Using the ultrafast pump-probe transient absorption spectroscopy, the femtosecond-resolved plasmon-exciton interaction of graphene-Ag nanowire hybrids is experimentally investigated, in the VIS-NIR region. The plasmonic lifetime of Ag nanowire is about 150 ± 7 femtosecond (fs). For a single layer of graphene, the fast dynamic process at 275 ± 77 fs is due to the excitation of graphene excitons, and the slow process at 1.4 ± 0.3 picosecond (ps) is due to the plasmonic hot electron interaction with phonons of graphene. For the graphene-Ag nanowire hybrids, the time scale of the plasmon-induced hot electron transferring to graphene is 534 ± 108 fs, and the metal plasmon enhanced graphene plasmon is about 3.2 ± 0.8 ps in the VIS region. The graphene-Ag nanowire hybrids can be used for plasmon-driven chemical reactions. This graphene-mediated surface-enhanced Raman scattering substrate significantly increases the probability and efficiency of surface catalytic reactions co-driven by graphene-Ag nanowire hybridization, in comparison with reactions individually driven by monolayer graphene or single Ag nanowire. This implies that the graphene-Ag nanowire hybrids can not only lead to a significant accumulation of high-density hot electrons, but also significantly increase the plasmon-to-electron conversion efficiency, due to strong plasmon-exciton coupling. The hot carriers then redistribute their energy through electron–electron scattering processes on a timescale τel ranging from 100 fs to 1 ps352 hybrid had been used to study the plasmon-driven chemical reaction10122 nanoparticles, by using femtosecond transient absorption spectroscopy with an IR probe2 nanoparticles, and the reaction time was within 240 fsSurface plasmon resonance (SPR) is the resonant collective oscillation of conduction electrons at the interface between materials with negative and positive permittivities, which is stimulated by incident light3Graphene, a single atomic layer of graphite, has attracted vast interest owing to its unique properties since its discovery in 200415171819202122232526In this paper, novel optical graphene-Ag nanowire hybrid materials are fabricated, and the advantages of this hybrid device are shown through ultrafast pump-probe transient absorption spectroscopy and surface catalytic reactions on this hybrid nanostructure. The experimental results indicate that graphene can strongly harvest hot electrons generated from Ag plasmon decay, which can not only lead to a significant accumulation of high density hot electrons, but also prolong the lifetime of these hot electrons from femtosecond to picosecond, through Ag plasmon-enhanced graphene plasmon. Furthermore, the surface-plasmon-to-hot-electron conversion efficiency is significantly enhanced. Our results show that the graphene-Ag nanowire hybrids with plasmon-exciton interaction can act as a good candidate in spectral censoring, catalysis, and potential applications in nano-photonic devices, energy and environments.2/Si substrate is shown in 2D/IG ≈ 2.23)The SEM image of the graphene on SiO2 nanoparticles in IR regionThe SEM image of a single Ag nanowire coated by monolayer graphene can be seen in F < ε < EF + hv in the system, where EF is the Fermi energy level.We also measured the time of charge transfer between Ag nanowire and single layer graphene in the NIR region, as shown in 32There are two dynamic processes with different time scales in the NIR region. The fitted time of hot electrons transferring from Ag nanowire to graphene is about 780 ± 92 fs. It is found that the reaction time of hot electron from Ag nanowire to graphene in the NIR region is slower that in the VIS region, due to lower kinetic energy in the NIR region. The fitted curve in One of the advantages of graphene-Ag nanowire hybrids is that, graphene plasmon enhanced by metal plasmon can be used to drive chemical reactions on this hybrid nanostructure The surface catalytic reaction of 4-nitrobenzenethiol (4NBT) to p,p’-dimercaptoazobenzene (DMAB) without graphene has already been clearly investigated−1 to 1500 cm−1 in To highlight the advantages of plasmon-graphene hybrids for the catalytic reactions, we first measured the Raman spectra of 4NBT and DMAB powders, and SERS spectra of 4NBT at the junction of tip-to-tip Ag nanowires  cannot be observed because of the strong background of graphene. We propose that photo-induced hot electrons generated from graphene provide the kinetic energy and the electrons for the reduction reactions from 4NBTs to DMAB. The density and efficiency of hot electrons provided by the graphene are strongly dependent on the laser power. However, the intensity of Raman spectra in the chemical reactions is weak, compared with the strong graphene Raman signal. More electrons with higher kinetic energy are needed to increase the reaction probability and efficiency of graphene-driven reduction reactions when excited with weak laser power.Now we present the results of our investigation on the graphene-driven reduction reactions of 4NBTs to DMAB on the GERS substrate. The SEM image is shown in −1 corresponding to the N-O2 vibrational modes of 4NBT almost disappears completely), which exhibits that the plasmon-graphene hybrids can provide a high-density hot electrons, and thus that leads to a highly efficient catalytic reaction. It is clearly observed that the intensities of Raman peaks of graphene at 1595 and 2687 cm−1 gradually increase with the increase of the laser power. When the laser power reaches 100% (11 mW), the Raman intensity at 2687 cm−1 becomes larger than that at 1595 cm−1, I2D/IG ≈ 1.13. Thus, we conclude that 1) the catalytic reaction is already highly efficient at weak laser power, and the reaction probability can reach 100% by increasing the laser power; 2) the intensities of the graphene Raman peaks at 1595 and 2687 cm−1 also increase gradually, and for sufficiently high laser intensities, the Raman intensity at 2687 cm−1 become larger than that at 1595 cm−1, which demonstrates that the catalytic reaction indeed occurs on the monolayer graphene-coated Ag nanowires. Note that Raman intensities of DMAB are weaker compared with the Raman intensities of graphene, the reason is that some of the DMAB may be desorbed from the surface of graphene when radiated at enough strong laser. There are two superposed Raman peaks between graphene and DMAB around 1595 cm−1, which leads to I2D/IG > 1, but is less than 2.3, when the laser power is 100%.The SEM image of graphene-Ag nanowire hybrids is presented in Novel graphene-Ag nanowire hybrid optical materials are fabricated and probed using the ultrafast pump-probe transient absorption spectroscopy, and then exploited as the G-SERS substrates to investigate the typical surface reductions of 4NBTs to DMAB. Firstly, single-layer graphene can significantly improve the surface-plasmon-to-hot-electron conversion efficiency. Secondly, with the assistance of graphene, the lifetime of hot electrons is greatly increased from femtosecond to picosecond. As a consequence, this G-SERS substrate significantly increases the probability and efficiency of the surface catalytic reactions of 4NBTs to DMAB co-driven by graphene-Ag nanowire hybridization, compared with the same kinds of reactions but driven individually either by graphene or single Ag nanowires. The graphene-Ag nanowire hybrids, with higher conversion efficiency and longer lifetime of plasmon-induced hot electrons, can be used as a source of hot electrons for catalysis and might be potentially applied as an analytical tool for observing such plasmon-driven reactions.2 gas at 1000 °C for 5 min. In the initial stage of the reaction, the reaction gaseous mixture Ar:H2:CH4 with a flow rate of 960: 40: 20 sccm was introduced into the tube. The reaction took place for 5 min at 1000 °C. Finally, the CVD tube was cooled to 150 °C under an Ar:H2 environment.Large-area monolayer graphene was synthesized on copper foils  using cold-wall low-pressure chemical vapour deposition (CVD)2/Si substrate and then dried in the air at room temperature.The silver nanowires were prepared through chemical fabrication2/Si substrate. First, a PMMA layer was spin-coated  on the graphene-covered copper foil. The copper foil was then etched away by ferric chloride solution (0.5 M) for 4 h. The PMMA film with graphene was soaked in deionized water four times and then transferred from the water to the substrate with Ag nanowires. Finally, the PMMA was dissolved in an acetone solution and then the sample was washed in an ethanol and deionized water.Polymethyl methacrylate (PMMA) was used to help transfer the graphene from the copper foil to Ag nanowires on the SiO−5 M 4-NBT ethanol solution for more than 12 h. The substrates were then washed with ethanol for 5 min, and dried with N2 gas. All SERS spectra were measured by using a Renishaw inVia confocal Raman spectrometer with a 532 nm laser  as the excitation light. Each Raman spectrum was recorded with an accumulation time of 10 s.The G-SERS substrates were immersed in a 5 × 10ex = 400 nm) of the fundamental laser by a 0.5 mm BBO . The 400 nm beam was attenuated to a power of approximately 3 μJ per pump pulse at the sample. The excited states were probed by using the other beam generated to pass through a controlled delay line  and focus onto a sapphire plate to generate a sub-picosecond white-light super-continuum. The pump and probe beams were incident on the sample at a small angle (θ ≤ 5°). Samples were placed in the beam path where the beam diameter was 300 μm. All of the above experimental measurements were performed at room temperature (295 K). The sample of Ag nanowire-graphene was placed on the quartz substrate for the transient absorption spectroscopy.The morphologies of the substrate system consisting a single-layer graphene-Ag nanowire were characterized by using a scanning electron microscope . The setup of the transient absorption spectroscopy has been reported in the previous paperHow to cite this article: Ding, Q. et al. Ultrafast Dynamics of Plasmon-Exciton Interaction of Ag nanowire-Graphene Hybrids for Surface Catalytic Reactions. Sci. Rep.6, 32724; doi: 10.1038/srep32724 (2016).


The APOBEC3 family of cytosine deaminase enzymes are able to restrict replication of retroelements, such as LINE-1. However, each of the seven APOBEC3 enzymes have been reported to act differentially to prevent LINE-1 retrotransposition and the mechanisms of APOBEC3-mediated LINE-1 inhibition has not been well understood. The prevailing view for many years was that APOBEC3-mediated LINE-1 inhibition was deamination-independent and relied on APOBEC3s blocking the LINE-1 reverse transcriptase DNA polymerization or transport of the LINE-1 RNA into the nucleus. However, recently it was shown that APOBEC3A can deaminate cytosine, to form uracil, on transiently exposed single-stranded LINE-1 cDNA and this leads to LINE-1 cDNA degradation. In this study, we confirmed that APOBEC3A is a potent deamination-dependent inhibitor of LINE-1 retrotransposition, but show that in contrast, A3H haplotype II and haplotype V restrict LINE-1 activity using a deamination-independent mechanism. Our study supports the model that different APOBEC3 proteins have evolved to inhibit LINE-1 retrotransposition through distinct mechanisms. Transposable elements can be grouped into two major classes based on whether they mobilize via a DNA intermediate  or an RNA intermediate 2. DNA transposons, although not being able to mobilize in the current human genome, have played a major role during the evolution of the eukaryotic genome6. Retrotransposons transpose through mRNA intermediates followed by reverse transcription, synthesis of double-stranded (ds) DNA, and integration at new genomic locations. Retrotransposons can be divided into two classes: Long Terminal Repeat (LTR) retrotransposons  and the non-LTR retrotransposons. The replication strategy of endogenous retroviruses resembles those of retroviruses transmitted person to person, except that they remain in one host and are transmitted vertically2. The present day endogenous retroviruses have acquired so many mutations that they cannot produce infectious virus2.Transposable elements are DNA sequences that can move from one location of the genome to another. Approximately 45% of the human genome is recognized as being derived from transposable elements8. Although the vast majority of L1s are inactive due to accumulated mutations or 5′ truncations, the human genome still contains ~80–100 copies of intact L1s that are retrotransposition competent10. Full-length L1 contains a 5′ UTR, two Open Reading Frames (ORFs) that are separated by a 63 nt inter-ORF region, and a 3′ UTR that ends with a polyadenosine-rich sequence (poly(A) tail)11. Proteins encoded by ORF1 and ORF2 are both required for efficient L1 retrotransposition. ORF1 encodes a nucleic acid binding protein with demonstrated nucleic acid chaperone activity; ORF2 encodes proteins with endonuclease (EN) activities and reverse transcriptase (RT) activities14. L1-encoded EN preferentially cleaves ssDNA at an AT-rich consensus sequence . L1 RT is a DNA/RNA dependent DNA polymerase, but lacks RNaseH activity, a trait that distinguishes itself from the retroviral RTs15. As a result, during cDNA synthesis the RNA strand of the resulting L1 RNA/DNA hybrid could be either degraded by a yet unidentified cellular protein or displaced during (+) strand cDNA synthesis16. Cellular RNaseH2 cooperates with flap endonuclease 1 (FEN-1) to remove RNA primers during lagging strand DNA synthesis and serves as a good candidate17. Unlike retroviruses and LTR retrotransposons which undergo reverse transcription in cytoplasm, L1 RNA is reverse-transcribed in the nucleus19. L1 relies on a mechanism termed the Target Primed Reverse Transcription (TPRT) for retrotransposition20.Non-LTR retrotransposons are found throughout the eukaryotes, and examples of non-LTR retrotransposons include LINE-1  and SINE (Short Interspersed Nuclear Element). Human L1s account for ~17% of the genomic DNA with ~500,000 copies identified, and they represent the only autonomous transposable elements that are currently active in humans10. The retroelement suppression mechanisms include DNA methylation22, RNA interference24, and cellular DNA repair factors26. Also, studies suggest that several individual proteins are involved in L1 restriction. Examples of these proteins include: Three primer repair Exonuclease 1 (TREX1)27, Zinc finger Antiviral Protein (ZAP)28, RNA helicase Mov1029, RNaseL30, SAM domain and HD domain-containing protein 1(SAMHD1)32, and the APOBEC family of cytosine deaminases41.Despite the positive effects of retroelement mobilizations, such as contributing to genome evolution and gene diversity, excessive transposition events need to be suppressed, as several genetic diseases have been associated with L1-mediated insertional mutagenesis in germline and somatic cells43. The most ancient APOBEC family members include Activation Induced cytidine Deaminase (AID) and APOBEC2 (A2) proteins that are identified in jawless and cartilaginous fish44. AID initiates class-switch recombination and somatic hypermutation, two processes that occur during antibody maturation, by deaminating cytosine in single-stranded (ss)DNA to form promutagenic uracil. The ssDNA is available during transcription and initiates an error-prone DNA repair process that diversifies the antibody genes and enables a specialized recombination to occur for immunoglobulin class switching45. The A2 enzyme is involved in cardiac and skeletal muscle development47. APOBEC4 (A4) and APOBEC5 (A5) then emerged in amphibians, although their functions are yet to be identified49. During tetrapod evolution, duplication of AID led to the emergence of APOBEC1 (A1)50. A1 plays an important role in lipid transport by editing the apolipoprotein B mRNA in intestinal cells by introducing a uracil that results in a stop codon and truncated version of Apolipoprotein B that has a different function than the full length form51. A1 can also deaminate cytosine in ssDNA52. In placental mammals the APOBEC3 (A3) locus evolved and greatly expanded, which is thought to partly be due to the increasing number of retrotransposons in the genome43. While only one A3 gene is present in mice, up to seven A3 genes  are present in primates43. A3 genes have likely acquired the anti-L1 activities from the Activation Induced Cytidine Deaminase (AICDA)/A2-like gene from which they have evolved. Several studies have demonstrated that in a cell culture system that A3 family members could inhibit a number of mammalian L1s41. Since A3 genes are only limited to mammalian lineages, but L1s are far more ancient, dating back to the emergence of vertebrates, it was not surprising to find that APOBEC proteins, such as pre-mammalian AID and reptilian A1 from green anole lizard, are indeed capable of inhibiting L1 retrotransposition53.The APOBEC enzyme family emerged at the origin of vertebrates and have diversified throughout the vertebrate lineage57. For a deamination-dependent mechanism, during the reverse transcription of the L1 RNA, the (-)cDNA gets transiently exposed after RNA template degradation, which renders it susceptible to A3-mediated cytosine to uracil (C → U) deaminations57. The uracils present on L1 cDNA could trigger cDNA degradation through the actions of the host base excision repair pathway. Specifically, cellular uracil DNA N-glycosylase (UNG) excises the uracils from ssDNA, generating abasic sites and apurinic/apyrimidinic endonuclease (APE) produces nicks on the DNA which would induce L1 cDNA degradation. Alternatively, a uracil-containing cDNA may escape the degradation, become replicated to form a dsDNA, and integrate into the host chromosome, but be functionally inactivated through A3 induced C/G → T/A transition mutations that result from the polymerase using uracil as a template. For a deamination-independent mechanism of L1 restriction, the A3s can sequester L1 ribonucleoprotein (RNP) complexes to cytoplasmic compartments such as stress granules or physically interact with L1 RT and inhibit DNA polymerization during TPRT59. There is no clear agreement in the literature as to whether A3s utilize the deamination -dependent or -independent mode for L1 restriction. These ambiguities arose because no G → A mutations on the coding strand, which would indicate the occurrence of C → U deamination on the L1 cDNA, could be detected. However, none of these cell culture studies blocked the action of UNG60. Richardson et al., reasoned that the unsuccessful attempts to uncover A3-induced mutations were likely due to the degradation of uracil-containing cDNA intermediates by the UNG and APE-mediated DNA repair proteins57. In their study, using the Uracil Glycosylase Inhibitor (UGI) protein of Bacillus subtilis bacteriophage PBS161, they demonstrated that A3A restricted L1 through a deamination-dependent mechanism, suggesting that the mechanism by which A3 enzymes restricted L1 needed to be readdressed. These data also explained how mammalian L1 sequences analyzed on larger genome-wide scales could show more evidence of cytosine deamination and is in agreement with the known restriction mechanism of several endogenous retroviruses that show a G → A mutation bias65.A3s have been reported to restrict L1 by both deamination -dependent or -independent mechanisms70. Since haplotype II (hap II) and haplotype V (hap V) comprise the majority of the stable haplotypes we examined how they restricted L1 retrotransposition. Previous research has demonstrated that stable A3H could inhibit L1, however, a mechanism was not identified71. In this study, we demonstrate that both A3H hap II and hap V suppress L1 to a similar level but the inhibition of L1 does not involve DNA deamination, in contrast to A3A.To address whether the deamination-dependent mechanism of L1 inhibition is unique to A3A or common to other A3s, we examined how A3H restricted L1 retrotransposition in cell culture. There are at least seven A3H haplotypes that exist in humans. Three of the haplotypes are stable and catalytically active , three are thermodynamically unstable and have no discernable activity , and one has an intermediate stability and is catalytically active (I)54.To establish a context with which to interpret our results with A3H, we also included A3A and A3G in our study. These additional A3s were chosen based on previous publications indicating that A3A was a strong inhibitor of L1 retrotransposition and A3G was unable or weakly able to inhibit of L1 retrotransposition73  reporter cassette, which, when expressed, can convey resistance to G418. The neo reporter cassette has its own promoter and polyadenyation signal, and it consists of an antisense neo gene disrupted by an intron of the γ-globin gene, which is in the same transcriptional orientation as the L1 element  cells will only arise when a transcript initiated from the L1 promoter is spliced, reverse transcribed, its cDNA is re-inserted into the host chromosome, and the neo gene is transcribed from an internal promoter 38, can accommodate high levels of L1 retrotransposition events from ectopically expressed L1 reporter plasmids74 and do not express endogenous A3A, A3G and A3H proteins38.To study the influence of A3 proteins on L1 retrotransposition, a pre-established cell line based retrotransposition assay was performed73 Fig. . To deteent Fig. . Becauseter Fig. . The L1 R colonies were stained and counted. Transfection of L1 vector alone with no A3 expression plasmid generates a quantifiable amount of G418R colonies  and A3G  according to the previously published catalytic mutants78. In this experiment we tested only A3H hap II since both hap II and hap V restricted L1 retrotransposition similarly , although this has been observed under other experimental conditions81, and that indeed the mutations were acquired during integration of the L1 by TPRT, consistent with Richardson et al.57. Because A3A prefers to deaminate C → U within a 5′TC or 5′CC context (underlined C is deaminated)83, we determined whether these G → A mutations on the genomic strand occurred within the preferred deamination motif as a confirmation of A3A deamination activity. Deamination of these motifs in the cDNA will result in 5′GA → 5′AA or 5′GG → AG mutations in the coding strand. Out of the 16 G → A mutations we recovered in the neo gene, two mutations were within the 5′GA → 5′AA context and 1 mutation was within the 5′GG → AG context (data not shown). In addition, other mutations at 5′GC → 5′AC and 5′GT → 5′AT motifs were observed that may or may not be due to A3A catalytic activity (data not shown). However, in the presence of UGI, the majority of the G → A mutations occurred within the 5′GA → 5′AA context, confirming that UGI blocked removal of uracils formed by A3A catalytic activity 66. A3H hap II and hap V also did not appear to be able to deaminate L1 mRNA, since no C → T mutations with the correct sequence context were found cDNA or physically impair RT’s activity86. Based on these observations and that both A3H Hap II and Hap V are primarily cytoplasmic proteins67, we hypothesize A3H could inhibit L1 mobility by binding to the L1 RNP in the cytoplasm, similar to what has been reported for A3C and A3D59.We found A3H hap II and hap V are able to suppress L1 retrotransposition to a moderate level and there were no apparent differences in their L1 restriction abilities Fig. . Three ld84 Fig. . However 85 Fig. . CytoplaThe analysis of the L1 restriction ability of A3A, A3G, and A3H hap II and hap V have demonstrated that each of the A3s has a unique effect on L1. In particular, although L1 restriction by A3s can be deamination-dependent, as shown for A3A, this does not appear to be applicable to all A3 enzymes. These data suggest that reanalysis of A3-mediated L1 restriction in the presence of UGI for all A3 family members is warranted to fully understand their mechanism of activity.88 and were cloned into pcDNA6-V5/His.A using XbaI and XhoI cloning sites giving the enzymes a C-terminal V5-His fusion tag. Mutants of A3 enzymes  were made using the wild type construct for site-directed mutagenesis . The Uracil DNA glycosylase inhibitor gene was synthesized by GenScript with codon optimization for human cells and subcloned into pcDNA6, but was expressed without the tags for the experiments or subcloned into pcDNA3.1 with a C-terminal V5 tag for checking expression by immunoblotting. All constructed plasmids were verified by sequencing.L1 expression plasmid pJM101/L1.3-Neo was kindly provided by Dr. John V. Moran (University of Michigan). The cDNA of A3A, A3G and A3H have been previously reported5 cells/well in Dulbecco’s Modified Eagle Medium (DMEM) with 10% FBS. Approximately 22 h after plating, Hela cells were co-transfected with 1 μg of L1 retrotransposon encoding plasmid (pJM101/L1.3) and 1 μg of an A3 encoding plasmid or empty vector. Transfections were performed using GeneJuice (EMD Millipore) according to the manufacturer’s protocol. Media was replaced the following day. Approximately 48 h post-transfection, cells were re-seeded into 6-well plates (at 1 in 5 dilution) and the next day attached cells were subjected to selection with 400 μg/mL G418 (Life Technologies). During the reseeding process, cells were checked for viability by trypan blue staining. We consistently observed a cell viability > 95% indicating that expression of A3A, A3G, or A3H did not cause cellular toxicity. G418 selection was carried out for 11 days and the selection media were replaced every other day. Afterwards, G418R colonies were washed twice with PBS, fixed with 2% paraformaldehyde/0.4% glutaraldehyde, and stained with 0.1% crystal violet solution.HeLa cells were seeded in each well of a 6-well tissue culture plate at a density of 2 × 10To detect A3 expression levels, cells were washed in 1x PBS, harvested in Laemmli sample buffer (58 mM Tris (pH 6.8), 5% glycerol, 2% sodium dodecyl sulphate (SDS), 1.5% dithiothreitol (DTT)), and 40 μg of total protein was resolved by SDS-PAGE. Proteins were transferred to a nitrocellulose membrane and A3 or UGI in the cell lysates were probed with primary antibodies  and rabbit monoclonal α-tubulin, 1:1000 (Sigma)). After incubation with the secondary antibodies  the tagged proteins and loading control was detected simultaneously on the same membrane by using the Licor/Odyssey system.neo gene using the forward primer (G418Neoseq For) 5′-TCGGGAGCGGCGATACCGT-3′ and the reverse primer (G418Neoseq Rev) 5′-CGGTGCCCTGAATGAGCTTCA-3′ with the Q5 High-Fidelity polymerase (New England Biolabs). The PCR cycle used had an initial denaturation step at 98 °C (3 min) followed by 35 cycles of amplification . PCR products were electrophoresed to separate unspliced (of plasmid origin) and spliced (of integrated L1 origin) PCR products. The spliced product was gel purified with the GenElute Gel Extraction kit (Sigma) and cloned into pJET1.2/blunt vector . After transformation into Escherichia coli DH5α cells, clones were picked and sent for sequencing using a kit-specific primer (pJET1.2 Forward) at the National Research Council DNA sequencing Facility . Sequence alignments were performed using Clustal Omega.Hela cells were co-transfected with 1 μg of A3 and L1 plasmids in the presence or absence of 1 μg UGI vector as described above. Cellular DNA from the transfected cells was extracted 72 hr post-transfection using DNAzol reagent (Thermo Fisher). Extracted DNA was used as a template for PCR amplification of the spliced E. coli (New England Biolabs) was used as a control. For DNA visualization the samples were mixed with an equal volume of formamide/EDTA loading dye, resolved on a urea denaturing 16% v/v polyacrylamide gel, and scanned using a Typhoon Trio multipurpose scanner .HeLa cells were transfected with 1 μg of L1 retrotransposon encoding plasmid (pJM101/L1.3) and either 1 μg empty vector or UGI vector as described above. Cells were harvested and lysed 72 h after the transfection in buffer containing 50 mM Tris-Cl pH 7.4, 1% Nonidet-P40, 0.1% sodium deoxycholate, 10% glycerol, 150 mM NaCl, 1 mM DTT, 20 μg/mL RNaseA and EDTA-free protease inhibitor (Roche). To test for UGI activity, 8 μL of the lysate was incubated with 100 nM of a 44 nt ssDNA oligonucleotide with a single deoxyuridine and a 5′ Fluorescein (5′ (Fluorescein)-AAA GAG AAA GTG ATA AAC AAA GAG TAA AGU AGA TAG AGA GTG ATA 3′) for 15 min at 37 °C. To cleave the DNA at abasic sites generated by the action of UNG, the DNA was then heated at 95 °C for 10 min in the presence of 0.2 M NaOH. Purified UDG from No datasets were generated or analyzed during the current study.


This nanoflower like architecture was decorated on 3D-graphene on Graphite electrode to design the solid-state-supercapacitor prototype device of dimensions of 23.6 × 22.4 × 0.6 mm3 having considerable high Csp of 58.0F/g and energy density of 24.59 Wh/Kg, and power density of 8.8 W/Kg. Four fabricated supercapacitors were connected in series for real state practical demonstration using the light emitting diode that remains enlightened for 40 s by charging it only for 25 s. This study demonstrates the 3D-graphene/MoS2 nanohybrid has a quite high overall potential window nearly about 2.7 V (−1.5 to +1.2 V) in KOH-PVA medium which can be used for the development of solid-state supercapacitors thereby completely eliminating the need for any expensive ionic liquid mediums thus building an exciting potential for high-performance energy storage/transfer devices.Portable and matured energy storage devices are in high demand for future flexible electronics. Flowery shaped MoS Due to the increase in demand for energy, the non-renewable sources like fossil fuels are continuously decreasing and will automatically come to an end in the near future. To meet this demand, alternate renewable energy sources has been projected like solar, wind and hydropower. However, a suitable storage device like Li-ion batteries and Na-air batteries and so on are currently lagging behind with a storage capacity of only 1% of the total renewable energy with longer charging time. Presently, batteries are used in wide variety not only for energy storage but also for the energy supply devices2. The use of a battery in large scale sophisticated electronic devices is limited due to its heavy weight, portability, flexibility and other issues4. Currently, researchers are looking for alternative energy storage devices that effectively store and provide energy as per requirement. Supercapacitors are portable, flexible5 and transparent unlike conventional batteries that draw the attention of the researchers to extract its interesting properties. Along with these physical advantages, supercapacitor has high charge storage capacity, high power density, low energy density, longlife time and short charging time as compared to traditional batteries8. A notable improvement in performance has been achieved through recent advances in understanding charge storage mechanisms and the development of advanced nanostructured materials in supercapacitor devices9. Supercapacitors are classified mainly in two categories such as, pseudocapacitor and electrical double layer capacitance (EDLC)8. In case of pseudo capacitor, the electrical energy is stored via faradaic reaction in electrolyte and electro-active species on the electrode surface. There are several reports in the literature based on pseudocapacitors made of various nanomaterials as well as their composite, such as SnO2-V2O5, Cobalt pyrophosphate, Metal oxide-CNT, RGO-metal sulfide, MnO2-veritcally aligned graphene, etc. having a specific capacitance in the range of 190–3480F/g with energy density ranging from 14–89 Wh/kg12. It has been reported that the capacitance due to faradaic peaks increases the specific capacitance of supercapacitor 10–100 times higher than the electrostatic capacitance of an EDLC. Recently, Ko et al.13. has demonstrated porous graphitic carbon with Ni2P2O7, heterostructure has shown high specific capacitance of 1893 F/g and Liao et al.14. has reported ultrahigh supercapacitance of 3480F/g using Co3O4 nanoparticles on vertically aligned graphene sheets supported in carbon fibers. Although pseudocapacitors are shown to have high initial capacitance, but due to limited durability, lack of stability during cycling and poor power density, its practical use in solid-state energy storage devices is limited16. In addition to this, crystallinity and morphology also play an important role in specific capacitance of pseudo capacitors, lower the crystallinity higher is the specific capacitance, which is due to the availability of chemically active dangling units which can easily take part in oxidation-reduction cycle8. To overcome these limitations, there is an extensive drive to develop EDLC type supercapacitor that carries various advantages over pseudo capacitor. In case of EDLC, electrical energy is stored over the layer of current collector without any redox couple. Due to the absence of faradaic current, EDLC type supercapacitors are more reliable and have shown long term durability with minimum loss of capacitance under multiple charging-discharging cycles.Current economy and population growth rates project a demand for energy of 40 terawatts (TW) with a total population of 10 billion by 2050et al. have fabricated graphene hydrogel for the EDLC type supercapacitor where storage performance is related to the high surface area17. Sundaram et al. have fabricated MnO2 nanostructures for EDLC type supercapacitance in a lower applied potential window having Csp of 50F/g18. Various carbonaceous materials till now have been developed for the testing of EDLC type supercapacitors due to its cost efficiency, high surface area, porous nature and versatile existing forms20. Significant enhancement in capacitive performance is not only governed by the properties of electroactive materials but also by the separators and electrolytes. Lin et al. have fabricated graphene based supercapacitor (Csp of 130F/g) in ionic liquid electrolyte21 where ionic liquids shows high chemical stability as compared to conventional electrolytes at a wide potential range22. Further development of the storage device lies on the various tricky parameters such as, proper choice of electrolyte, electrochemically stable current collector and thin porous spacer matrix between the electrode materials etc24. The current developmental trend of miniaturized autonomous electronic equipment such as implantable medical devices and active radio frequency identification (RFID) tags have raised the demand for solid-state-supercapacitors25. Significant efforts have been devoted to improve flexibility and energy storage/transfer capacity of solid-state supercapacitors based on various carbonaceous materials and their composites26, out of which graphene shows a promising choice of material due to high conductivity, flexibility, transparency, large surface area and layer like structure that drags attention to various scientific communities for its large scale application in energy storage devices24. In most of the cases, graphene based solid-state-supercapacitors demonstrate very low specific capacitance which is mainly due to the parallel restacking of graphene sheets resulting in decrease of the specific capacitance28. Recently, 3D graphene proves to be more prominent material for solid state supercapacitor due to the partial stacking of graphene sheets in random orientation in three dimensions resulting in formation of micro porous structures17. This unique orientation of graphene not only helps to improve the charge storage capacity of graphene but helps the electrolytic ions to move freely via porous geometry. To further improve the response of the 3D graphene as solid-state-supercapacitor various layer/porous nanomaterial hybrids have been reported which develops the charge storage capacity of the 3D graphene via increase in surface area and porous nature of the hybrid materials33. It is already reported that graphene based hybrid material possess superior charge storage capacity by providing enhanced surface area, electrical conductivity, thermal stability and mechanical strength to graphene34. Out of the various nanomaterials, 2D transition-metal dichalcogenides have been a perfect candidate for charge storage applications due to their layer like structures that shows weak Van der Waal force of attraction with graphite or graphene, especially 1T-MoS2 nanostructures41. Due to their band gap of ~1.9 eV, which reveals the semi-insulating nature, they are thus not immediately thought to be an electrode material for energy storage application42. But still MoS2 nanosheets display an excellent performance in wide, negative potential in neutral electrolytes, such as high specific capacitance and rate performance, which promotes the MoS2 nanosheets to be a promising electrode material for supercapacitor applications43. Till date various MoS2 based supercapacitor have been developed but only handful reports have been presented which shows the practical applicability of solid state supercapacitor46. The main research challenges associated with electrochemical supercapacitors are to develop new electrode materials, to enhance specific capacitance by modification of the electro- active material37.Xu 2 nanoflowers with 3D graphene heterostructure as an active material for energy storage device. The real demonstration of the solid state supercapacitor has been performed here using the 3D graphene-MoS2 hybrid over graphite current collector. Herein, the specific capacitance of bare MoS2 nanostructures comes out to be 169.37F/g with an energy density of 28.43 Wh/kg and power density of 10.17 W/kg. The capacitive performance of 3D graphene-graphite electrode has been improved significantly when the current collector is incorporated with MoS2 nanoflowers. This remarkable supercapacitor performance of the hybrid could be attributed to the combined effect between layered MoS2 and 3D graphene which could have some real phase application in modern energy storage devices.In this work, we report the simpler and cost effective process via chemical route of synthesis of hybrid MoS2 nanoflowers were characterized by XRD, SEM, and Raman spectroscopy. Figure 2 nanoflowers. It shows the diffraction patterns at 2θ = 13.55, 33.06,35.22, 42.5, 49.82, 58.01, and 69.77 which arises from the , (1 0 2), (006), (105), (110) and (201) planes of MoS247. The observed diffraction patterns are broadened in nature with a slightly lower diffraction angle shift as compared to the bulk 2H-MoS2, suggesting the presence of randomly stacked layers of MoS2 which may be due to the intercalation of NH3 in between the layers during the synthesis48. The XRD pattern well matches with the already common works on randomly stacked MoS2 nanostructures50. No peak other than MoS2 has been observed in the XRD pattern revealing that the synthesized MoS2 nanoflowers are highly pure in nature. Figure 2 nanoflowers at a low magnification, it is observed that the particles are spherical in nature with porous nanostructure in the order of nanometers. It is well established in the literature that porous nanomaterials possess a superior charge storage capacity as compared to that of their bulk counterpart51. The formation of the porous MoS2 nanoflowers is due to the reaction conditions as well as the precursors used in the reactions. Coagulation into a sphere is most likely due to the hydrothermal reaction conditions and the precursors used. During the reaction, ammonium molybdate releases MoO4−. These ions, having a layered structure, react with sulfide ions and intercalation by the residual ammonia prevents the stacking of MoS2 layers which leads to the self-assembly of a sphere-like morphology48. The Raman spectrum of MoS2 nanostructure was carried out at room temperature , arises due to S atom in the basal plane. Band appearing at 347.86 cm−1 (E2g symmetry) arises due to intralayer vibrational mode of Mo and S atoms in the basal plane. The A1g mode which appears at 390 cm−1 is due to the intralayer mode involving the motion of S atoms53. Figure −1, respectively56. The appearance of D band arises due to the activation of the first order scattering process of sp3 hybrid carbons, which is attributed to dislocation defects in graphene sheets57. Similarly, the SEM studies of as-synthesized 3D graphene were carried out, see  planes of MoS2 and multilayer graphene59.The MoSial Fig.  demonstr2 modified glassy carbon electrode in 0.1 M KOH solution from low (10 mV/s) to higher (300 mV/S) scan rate. From the CV it is clear that no significant redox peak was observed which reveals that MoS2 nanostructure behaves as typical electrical double layer capacitance which is similar to already published in the literature60. Also the working potential of MoS2 nanoflowers were quite high i.e. −0.9 to 0.2 i.e. 1.1 V of working potential was observed. The specific capacitance of MoS2 was calculated using given equation:61. By this equation, the specific capacitance of MoS2 nanostructure comes out to be 145.73F/g (100 mV/s).Figure 62:The energy density of the present system was calculated by using equation2 nanospheres in 0.1 M KOH solution at various scan rates. As seen from the figure the approximately rectangular shape with high symmetry is an indicator to double layer capacitance with fast charge-discharge rates. These results i.e. electrical double layer formation, are in good agreement with the previously published literature63. For various scan rates i.e. 100, 150, 200, 250, 300 mV/s, the specific capacitance using equation  in 0.1 M KOH. By comparing their respective cyclic voltammograms, it is observed that the hybrid material not only increases the working potential window but also increases the current response, Csp and energy density for graphite, 3D graphene over graphite and of 3D graphene-MoS2 hybrid comes out to be 7.83, 46.82, 58.0 F/g and 1.09, 13.0, 24.59 Wh/Kg, respectively which shows that the hyrid material has more supercapacitive performance as compared to both 3D graphene and graphite material. The complete values of specific capacitance, energy density, and power density was given in Tables The specific capacitance obtained by from CV curves is about 169.37, 95.66, 62.52 and 46.17F/g for 1, 2, 3 and 4 A/g respectively. This observed specific capacitance shows a significant improvement over previously reported valueseet Fig. . Green, 2 was scotch taped and then put in teflon line stainless steel vessel, 5 mg/mL solution of graphene oxide was added along with 2 M ascorbic acid solution and afterwards hydrothermal was sealed. The hyrothermal bomb was then kept at 120 °C for 4 h resulting in the formation of hydrogel of 3D graphene over graphite piece. This modified graphite was kept in 5 mg/mL solution of MoS2. Similiary other plate of supercapacitor was fabricated using same protocol. After this the two plate was assembled by using filter paper soaked with PVA/KOH solution.Figure 2 comes out to be 74.94F/g and the specific capacitance of 3D graphene-MoS2 hybrid in solid state device comes out to be 58.0F/g  fully opens the stacked 3 D graphene as well as MoS2 nano flaks which allow ion intercalation and creates abundant ion-accessible sites for adsorption/intercalation, and dramatically increases the cell’s capacitance68.Successful implementation of the supercapacitor in solid-state without a loss in performance can be inferred. Figure see Fig. . This is2 3D graphene hybrid over graphite. TGA spectrum shows two weight losses one at ~350 °C and other at around 500 °C. Weight loss i.e. 5% at 350 °C corresponds to 3D graphene69. While the weight loss i.e. 2.5% at around 500 °C corresponds to MoS2 nanoflowers59. From the TGA calculation, the weight of the active material comes out to be 1.1 mg, which has been used to calculate the specific capacitance  is performed by electroactive materials to analyze the kinetic feature of the ion transportation and ion diffusion in the porous hybrid electrode in aqueous electrolyte system. Figure s) for MoS2-3D graphene hybrid is 18.66 ohm. The corresponding equivalent circuit has been shown in Figure 70. The enchantment in energy density is found to be attributed to the enlarge operating potential voltage of 2.7 V in the gel electrolyte (PVA with KOH) and the relatively high specific capacitance of both electrodes, especially the negative electrode, was found which is very important for the solid state devices. The stability curve strongly indicates that about 90% of the initial capacitance can still be retained after 1400 cycle at the scan rate of 100 mV/s  in KOH-PVA medium without utilizing any expensive ionic liquid mediums. Recently a solid state architecture has been designed using a similar heterostructure66, however a costlier growth technique has been adopted where for the graphene growth was performed in 1000 °C in CVD setup. In contrast our study shows a very simpler and cost effective process via chemical route to design heterostructure. In comparison to the device performance, they report the specific capacitance of 19.44 F/cm3 in contrast to our device as 58.0 F/g. Here, it is noticed that the straight forward comparison is not feasible because the analytical approaches are entirely different.The Table 2 was successfully paired with 3D graphene to fabricate a flexible, solid-state supercapacitor with outstanding performance. The facile method of fabrication can easily be scaled up at minimal cost to create a large array of supercapacitors which is lightweight and reliable. An immediate application can be found in solar energy storage due to its large energy storage capacity in a short span of time in purely EDLC type nature. The specific capacitance of the solid state supercapacitor comes out to be 58 F/g with energy density of 24.59 Wh/Kg and power density of 8.8 W/kg. It can be a partial replacement of conventional batteries and can be easily incorporated into flexible and thin electronics due to its high stability, thinness and flexibility. The operating potential window can be further enhanced by using an ionic-liquid gel-based electrolyte. Other transition metal dichalcogenides can also be investigated along with MoS2 for their supercapacitor performance on top of 3D graphene matrix.MoSGraphite, ammonium molybdate, ascorbic acid, isopropyl alcohol, thiourea were purchased from sigma aldrich. All the solutions were prepared in deionized water. KOH was purchased from TCI chemicals.2 nanoflowers were synthesized by a hydrothermal method using ammonium molybdate and thiourea as a starting precursor. Briefly, 1 M solution of ammonium molybdate and 5 M solution of thiourea were prepared separately and transferred into a teflon-lined stainless steel autoclave (100 mL capacity) and hydrothermal reaction was carried out at 180 °C for 24 h. Black precipitate was obtained followed by washing with deionized water and absolute ethanol. The washing procedure is repeated several times until the final product is free of any trace amount of residual ions. The final product was dried at 80 °C for 12 h.The MoS2 nanospheres were coated on a polished GC electrode surface  to modify the Glassy Carbon (GC) electrode. For the coating, a solution of 1 mg/mL of MoS2 was prepared in isopropyl alcohol from which 5 µL was drop casted over the GC electrode surface. The prepared electrode was kept in a vacuum desiccator for 24 h to form a uniform layer over the surface. Cyclic voltammetry was carried out using a CHI electrochemical workstation with a three electrode system. While the MoS2-modified electrode was the working electrode, Ag/AgCl in sat. KCl was used as the reference electrode and platinum electrode was the counter electrode. These were dipped in 0.1 M KOH electrolyte.The as-synthesized MoS2 in isopropyl alcohol for 12 h. The graphite pieces were finally taken out and dried in an oven at 60 °C for 12 h.We followed the modified hydrothermal reduction method for the deposition of 3D graphene onto the graphite electrodes. Briefly, two rectangular pieces were taken from a graphite sheet and they were dipped into 10 mL of 5 mg/mL aqueous dispersion of graphene oxide (GO). This was freeze dried at −20 °C for 12 h. After the solution temperature was brought to room temp, 0.6 ml of 2 M ascorbic acid (AC) was added to the graphene oxide aqueous dispersion and the mixture was sealed in a teflon-lined autoclave and heated in an oven at 120 °C for 4 h, followed by drying of the attached graphite pieces in an oven set at 60 °C for 6 h. Once the drying was completed, the graphite pieces were dipped into a 5 mg/mL dispersion of MoS0.5 g of poly-vinyl alcohol (PVA) was added in small amounts to 9 mL of boiling de-ionized water under constant stirring with a magnetic heater-stirrer. Once the PVA gel reached a uniform consistency and bubbles started to form, 1 ml of 0.5 mg/mL aqueous solution of KOH was added into the gel drop wise under constant stirring. On attaining homogeneity, a piece of whatman filter paper was cut, soaked in the gel and placed in between the two electrode pieces. This arrangement was left to dry for 12 h and hence, the final device was completely fabricated.Supplementry videoSupplementry Information


Graphene has been intensively studied for applications to high-performance sensors, but the sensing characteristics of graphene devices have varied from case to case, and the sensing mechanism has not been satisfactorily determined thus far. In this review, we describe recent progress in engineering of the defects in graphene grown by a silica-assisted chemical vapor deposition technique and elucidate the effect of the defects upon the electrical response of graphene sensors. This review provides guidelines for engineering and/or passivating defects to improve sensor performance and reliability. Graphene, a two-dimensional (2D) zero-gap semiconductor, has drawn great interest as a promising platform for novel electronic, optoelectronic, and energy harvesting systems –9. In pa− ions to edge defect sites, but the binding characteristic was not thoroughly determined. Nevertheless, existing methods for introducing defects in graphene entail difficulties in controlling the quantity of the defects and/or avoiding contamination from external substances . I. I30]. I3 atmosphere. In the drain current versus gate voltage (Id–Vg) curves of graphene mesh FETs, the Dirac point was shifted greatly in the negative direction after N doping. During the annealing process, physisorbed molecules such as O species [Because the graphene meshes are directly synthesized, their edges are less vulnerable to contamination than those produced by lithographic methods. To further examine the characteristics of the edge defects, doping of graphene meshes was explored by high-temperature thermal annealing under NH species , 53 were2, even at room temperature [2 sensing. However, rigid Pd films undergo structural degradation during reaction with H2 [Pd shows high reactivity and resistance change upon exposure to Hperature –57, maki with H2 , 57–60.  with H2 , Pd-Si [ with H2 , and Pd- with H2 –65. In t with H2 , 67. Acc2 gas sensors have been fabricated with normal Pd-Gr and with Pd-GM [2 gas, and the continuous changes in resistance were monitored at room temperature. The relative resistance changes were greater for Pd-GM sensors upon exposure to H2 gas of concentrations ranging from 2 to 15 ppm /R0, where R0 and R respectively denote the channel resistances before and after exposure to H2 gas, improved by 18.2–28.8 % when graphene was replaced with graphene mesh  showed that Pd-GM sensors responded slightly faster than Pd-Gr to H2 gas  provide binding sites for the H+ ions, thereby further increasing the Dirac point shifts in the GM-FETs.To investigate the effect of defects in graphene-based FET pH sensors, electrolyte-gated graphene-FET (Gr-FET) and graphene mesh–FET (GM-FET) devices have been tested . Both se+ ion binding upon the cycling behaviors of graphene-based FET sensors. During cyclic tests in which GM-FET sensors were repeatedly exposed to conditions of various pH from 8.25 to 6.55, large Dirac point shifts of 90 mV were observed in the first cycle (Fig. d–Vg characteristics in response to pH became reversible, with corresponding sensitivities of ~7 mV/pH (Fig. Further analysis has been carried out to examine the influence of defect–H+ ions, whereas the increased number of defects at the mesh edges enhanced the pH response of the GM-FETs during the initial cycles. Furthermore, the irreversible response is believed to be caused by the direct adsorption of ions onto defects, which presumably involved strong chemisorption. It has been reported that H+ ions were attached so strongly to the edge defects and have only been detached after high temperature annealing [Gr-FETs have shown results consistent with those of GM-FETs: there was an irreversible portion of the response that gradually decreased with cycling, and only a reversible component remained after five cycles Fig. d–f. The nnealing . Such st+ ions at edge defects, which results in increased sensitivities that sometimes exceeded the Nernst limit. Unfortunately, such direct interactions involve irreversible covalent bonding, and therefore are not preferred in multiple-cycle sensor operations. For graphene mesh pH sensors, this issue has been solved through a simple process of passivating the edge sites. These results suggest how graphene mesh edge defects can improve sensors’ sensitivity and response time and also enable stable multiple-cycle operation through indirect carrier injection. As an example, biological sensors can be constructed by including graphene mesh for enhanced sensing characteristics, while improving stability by attaching appropriate receptors to the graphene edge sites to inhibit direct carrier injection.In this review, we investigated the defects of graphene meshes and their influence upon various sensor applications. Direct growth of graphene mesh by silica-assisted CVD is an excellent way to produce graphene with contamination-free defect sites. When these defects are introduced to Pd-GM gas sensors, the lowered energy barrier at the junctions between Pd nanoparticles and edge defects enhance indirect charge carrier injection into the graphene channel. As a result, the sensitivity and response time of Pd-GM gas sensors have been greatly improved over those of other graphene-based sensors. In contrast, graphene mesh pH sensors respond directly to H


This systematic analysis aims to assess the efficacy of PD-1/PD-L1 blockades compared with non-PD-1/PD-L1 therapy and investigate the potential predictive factors in epithelial carcinoma patients.p < 0.001 for OS and 0.66 , p < 0.001 for PFS. In subgroup analyses, HRs were 0.58 , p < 0.001 in PD-L1 ≥ 1% group, 0.75 , p = 0.001 in PD-L1 < 1% group for OS and 0.59 , p < 0.001 in PD-L1 ≥ 1% group, 0.80 , p = 0.136 in PD-L1 < 1% group for PFS. The p values of pooled HRs for OS in different age, sex and ECOG score groups were less than 0.001. In NSCLC patients, aggregated HRs for OS were 1.40 , p = 0.114 in EGFR mutant group and 0.88 , p = 0.536 in never smokers.A total of 11 trials with 6716 patients of melanoma, non-small cell lung cancer (NSCLC) and renal cell carcinoma (RCC) were included. The pooled HRs (95%CI) were 0.67 , A systematic search from January 2010 to April 2016 was conducted for eligible clinical trials. Based on the data of hazard ratios (HRs) and 95% confidence intervals (CIs) for overall survival (OS) and progression-free survival (PFS), we assessed the pooled HRs and proposed the subgroup analyses.EGFR wild type were associated with extended OS in NSCLC patients.PD-1/PD-L1 blockades prolonged OS and PFS in epithelial carcinoma patients. PD-L1 expression was a predictive factor for PFS but not predictive for OS. Age, sex and ECOG score were excluded to predict any of the efficacy endpoints. Smoking history and  The checkpoint immunotherapy has been increasingly understood and used to unleash the immune system to fight against cancer . These yAs one of the most critical checkpoint immunologic treatments, PD-1/PD-L1 blockade has become a promising focus of immunotherapy in cancer treatment . Two antAlmost each of PD-1/PD-L1 blockades has satisfying overall response rates in treating different types of epithelial carcinoma , 7. HoweEGFR mutation (within NSCLC patients) and BRAF mutation (within melanoma patients) are also conducted to provide further evidence for clinical treatment.In consideration of predictive factors, we analyze different membranous PD-L1 expression levels, because PD-L1 expression of tumor cells is most abundant in epithelial carcinoma and most closely correlated with response to anti-PD-1/PD-L1 agents , 8, 9. BCochrane handbook for systematic reviews of interventions. The study selection process was presented in Figure According to the outlined search strategy, a total of 820 records were obtained, of which 371 duplicates were removed. After screening, 484 articles including reviews, case reports and non randomized controlled trials were excluded. Of the rest 19 records, 8 studies did not report the relevant data. Upon the remaining 11 studies, the two reviewers had the perfect agreement on their eligibility and assessed the quality of included studies independently by the scoring criteria stated in n = 3510), 4 in NSCLC patients (n = 2385), and 1 in RCC patients (n = 821). 3 of the trials were phase 2 trials, 1 was phase 2/3 trial, and 7 were phase 3 trials.The analyses were based on data from a total of 6716 patients enrolled in 11 randomized controlled trials. The experimental treatment drugs of those trials were PD-1/PD-L1 blockades, including nivolumab, pembrolizumab and atezolizumab, while the controlled interventions were standard chemotherapy , targeted therapeutic agents (everolimus) and other form of immunotherapy (ipilimumab). According to currently completed trials focused on epithelial carcinoma, all randomized controlled trials were conducted within melanoma, NSCLC and RCC patients. 6 of the enrolled trials were in melanoma patients  for OS and PFS of patients. For the PD-L1 expression evaluation, the immunohistochemistry assays of PD-L1 employed in the selected studies contained Dako, clone 28-8 (Epitomic) and 22C3 antibody (Merck). We retrieved the corresponding HR estimates with the cut-off of 1%, which meant membranous PD-L1 staining in at least 1% of tumor cells. The information of included studies' authors, cancer types, numbers of patients, interventions, basic characteristics of patients, and HRs for OS and PFS of PD-1/PD-L1 treatment p < 0.001) without significant heterogeneity (I2 < 0.1%), which reflected that compared to non-PD-1/PD-L1 therapy, PD-1/PD-L1 blockades reduced 33% in risk of death among epithelial carcinoma patients. This benefit had met the criteria of treatment superiority.The data available on OS pooling were from 10 observations. The pooled HR for OS Table  was 0.67p < 0.001). However, a considerable heterogeneity with I2 = 79.7 % was observed with the random effect model  for PFS in melanoma, NSCLC and RCC were 0.54 , 0.84 , 0.84  respectively.All studies reported the data on PFS, and the combined HR for PFS with 15 records was 0.66,  in PD-L1 ≥ 1% group, 0.75,  in PD-L1 < 1% group for OS and 0.59,  in PD-L1 ≥ 1% group, 0.80,  in PD-L1 < 1% group for PFS with not important heterogeneity within all subgroups .To investigate predictive role of PD-L1 expression, we computed the aggregated HRs for OS and PFS in different levels of PD-L1 expression  groups and generated the forest plot Figure . Pooled We also analyzed the baseline data such as age, sex and ECOG score of 0 or 1  to further explore other factors. The results of pooled HRs for OS and PFS corresponding to these factors were listed in Table p < 0.001) and non-squamous patients ; p < 0.001) had more extended OS when compared with non-PD-1/PD-L1 therapy. In EGFR wild type patients, the HR for OS was 0.66, ; p < 0.001, indicating the better efficacy of PD-1/PD-L1 blockades in those patients. However, in patients with EGFR mutant type, the HR for OS was 1.40,  and p was 0.114 (I2of heterogeneity = 67.3%), suggesting that anti-PD-1/PD-L1 therapy functioned not distinctly better than the control group treatment. Similarly, the never-smokers were verified not to have the expectedly longer OS with HR pooled as 0.88, ; p = 0.536 with I2 = 39.0%. The HR for OS in smokers was 0.71, ; p < 0.001 with the meaning of better outcomes due to the use of anti-PD-1/PD-L1 agents. Besides, in melanoma patients, both the BRAF wild type and BRAF mutant patients benefited in OS, and HRs (95%CIs) were 0.51, , p < 0.001; 0.55, , p < 0.001 respectively with not important heterogeneity   = 0.67, ; %CI = 0.6All reported data of the meta-analyses results were listed in Table The sensitivity analyses were conducted by excluding studies one by one. The results of those analyses showed no significant differences when compared to the former summary estimates and had excellent stability.p < 0.05) in our main analyses. Nevertheless, two possible biases with p < 0.05 by Egger's test were observed in the subgroups of Age < 65 and Male patients in the pooling process of HR for PFS.We assessed the publication bias of included data for pooled analyses using the Egger's test, and the p values were listed in Table It was acknowledged that PD-1/PD-L1 blockade, as a type of immune checkpoint inhibitors, had remarkable response rate and clinical results in patients with different kinds of cancer, especially those with epithelial-originated malignancies. Nonetheless, it remained unclear whether anti-PD-1/PD-L1 therapy, in contrast with other therapy, functioned better to extend OS and PFS and which subgroups of patients would benefit from the treatment. Our meta-analysis integrating all data from relevant trials were requested to solve the problem. To our knowledge, the meta-analysis was the first study to investigate the outcome and predictive biomarkers for PD-1/PD-L1 therapy in epithelial carcinoma patients within solely randomized controlled trials by assessing the two primary endpoints of OS and PFS.Our results provided convincing evidence that the OS of patients given PD-1/PD-L1 inhibitors was significantly longer than patients given other drugs as those individual studies reported (only one study comparing nivolumab with ipilimumab (CTLA-4 antibody)  excludedIn PD-L1 expression investigation, we found it not practicable to use PD-L1 as a biomarker to predict OS benefit when comparing PD-1/PD-L1 blockades with other control therapy, for both the higher (PD-L1 expression ≥ 1%) and lower (PD-L1 expression < 1%) expression groups could gain obvious clinical benefit from PD-1/PD-L1 blockades. Be that as it may, the higher expression group had the better PFS outcome, but the lower expression group was associated with insubstantial improvement of PFS. The immunohistochemistry (IHC) cut-off value (ranging from 1% to 50%) we chose to define the PD-L1 positivity was 1%, in that the clinical data assessed by this point were the most abundant in the included 11 trials. Besides, cutting off by the lowest expression level enabled to include the most possible patients who could benefit from the therapy. However, the assessment of PD-L1 expression was much complicated because the tumor PD-L1 expression was not constant which was associated with activated tumor antigen-specific T cells  and coulBRAF mutant or BRAF wild type patients in melanoma group, and the squamous cancer patient or non-squamous cancer patients in NSCLC group had gained better outcome of survival. But there was no significant improvement for OS in patients without smoking history and patients with EGFR mutations. Whilst, the present or previous smoker, patients with EGFR wild type showed longer survival time, implicating the smoking history and EGFR wild type might be considered as the potential predictive factors for anti-PD-1/PD-L1 treatment. In our speculation, the association of mutations and other exposures to mutagens like smoking with the efficacy of PD-L1 blockades was possibly because the tumor antigen, considered as the target of T cell activated by checkpoint blockade, was related to the consequence of somatic mutations [The next novel finding of our analyses was in different subgroups. We found that the utations , 21, 22.Finally, OS had been improved in the overall patient regardless of the age, sex and ECOG score. Thus, it was persuasive that those factors were not meaningful indicators for the eligibility of anti-PD-1/PD-L1 treatment.EGFR wild type were potential indicators for prolonged OS in NSCLC patients. There were multiple clinical trials ongoing and many other antibodies targeting PD-1/PD-L1 under early-stage development currently, more comprehensive data from future clinical trials focused on this field were still needed for further investigation.In conclusion, the aggregated HRs for OS and PFS summarized in our systematic analyses revealed that in the comparison of anti-PD-1/PD-L1 agents with other control therapy, the PD-L1 expression was not an appropriate factor to predict the benefit of OS in epithelial carcinoma patients, but could be predictive for PFS. Age, sex and ECOG score were excluded to predict any of the outcome endpoints. Smoking history and We searched for the articles of clinical trials from PubMed, Embase, Web of Science, and the Cochrane Library from January 2010 to April 2016. Records at American Society of Clinical Oncology (ASCO), the European Society for Medical Oncology (ESMO), the world Conference of Lung cancer (WCLC) were also reviewed. The following search terms were used: “pembrolizumab”, “Nivolumab” “atezolizumab”, “Tremelimumab”, “AMP-224”, “MDX-1105”, “pidilizumab”, and “cancer/carcinoma”.All relevant articles underwent evaluation for eligibility by two investigators independently and we selected the articles according to the following criteria: 1) articles with randomized controlled trials (RCTs); 2) at least one of the two endpoints  reported; 3) published in English; 4) the full text available. Our exclusion criteria were as below: 1) letters, expert opinions, case reports and reviews; 2) articles without available data; 3) duplicate publications.Cochrane Handbook for Systematic Reviews of Interventions , and evaluated the random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, selective reporting and other bias to ensure the low-risk of bias of the studies included.We assessed the quality of involved randomized controlled clinical trials according to the criteria presented in the The data of study identification, the intervention of experimental treatment and control group, numbers of enrolled patients in each trial, patients' detailed information , hazard ratios(HR) with their 95%CIs and p values for OS and PFS were extracted by two individual investigators independently. We also collected the relevant information in every subgroup we set to render sufficient data to our subgroup analyses.2 statistic were used to indicate the heterogeneity. A p value less than 0.05 in the Q test or an I2 value greater than 50% in the I2 statistics suggested the significant heterogeneity. If the heterogeneity was significant, we used the random effect model of pooling instead of fixed effect model and designed subgroups analyses to clarify the between-study heterogeneity. To test the publication bias of the included studies, the Egger's test was chosen. All the statistical analyses were performed with STATA/SE software version12.0 .We calculated pooled HRs and their 95%CIs for OS and PFS which were considered to be the primary outcome of the meta-analyses and generated the forest plots accordingly. The chi-square Q test and I


SQSTM1) and valosin-containing protein (VCP) are 2 key genes associated with several neurodegenerative disorders but have yet to be thoroughly investigated in sIBM. A candidate gene analysis was conducted using whole-exome sequencing data from 181 sIBM patients, and whole-transcriptome expression analysis was performed in patients with genetic variants of interest. We identified 6 rare missense variants in the SQSTM1 and VCP in 7 sIBM patients (4.0%). Two variants, the SQSTM1 p.G194R and the VCP p.R159C, were significantly overrepresented in this sIBM cohort compared with controls. Five of these variants had been previously reported in patients with degenerative diseases. The messenger RNA levels of major histocompatibility complex genes were upregulated, this elevation being more pronounced in SQSTM1 patient group. We report for the first time potentially pathogenic SQSTM1 variants and expand the spectrum of VCP variants in sIBM. These data suggest that defects in neurodegenerative pathways may confer genetic susceptibility to sIBM and reinforce the mechanistic overlap in these neurodegenerative disorders.Genetic factors have been suggested to be involved in the pathogenesis of sporadic inclusion body myositis (sIBM). Sequestosome 1 ( Muscle SQSTM1 had not been investigated in sIBM until a recent study using targeted next-generation sequencing in a group of 79 patients  (VCP) gene are known to cause an inherited form of IBM with Paget disease and frontotemporal dementia (IBMPFD) , has been recognized as a strong biomarker in muscle with a high sensitivity and specificity for sIBM . Geneticpatients . In thatmyopathy . In addiia (FTD) . Mutatio(IBMPFD)  and have(IBMPFD) . Two misdementia .SQSTM1 and VCP genes in sIBM, we investigated these 2 genes using whole-exome sequencing data from 181 sIBM patients, which was produced as a part of an International IBM Genetics Consortium.These findings, along with denervation in muscle electromyography of sIBM patients, suggest a possible genetic overlap between sporadic and IBM-like myopathies and also neurodegenerative diseases. To thoroughly investigate the contribution of 22.1N = 235) aged >60 years were used as an internal aged control group to compare with our sIBM cohort. The study was approved by the National Research Ethics Service Committee London—Queen Square (research ethics committee reference: 12/LO/1557).This study is part of the International IBM Genetics Consortium, a Muscle Study Group–endorsed project, which currently has members from 17 specialized centers in 7 countries around the world. For this whole-exome sequencing Consortium study, DNA samples from a total number of 181 sIBM patients were collected from 11 centers. Patients diagnosed with sIBM had to have an sIBM diagnosis according to a muscle diseases expert and also had to fulfil the Griggs criteria , the Eur2.2SQSTM1 and VCP genes. We excluded all synonymous variants, and all common variants with a population frequency >1% were identified in the 1000 Genomes project (www.1000genomes.org/), in the Exome Variant Server (EVS) database (evs.gs.washington.edu/EVS/), in the Exome Aggregation Consortium (ExAC) Browser (exac.broadinstitute.org/), and in the internal aged controls, as these variants less likely play a role in a rare disease. The filtered variants were confirmed by the conventional Sanger sequencing. The allele frequency of each variant found in sIBM was compared with the ExAC database using Fisher test. The pathogenicity of these variants was evaluated using the following in silico prediction tools: SIFT , and the concentration, purity, and integrity of each RNA sample were assessed as previously described , 2 patients with VCP variants (cases 6 and 7), and 5 age- and gender-matched controls. Raw expression data were log2 transformed and quantile normalized, and differential expression analysis  was performed using the limma Bioconductor package  pathways, and Human Phenotype Ontology terms was performed using g:Profiler (biit.cs.ut.ee/gprofiler/). Among the upregulated genes, 3 genes associated with inflammation markers  were selected for validation using real-time quantitative polymerase chain reaction (RT-qPCR). Briefly, total RNA (600 ng) from the 6 cases and 8 controls was reverse transcribed into complementary DNA using random primers from High-Capacity cDNA Reverse Transcription Kit . Three replicates per sample were assayed for each target gene using Fast SYBR Green PCR Kit (Applied Biosystems) and run in a QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems). Details on primers and RT-qPCR conditions are available on request. Cyclophilin (PPIA) was selected as the reference gene. The 2−ΔΔCt method .Whole-genome expression profiling was performed using the Illumina HumanHT-12 v4 Expression BeadChip  on 3 patients with  package . Genes wt method  was used3From the entire cohort of 181 sIBM patients, 150 (82.9%) were Caucasians and 16 (8.8%) were from other ethnicities, including Asian Chinese, the Indian subcontinent, and Black Africans (ethnicity information was unavailable for 15 patients). The majority of sIBM patients were male (65.7%). Age of onset, which was collected retrospectively, ranged from 31 to 85 years (mean 59.6 ± 9.6 years). The mean age of the 235 healthy aged controls was 79.1 ± 8.5 years, ranging from 60 to 102 years, and similarly to the sIBM cohort, the majority were also male (61.7%).SQSTM1 gene (ENST00000389805) were found in 4 patients (VCP gene (ENST00000358901) were found in 3 sIBM patients. Of note, the frequency of the variants SQSTM1 p.G194R and VCP p.R159C was significantly higher in our sIBM cohort compared with the ExAC database . These 2 variants were absent in the other population databases and in our aged control group. From the 6 rare variant we found, 4  from patients with VCP variants prevented statistical analysis of this sIBM patient group. The expression of SQSTM1 and VCP did not show significant differences between any patient group and controls, but a significant upregulation of MHC genes (class I [HLA-A] and class II [CD74 and HLA-DRA]) was seen in the group of patients carrying SQSTM1 variants , which is a multifunction protein participating in a number of different biological pathways  and II (HLA-DRA) genes were significantly upregulated in patients compared with controls by RT-qPCR, which is consistent with a previous study (CD74) was significantly upregulated only in the SQSTM1 patient group. The statistical analysis could not be carried out for the VCP patient group because of small sample size, but there was also a trend for the upregulation of these inflammation markers. The more pronounced upregulation observed in the SQSTM1 sIBM patient group than in the VCP patient group. This is the first time that different expression level of inflammation markers between patients with sIBM is suggested. Although further cases should be analyzed, the MHC expression could be a potential differentiating factor that directs the clinical phenotype of SQSTM1 or VCP toward sIBM as opposed to the other neurodegenerative conditions. Additional analysis in muscle and other tissues of patients carrying SQSTM1 or VCP mutations without symptomatic sIBM but other phenotypes is also necessary in the future to confirm this hypothesis.The MHC class I (us study , and theSQSTM1 variants and expand the spectrum of VCP variants in sIBM. Our findings suggest that variants in these genes constitute genetic susceptibility factors for sIBM and for other multisystem proteinopathy phenotypes. The findings from this study also expand the clinicopathologic spectrum of diseases associated with SQSTM1 and VCP genes, and the overlap between sIBM and IBMPFD, ALS, and/or FTD suggests that muscle and brain diseases share similar pathogenic pathways that may be important for further biomarkers, genes, and therapeutic target discovery. Further investigation of the sIBM whole-exome sequencing data is still ongoing and data from this international collaboration will likely reveal further findings.In conclusion, we report for the first time likely pathogenic Declaration of interests: All authors have no competing financial interests. Authors' contributions: Qiang Gang contributed to sample collection, all the experimental work, data analysis, and drafting the first version of the manuscript; Conceição Bettencourt contributed to the experimental plans of microarray and RT-qPCR, preliminary analysis of microarray data, and statistical plan; Pedro M. Machado contributed to overall coordination of the study, namely worldwide sample collection and liaising with all the study collaborators; Conceição Bettencourt, Pedro M. Machado, and Henry Houlden also contributed to drafting the first version of the manuscript; Janice L. Holton contributed to the review and photography of the muscle biopsies; Alan M. Pittman and Deborah Hughes contributed to the generation of whole-exome sequencing data; Stefen Brady, Janice L. Holton, and Boel De Paepe contributed to immunotyping of muscle biopsies; and Andrew B. Singleton contributed to the exome data from control individuals. All the authors from International IBM Genetic Consortium and Muscle Study Group contributed to acquisition of clinical data and sample collection. Michael G. Hanna, Henry Houlden, and Pedro M. Machado are principal investigators of the International IBM Consortium Genetics Study. All the authors contributed to the critical revision of the manuscript and approved the final version.


Radiation therapy is critical for the control of many tumors and lung is an important dose-limiting organ that impacts radiation dose prescribed to avoid irreversible pulmonary fibrosis in cancer survivors. Idiopathic pulmonary fibrosis (IPF) is a chronic, irreversible lung disease caused by aberrantly activated lung (myo)fibroblasts. The presence of pro-fibrotic, apoptosis-resistant fibroblasts in IPF promotes progressive fibrosis and may have a role in other diseases, if these resistant cells are selected for as a consequence of treatment. However, the pathological response of IPF fibroblasts to radiation compared to non-IPF lung fibroblasts is not known. To address this, we examined fibroblast viability following radiation in lung fibroblasts from IPF and non-IPF patients and the underlying mechanism that protects IPF fibroblasts from radiation-induced death. IPF fibroblasts are significantly more resistant to apoptosis compared to non-IPF lung fibroblasts, suggesting that resistance to radiation-induced cell death is a predominant mechanism leading to lung fibrosis. Analysis of γH2AX induction demonstrated that radiation-induced DNA damage is reduced in IPF fibroblasts and correlates to the activation of the transcription factor forkhead box M1 (FoxM1) and subsequent upregulation of DNA repair proteins RAD51 and BRCA2. FoxM1 activation occurs secondary to FoxO3a suppression in IPF fibroblasts while restoration of FoxO3a function sensitizes IPF fibroblasts to radiation-induced cell death and downregulates FoxM1, RAD51, and BRCA2. Our findings support that increased FoxO3a/FoxM1-dependent DNA repair may be integral to the preservation of death-resistant fibrotic fibroblasts after radiation and that selective targeting of radioresistant fibroblasts may mitigate fibrosis. Rates of pulmonary fibrosis can be as high as 70–80% in high dose regions of irradiated lung3. It is currently unclear whether radiation-induced lung fibrosis (RILF) results from failure of normal healing after pneumonitis or is a separate, complicating event5. Thus, it is difficult to predict the true risk of RILF, for which there are no effective treatment strategies7. Recent work in idiopathic pulmonary fibrosis (IPF), a progressive and lethal lung disease, showed that fibroblasts derived from IPF patients maintain an apoptosis-resistant phenotype in response to various stressors14. Elucidation of this mechanism is crucial in understanding fibrotic disease, regardless of the inciting cause.Radiation therapy is prescribed in over 50% of patients receiving cancer treatment. Radiation-induced toxicities are relatively common following radiation when normal lung is in close proximity to tumor. While pneumonitis is an early and potentially reversible toxicity, pulmonary fibrosis is delayed, progressive and can impair normal lung function15. Fibroblasts become activated and produce collagen-rich extracellular matrix during repair of basement membranes15. Contrary to normal healing, thoracic radiation inappropriately activates myofibroblasts, which promote the deposition of type I collagen that destroys parenchyma and establishes a niche for ongoing fibrosis17. To further compound this injury, alveolar epithelial cells may undergo trans-differentiation into myofibroblasts in IPF and RILF18. The most lethal event following radiation to non-hematopoietic cells is the induction of DNA double-strand breaks (DSB), which induces mitotic catastrophe and apoptosis after 2–6 days23. Under normal physiologic conditions, DNA DSB trigger a cascade of events that encourage repair at the site of DNA damage24. Homologous recombination (HR) repair following DNA DSB is a primary, high-fidelity mechanism of radiation repair in human cells. An important step in HR is recruitment of the repair protein RAD51 by breast cancer-associated gene 2 (BRCA2) to the damaged DNA sites; the alteration of these proteins renders cells resistant to cytotoxic damage26. FoxM1, a member of the Forkhead family of transcription factors, is known to upregulate DNA repair proteins such as RAD51 and BRCA2, thereby protecting cells from radiation-induced DNA damage28. FoxM1 was increased in irradiated murine lung tissue and in human IPF fibrotic lesions; moreover, the conditional deletion of FoxM1 prevented lung fibrosis29. FoxM1 activation occurs following suppression of FoxO3a, indicating a negative feedback loop exists between these two family members30, and FoxO3a is aberrantly suppressed in IPF fibroblasts and patient IPF lung tissues32. We therefore sought to examine the FoxO3a/FoxM1-dependent pathway in IPF cells in response to ionizing radiation. We found that IPF fibroblasts sustain less radiation-induced DNA damage compared to non-IPF lung fibroblasts and are highly viable after radiation. We further demonstrated that enhanced FoxM1 in IPF fibroblasts leads to increased RAD51 and BRCA2 gene expression. Following FoxO3a over-expression or FoxM1 silencing, RAD51 and BRCA2 expression is abrogated and sensitizes IPF fibroblasts to radiation-induced death. We propose that FoxM1 may be pivotal in selecting apoptosis-resistant fibrotic fibroblasts within irradiated lung tissue, contributing to the development and progression of RILF. FoxM1 may therefore represent a therapeutic target as it regulates fibroblast survival following radiation damage.Ionizing radiation initially injures pulmonary epithelial cells, releasing pro-inflammatory cytokines that recruit inflammatory cellsAs radiation is a risk factor for organ fibrosis, irradiated fibrotic fibroblasts likely also acquire pathological properties and become resistant to death. To test this, radiation dose-response viability was measured in IPF and control lung fibroblasts in the presence or absence of collagen matrix. There was no significant difference between IPF and control fibroblasts when cells were cultured on a tissue culture plate Fig. . However34. We measured γH2AX as an indirect indicator of DNA DSB, which correlates to radiation-induced cell death36. Radiation upregulated γH2AX expression in both IPF and control fibroblasts at 1 h  molecules form around the break site to encourage accumulation of DNA repair proteins1 h Fig. , left, h1 h Fig. , right. 1 h Fig. . To furt1 h Fig. , we trea1 h Fig. , left. E1 h Fig. , right. 29. FoxM1 regulates the expression of several DNA repair proteins including RAD51 and BRCA2, which are important for proper DNA DSB repair28. We therefore examined the role of FoxM1 on the expression of RAD51 and BRCA2 in irradiated IPF fibroblasts. Increased FoxM1 levels were found in IPF fibroblasts between 1 and 6 h compared to FoxM1 levels of irradiated control fibroblasts . The tissue samples were stripped of all identifiers and designated as waste (exemption 4). Written informed consent was obtained on all patients prior to the procedure being performed. Use of human lung tissues was approved by the Institutional Review Board (IRB) at the University of Minnesota. The diagnosis of IPF was supported by history, physical examination, pulmonary function tests, and typical high-resolution chest computed tomographic findings of IPF. In all cases, the diagnosis of IPF was confirmed by microscopic analysis of lung tissue that demonstrated the characteristic morphological findings of interstitial pneumonia52. Removed lung tissues were chopped to 5 mm3 and cultured in Dulbecco’s modified Eagle’s medium  supplemented with 20% fetal calf serum  and 2% antibiotics for 4–5 weeks at 37 °C in a 5% CO2 humidified incubator to prepare IPF and non-IPF (control) lung fibroblasts. They were used at passage numbers 3–9 for all experiments.Lung tissues removed at the time of transplantation or death from non-IPF and IPF patients , 10% 10× DMEM (MilliporeSigma) and 10% 1×DMEM, and the pH was adjusted to 7.2 with 0.1 M NaOH. Cell culture dishes or 96-well plates were coated with collagen and incubated for a minimum of 2 h at 37 °C prior to use. Following 24 h, serum-free DMEM (SF DMEM) was used for experiments. Human Bronchial Epithelial Cell3-KT (HBEC3-KT) cell line was obtained from American Type Culture Collection  and cultured in airway epithelial cell basal medium (ATCC) supplemented with 1% antibiotics and bronchial epithelial cell growth kit (ATCC) at 37 °C.Radiation was delivered using 6 MV photons from a clinical linear accelerator  routinely calibrated and maintained to clinical standards. Cells were treated with increasing radiation doses (0–15 Gy). Culture plates were placed on 1.5 cm of tissue equivalent material and the gantry rotated to 180 degrees to ensure electronic equilibrium at the level of cells. Radiation was prescribed using a single source-to-surface distance technique.n = 8) and IPF fibroblasts (n = 8) were cultured on collagen-coated or non-collagen-coated 96-well plates in SF DMEM prior to radiation or bleomycin . Cells were then incubated for 3 h with 20 μL CellTiter Blue reagent  to assess viability or with 20 μL CellTiter 96 Aqueous One Solution (Promega) to assess proliferation for 3 h. Cell viability and proliferation were measured at 560 nm (excitation)/590 nm (emission) of fluorescence and at 490 nm of absorbance using a 96-well plate reader , respectively.Randomly selected non-IPF control lung fibroblasts (Deoxynucleotidyltransferase (TdT)-mediated dUTP nick-end labeling (TUNEL) assay was conducted using In Situ Cell Death Detection kit  according to manufacturer’s instruction. Briefly, 4 of each control and IPF fibroblasts plated on collagen matrix coated cover slips  in SF DMEM were irradiated with 15 Gy and cells were then incubated for an additional 24 h. Control cells showing low cell viability and IPF cells showing high cell viability in response to the radiation were chosen for the current assay. After the incubation, cells on cover slips were washed with phosphate buffered saline  and fixed with 4% paraformaldehyde solution  at 4 °C over night. Cells on cover slips were then permeabilized with Proteinase K  at room temperature for 20 min. After the washing with PBS, cells were incubated with TUNEL reaction mixture at 37 °C for 2 h and washed with PBS twice. In addition, the fixed control cells on cover slips was incubated with Nuclease  at 37 °C for 25 min before incubating in TUNEL reaction mixture to be used as a positive control. For fluorescence microscopic analysis, the cover slips were washed with PBS and sequentially moved on slide glasses having mounting solution with DAPI . The images were taken from two different areas on each cover slip using a confocal fluorescence microscopy  at ×200 magnification, and the images were processed using Progres Capture Pro V2.8.8 software . Images analysis was conducted using ImageJ program .n = 8, each) used in cell viability assay (6 × 105 cells/60 mm cell culture dish) were irradiated with  9 Gy and incubated for the assigned time periods. After the incubation, cells were lysed with 1×cell lysis buffer  containing protease inhibitor (Roche Applied Science) and phosphatase inhibitor . Cell lysates were collected, sonicated on ice for 15 s, and then denatured using 5×Laemmli buffer at 95℃ for 10 min. The lysates were separated by a gradient polyacrylamide gels having 5–20% polyacrylamide concentration and proteins on the gel were electrically transferred to a PVDF membrane  using a Protean III tank transfer system (Bio-Rad). After blocking step using 5% skim milk in TTBS  for 1 h at room temperature, the membrane was incubated with γH2AX , H2AX , FoxM1 , FoxO3a , RAD51 , BRCA2 , GAPDH , Lamin A/C  or β-actin  antibody diluted in TTBS containing 5% bovine serum albumin (BSA) at 4℃ for 24–48 h. After washing with TTBS three times, membranes were incubated with anti-rabbit or mouse IgG antibody conjugated with horseradish peroxidase (MilliporeSigma) diluted in TTBS containing 5% skim milk for 1 h at room temperature. The protein bands on a membrane were detected by ECL solution (ThermoFisher Scientific) using Chemi Doc-IT2 image analyzer , and quantified using VisionWorks LS program (UVP BioImage systems).Same control and IPF fibroblasts (n = 8) and IPF (n = 8) fibroblasts using NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (ThermoFisher Scientific) according to the manufacturer’s instructions. Briefly, control and IPF fibroblasts (1.6 × 106 cells/10 cm cell culture dish) cultured on collagen in SF DMEM were incubated for 6 h after 9 Gy radiation. Cells were lysed with Cytoplasmic Extraction Reagent I and II. Cytosolic fraction was collected by centrifugation at 16,000×g for 5 min and the remaining pellet was lysed with Nuclear Extraction Reagent. Nuclear fraction was then isolated by  centrifugation at 16,000×g for 10 min. Both cytosolic and nuclear fractions were denatured with 5×Laemmli buffer at 95 °C for 10 min, and FoxM1 and FoxO3a protein expression was measured using Western analysis. Lamin A/C and GAPDH were used as a nuclear and cytosolic protein marker, respectively.Nuclear protein fractions were isolated from control  cultured (3 × 105 cells/35 mm cell culture dish) on collagen was extracted at specified time points following 9 Gy radiation using TRIGent . One µg of total RNA was synthesized into complementary DNA, and PCR was conducted using Step One Plus real-time PCR system  and Power SYBR Green PCR master mix (Applied Biosystems) under the following conditions: initial denaturation at 95 °C for 10 min then amplification by cycling 45 times at 95 °C for 15 s, 55 °C for 30 s, then 68 °C for 45 s. GAPDH expression was examined to normalize the copy numbers of each gene. Primer Sequences for PCR: FoxO3a , FoxM1 , RAD51 , BRCA2  and GAPDH .Total RNA from control or IPF fibroblasts (n = 4) were transfected with FoxM1, BRCA2, RAD51, or control siRNA  using Lipofectamin RNAiMAX (Life technologies). For overexpression of FoxM1, control fibroblasts (n = 4) were transfected with 2 µg empty pCMV3 or FoxM1-pCMV3 vector  using Lipofectamin 3000 (Life technologies) in Opti-MEM . For FoxO3a overexpression, IPF fibroblasts (n = 4) were infected with either 2 × 106 PFU of ad-GFP-FOXO3a  or Ad-GFP  according to the manufacturer’s instructions. For co-overexpression experiments, IPF fibroblasts (n = 4) infected with ad-GFP-FOXO3a were transfected with FoxM1-pCMV3 vector as described above. Cells were then cultured on collagen in SF DMEM for 24 h followed by irradiation (9 Gy). FoxM1 overexpression or silencing by siRNA was verified by measuring FoxM1 mRNA  cultured on collagen in SF DMEM were irradiated at 9 Gy followed by incubation for an additional 6 h at 37 °C. Cells were then fixed with 4.0% paraformaldehyde in PBS and permeabilized in 0.1% Triton-X (MilliporeSigma) in PBS for 15 min. The cells were incubated with the anti-RAD51 antibody  diluted 1:300 in PBS at 4 °C for 24 h. Cells were then incubated with an anti-rabbit Alexa Fluor 488 conjugated secondary antibody  diluted 1:250 in PBS for 20 min. Cells on collagen matrix were cover slipped with ProLong Gold Antifade Mountant with DAPI (ThermoFisher Scientific). From each sample, 3 of ×20 magnified images were analyzed using an upright microscope . Images analysis was conducted using ImageJ (NIH).Control and IPF fibroblasts , showing lowest, lower quartile, median, upper quartile and highest expression levels. Two-dimensional column graphs were prepared with Microsoft Excel. Comparison between unirradiated and irradiated fibroblasts or comparison between irradiated IPF and control fibroblasts as a function of time was conducted using two-tailed Student’s Supplementary Figure 1Supplementary Figure 2Supplementary Figure 3Supplementary Figure 4Supplementary Figure 5Supplementary figure legends