral blood mononuclear cells (PBMC) in the patients, multicolor fluorescence-activated cell sorting (FACS) was performed using a FACS caliber flow cytometer (BD Biosciences). The human cytotoxic T cells subset characterized as CD3The suppressive function of tumor cells was measured by their ability to inhibit the proliferation of autologous T cells in the following Suppression Assay, as described previously . The isoThe significance of differences between samples was determined using Student's t-tests. Data are presented as the means ± standard error of the mean (SEM). All experiments, comprising three replicates, were performed at least twice independently. A probability level of p < 0.05 was adopted throughout to determine statistical significance, unless otherwise stated.


Human cancer cells may suppress the adaptive immune response by expressing PD-L1 and down-regulating T cell activity through PD-L1/PD-1 and PD-L1/B7.1 interactions. Disruption of PD-L1 signaling restores anti-tumor immunity, resulting in durable responses across multiple human tumor types. Here we describe the clinical activity and development of predictive biomarkers for MPDL3280A, a human monoclonal antibody with an engineered Fc-domain designed to optimize safety and efficacy, that targets PD-L1 and prevents binding to receptors PD-1 and B7.1.We evaluated a multitude of biomarkers from pretreatment tumor specimens collected during clinical study of MPDL3280A. MPDL3280A has been administered as a monotherapy in over 300 pts with locally advanced or metastatic solid tumors in a phase 1 study.To date, MPDL3280A has been well tolerated across multiple dose levels and no G3-5 pneumonitis has been reported. Responses were observed in multiple tumor types, including NSCLC, RCC, and melanoma, with ongoing responses seen in the majority of responders. We report the association between pretreatment immune-related markers and response to MPDL3280A.Overall, our assessment of tumor specimens not only has resulted in markers that may potentially identify response to MPDL3280A but also has furthered our understanding of the biologic activity of PD-L1 inhibition on treatment.


The chemical vapor deposition (CVD) method to obtain tailored graphene as a transparent and flexible gas barrier has been developed. By separating nucleation step from growth, we could reduce early graphene nucleation density and thus induce better stitching between domain boundaries in the second growth step. Furthermore, two step growth in conjunction with electrochemical polishing of Cu foils achieved large graphene domains and improved graphene quality with minimized defects. The performance of resulting graphene as a gas barrier was superior to the graphene obtained by one-step growth on polished or unpolished Cu foils. The CVD graphene reported here could open up the possibility for exploring graphene-based gas barrier due to the minimized density of defect area. As a consequence, unfavorable gas and aerosol molecules can be transferred through these defects, causing local corrosive change in the surface and degradation of the protection ability of the graphene18212223242526272812et al.For practical applications as protective barrier, preparation of large area, defect-free graphene and a suitable method for its integration on polymeric or other substrates are important requirements. Among many preparation methods, chemical vapor deposition (CVD) is the most effective method to reliably produce high-quality, wafer-scale monolayer graphene films. By virtue of this, CVD-grown graphene has been investigated as a protective barrier to prevent transport of gas molecular between the environment and the arbitrary surface such as polymers or metal layer9101112131415161284/H2 mixture which act as nucleation seeds and can influence the gas barrier properties of graphene26Here, we use a combined approach of electro-chemical polishing (ECP) and two-step growth methods to improve the gas barrier property of graphene without an increase in the layer numbers or additional fabrication process. ECP is known as a highly effective step to reduce the surface roughness of Cu foil and remove the impurity particles4 to H2 flow ratio, total pressure, temperature, and Cu foil treatments are crucial parameters that influence the graphene domain shape21222324252, twice or four times larger than the graphene domains obtained by one-step growth on ECP and unpolished Cu foils, respectively, as illustrated in 6/cm2 in the case of two-step growth on ECP Cu. It is well known that steps, wrinkles, defects, and grain boundaries in Cu foils have much lower nucleation barriers than flat surface. These could act as nucleation sites, triggering the formation of graphene nuclei and eventually give rise to small single-crystal graphene domains. On the other hand, ECP process reduces the surface roughness as well as defects and impurities that subsist on Cu foils, leading to the suppression of nucleation sites and hence reduced domain density of graphene. In addition, during the early stage of two-step growth, the low CH4 concentration also mitigates the graphene nucleation and provides space for the growth of large graphene domains. As a result, two-step growth of graphene on ECP Cu foil gives large-size single crystalline graphene domains with a reduced domain density.The initial stage of growth is critical to obtain large size domains and high quality graphene layer. In this study, three different graphene layers are grown as follows: The first graphene was grown on as-received Cu foil without any treatment by one step growth (1step-w/o ECP), the second graphene was grown on electrochemically polished Cu foil by one step growth (1step-ECP) and the third graphene was grown on ECP Cu foil by two step growth (2step-ECP). 21222/Si substrate. −1 and 2690 cm−1, respectively. However, the G-band position is found to be blue shifted compared to the value 1582 cm−1 reported in the literaturep-type doping of graphene caused by the redox reaction between oxygen from air and adsorbed water at the surface of SiO2/Si substrate having hydrophilic property−1 and the intensity ratio of 2D to G greater than 2. Also, the so-called defects or disorder-induced D-band peak is seen around 1350 cm−1 at about half of the frequency of the 2D band with negligible intensity.To investigate the quality and number of graphene layers, Raman measurements were performed on representative graphene films after being transferred on to SiOEven though the transmittance of 2step-ECP graphene films shows no difference compared with that of 1step-w/o ECP or the 1step-ECP graphene films, sheet resistance change reflects graphene quality rather than thickness. In 30352-day, which is 57.9% less than that of the value of 1.58 ± 0.029 g/m2-day measured for the bare PET. −14, 5.806 × 10−14 and 4.224 × 10−14 mols−1 m−1atm−1, respectively. It is evident that the lowest WVTR and P values are obtained for monolayer graphene formed by two-step growth on ECP Cu foil without any additional treatment or control of graphene number.4)2S2O8], is dropped onto graphene synthesized on Cu foils. After waiting for about 5 sec, the dropped etchant is removed through rinsing process with deionized water and subsequently the surface is examined by atomic force microscope (AFM), as shown in −7 ± 3.3 × 10−6/cm2, 2.3 × 10−7 ± 2.8 × 10−6/cm2, and 2.2 × 10−6 ± 6.0 × 10−5/cm2, respectively. It is evident that the graphene grown by two-step method on ECP Cu has the lowest etch pit density, signifying that the two-step growth method reduces the defect density to a great extent compared to conventional one-step growth. PET) and etch pit density and etched to the total area ratio (Δ) obtained by a commercial image processor  from corresponding AFM images. The Etch pit density shows a linear decrease from the first sample (1step-w/o ECP) to the third one (2step-ECP), which is in accordance with the SEM results shown in Though Raman analysis can well assess the defect distribution and quality of graphene through the G-to-D band intensity ratio, it is hard to measure the spatial distribution of defects that spread across over large area. To give a further insight into the structural quality of graphene on large scale, a film-induced frustrated etching (FIFE) is carried out, which is a simple and facile method to observe the structural defects spread on large area of grapheneThe solution of the Fick’s second law of diffusion is used to fit the measured WVTR, by assuming the water vapor concentration is close to zero at the exit side of the film as given belowP0 = Jd, P = Jsd = SD, J and Js are the water vapor molar flux at time t and at steady state, d is the sample thickness, P is permeability, D is diffusivity, and S is solubility. If the whole area of PET film is fully covered by impermeable graphene layer, the permeability should be zero. However, the CVD graphene layer consists of defect-holes through which water molecules may diffuse. The water molecules in the PET film may diffuse out through the defect-holes. The surface defect-hole density, ψ, can be defined as the ratio between the defect-hole area and the total area. Inside of the defect-holes, the inhibition of water diffusion by graphene layer may be negligible, when the sizes of the holes are much larger than those of the water molecules and the thickness of hole is much smaller than the mean free path of water molecules. In this case, the permeability can be simply expressed aswhere Peff , PG and P° represent the permeability of graphene layer/PET film, bare graphene film, and pure PET film, respectively. However, PG is not zero but a tiny value due to undetected defects, and thus the equation can be approximated in the form.because graphene without defects can be assumed as an impermeable membrane in the simple model where B is a correction constant arising from undetected defects by the FIFE. It is reasonable to assume that the Δ is proportional to ψ such as ψ = AΔ. Then the reduced permeability can be related to Δ as follows;where −14 ± 2.205 × 10−16, respectively. Thus, the ψ value of graphene grown by 2step-ECP on PET is found to be 0.624 ± 0.0081. This result indicates that the formation of graphene layer on PET is an effective way to improve the water vapor barrier property of the polymer film. If barrier performances of graphene are further improved by optimizing growth conditions, it can be used as transparent and flexible gas barrier in display devices.where A is an arbitrary constant. The permeability of graphenes on PET with Δ examined in In conclusion, a combination of electrochemical polishing and two-step growth is demonstrated to obtain high-quality monolayer graphene by low pressure CVD. This method allows achieving large size graphene domains by controlling the nucleation density of graphene on Cu foils, a crucial starting step for the realization of high-quality graphene. The characteristics of the graphene were compared to that of graphene produced by one-step growth on both unpolished and electrochemically polished Cu foils. Two-step growth on electrochemically polished Cu also led to improved permeability property. This is attributed to the decrease in structural defects and suppression of nucleation of graphene at the initial stage. A simple and accurate model has been proposed to examine the barrier property of CVD monolayer graphene. Our study also brings into picture that the effective permeability of CVD monolayer graphene is proportional to defect area rather than defect density.3PO4:H2O acid solution at a constant voltage of 2.0 V for 12 min using Cu plate as cathode and Cu foil (80 mm × 80 mm) as working electrode.ECP was performed in diluted 3H2 gas was flown through the reactor at 15 standard cubic centimeters per minute (sccm) during the temperature ramp-up up to 1030 °C. Then, the Cu foils were annealed for 50 min. In the one-step growth, CH4 of 13 sccm and H2 of 15 sccm were supplied at 1030 °C for 23 min under a growth pressure of 0.160 Torr. In the case of two-step growth, at first, large size graphene domains were formed on Cu foil in a mixed CH4/H2 (5 sccm/100 sccm) ambient for 1 min at 1030 °C under a growth pressure of 0.365 Torr. This is the optimal condition for obtaining uniform and large domains of graphene, as shown in the 4 and growth time were increased to 13 sccm and 8 min, respectively, while maintaining the H2 flow rate and temperature unchanged. Finally, the samples were cooled down rapidly to room temperature with a cooling rate of 30 °C min−1 at 15 sccm of H2 flow by moving the furnace out of the substrate region.Large-scale graphene layer studied in this work was synthesized on 35-μm-thick Cu foils (Nippon Mining) by low pressure chemical vapor deposition (LPCVD). The Cu foil was then set into a 2 inch quartz tube and heated by split-tube furnace. Meanwhile, quartz tube was pumped down to 0.072 Torr, and H2 plasma etching. Then, the Cu foil with PMMA-covered graphene was dipped in 0.1 M Ammonium Persulfate [(NH4)2S2O8] solution for 4 h to etch the Cu foil. Thereafter, PMMA/graphene layer was transferred onto various substrates, such as 300 nm SiO2/Si substrate, PET film, and glass, to investigate the opto-electrical and gas barrier properties. The PMMA was removed by using acetone and the sample was annealed in an Ar/H2 gas mixture at 400 °C for 3 h to eradicate remained PMMA residues.Polymethyl methacrylate (PMMA) was spin-coated onto graphene surface at 4200 rpm for 50 s to protect and transfer the graphene layer onto other substrates of interest. Prior to graphene transfer, graphene unintentionally formed on the back side of the Cu foil was removed by O4)2S2O8], was dropped on to graphene synthesized on Cu foils. After 5 sec, the dropped etchant was removed by rinsing with deionized water.Commercial copper etchant, ammonium persulfate [ was used to evaluate the surface morphology of the graphene samples studied in this work. The surface topography of graphene/Cu foils after FIFE test was probed by atomic force microscope  in tapping mode. The Raman spectra were recorded using 514 nm-line of an Ar ion laser as an excitation source. In order to observe the change in electrical conductivity as a function of graphene domain size, the sheet resistance was measured by Van der Pauw method using Hall measurement system (Lakeshore 7500 serises).How to cite this article: Seo, T. H. et al. Tailored CVD graphene coating as a transparent and flexible gas barrier. Sci. Rep.6, 24143; doi: 10.1038/srep24143 (2016).


Recent clinical studies show that tyrosine kinase inhibitors slow the rate of lung function decline and decrease the number of acute exacerbations in patients with Idiopathic Pulmonary Fibrosis (IPF). However, in the murine bleomycin model of fibrosis, not all tyrosine kinase signaling is detrimental. Exogenous ligands Fibroblast Growth Factor (FGF) 7 and 10 improve murine lung repair and increase survival after injury via tyrosine kinase FGF receptor 2b-signaling. Therefore, the level and location of FGF/FGFR expression as well as the exogenous effect of the most highly expressed FGFR2b ligand, FGF1, was analyzed on human lung fibroblasts.FGF ligand and receptor expression was evaluated in donor and IPF whole lung homogenates using western blotting and qPCR. Immunohistochemistry for FGF1 and FGFR1/2/3/4 were performed on human lung tissue. Lastly, the effects of FGF1, a potent, multi-FGFR ligand, were studied on primary cultures of IPF and non-IPF donor fibroblasts. Western blots for pro-fibrotic markers, proliferation, FACS for apoptosis, transwell assays and MetaMorph analyses on cell cultures were performed.Whole lung homogenate analyses revealed decreased FGFR b-isoform expression, and an increase in FGFR c-isoform expression. Of the FGFR2b-ligands, FGF1 was the most significantly increased in IPF patients; downstream targets of FGF-signaling, p-ERK1/2 and p-AKT were also increased. Immunohistochemistry revealed FGF1 co-localization within basal cell sheets, myofibroblast foci, and Surfactant protein-C positive alveolar epithelial type-II cells as well as co-localization with FGFR1, FGFR2, FGFR3, FGFR4 and myofibroblasts expressing the migratory marker Fascin. Both alone and in the presence of heparin, FGF1 led to increased MAPK-signaling in primary lung fibroblasts. While smooth muscle actin was unchanged, heparin + FGF1 decreased collagen production in IPF fibroblasts. In addition, FGF1 + heparin increased apoptosis and cell migration. The FGFR inhibitor (PD173074) attenuated these effects.Strong expression of FGF1/FGFRs in pathogenic regions of IPF suggest that aberrant FGF1-FGFR signaling is increased in IPF patients and may contribute to the pathogenesis of lung fibrosis by supporting fibroblast migration and increased MAPK-signaling.The online version of this article (doi:10.1186/s12931-015-0242-2) contains supplementary material, which is available to authorized users. Idiopathic pulmonary fibrosis (IPF) is a rare interstitial lung disease of unknown origin, with prevalence rates ranging from 2-4/10000 , and theBoth Fgf7 and Fgf10 are mesenchymal-derived growth factors that signal in a paracrine manner to bind with high affinity to epithelial expressed Fgfr2 b-isoform . OverexpFgf/Fgfr-binding is stabilized by heparin and transduced by a phosphorylation cascade, mediated by fibroblast growth factor receptor substrate (Frs2)  which acUntil now, studies investigating the activity of heparin + FGF1 in the context of lung fibrosis have been carried out exclusively on cell lines. Treatment of lung fibroblast cell-line N12, with heparin + FGF1 had no impact on proliferation but induced apoptosis and decreased smooth muscle actin production . MoreoveThe study protocol for tissue donation was approved by the Ethics Committee of the Justus-Liebig-University School of Medicine (AZ 111/08). Informed consent was obtained from each individual patient or next of kin.n = 36) or donors (n = 15) were obtained during transplantation at the Department of Cardiothoracic Surgery, University of Vienna, Austria or University Hospital Giessen-Marburg, Giessen, Germany and processed as previously described [Lung homogenates from IPF n = 6 or donoLoading buffer was added to protein samples from cell extracts (5 % SDS in bromophenol blue and β-mercaptoethanol) denatured for 5 min at 95 °C and cooled on ice. At least 10 μg of sample was loaded on a 10 % polyacrylamide gel and run at 25 mA for 2 h then transferred to a polyvinylidene fluoride (PVDF) membrane (Amersham) by semi-dry electro-blotting  for 90 min. The membrane was blocked with 5 % milk in TBS-blocking buffer at RT for 1 h followed by 4 °C incubation with primary antibody overnight. Membranes were washed in 1X TBST buffer four times and incubated with HRP-labeled secondary antibody at RT for 1 h followed by washing with 1X TBS-T. Bands were detected by ECL  treatment, followed by exposure of the membrane. . cDNA was diluted to 20 ng/μL. Primers were designed to span introns using Roche Applied Sciences online Assay Design Tool. Sybr Green Master Mix (Applied Biosciences 4309155) was used for qPCR with a Roche LightCycler480 machine. Samples were run in triplicates using 3. cDNA wHuman lungs were placed in 4 % (w/v) paraformaldehyde for 12-24 h, and processed for paraffin embedding. 3 μm sections were cut and mounted on slides . Paraffin-embedded tissue sections of donor and IPF-lungs were deparaffinized in xylene and rehydrated in graded alcohol. Antigens were retrieved by microwave antigen retrieval (800 W); in 10 mmol/L freshly prepared citrate buffer (pH 6.0) for 5 min. For immunostaining, the streptavidin-biotin-alkaline phosphatase (AP) - or the streptavidin-biotin- horseradish peroxidase (HRP) method was used with the ZytoChem-Plus AP Kit (Fast Red), Broad Spectrum  according to the manufacturer’s protocol. Sections were counterstained with hemalaun  and mounted in Glycergel (DakoCytomation). Control sections were treated with 2 % BSA in PBS alone or with rabbit or mouse primary antibody isotype control  to determine the specificity of the staining. Lung tissue sections were scanned with a Mirax Desk slide-scanning device , and examined histo-pathologically at 50×, 100×, 200×, 400×. IHC for mentioned antibodies was performed in at least 8 IPF and 5 control-lung samples. A complete list of antibodies and dilutions is provided in the online supplement.2 flask and grown out at 37 °C, 5 % CO2 for up to one month with weekly media changes according to a previously published protocol [5 cm lung cubic biopsies of human lung tissue were washed in PBS and cut into small pieces in growing culture medium: Dulbecco's Modified Eagle's Medium (DMEM) with 10 % Fetal calf serum (FCS) 1 % glutamine, and 1 % Penicillin-Streptomycin. Pieces were seeded initially in a large 75 cmprotocol . No enzyThe affymetrix APC annexin V and propidium iodide staining kit was used to perform an apoptosis assay according to manufacturers instructions (eBioscience #88-8007-74) on an Accuri C6 flow cytometer (BD Biosciences). Briefly, cells were washed twice in cold PBS, then trypsinized at 37 °C for 5 min. 2 wells of fibroblasts grown in 6-well plates were gently collected, combined and pelleted. Pellets were washed in PBS, followed by binding buffer, and incubated first with kit antibody, then propidium iodide in the dark for 10 min. At least 30,000 cells were counted per FACS experiment. Gating was established based on plots of propidium iodide alone and annexin V alone.Primary lung fibroblasts were starved for 24 h and seeded  in the upper chamber of the transwell  containing serum free DMEM F-12 medium and the lower chambers with various experimental conditions. The system was incubated at 37 °C, 5 % CO2 to allow the migration of cells through the membrane (8.0 μm). After 16 h, media was removed using a gentle suction and cells were washed with 1X PBS, fixed with methanol and stained with crystal violet. Next, the transwell was swabbed to remove the non-migrated cells and the total number of migrated cells was quantified using phase contrast microscope.2 and 37 °C environment. Random regions were marked, and Leica Live Imaging Software snapped a photo of these regions every 5 min for 20 h. LIF files were exported and analyzed in Leica MetaMorph software version 1.5.0. The average total distance traveled of at least 6 random cells per group were tracked and recorded.Fibroblasts from at least 3 biologically unique samples were seeded at low density, in a 24-well-plate, starved 24 h, and underwent various treatments. The plate was set on a motorized stage in a 5 % COt-Test was performed on the log-transformed value of the qPCR fold changes. For western blots, t-tests were performed on the probit values. One-way ANOVAs with Dunnet’s test (untreated groups served as controls) were performed on Transwell and MetaMorph data.A Student’s , FGF7, and FGF10. A significant increase in FGF1 was observed in IPF patients  and Collagen 1a1 (COL1A1) transcripts  of donor lungs  which assemble into ‘sheets’,  Fig. 3,,3, b1,2.Keratin 5-staining was also used to identify bronchial epithelium of IPF lungs Fig. ; 1,2. InGiven the strong expression of FGF1 and FGFR1/2/3/4 in regions of usual interstitial FF, as well as their co-localization with the migratory marker Fascin, and the myofibroblast marker α-SMA, the effect of exogenous FGF1 on IPF lung fibroblasts was addressed.IPF and non-IPF, donor fibroblasts  were harvested and cultured as previously described . Cells wHeparin + FGF1 treatment of IPF fibroblasts resulted in a trend towards decreased collagen production by IPF fibroblasts while no effect was observed on non-IPF fibroblasts Fig. 5,d.Fig. 5The trend towards decreased collagen production in IPF fibroblasts treated with heparin + FGF1 may be in part regulated via activation of p-ERK1/2 signaling. The p-ERK1/2 signal was significantly increased in both FGF1 alone and FGF1 + heparin treated IPF fibroblasts Fig. ,f,g. TheIn summary, the FGFR inhibitor PD173074 efficiently mitigated both endogenous and exogenous FGFR mediated p-ERK signaling. Furthermore, FGF1 + heparin did not significantly attenuate α-SMA production nor did it significantly regulate Fascin. However, COL1a1 production trended towards a decrease in this group. Due to the substantial activation of p-ERK signaling following heparin + FGF1 treatment, whether proliferation and apoptosis were influenced was tested next.Flow cytometry was performed on treated donor and IPF fibroblasts for Annexin V and propidium iodide. In accordance with previous reports , 28, theTo further investigate potential functional effects of exogenous FGF1 + heparin on primary lung donor and IPF fibroblasts, both transwell migration experiments and MetaMorph analyses of cell cultures were performed. The FGFR inhibitor alone (0.1 μM) did not inhibit migration through the transwell Fig. . The addMetaMorph analyses of low-density fibroblasts cultures revealed that heparin + FGF1 stimulated IPF fibroblasts to travel longer distances than non-stimulated fibroblasts Fig. . In thisIn summary, heparin + FGF1 stimulated migration of both IPF and donor fibroblasts and this effect could be attenuated by the simultaneous addition of FGFR inhibitor.In the rodent bleomycin-model, enhanced Fgfr2b-signaling on alveolar epithelial cells via the exogenous application or induction of FGF10 or FGF7, conferred increased survival and reduced lung fibrosis . On the FGF1 emerged as the most highly expressed FGFR2b-binding ligand in IPF lung homogenates. FGF1 is expressed by both mesenchymal and epithelial cell types in the lung  and bindIn this study, FGF1 and FGFR1/2/3 were increased at the protein level in whole lung homogenates taken from end-stage IPF patients compared to non-IPF lung homogenates. IHC analyses revealed robust expression of FGF1 in regions of irregular lung architecture particular to IPF, including: basal cell sheets or basal cells of hyperplastic bronchioles Fig.  and SMA+Former in vitro studies suggest that FGF1 may have an anti-fibrotic effect on lung fibroblasts. For example, heparin + FGF1 was found to decrease smooth muscle actin production and had a pro-apoptotic effect on a normal lung fibroblast cell line . In addiFGF1 and FGFR co-localization with the motility and invasion marker Fascin was observed in usual interstitial fibroblastic foci. In addition, a preliminary scratch assay experiment revealed that FGF1 + heparin treated IPF fibroblasts closed a scratched area of confluent fibroblasts faster than untreated cells (data not shown). Although Fascin expression was not observed to be significantly regulated by heparin + FGF1, the failure of heparin + FGF1 to reduce SMA expression, the preliminary scratch assay data, the absence of FGF1 from condensed regions of smooth muscle cells, as well as previous studies indicating that FGF1 influences cell migration , was ratThe addition of heparin + FGF1 stimulated significant migration of both IPF and non-IPF fibroblasts through a transwell filter compared to starved, untreated cells. When heparin + FGF1 were added in the presence of the FGFR inhibitor, this effect was mitigated. Interestingly, while the addition of FGF1 alone or heparin alone did not stimulate migration in donor fibroblasts, these conditions stimulated migration in IPF fibroblasts, but to a lesser extent than heparin + FGF1 together. These results suggest that IPF fibroblasts may be primed to receive chemotactic signals. Faster migration by IPF fibroblasts may also be due to enhanced p-ERK1/2 signaling as stronger activation of p-ERK1/2 by FGF1 alone and heparin + FGF1 was also observed in IPF fibroblasts compared to donor, non-IPF fibroblasts used in this study.In addition, MetaMorph analyses of low-density cultures of fibroblasts revealed that heparin + FGF1 stimulated IPF fibroblasts to travel longer distances while no effect was observed in non-IPF, donor fibroblasts. Unlike the transwell experiments, addition of the inhibitor alone reduced overall distance travelled in both donor and IPF fibroblasts compared to untreated cells. However, as in the transwell experiments, the FGFR inhibitor efficiently attenuated the effect of exogenous heparin + FGF1 in IPF fibroblasts when added simultaneously. Lastly, unlike the transwell assay, FGF1 alone had no effect on IPF fibroblasts, while heparin alone reduced distance travelled of IPF fibroblasts. The discrepancies between the transwell and live imaging experiments may be due the lack of a gradient formation in the 24-well plates used for live imaging. In summary, MetaMorph analyses of live imaging experiments support the conclusion that the migration of IPF fibroblasts was enhanced following exposure to heparin + FGF1.In summary, this study described strong expression of FGF1 and FGFR1/2/3/4 receptors in pathogenic areas of IPF lungs and identified FGF1-FGFRs as potential contributors to increased MAPK-activity in IPF. Though IPF lung fibroblasts responded to heparin + FGF1 treatment by attenuating COL1a1 expression and increased apoptosis, increased p-ERK1/2 signaling along with enhanced cell migration was also observed reflecting a potentially dual nature of FGF1/FGFR in the context of lung fibrosis. Though tyrosine kinase inhibitors have recently been approved for the treatment of IPF, given the multi-faceted nature of FGF1-FGFR signaling, further studies should be designed to identify targets of growth factor signaling that mediate specific cellular functions such as fibroblast apoptosis and migration.


Patients were prospectively followed up for 40 months. Receiver operating characteristics curve analysis determined cut-off points associated with mortality, and Cox proportional hazard ratio (HR) were conducted. Thresholds for increased mortality risk in IPF patients were determined as IPAQ ≤ 417 metabolic equivalent task (METS)-min/week, p = 0.004 , p = 0.027), and ∆SpO2 ≥ 10%, p = 0.002, , p = 0.025). This study demonstrated a significant association of PA and ED with mortality in IPF patients. The findings emphasize the clinical importance of PA and ED assessments to aid in IPF risk stratification, prognosis prediction, and in providing early appropriate treatments, such as pulmonary rehabilitation, PA consultation, oxygen supplementation, and lung transplantation referral. These results underscore that even low levels of PA corresponding to 100–105 min/week were associated with a reduced mortality risk and better survival in IPF.Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease that manifests in hypoxemia, inactivity, and poor prognosis. This study aimed to assess the prognostic role of physical activity (PA) and exertional desaturation (ED) with mortality in IPF. At baseline, 34 IPF patients (68 (50–81) years) were interviewed using the International Physical Activity Questionnaire (IPAQ), and SpO Exey in IPF ,4. In ady in IPF . Worldwiy in IPF ,8,9. Mory in IPF . Neverthy in IPF ,12.2 < 88% may be different for patients with various resting saturation levels (with and without resting hypoxemia), and possibly has less prognostic sensitivity for all spectrums of disease severities. Secondly, physical inactivity is a well-established, independent risk factor for mortality in the general population [2 pre- to post-6MWT (∆SpO2)) with mortality among patients with IPF.In reviewing the literature addressing IPF, we detected some clinically important gaps related to levels of physical activity (PA) and exertional desaturation (ED) with mortality risk. Firstly, the previous proposed exertional nadir of absolute SpOpulation  as well pulation . NonetheThis observational prospective follow-up study was conducted at the Pulmonary Institute, Rabin Medical Center, Beilinson Hospital, Petach-Tikva, Israel. The study protocol was approved by the Ethics Committee of the hospital and was registered with clinicaltrials.gov NCT01499745). The study was conducted in accordance with the Declaration of Helsinki and written informed consent was obtained from each subject prior to participation. Patients were included if they were diagnosed with IPF according to the accepted clinical-radiological criteria of the ATS/ERS and were clinically stable in previous 3–6 months [499745. Tn = 34) were used to determine the prognostic predictors of survival. Subjects were tested as described below and followed up for 40 months from baseline:In the current study, subjects from our previous exercise training study  were fol600 METS-min/week (150 min/week * 4METS of moderate intensity PA) was considered a threshold for meeting the general PA recommendation [Self-report 7-day short form International Physical Activity Questionnaire (IPAQ) was used for all patients ,23. Eachendation ,26,27,282%; Pulse Oximeter 2500 30 EM, Nonim Medical, Minneapolis, MN, USA) were obtained prior and immediately after the test [2% pre- to post-6MWT (∆SpO2%).The 6MWT was conducted according to ATS guidelines in a 35-meter corridor at the pulmonary unit within the hospital . Borg dythe test . Oxygen Pulmonary function tests, including spirometry total lung capacity, maximal voluntary ventilation, and diffusion capacity for carbon monoxide were performed according to standard techniques and ATS/ERS guidelines  ,32. All p < 0.05. Demographics, clinical, and physiological data of the participants are presented as median and (range). Categorical variables are presented in absolute values and (%) of group. Receiver operating characteristics (ROC) curve analysis for cut-off points’ ˃70% sensitivity and ˃70% specificity to detect mortality was performed for IPAQ and ∆SpO2. A multivariate Cox proportional hazard model using significant cut-off points determined from ROC was analyzed for IPAQ and ∆SpO2. The model was adjusted for gender, age, forced vital capacity %predicted, diffusion capacity for carbon monoxide %predicted, and Prednisone usage. Kaplan–Meir curves were used for survival analysis. Subjects who underwent lung transplants were considered as fatalities in the statistical analysis, as has been reported previously [2) with 6 min walking distance (6MWD) [The statistical analyses were conducted using SPSS v.23 software . The significance level was set at eviously ,35. Exple (6MWD) . PA and The study population consisted of 34 IPF subjects from the Pulmonary Institute who were recruited for the pulmonary rehabilitation study . All sub2 were determined as prognostic predictors using ROC analysis , p = 0.027 and ∆SpO2 ≥ 10%, p = 0.002, HR; 23.3, CI 95% (1.5–365), p = 0.025.  but not for ED and 6MWD . Mortality was more prevalent among subjects with IPAQ ≤ 417 METS-min/week and ∆SpO2 ≥ 10% . Two of the 11 patients were at end-stage IPF and underwent lung transplants, and were considered as fatalities in the analysis. In the current study, significant cut-off points for IPAQ and ∆SpOanalysis . Patient= 0.025. . ExploraO2 ≥ 10% .2 ≥ 10% in 6MWT were significantly associated with mortality in patients with IPF  between PA and 6MWD, which strengthens the findings, since the latter is a well-established prognostic marker in IPF [This prospective follow-up study sought to evaluate the prognostic utility of PA and ED among IPF patients. We found that PA levels ≤417 METS-min/week and ∆SpOwith IPF . In thisr in IPF ,38,39. T2 < 88% during 6MWT as a strong prognostic marker in IPF [2 levels. In this regard, desaturation of 10% or more during 6MWT was significantly associated with mortality, which overcomes the variation in resting SpO2 levels across patients. Furthermore, the findings demonstrated for the first time in an IPF group that PA levels assessed by IPAQ are associated with mortality, and even low activity levels  revealed a significant reduced risk for death [2 ≥ 10%) that can enhance the overall risk stratification and add important information in the process of decision making and treatment options for patients with IPF, which further studies using large sample sizes should explore more extensively. The results of the current study provide novel evidence on the importance of PA and ED for survival in IPF. These findings are consistent with a previous large cohort study showing mortality risk reduction with only 15 min of daily PA in a general population . Our finr in IPF ,42. Howeor death ,43,44. Tor death ,46. MorePossible mechanistic explanations for these findings can be related to the fact that physical inactivity is a well-established all-cause cardiovascular and respiratory mortality risk factor ,47,48,492 ≥ 10% is a good sensitive threshold for detecting high risk IPF patients, and as such may be considered for implementation in clinical and research settings, due to the low cost, ease of assessment, and practical availability. However, despite the strong association between PA and ED with mortality observed in the current study, given the multifactorial manifestation of the disease and the diversity of predictors, we believe that prognosis should be based on a combination of predictors rather than on a single variable, and future trials should address this issue using large cohorts and multivariate analyses.ED was also clearly demonstrated as a significant risk factor for death and was associated with more severe conditions of IPF disease ,4. HigheA few possible limitations in our study are related to the small sample size and the level of IPF severity among patients ; 68% predicted), thus caution should be taken with generalization of our findings. Despite these limitations, the study has a number of strengths: collecting data prospectively, analyses using various statistical methods for the detection of sensitive thresholds, and calculating the risk for mortality and survival analysis. Moreover, the study found novel, sensitive, and practical indicators that can be easily implemented in clinical practice. The number of participants in our cohort is comparable to a previous study of PA and prognosis in IPF ,23,24,522 ≥ 10% in 6MWT were significantly associated with increased risk of mortality. This study strengthens the evidence for assessing PA and ED for the detection of high-risk IPF patients and aids in addressing appropriate treatments, such as PA consultation, pulmonary rehabilitation programs, oxygen supplementation, and referral for lung transplantation. The findings also underscore that even low levels of PA seem to have prognostic benefits for IPF patients. We suggest considering physical inactivity as a new independent modifiable prognostic risk factor for IPF. Further research with large cohorts is warranted to encourage implementation and the establishment of PA assessments in standard clinical practice.In summary, this prospective follow-up study originates new, simple, and practical prognostic cut-off points of PA levels and ED in IPF subjects. The results demonstrated that PA levels ≤417 METS-min/week and ∆SpO