Pteropus vampyrus, and found a pattern of periodic L1 expansion and quiescence. In contrast to the well-characterized L1s in human and mouse, megabat genomes have accommodated two or more simultaneously active L1 families throughout their evolutionary history, and major peaks of L1 deposition into the genome always involved multiple families. We compared the consensus sequences of the two major megabat L1 families at the time of their extinction to consensus L1s of a variety of mammalian species. Megabat L1s are comparable to the other mammalian L1s in terms of adenosine content and conserved amino acids in the open reading frames (ORFs). However, the intergenic region (IGR) of the reconstructed element from the more active family is dramatically longer than the IGR of well-characterized human and mouse L1s. We synthesized the reconstructed element from this L1 family and tested the ability of its components to support retrotransposition in a tissue culture assay. Both ORFs are capable of supporting retrotransposition, while the IGR is inhibitory to retrotransposition, especially when combined with either of the reconstructed ORFs. We dissected the inhibitory effect of the IGR by testing truncated and shuffled versions and found that length is a key factor, but not the only one affecting inhibition of retrotransposition. Although the IGR is inhibitory to retrotransposition, this inhibition does not account for the extinction of L1s in megabats. Overall, the evolution of the L1 sequence or the quiescence of L1 is unlikely the reason of L1 extinction.Although L1 sequences are present in the genomes of all placental mammals and marsupials examined to date, their activity was lost in the megabat family, Pteropodidae, ∼24 million years ago. To examine the characteristics of L1s prior to their extinction, we analyzed the evolutionary history of L1s in the genome of a megabat,  Most of a typical mammalian genome is occupied by transposable elements, which have played an important role in shaping these genomes, and L1s account for approximately half of this transposable element load. Mammals have evolved several mechanisms to control L1 retrotransposition, and yet L1s remain active in almost all mammalian lineages. However, L1s were found to have gone extinct in the megabat family ∼24 million years ago. We were able to trace megabat L1s to the ancestral L1 families shared by all mammals as well as identify bat-specific L1 families. Unlike most well-characterized mammals which have a single active L1 lineage, multiple L1 lineages have persisted in megabats throughout their evolutionary history. When the L1 extinction occurred in megabats, two active lineages lost their ability to retrotranspose almost simultaneously after a burst of activity. We synthesized the L1 from the most active family at the time of extinction and found a long intergenic spacer between its two protein coding genes. Tissue culture assays of the reconstructed megabat L1 revealed that both genes supported retrotransposition, but that the spacer is inhibitory. Despite the inhibition, this family accounted for 18% of the L1s detected in the megabat genome. Functional L1s are 6,000–7,000 bp long and made up of a 5′ untranslated region (5′UTR), two non-overlapping open reading frames (ORFs) known as ORF1 and ORF2, an intergenic region (IGR) usually less than 100 bp and a 3′UTR followed by a poly-adenosine sequence There is considerable evidence that transposable elements, including L1s, have significant effects on the genome. L1 retrotransposition is one of the major sources of mutagenesis and genome instability L1s have been coevolving with their mammalian host genomes since before the eutherians and metatherians diverged One possible reason for this unusual pattern of L1 evolution is that L1s are epigenetically silenced Pteropus vampyrus, the first publicly available genome trace files of the megabat family. At the time of L1 extinction, P. vampyrus contained two active L1 lineages. We determined that these lineages likely diverged before the origin of bats. We reconstructed the master element of the more active lineage at the time of L1 extinction and compared its structure to other active L1s, noting particularly that the IGR between the two ORFs is dramatically longer than that of the well-characterized L1s of human and mouse. Finally, we created chimeric L1s between the reconstructed megabat L1 and a human L1 to test the ability of the extinct megabat L1 to support retrotransposition in tissue culture and we manipulated the IGR to explore its effect on retrotransposition.In this study, the evolutionary history of L1s prior to their extinction in megabats was explored by data-mining the unassembled genome of subfamilies. Closely related subfamilies are grouped into families that represent a window of L1 deposition into the genome. These families replace each other sequentially within a clade to form a lineage.To be clear about nomenclature used in this paper, we refer to clades of closely related L1s identified by shared, co-segregating sites as To investigate the history of L1 retrotransposition in the megabats, we identified subfamilies using COSEG in RepeatMasker et al.Previous work indicated that two major lineages of L1 were active at the time of L1 extinction in megabats P. vampyrus genome (Baylor College of Medicine) and assigned them to one of the subfamilies described above based on sequence similarity. The age of each sequence was approximated by its percent identity to the subfamily consensus – the higher the percent identity, the younger the sequence. Subfamilies were combined into their designated families as determined by phylogenetic analysis (described above) and the age distribution was determined for each family. Taking all families together, we observed periodic fluctuations in the number of L1s fixed in the genome . However, the mean of each family is 1–2% larger than the peak, indicating that the variance of the distribution is higher than that of a Poisson distribution. This increased variance could be due to sequence differences between active L1s in the same subfamily at the time of transposition, a wave of retrotransposition over an extended period of time, errors introduced during L1 retrotransposition, technical problems with the analysis, or some combination of these. Technical issues might include false detection by RepeatMasker, incorrect assignment of some elements to their lineage or combining small lineages with larger ones, for example. Interestingly, the highest copy number peak is for family 1A, one of the two youngest detectable lineages active just prior to L1 extinction. This peak accounts for 18% of the total L1s detected in the megabat genome.The dynamics of families within lineages 1 and 2 are not perfectly consistent with short bursts of retrotransposition followed by long periods of quiescence. Given the evolutionary pattern of L1 as master lineages, most L1 sequences evolve neutrally after their insertion into the genome. Therefore, the distribution of mutations in elements inserted at the same time should follow a Poisson distribution  of the more active L1 lineage in megabats at the time of L1 extinction, synthesize it and test its activity in a tissue culture assay. It was not possible to reconstruct the less active lineage with confidence because the copy number, especially in the 5′ end, is too low. Since the extinction of megabat L1 retrotransposition happened in the common ancestor of the family, the retrotransposition history of L1 in P. vampyrus genome using a consensus-based method, with curated correction of CpG sites. We performed this reconstruction independently, without reference to RepBase http://www.girinst.org/2010/vol10/issue3/L1-2_PVa.html) at the nucleotide level, with six differences (two in ORF1 and four in ORF2) at the amino acid level. The amino acid differences were examined individually in the original alignments: three resulted from ambiguous nucleotides or frame shifts in the RepBase reconstruction, one from CpG site correction and two from variable sites which we called differently than RepBase. None of these differences were at sites of conserved amino acids (see below). Note that although RepBase designation L1-2_PVa suggests that this sequence falls within lineage 2, we follow the precedence of Cantrell et al.Reconstruction was conducted on the Oryzomys palustris. As noted in the We compared the reconstructed L1 to the most recently active consensus sequences from 31 diverse mammalian species  and S2. We investigated the adenosine content of the reconstructed terminal members of megabat lineages 1 and 2 and 31 additional L1 consensus sequences from the mammalian species listed in To our surprise, the length of the megabat L1 IGR set it apart from the well-characterized L1s of rodents and primates. The IGR lengths of the surveyed L1 sequences from 31 species are listed in et al.To ask whether the reconstructed megabat L1 is capable of supporting retrotransposition, we synthesized it and assessed its activity in a retrotransposition rate assay derived from the work of Moran et al.neo cassette  reduces the retrotransposition rate ∼26-fold, while introducing the human L1 IGR into the reconstructed megabat L1 (BBB→BHB) increases the retrotransposition rate ∼40-fold . In a miTo further investigate the inhibitory effect of the reconstructed megabat IGR on retrotransposition and its interaction with the L1 ORFs, we manipulated the megabat IGR and tested variants in the chimeric L1 context. Manipulation of the IGR included truncated versions of the full-length IGR, a shuffled version with the same nucleotide composition (GenBank accession number KF796624) and an IGR with the sense-oriented AUG codons in all three reading frames mutated to AGU. We tested these variant IGRs in all four ORF contexts . We found that while the absolute level of transposition was affected by whether human or megabats ORFs were framing the IGR, the relative decrease in retrotransposition was comparable in all ORF contexts. Therefore, the effect of the manipulated IGR on retrotransposition is shown only in the human L1rp context, HXH, in To determine whether the inhibitory property of the megabat IGR is due solely to its length, we truncated one-third or two-thirds of the IGR from either the 5′ end, the 3′ end or both . All theAlthough the megabat L1 IGR is inhibitory to retrotransposition compared to its human counterpart, we would expect to see that at this length, the reconstructed IGR still supports retrotransposition better than a randomized version with the same nucleotide composition. The randomized IGR with the same nucleotide composition reduces the retrotransposition rate ∼8.8-fold . Either of these scenarios could be successful in the evolutionary sense as mechanisms to avoid competition while still resulting in insertions that can be inherited by the next host generation. It is also possible that the L1 regulation mechanisms of the host are specific towards a certain lineage. Under that scenario, one lineage could dominate while the other is relatively quiescent, and eventually the second lineage could escape control and the first lineage be silenced. In other words, there would be no reason to expect that lineages would have the same peaks of increased retrotransposition. The fact that distinct lineages experienced fairly synchronized periods of activity and quiescence could suggest global rather than lineage-specific regulation of L1 retrotransposition. Peaks of L1 copy number are generally assumed to indicate transpositional bursts attributable to L1 activity, but other factors might account for peaks of L1 fixation in the genome. For example, host population bottlenecks could account for an increase in the rate of L1 fixation in the genome if there is selection against L1 Where multiple lineages are maintained, it is possible that they are specialized on different tissue types  is also slightly above the average level (29.5%) of the species surveyed . The A-ret al.et al.et al.et al.The most unexpected feature of the reconstructed megabat L1 is its long IGR. Alisch de novo insertion, as a backbone et al.To determine whether the reconstructed megabat lineage 1 element was active, we made chimeric sequences using human L1rp, a highly active Although exchanging the L1rp ORFs with either or both of the corresponding megabat counterparts lowers the retrotransposition rate considerably, the activity of chimeric L1s is comparable to the majority of full-length human L1s. The retrotransposition rate of chimeric constructs containing megabat ORFs is much lower than the retrotransposition rate of the most active ‘hot’ L1s, but more active than 82% of full-length L1s in the human reference genome et al.There are some caveats relevant to this comparison. First, the retrotransposition assays of Brouha et al.Demonstrating activity of a reconstructed element in a tissue culture assay is the ultimate test of the quality of the reconstruction. To our knowledge, this is the first L1 element from a species that does not carry currently active L1s to be resurrected and tested for activity. However, ancestral L1s have been extensively reconstructed et al.et al.RepBase have been actively reconstructing and hosting reconstructed ancestral transposable elements since its establishment et al.et al.et al.et al.The most striking feature of the reconstructed megabat L1 is the long IGR, which is is co-adapted with the ORFs to support retrotransposition. This is most evident in the comparison of the randomized IGR with the intact version , where rPerhaps the most difficult aspect to reconcile about the long IGR in lineage 1 is its evolutionary persistence. An active element that deleted this long IGR would be expected to dramatically increase its retrotransposition rate and, thus, to dominate future retrotransposition. That is to say, there should have been strong selection favoring the deletion of the IGR. One might expect such a deletion to be ‘easy’ from an evolutionary perspective since it need not maintain a reading frame, and yet this did not happen.The tempo of L1 retrotransposition in megabats directly preceding L1 extinction is also noteworthy. A significant burst of retrotransposition occurred just prior to L1 extinction in megabats, contributing 25% of the detectable L1s to the genome. Family 1A accounts for the bulk of this activity – 18% of the total detectable elements in the genome – despite the demonstrated inhibitory effect of the long intergenic spacer on this family. The IGR has a long evolutionary history in this L1 lineage and likely preceded the evolution of megabats. Thus, despite its inhibitory effect on retrotransposition, it is unlikely that it contributed to L1 extinction.There are some characteristics of bat genomes that make them unique among the mammals. Bats, and especially megabats, have much smaller genomes than other mammals Since transposable elements are the major contributor to mammalian genome size P. vampyrus  to find detectable sequences with >60% identity and >90% coverage of the query. Using 2000 random sequences from the CENSOR run, subfamilies were identified based on shared sequence variants (co-segregating mutations) with COSEG 0.2.1 (http://www.repeatmasker.org/COSEGDownload.html) P. vampyrus genome. All identified L1 sequences from the second CENSOR run were used for a second round of COSEG, which required the additional parameter of at least 250 sequences to form a subfamily. Consensuses of the 64 subfamilies thus generated were used as query sequences to run CENSOR for a third time. Each hit's percent identity to the corresponding query was used to assign it to a L1 subfamily, and the copy numbers in each subfamily were counted. Seven subfamilies containing less than 250 sequences were removed. Consensuses from each of the remaining 57 subfamilies were used as query sequences to run CENSOR for a fourth time and all detected L1s were assigned to their subfamilies by the percent identity of each hit to its query. The 57 subfamily consensuses were aligned with ancestral mammalian L1s from RepBase et al.et al.Since the large majority of L1s are truncated at the 5′ end Histograms of L1 age distribution were generated by the R http://www.perl.org/), Ruby (https://www.ruby-lang.org/en/) and Bash (http://www.gnu.org/software/bash/) scripts. The pipeline queried, filtered and extracted data from the genome of P. vampyrus. An individual step resulted in the addition of 100–500 bp of sequence to the consensus, depending on the quality of the alignment at the ends, which was then used in the next step of the walk and in the final L1 reconstruction. Candidate sequences were identified in the database using BLAST with default parameters and an e-value of 1×10−50, parsed through the BioPerl SearchIO module (http://www.bioperl.org) and screened based on their similarity to the input sequence. Only hits with at least 92% identity were retained to assure that the reconstruction did not include older lineages, and then a Ruby script extracted those sequences with overhangs of at least 100 bp. Alignments for each end were created and hand-edited to yield consensuses of clean read which were aligned into a master alignment. A 300–500 bp region from each end was selected to act as the seeds for the next step in the walk. The process was repeated until the entire element was reconstructed. Upon completion of the full-length L1, a 500 bp seed was chosen arbitrarily from the final consensus and the pipeline was run again to verify the reconstruction. Methylated CpG sites evolve rapidly and must be corrected in the final consensus. CpG sites were identified by their high variation and the presence of dinucleotide sequence CG, CA, TG or TA; these were examined, manually edited and designated as CG in the final consensus. This pipeline also reconstructed the most recently active L1 lineage of four additional species listed in A full-length consensus sequence of the most recently active L1 from megabat lineage 1 was reconstructed by a series of progressive steps. The seed for the reconstruction was a conserved 575 bp region in the 3′ half of ORF2 . This reTo compare the reconstruction of the extinct L1 to other L1s, sequences from a range of mammalian species were either reconstructed as described above, or selected from the RepBase report of February 2013 −  backbone. A puromycin resistance gene and its affiliated promoter pPGKpuro  were cloned into pL1PA1tag, creating plasmid pLY1004. The L1 insert of pLY1004 was removed by NheI and EcoRI digestion, creating the final plasmid backbone . Reconstructed L1s were synthesized in two blocks consisting of ORF1+IGR and ORF2. The manipulated IGRs were synthesized separately or in combinations containing distinct cloning sites. The synthesized sequences were cloned into pUC57 with flanking ends compatible to the linearized pLY1004 backbone and with et al.neo cassette, granting the cell resistance to the antibiotic G418.Retrotransposition rates were tested in an assay derived from Moran et al.The HeLa cell line (ATCC CCL-2) was a gift from Dr. Wenfeng An and maintained in Dulbecco's Modified Eagle Medium with 4500 mg/L glucose and 110 mg/L sodium pyruvate (ThermoFisher Scientific) supplemented by 10% fetal bovine serum , 2 mM l-alanyl-l-glutamine dipeptide and 100 units/mL Penicillin-Streptomycin (ThermoFisher Scientific). The assay was conducted as described by An et al.neo cassette so that cells hosting retrotransposition events, and the corresponding spliced cassettes, yield 653 bp PCR products. pLY1101, a self-ligated version of the linearized pLY1004 without a L1 insertion, was constructed as a positive control; genotyping PCR of pLY1101 yields a 1556 bp construct corresponding to the unspliced neo cassette.To confirm retrotransposition, two retrotransposition-positive colonies of each chimeric L1 construct were isolated with cloning rings, dissociated with trypsin (ThermoFisher Scientific), seeded on T75 flasks and allowed to grow into confluence. Cells were harvested and their genomic DNA was extracted with the QIAamp DNA mini kit . Genotyping PCRs were conducted with primers bracketing the intron of the G418 reporter gene as described by An Figure S1Maximum likelihood tree of the detected megabat L1 subfamilies. Selected ancestral mammalian L1 families, labeled L1MXX, are included to facilitate comparison. The tree was constructed using PhyML (TIF)Click here for additional data file.Figure S2neo cassette from two surviving colonies. Genomic DNA was extracted and used as template. Genotyping PCR primers were designed to amplify the neo cassette so that cells hosting retrotransposition events, and thus the spliced cassette, yield 653 bp PCR products. PCR of positive control construct pLY1101, identical to backbone pLY1004 but with no L1 insertion, yields a 1556 bp product that corresponds to the unspliced neo cassette. The 653 bp band was detected from all colonies. Non-specific bands were detected in a few cases; these were not further characterized.Confirmation of retrotransposition. Retrotransposition was confirmed for each construct by PCR of the (TIF)Click here for additional data file.Figure S3Effect of IGR on retrotransposition rate. Results are shown for all chimeric backgrounds on representative retrotransposition assay plates. Columns represent the various genetic contexts of ORF1/IGR/ORF2; H indicates human L1rp sequence, B indicates reconstructed megabat L1 and X corresponds to the IGR manipulation assayed in each row. Characters to the left of the rows indicate the truncation of the megabat IGR as represented in (TIF)Click here for additional data file.Table S1Summary of megabat L1 families. Families are based on <3.5% distance among the corresponding subfamilies identified by COSEG and shown in (DOCX)Click here for additional data file.Table S2Sequences from RepBase are indicated with an X; other sequences were constructed from genomic trace files. Adenosine content is compared between genomic DNA and L1 segments. AT content from the NCBI genome database was divided by two for Genomic %A and does not take into account any strand bias in coding regions. L1 %As were determined from the coding strands. Numbers in parentheses in the IGR length column indicate IGR lengths from alternative ORF2 starts. Average %As and IGR length are in the bottom row.(DOCX)Click here for additional data file.Text S1Alignment of L1 ORF1 sequences. Protein alignment of the homologous region of ORF1, amino acids 123–321, bp 1273–1869 of L1rp (GenBank accession number AF148856), including the reconstructed megabat L1 lineage 1 (L1-2_PVa), megabat L1 lineage 2 (L1-1_PVa), 26 RepBase-reconstructed L1 consensuses and four L1s reconstructed by us as described in (PDF)Click here for additional data file.Text S2Alignment of L1 ORF2 sequences. Protein alignment of the homologous region of ORF2 spanning the full length L1rp ORF2 , including the reconstructed megabat L1 lineage 1 (L1-2_PVa), megabat L1 lineage 2 (L1-1_PVa), 26 RepBase-reconstructed L1 consensuses and four L1s reconstructed by us as described in (PDF)Click here for additional data file.


Rapidly proliferating tumors attempt to meet the demands for nucleotide biosynthesis by up-regulating folate pathways that provide the building blocks for pyrimidine and purine biosynthesis. Reduced folates are carriers of one carbon units required for the synthesis of purines, thymidylate and methionine, derived from serine, glycine and formate. As folate metabolism plays a key role in cell proliferation, the folate-requiring enzymes dihydrofolate reductase and thymidylate synthase have long been key targets for treatment of cancer. Recent studies show that the mitochondrial folate enzymes are also critical, in that they enable mitochondria to produce additional one carbon units for purine synthesis to allow for rapid growth. In transformed cells, methylene tetrahydrofolate dehydrogenase MTHFD2 is often reactivated and expressed along with other members of the serine synthesis, one carbon (folate) metabolism and glycine cleavage system, allowing for enhanced production of purines, ATP and NADPH, fueling cell proliferation . More reUsing gene expression arrays, we have shown that overexpression of mitochondrial enzymes, particularly MTHFD2, is associated with both high proliferation rates and cMYC overexpression ; this keThe New Twist. It has recently been shown that MTHFD2 can have an impact on proliferation independent of its enzymatic activity . In thes


Due to its adjustable electronic properties and effective excitation of surface plasmons in the infrared and terahertz frequency range, research on graphene has attracted a great deal of attention. Here, we demonstrate that plasmon modes in graphene-coated dielectric nanowire (GNW) waveguides can be excited by a monolayer graphene ribbon. What is more the transverse resonant frequency spectrum of the GNW can be flexibly tuned by adjusting the chemical potential of graphene, and amplitude of the resonance peak varies linearly with the imaginary part of the analyte permittivity. As a consequence, the GNW works as a probe for capturing the molecular spectrum. Broadband sensing of toluene, ethanol and sulfurous anhydride thin layers is demonstrated by calculating the changes in spectral intensity of the propagating mode and the results show that the intensity spectra correspond exactly to the infrared spectra of these molecules. This may open an effective avenue to design sensors for detecting nanometric-size molecules in the terahertz and infrared regimes. The real part of β relates to the surface plasmons’ wavelength λgraphene .m = 0 mode is cutoff-free and the effective mode indexes of all modes decrease monotonically with decreasing frequency. It also indicates a larger proportion of the mode energy resides inside the GNW near the graphene-nanowire at high frequencies, while at low frequencies, they are mainly localized outside the GNW.1 = 2.1ε0 and magnetic conductivity μ0. The studied structure is embedded in air. The complex conductivity of the thin layer graphene colored in gray is given by the Kubo formula [k, chemical potential μc = 0.5 eV, and the carrier scattering rate Γ = 1.32 meV represents the carrier scattering rate.The 3D geometrical structure of the GNW waveguide excited by a graphene ribbon is exhibited in  formula ,36. In tR1 = 190 nm, the thickness of the graphene h = 0.5 nm, while the coupling distance between the graphene ribbon and the outer surface of the GNW is set to be g = 10 nm. The excitation source of unit amplitude, with magnetic vector parallel to the z-axis is set at port1 of the graphene ribbon. A radiation boundary condition is used to truncate the computational domain. The whole space is meshed by triangle grids except for the graphene ribbon and the graphene layer of the GNW, where smooth mapped mesh grids are adopted to ensure the simulation accuracy. The magnetic field distribution in the vicinity of the GNW is shown in m = 1, 2, 3, 4, 5, 6, 7, 8 can be observed in the frequency range of 7.13 THz to 49.36 THz. It is seen that the magnetic fields are tightly confined to the surface of the GNW. Therefore, this area will be quite sensitive to the dielectric environment.The transverse resonance characteristics of the GNW are studied based on the commercial finite element method (FEM) software COMSOL Multiphysics. In the simulation, the radius of the dielectric core is set to be z| with frequency. From left to right, the spectral lines correspond to modes 1, 2, 3, 4 and 5. The resonant frequency of the GNW is calculated and compared with the theoretical results, as shown in ef) and the resonant frequency (rf) confirms the effectiveness of the GNW.In cVg)1/2 and Vg is the gate voltage [In order to investigate the tunability of the resonant frequency of the GNW, frequency spectra with respect to the change of the chemical potential of graphene are simulated. The chemical potential in graphene can be controlled with potentially fast speed using a FET structure , where t voltage . It has  voltage ,39, so wk) of 8 nm is attached to the surface of the graphene, as shown in ε2, and the surrounding medium is air. In order to investigate the sensing performance of the GNW, resonant frequency spectra with respect to the change of analyte permittivity were simulated. Firstly, we suppose that the analyte is lossless and the real part of the relative permittivity varies from 1.0 to 1.5 with an interval of 0.1. Taking mode 4 as an example, simulation results are shown in To explore the sensing properties of the GNW, we suppose that a layer of analyte with thickness  , due to z direction is −z/LI = e. Here, z denotes the length of the GNW which is put along the z axis as shown in L/λ0 = 1/Im(effn). λ0 is the free space wavelength, effn is the effective mode index. We set molI and I0 to indicate the intensities with and without molecules deposited on the graphene surface. In what follows, we perform mode analysis to demonstrate the capture of molecular spectrum with the GNW. The absorption spectrum of the analyte used in the simulation is obtained from the National Institute of Standards and Technology  . The intmolI/I0) are illustrated in Taking the liquid ethanol molecule as an example, the normalized intensity spectra (i.e., confinement to the surface of plasmonic materials and existing in an extended frequency band, within which the molecular vibrational modes appear. To demonstrate the tunability features of the SPP mode, liquid toluene molecules of which the characteristic absorption spectrum is localized in the frequency range of 18–24 THz is adopted in the simulation. Similarly, an intensity change which directly maps the fingerprint of the molecule is obtained, as shown in z direction to induced a 3 dB change in mode intensity. z direction to induced 3 dB change in mode intensity. Taking again ethanol as an example, the variation of intensity spectra with respect to analyte thickness is shown in To achieve such a wide band detection, the surface plasmon modes should have two features, In summary, we demonstrate that plasmon modes of the GNW waveguide can be excited when coupled to a graphene ribbon. The GNW works as a probe for capturing a molecular spectrum. This is due to the factor that the resonant spectra of the GNW sensor can be flexibly tuned by adjusting the chemical potential of graphene, and the peak values of the spectra decrease linearly with the increase of analyte loss. Mode analysis reveals that intensity spectra of the GNW maps well with the absorption spectra of the molecules deposited on the surface of the graphene ring, which further verifies the broadband molecular sensing capabilities.


Despite major research efforts leading to the recent approval of pirfenidone and nintedanib, the dismal prognosis of idiopathic pulmonary fibrosis (IPF) remains unchanged. The elaboration of international diagnostic criteria and disease stratification models based on clinical, physiological, radiological, and histopathological features has improved the accuracy of IPF diagnosis and prediction of mortality risk. Nevertheless, given the marked heterogeneity in clinical phenotype and the considerable overlap of IPF with other fibrotic interstitial lung diseases (ILDs), about 10% of cases of pulmonary fibrosis remain unclassifiable. Moreover, currently available tools fail to detect early IPF, predict the highly variable course of the disease, and assess response to antifibrotic drugs. Recent advances in understanding the multiple interrelated pathogenic pathways underlying IPF have identified various molecular phenotypes resulting from complex interactions among genetic, epigenetic, transcriptional, post-transcriptional, metabolic, and environmental factors. These different disease endotypes appear to confer variable susceptibility to the condition, differing risks of rapid progression, and, possibly, altered responses to therapy. The development and validation of diagnostic and prognostic biomarkers are necessary to enable a more precise and earlier diagnosis of IPF and to improve prediction of future disease behaviour. The availability of approved antifibrotic therapies together with potential new drugs currently under evaluation also highlights the need for biomarkers able to predict and assess treatment responsiveness, thereby allowing individualised treatment based on risk of progression and drug response. This approach of disease stratification and personalised medicine is already used in the routine management of many cancers and provides a potential road map for guiding clinical care in IPF. Hopefully, this is now beginning to change. Although the etiology and the pathogenesis of IPF are still incompletely understood, two antifibrotic drugs, pirfenidone and nintedanib, have recently been proven to be effective in slowing disease progression and are now approved as treatments in the United States and Europe4.Idiopathic pulmonary fibrosis (IPF) is typically introduced as a chronic progressive and inevitably fatal scarring lung disease with a prognosis worse than that of numerous cancers5 and have enormous potential in offering new insights into the understanding and management of pulmonary diseases6, including IPF.The recent development of affordable, high-throughput -omics technologies has opened the era of systems biology and has enabled the emergence of stratified and personalised medicine. These approaches are becoming routine practice in oncology7 and will not be discussed in this current review.This article aims to provide an overview of recent developments in disentangling the complex interrelated mechanisms involved in the pathogenesis of IPF with a particular focus on those that may lead to improved diagnosis, stratification of disease behaviour, and identification of potential novel therapeutic targets and predictors of response to treatment. Considerations concerning the past, present, and future pharmacotherapy of IPF were addressed in the March 2014 issue of this journal8 and enshrine the place of multidisciplinary discussion among experienced clinicians, radiologists, and pathologists as the gold standard method for establishing a diagnosis of IPF. Using the current guidelines, in about two-thirds of the cases, a confident diagnosis of IPF can be achieved based on an appropriate clinical history in association with a typical high-resolution computed tomography (HRCT) pattern of usual interstitial pneumonia (UIP)  is assigned to about 10% of patients who present with progressive pulmonary fibrosis10.The current approach to IPF diagnosis was first described in international guidelines published in 2001, which were recently updated. These guidelines define precise diagnostic criteria based on clinical, radiological, and histopathological featuresa (UIP) . When cla (UIP) . However11. This minimally invasive technique represents an attractive alternative to SLB and may, pending further studies, be included in the diagnostic algorithm of IPF and other fibrotic ILDs in the near future.In a recent study of 117 patients with fibrotic ILDs, bronchoscopic lung cryobiopsy has proven to be safe and effective in providing adequate lung tissue samples, which enabled increased diagnostic confidence in the multidisciplinary diagnosis of IPF12. This multiple-pathway model probably explains the disappointing results of therapies targeting single receptors or pathways in IPF. Future treatment strategies in IPF are likely to focus on combinations of therapies targeting multiple pathogenic pathways simultaneously, as is currently used in the treatment of many cancers13.The heterogeneity in radiological and histopathological appearances, rate of progression, and treatment response observed in individuals with IPF suggests that fibrosis arises as a consequence of multiple co-activated pathogenic pathways, all of which are influenced by complex interactions between endogenous and environmental factors14. Multiple studies have shown that alveolar epithelial cell (AEC) apoptosis secondary to injury is followed by extravascular coagulation, immune system activation, and aberrant persistent activation of AECs, even in the absence of the primary stimulus15. These cells, in turn, induce the migration and proliferation of local fibroblasts, recruit circulating fibrocytes to areas of injury, and promote differentiation of fibroblasts into myofibroblasts. This results in the formation of myofibroblast foci, the histologic hallmark of UIP, in which persistently activated myofibroblasts secrete excessive amounts of extracellular matrix (ECM) proteins. Disordered deposition and accumulation of ECM components within the interstitium and alveolar spaces lead to established fibrosis with progressive destruction of lung architecture and loss of function.Until 15 years ago, the prevailing pathogenic paradigm in IPF was one of chronic inflammation being the precursor to progressive fibrosis. This has shifted over the last decade to a model of abnormal wound healing response driven by persistent or recurrent alveolar epithelial microinjuries  in individuals rendered susceptible by ageing or genetic predisposition19. Cardinal among these is transforming growth factor-beta (TGF-β), a potent profibrotic mediator involved in cell recruitment, myofibroblast differentiation, and induction of ECM production19  and diffusion capacity for carbon monoxide (DLCO) are more predictive of prognosis than baseline values. Thus, a 5–10% decline in FVC at 6 months is associated with a more than twofold increase in the risk of mortality over the subsequent year22. Using relative change in FVC instead of the absolute change enables earlier detection of progression with similar prognostic accuracy23. Also reported as independent predictors of mortality are baseline 6-minute walk distance (6MWD) and change in 6MWD at 6 months24.Several clinical, physiologic, radiographic, and pathologic variables enable a certain degree of mortality prediction in IPF. Older age, male sex, smoking history, low body mass index (BMI), pulmonary hypertension, and concomitant emphysema are clinical predictors of worse survival29  may provide a novel method for evaluating disease activity in IPF. Areas of established honeycomb fibrosis appear to be highly metabolically active, as shown by increased18F-FDG uptake on PET/CT32. More importantly, increased18F-FDG uptake is also observed in areas of radiologically normal lung parenchyma on HRCT, suggesting that PET/CT may have a higher sensitivity than HRCT in detecting early disease in IPF and may thus represent a potential useful tool in monitoring disease activity and response to treatment33, albeit one which is limited by radiation exposure.Interestingly, the development and greater accessibility of6. Such multi-scale modelling should permit mapping of the considerable phenotypic heterogeneity of IPF and may enable the identification of specific molecular phenotypes associated with clinical outcomes that could be used to improve diagnosis accuracy and disease stratification21  study in the United States38 and the PROFILE  study in the United Kingdom40. The latter is the largest prospective cohort study of incident IPF with over 550 patients recruited, all of whom were naïve for antifibrotic therapy at the time of inclusion.Ideal molecular biomarkers should reflect key pathological pathways, be easily and accurately measured, have been validated, and offer added value to currently used approaches42. A single nucleotide polymorphism (SNP) in the promoter region of theMUC5B gene, encoding a mucin involved in airway host defence43, is significantly associated with sporadic and familial IPF44 and, paradoxically, with improved survival45. ThisMUC5B promoter polymorphism is not associated with lung fibrosis in scleroderma or sarcoidosis and thus appears to be specific to IPF46. Similarly, several SNPs conferring susceptibility to IPF have been identified within theTOLLIP locus42. TheTOLLIP gene encodes for a protein with reduced expression in patients with IPF and that regulates part of the innate immune system mediated by Toll-like receptor and TGF-β signalling pathways. Surprisingly, the minor allele rs5743890 inTOLLIP appears to be protective against the development of IPF but when present tends to be associated with increased mortality.Two large genome-wide association studies (GWAS) have identified several common genetic variants associated with susceptibility to IPF and risk of disease progression. The genes identified are involved in host defence, cell-cell adhesion, and DNA repairSFTPC) and A2 (SFTPA2), and in several genes linked to telomere function, such asTERT . Short telomere length as well as evidence of lung parenchymal remodelling and epithelial dysfunction have been identified in asymptomatic first-degree relatives of familial IPF patients and may represent the earliest stages of IPF48. Even in the absence ofTERT polymorphisms, short telomeres in peripheral blood mononuclear cells (PBMCs) or in AECs are also frequently found in IPF patients and portend a poorer prognosis49. This suggests that both genetic variants and environmental factors such as cigarette smoke play a role in telomere shortening.Studies based on familial IPF have identified rare genetic variants in genes encoding surfactant proteins, including surfactant protein C , a cardiolipin-remodelling enzyme, in PBMCs of IPF patients appeared to be strongly correlated with lung function parameters and survival56.Whole RNA microarray analysis of lung tissue from patients with different ILDs has identified disease-specific gene expression signatures that permit UIP to be identified from non-UIP samplesThe identification of these diagnostic or prognostic gene expression signatures is a first step towards the development of molecular tests that could be applied to bronchoscopy samples or peripheral blood, thus allowing less invasive approaches to the diagnosis of IPF and earlier identification of individuals at risk of rapid progression.58, histone modifications60, and noncoding microRNAs (miRNAs)61 are epigenetic mechanisms identified as contributing to differences in gene expression observed in IPF. These regulatory mechanisms are influenced by various factors including environmental exposures (cigarette smoke and infection), genetic profile, sex, and ageing62. A genome-wide DNA methylation analysis of lung tissue identified 2130 significantly differentially methylated regions in IPF samples compared to controls, of which about a third were associated with significant changes in gene expression, including genes identified as IPF-associated common genetic variants63. Thus, dysregulated gene expression in the IPF lung appears to result from complex interactions between genetic and epigenetic factors.DNA methylation64 or decreased65 levels of several regulatory miRNAs in IPF patients, thereby distinguishing the normal lung from the IPF lung and rapidly progressive from slowly progressive disease66. TGF-β seems to play a critical role in the upregulation of profibrotic miRNAs and downregulation of antifibrotic miRNAs67. For example, the direct inhibition of let-7d expression by TGF-β in AECs is associated with epithelial to mesenchymal transition and collagen deposition68. Similarly, several circulating miRNAs appear to be differentially expressed in the serum of IPF patients67. Moreover, the expression levels of miR-21, miR-155, and miR-101-3p in serum seem to be correlated with FVC and HRCT features of IPF69. Interestingly, in mice, intravenous injection of synthetic miR-29 during bleomycin-induced pulmonary fibrosis restored endogenous miR-29 function and was followed by decreasing collagen expression and reversal of pulmonary fibrosis70. These changes in miRNA expression in IPF patients suggest that they play an important regulatory role in lung fibrosis and may represent potential diagnostic and prognostic biomarkers as well as therapeutic targets.miRNAs influence protein expression by binding to mRNA. Aberrant expression of miRNAs has been described in the pathogenesis of many cancers. Lung tissue miRNA profiling identified significantly increased72, mucin 1 (KL-6/MUC1)73, CC-chemokine ligand 18 (CCL18)74, C-X-C motif chemokine 13 (CXCL13)76, periostin77, fibulin-178, matrix metalloproteinases MMP-1 and MMP-780, interleukin-8 (IL-8), intercellular adhesion molecule (ICAM)-180, and lysyl oxidase-like 2 protein (LOXL2)81. Elevated baseline serum levels of KL-6/MUC1 also appear to predict the risk of future acute exacerbation82. Similarly, some circulating cells have been associated with worse survival. Among cellular markers of rapidly progressive IPF are elevated circulating fibrocytes83 and semaphorin 7a+ regulatory T cells (Tregs)84.A growing number of studies have sought to identify protein- and cell-based predictors of IPF disease behaviour. Elevated serum levels of several proteins have been associated with worse prognosis in IPF, including surfactant protein A (SP-A) and D (SP-D)40. These results suggest that serial longitudinal measurement of circulating proteins have potential for use as prognostic or theragnostic biomarkers.Serial measurements of serum ECM protein fragments generated by MMP activity in 189 IPF patients recruited in the PROFILE cohort identified increased serum concentrations of these protein fragments in IPF patients compared to controls. More importantly, increasing neoepitope concentrations were associated with disease progression, and the rate of change over 3 months of 3 of these MMP-degraded ECM proteins predicted survival85, S100A9 protein86, and soluble annexin V87.Studies based on lung tissue or bronchoalveolar lavage fluid (BALF) analysis have also identified some candidate diagnostic and prognostic biomarkers of IPF, including αvβ6 integrinCO % predicted, and MMP-7 serum concentration, which accurately predicted mortality in their validation cohort80. Two other prediction models integrating SP-A and SP-D levels or MMP-7, SP-A, and KL-6/MUC1 levels have shown improved predictability of mortality compared with clinical predictors alone88.The value of these protein or cell biomarkers as diagnostic or prognostic factors in IPF needs to be further assessed. Furthermore, integrating validated molecular variables in multivariate risk prediction models could improve their accuracy in predicting outcomes in IPF. In view of this, Richards and colleagues formulated the personal clinical and molecular index (PCMI), integrating sex, FVC % predicted, DL89. Dysregulated metabolic mechanisms have also been highlighted in the pathogenesis of IPF. Increased levels of lactic acid in IPF lung tissue compared with controls appear to play a role in myofibroblast differentiation via a pH-dependent activation of TGF-β90. Recently, a metabolomic assay by Xie and colleagues demonstrated that augmented aerobic glycolysis, mediated by upregulated glycolytic enzymes, including PFKFB3, represented an early and sustained event during myofibroblast differentiation91. More importantly, PFKFB3 inhibition mitigated myofibroblast differentiation and dampened the profibrotic phenotypes of myofibroblasts isolated from IPF lungs. These data suggest that glycolytic reprogramming is important in the pathogenesis of lung fibrosis and therefore represents a potential therapeutic target. More research is needed in the field of metabolomics to clarify the role of these dysregulated pathways of cellular metabolism in the pathogenesis of IPF and to integrate them with available genetic, epigenetic, transcriptomic, and proteomic data.Metabolomics is the systematic analysis of the complete set of metabolites (the metabolome) within a biological system under given conditions. This approach offers the potential for a better understanding of dysregulated metabolic pathways underlying numerous diseases, including airway diseases such as asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis92, including familial IPF93, and is associated with a worse survival94. Some other environmental and occupational exposures, including wood, mineral, and metal dusts, agriculture, and livestock, have also been associated with IPF, although a formal causal link has not been established95. Furthermore, air pollution may also play a role in the pathogenesis of IPF. A recent study reported a significantly higher risk of acute exacerbation of IPF with increased ozone and nitrogen dioxide exposure over the preceding 6 weeks96.Smoking history has long been described as a prevalent risk factor for the development of IPF97. Anti-acid treatment in IPF has been associated in retrospective data with decreased radiologic fibrosis, longer survival, and smaller decrease of FVC at 30 weeks99. Despite growing evidence suggesting that GER and silent microaspiration might play a role in the pathogenesis of IPF, there is, to date, no confirmation that this association is causative. Consequently, the recently updated international guidelines on IPF treatment maintained a conditional recommendation for the use of anti-acid therapy100. A prospective randomised controlled trial is needed to further assess the role of GER and microaspiration in IPF and confirm the effectiveness of anti-reflux therapy. Gastroesophageal reflux (GER) is highly prevalent in IPF, though often asymptomatic, and confers an increased risk of microaspiration101. Whether this association is causative has not yet been proven. HHVs have the potential to induce endoplasmic reticulum stress and apoptosis102; it is therefore hypothesised that viral infection may act as a cofactor in the development of IPF through the reactivation of latent HHVs within the alveolar epithelium following exposure to a first injury103. Furthermore, a recent study found increased copy numbers of EBV and CMV DNA in BALF of IPF patients and, to a lesser extent, in first-degree asymptomatic relatives of familial IPF patients48. Thus, enhanced HHV replication may trigger epithelial cell stress and participate in disease initiation. A small clinical trial of ganciclovir in individuals with severe IPF with positive EBV-IgG serology showed a modest improvement in surrogate markers of disease progression104. It has recently been reported that influenza infection may also play a role in lung fibrosis by promoting collagen deposition via αvβ6 integrin-mediated TGF-β activation in epithelial cells105.Infectious processes may play a role in the initiation, progression, or exacerbation of IPF. Viral infections, particularly human herpes viruses (HHVs), including herpes simplex virus type 1 (HSV-1), Epstein-Barr virus (EBV), cytomegalovirus (CMV), HHV-7, and HHV-8, have been associated with IPF in several studiesStaphylococcus andStreptococcus genera in BALF38. Similarly, Molyneaux and colleagues found an increased bacterial load, consisting particularly ofHaemophilus,Streptococcus,Neisseria, andVeillonella spp., in BALF of IPF patients compared to healthy smokers, nonsmokers, and patients with moderate COPD106. More importantly, the total bacterial burden was an independent predictor of decline in lung function and mortality. Whether these differences in lung microbiome are a cause or consequence of IPF is unknown. A clinical trial of 12 months of co-trimoxazole in addition to standard treatment in 181 patients with fibrotic idiopathic interstitial pneumonia (about 90% of whom had IPF) showed a reduction in mortality but did not slow functional decline107. The exact role of viruses and bacteria in the pathogenesis of IPF has yet to be determined and the potential for antiviral or antibiotic treatments requires further evaluation.Recent data also suggest a putative role for bacteria and lung microbiome in IPF. An analysis of the COMET study showed an association between progression of IPF and the presence of specific members within theCurrently available therapies for IPF are of limited efficacy, and the prognosis associated with the condition remains poor. Recent advances in our understanding of the complex interrelated mechanisms underlying fibrosis in the lung are encouraging and pave the way towards an integrated approach to diagnosis, stratification, and treatment. It is becoming increasingly clear that genetic polymorphisms, whole blood transcriptomic profile, and lavage microbiome all predict groups of patients with differing disease behaviour and outcomes and potentially variable responses to treatment. Furthermore, prospective longitudinal cohort studies have started to identify blood biomarkers that have the potential to be used as early measures of treatment response. Considerable further research is required to deliver personalised medicine for IPF into the clinic, but at least now there is light at the end of what has been a very long tunnel.


Prevention of microbially induced corrosion (MIC) is of great significance in many environmental applications. Here, we report the use of an ultra-thin, graphene skin (Gr) as a superior anti-MIC coating over two commercial polymeric coatings, Parylene-C (PA) and Polyurethane (PU). We find that Nickel (Ni) dissolution in a corrosion cell with Gr-coated Ni is an order of magnitude lower than that of PA and PU coated electrodes. Electrochemical analysis reveals that the Gr coating offers ~10 and ~100 fold improvement in MIC resistance over PU and PA coatings respectively. This finding is remarkable considering that the Gr coating (1–2 nm) is ~25 and ~4000 times thinner than the PA (40–50 nm), and PU coatings (20–80 μm), respectively. Conventional polymer coatings are either non-conformal when deposited or degrade under the action of microbial processes, while the electro-chemically inert graphene coating is both resistant to microbial attack and is extremely conformal and defect-free. Finally, we provide a brief discussion regarding the effectiveness of as-grown vs. transferred graphene films for anti-MIC applications. While the as-grown graphene films are devoid of major defects, wet transfer of graphene is shown to introduce large scale defects that make it less suitable for the current application. Literature indicates that the annual costs for corrosion14535Metal coatings and alloys have been commercially379111213142O3 nanoparticles) caused a significant improvement in the corrosion resistance of monolayer grapheneGraphene (Gr), a two-dimensional sheet of sp1819202122242527The objective of this study was to investigate the effectiveness of nano-scale graphene coatings to prevent MIC of the immersed, metallic structure under galvanic conditions. More specifically, the MIC resistance of Ni was compared in presence of three different coatings: i) parylene-C coating on Ni (PA/Ni), ii) polyurethane coating on Ni (PU/Ni) and iii) graphene coating on Ni (Gr/Ni). A Ni foam−1) in Chemical vapor deposition (CVD- see methods section) was used to obtain a uniform coating of PA–C on the Ni surface . The thi3031The corrosion cells  were opeThe Nyquist plots  shows thct) and interfacial capacitance (Cm). The seepage of anolyte through the PU coating is responsible for the charge transfer reactions associated with the corrosion process. In order to model diffusion limitations of corrosion by-products through the porous electrode, a Warburg element (WO) is added in series to the corrosion impedance. Within the pore length, the electrolyte resistance is Rint, and the insulating part of the coating can be considered to be a capacitor Cdl which is in parallel with the impedance in the pore (el) is added in series with the previous impedance.the pore . The eleel), the anolyte/PA interfacial impedance and PA/Ni interfacial impedance (int). Similarly, the interfacial capacitance at the anolyte/PU interface is modeled as Cdl. The charge transfer reactions associated with Ni corrosion at the anolyte/PA interface were incorporated as a parallel combination of charge transfer resistance (Rct) and interfacial capacitance (Cint)34The thickness of the parylene coating in the PA/Ni system is ~3 orders of magnitude smaller than that of PU in PU/Ni. We therefore used an alternate equivalent circuit model based of the anolyte resistance (Rmpedance . At the ct of Gr/Ni (~35.8 kΩ.cm2) was ~10-fold higher than that of PU/Ni (~1.57 kΩ.cm2), and ~100-fold higher than that of PA/Ni (~0.38 kΩ.cm2)  increased linearly. In Gr/Ni, the corrosion rates remained in the 1.5–3 mg/L/day range in all the three cycles. The cell with Gr/Ni demonstrated an improvement in the corrosion resistance . The higher values of Ni concentrations in PU/Ni and PA/Ni cells during subsequent weeks denote an eventual failure of the polymer coatings. The lower corrosion rates in the Gr/Ni are likely due to a combination of the following: i) minimal defects in the Gr coating compared to PA and PU coatings, ii) limited diffusion of corrosion by-products from the Ni to the bulk liquid due to restricted access through the few-layered Gr coating, and iii) adhesion of polysaccharides and insoluble biomass to the hydrophobic graphene surface which plugs the few defect sites that may exist on the Gr film surface.Scanning electron microscopy (SEM) was used to observe the extent of corrosion-induced debilitation of the PA/Ni and PU/Ni surfaces. We found micron-length tears on the PU/Ni surfaces  while th373839in-situ on a copper foil by CVD, was transferred layer-by-layer onto SiO2 using wet chemistry based transfer methods2 (~80 μm × 80 μm).On having established that CVD-grown (few-layer) graphene offers superior resistance to MIC, we sought to characterize the defectiveness of the as-grown graphene coating when compared to graphene films that are transferred , as compared to the Gr films transferred onto SiO2 and half the value for monolayer Gr as-grown on Cu foil. This is visually represented in the Raman map for the few-layer graphene grown on Ni foam  sulfide-oxidizing bacteria convert HS− to sulfate that is quickly oxidized to sulfuric acid and abruptly decrease the local pH (<1.0) to enhance the Ni corrosion.Corrosion characteristics are determined by the corroding and the corroded species. In order to better understand the interactions between the microbial community and the coated nickel surfaces, a phylogenetic microbial community analysis was carried out and its results are presented in this section . The ana4243In our experiments, we observed a change of the anolyte color from yellow to black within 48 hours of initial operation  which ca502+ concentration  corrosion current and ii) Nintration  in the Gin-situ with minimimal defect density. Further, the few defects that may be present on the surface can be plugged by biofilms through the production of non-conductive polymeric debris. On exposure to similar harsh microbial environments, parylene C failed by extensive microbial degradation and polyurethane due to its non-conformal adhesion to the metallic surface. In order to study the feasibility of Gr coatings as MIC resisting elements on non-CVD surfaces, we study the effect of wet-transfer on the quality of graphene. The study revealed that transferred graphene films are significantly more defective than their as-grown counterparts, thereby warranting future research targeting defect-free transfer of graphene for surfaces that are not amenable to CVD growth.Graphene coatings have not been explored in detail for corrosion prevention in biotic environments. The current study shows that graphene coatings outperforms its polymer counterparts (parylene C and polyurethane) by offering 1 and 2 orders of magnitude better impedance against microbial corrosion. The shortcomings associated with defect based corrosion of graphene coatings in abiotic environments has been countered in this study by the use of 3–4 layer graphene films that are grown −). The cell configuration enables the Ni anode to be immersed in a 0.2 L electrolyte that supports the growth of a diverse set of mixed microbial communities. The Ni anode is coupled to a graphite cathode to promote the bimetallic corrosion. Further, a ferricyanide system  with a faster redox kinetics was used as an electron acceptor in the cathode , counter electrode (graphite cathode), and a reference electrode . The observed reflected light was used to determine ratios of the amplitude (ψ) and phase changes of the p and s components of polarized light (Δ)57Clean Ni foam and Si wafer samples (~2 × 2 cm) were placed in a Union Carbide chemical deposition chamber and pumped down to low vacuum conditions of 50 mTorr. A 0.5 g of PA C dimer  was sublimed at 150–170 °C and pyrolized at 650 °C. The pyrolized vapors were deposited on the Ni sample at room temperature. A NA Krylon clean polyurethane spray operation was used to establish the polyurethane coating on the Ni foam.2 and Ar atmosphere at 1000 °C to remove the layer of metal oxide. Methane was then introduced to the CVD chamber for 5 mins at ambient pressure. Next, the chamber was rapidly cooled to precipitate the dissolved carbon as a multilayer graphene on the Ni surfaceThe chemical vapor deposition (CVD) process was used to grow graphene on the Ni foam. The Ni foam was annealed in H2-transferred graphene (80 μm × 80 μm) and CVD-grown graphene (30 μm × 30 μm copper and nickel foam) samples. After data collection, Lorentzian curve fitting was used to fit the D, G and 2D peaks in order to create maps of the spectral features.A Raman microscope  with a confocal pinhole was used to obtain a Raman mapping of SiOPost experiments, the electrodes were immediately fixed using 2.5% glutaraldehyde buffered with 0.1 M phosphate buffer (pH 7.4). The fixed samples were refrigerated for 24 hours, dehydrated in ethanol series  for 5 minutes at each gradation. The sample was left in 100% ethanol in refrigerator overnight, and dried using an automated critical point dryer . Post dehydration, a thin layer of Platinum (few nm) was sputtered on the electrode. The samples were then examined in SUPRA® Focused Ion Beam (FIB) under EHT voltage ~1 kV and magnification of 10 kx. The Gr/Ni foam and the transferred Gr samples were viewed without any surface preparation at 3 kV EHT and at low magnifications upto 100x.The Ni foam samples (biotic and abiotic) were analyzed using Al K-alpha X-ray gun at 40W on a Phi Versaprobe® XPS system. After a basic background scan, a detailed scan was conducted with 20 eV pass energy. To confirm the presence of sulfur on a large surface area, a 100 μm × 100 μm sample was chosen with large acquisition time (~75 minutes).Voltage data was acquired periodically using DAQ/54 modules  across a resistance substitutor . Electrochemical impedance spectroscopy was used to analyze the corrosion specimens  with GAMRY ®Reference 3000™. A small AC RMS current (~1 mA) was applied as a perturbation signal in order to measure the impedance response of the anode in a frequency range of 0.1 Hz–10 kHz. The cyclic voltammograms were recorded in the voltage range of −0.8 V–1.0 V and at a scan rate of 25 mV/s.2+) in the three corrosion cells were quantified using 1-(2 Pyridylazo)-2-Napthol (PAN) indicator methodThe concentration of free Ni ions  following manufacturer’s guidelines. The 16S rDNA V4 region amplicons (single index) were produced by PCR and sequenced on the MiSeq platform (Illumina) using the 2 × 250 bp protocol yielding pair-end reads that overlap by ~247 bps. Following sequencing, raw BCL files were retrieved from the MiSeq platform and called into fastqs by Casava v1.8.3 (Illumina). The read pairs were demultiplexed based on unique molecular barcodes, filtered for PhiX using Bowtie2 v2.2.1, and reconstituted into two fastq files for each read using standard BASH. A barcodes file was generated from a raw fastq base called previously preserving the original barcode qualities associated per read cluster. Reads were merged using USEARCH v7.0.1001  and a barcodes file was generated for the merged set in the same manner they were generated for the raw reads. Processing of sequencing reads included quality-filtering using USEARCH v7.0.1001 (maximum expected error method). Sequences were demultiplexed using QIIME v1.8.0 and then clustered using the UPARSE pipeline. Operational taxonomic unit (OTU) classification was achieved by mapping the UPARSE OTU table to the SILVA database. Abundances were recovered by mapping the demultiplexed reads to the UPARSE OTUs. A custom script constructed an OTU table from the output files generated in the previous two steps. The OTU table was used to calculate alpha-diversity, beta-diversity, provide taxonomic summaries, and in a variety of other analyses built into QIIME that allowed for the characterization of individual and group of samples. Bacterial diversity in each sample was assessed by determining richness based on the calculation of Alpha diversity and Beta diversity in addition to computing diversity indices such as Chao, Simpson, and Shannon.How to cite this article: Krishnamurthy, A. et al. Superiority of Graphene over Polymer Coatings for Prevention of Microbially Induced Corrosion. Sci. Rep.5, 13858; doi: 10.1038/srep13858 (2015).


Clinical trials of immune checkpoints modulators, including both programmed cell death-1 (PD-1) and programmed cell death-ligand 1 (PD-L1) inhibitors, have recently shown promising activity and tolerable toxicity in pre-treated NSCLC patients. However the predictive role of PD-L1 expression is still controversial. This pooled analysis aims to clarify the association of clinical objective responses to anti PD-1/PD-L1 monoclonal antibodies (MoAbs) and tumor PD-L1 expression in pre-treated NSCLC patients.Data from published studies, that evaluated efficacy and safety of PD-1/PD-L1 inhibitors in pre-treated NSCLC patients, stratified by tumor PD-L1 expression status , were collected by searching in PubMed, Cochrane Library, American Society of Clinical Oncology, European Society of Medical Oncology and World Conference of Lung Cancer, meeting proceedings. Pooled Odds ratio (OR) and 95% confidence intervals (95% CIs) were calculated for the Overall Response Rate (ORR) , according to PD-L1 expression status.A total of seven studies, with 914 patients, were eligible. Pooled analysis showed that patients with PD-L1 positive tumors (PD-L1 tumor cell staining ≥1%), had a significantly higher ORR, compared to patients with PD-L1 negative tumors .PD-L1 tumor over-expression seems to be associated with higher clinical activity of anti PD-1/PD-L1 MoAbs, in pre-treated NSCLC patients, suggesting a potential role of PD-L1 expression, IHC cut-off point 1%, as predictive biomarker for the selection of patients to treat with immune-checkpoint inhibitors. Cancer immunotherapy is emerging as a very promising therapeutic strategy for several solid tumors, including non-small cell lung cancer (NSCLC). Differently from other treatment approaches directed against the tumor, such as chemotherapy or targeted therapy -6, targeOur search, according to the aforementioned criteria, performed in October 2015, identified 211 publications. Among these, after a careful selection procedure, only seven studies (914 patients) met our inclusion criteria and were included in the pooled-analysis  had a significantly higher ORR compared to patients with PD-L1 negative tumors  . The results of this work have shown that tumor PD-L1 positivity (PD-L1 tumor cell staining ≥1%) is associated with significantly higher ORR, suggesting a potential role of tumor PD-L1 over-expression as predictive biomarker for clinical setting. After the recent approval of Nivolumab by FDA for the second-line treatment of squamous NSCLC, searching for predictive biomarkers has become an urgent issue for clinical research. A validated biomarker would allow to select those patients who could benefit more from immune-therapies, sparing the others from ineffective high-cost treatments, as well as futile immune-related toxicities. In this scenario, tumor PD-L1 expression represents the predictive biomarker most evaluated in clinical studies. Although the majority of such studies suggested a significant association between tumor PD-L1 expression and anti-PD-1/PD-L1 MoAbs activity , 24, 27,Despite several limitations our pooled-analysis shows that tumor PD-L1 expression, IHC cut-off >1%, is associated with significantly higher ORRs to anti PD-1/PD-L1 MoAbs, in pre-treated NSCLC patients. These data suggest that PD-L1 at the lowest expression level, though still limited by varied testing approaches and definitions of positivity, has a potential role as predictive biomarker for clinical setting. Probably, as suggested by recent studies, a combination of tumor PD-L1 expression with other “immune-biomarkers” will enhance our ability to identify not only the best candidate to receive immune-therapies, but also the mechanisms of immune-evasion at a single patient level, and ultimately personalize the immune-treatments and combination strategies.We performed our pooled-analysis according to a predefined written protocol. We searched for all published studies, that report the ORR of patients with NSCLC treated with anti PD1/PD-L1 MoAbs, who received prior chemotherapy regimens, stratified by tumor PD-L1 expression status (IHC cut-off point 1%). Publications were identified by an electronic search in Medline, using PubMed online service, updated in October 2015. The search for publications was made by other databases including the Cochrane Library and EMBASE. However the search on PubMed allowed the widest collection of publications about this topic. The following search terms were used: “PD-1”, “PD-L1”, “lung cancer”, “NSCLC”, “non-small cell lung cancer”, “predictive biomarker”, “anti-PD1”, “anti-PD-L1”, “monoclonal antibodies”, “cancer immunotherapy”. The search was limited to human studies in the English language. The results were supplemented with manual searches of American Society of Clinical Oncology (ASCO), European Society of Medical Oncology (ESMO), and World Conference of Lung Cancer (WCLC), meeting proceedings, references of selected articles and published reviews. A systematic review on this topic in the Cochrane database of systematic reviews was not found.According to this search clinical trials were taken into account if they had to fulfil all the following inclusion criteria: 1) only patients with metastatic NSCLC were included; 2) only patients who received prior therapies were included 3) studies that report the ORR of NSCLC patients treated with an anti-PD1/PD-L1 MoAb as single agent; 4) studies that report the ORR, stratified according to the PD-L1 expression status (IHC cut-off point 1%).Two authors independently selected studies according to the aforementioned inclusion criteria. If these two authors could not reach a consensus, another author was consulted and a final decision was made by consensus. Information was carefully extracted by overall selected studies. The following data were collected and organized from eligible studies: first author name, journal and year of publication, study design, study treatment, baseline characteristics of patients , and ORR stratified according to tumor PD-L1 expression status. The proportion of patients for each outcome was calculated basing on the percentages reported in included trials, when it was not reported as absolute number. Data extraction was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines .Patients were stratified according to PD-L1 expression status into 2 groups: PD-L1 positive (PD-L1 tumor cell staining ≥1%) and PD-L1 negative. The outcome measure was ORR, defined as the percentage of patients who have a complete or partial tumor response according to World Health Organization (WHO) criteria or Response Evaluation Criteria in Solid Tumors (RECIST), calculated in PD-L1 positive over PD-L1 negative NSCLC patients. The number of events (i.e. objective responses) was extracted from each study or calculated from the percentage provided, and the proportion of patients was calculated for each group. The association between these endpoints and PD-L1 expression status was expressed as an odds ratio (OR) of PD-L1 positive over PD-L1 negative patients. Thus, an OR greater than 1 indicates that PD-L1 overexpression (PD-L1 tumor cell staining ≥1%) is associated with higher ORR in pre-treated NSCLC patients. A pooled-analysis of ORs was performed to calculate a pooled OR for such outcome, using a fixed-effect or random-effect, based on statistical significance of Q-test, according to Mantel-Haenszel method. The heterogeneity between trials was tested using the Cochran Q-test, with a predefined significance threshold of 0.1. Publication bias for ORR analysis were assessed using Begg's funnel plots and Egger's test. The level of significance was set at 5% . All statistical analyses were performed with Review Manager 5.3.5  and Comprehensive Meta-Analysis software .


A humidity microsensor integrated with a readout circuit on-a-chip fabricated using the commercial 0.18 μm CMOS  process was presented. The integrated sensor chip consists of a humidity sensor and a readout circuit. The humidity sensor is composed of a sensitive film and interdigitated electrodes. The sensitive film is titanium dioxide prepared by the sol-gel method. The titanium dioxide is coated on the interdigitated electrodes. The humidity sensor requires a post-process to remove the sacrificial layer and to coat the titanium dioxide. The resistance of the sensor changes as the sensitive film absorbs or desorbs vapor. The readout circuit is employed to convert the resistance variation of the sensor into the output voltage. The experimental results show that the integrated humidity sensor has a sensitivity of 4.5 mV/RH% (relative humidity) at room temperature. The conductivity of the humidity-sensing film changed upon adsorption/desorption of water vapor. Lazarus and Fedder [et al. [et al. [Humidity sensors are important devices for application in industrial and electronic equipment. Various traditional sensors have been miniaturized as microsensors using micromachining technology. Micromachined sensors have the benefits of high performance, low cost, small size, and easy mass-production . Many huet al.  used mic [et al.  develope [et al.  presente [et al.  manufact [et al.  proposed [et al.  employedd Fedder  fabricat [et al. , was fab [et al.  utilized [et al.  were not [et al. . In thisThe commercial CMOS process has been used to develop various microsensors and microactuators –14. Micr2.OP represents the operational amplifier, Vin is the input voltage of the circuit; Vout is the output voltage of the circuit; Rs is the resistance of the humidity sensor; R1–R7 are the resistances of the circuit.As shown in R1 = 5 kΩ, R2 = 5 kΩ, R3 = 30 kΩ, R4 = 30 kΩ, R5 = 20 kΩ, R6 = 30 kΩ, R7 = 10 kΩ and Rs = 100 kΩ are set, and the input voltage Vin is 2.5 V. The bias voltage of the amplifier is 3.3 V. The resistance of the humidity sensor varies from 400 to 100 kΩ. The simulated results revealed that the output voltage of the readout circuit decreased from 2.39 to 2.14 V as the resistance of the sensor changed from 400 to 100 kΩ. To characterize the relation between the output voltage and temperature for the readout circuit, the readout circuit is simulated at different temperatures. Vin is 2.5 V. The resistance of the humidity sensor is 120 kΩ, and the resistances are R1 = 5 kΩ, R2 = 5 kΩ, R3 = 30 kΩ, R4 = 30 kΩ, R5 = 20 kΩ, R6 = 30 kΩ and R7 = 10 kΩ. The temperature changes from 20 to 80 °C. The results showed that the output voltage of the readout circuit increased from 2.1485 to 2.1491 V as the temperature changes from 20 to 80 °C, in which the output voltage varies about 0.6 mV.The software HSPICE  is utilized to simulate the characteristics of the readout circuit. 3.3H7)4) precursor of 3 mL  was dissolved in isopropanol   with stirring for 30 min until the solution was mixed uniformly. Deionized water (40 mL) was added into the mixed solution with stirring for 1 h. Then, HNO3 solution of 5 mL  was added into the mixed solution and stirred for 20 min, followed by aging for 30 min. The slurry of titanium dioxide was filtered, and then rinsed with deionized water. Finally, the titanium dioxide was coated on the substrate with calcinations at 400 °C for 1 h.The sensitive film of the humidity sensor was titanium dioxide prepared by the sol-gel method ,26. The Scanning electron microscopy  was used to measure the surface morphology of the titanium dioxide film. 4.The commercial 0.18 μm CMOS process of Taiwan Semiconductor Manufacturing Company  was employed to manufacture the integrated humidity sensor chip. 5.The performances of the humidity microsensor were tested by a power supply, a test chamber , a LCR meter and an oscilloscope. The test chamber could supply a humidity range of 25–90 RH% and a temperature range of 0–100 °C. Temperature and humidity in the test chamber could be tuned separately and maintained at constant levels. The power supply was used to provide the bias voltage and the input voltage to the readout circuit.The humidity sensor without readout circuit was tested in order to characterize the resistance variation of the sensor. The humidity sensor without readout circuit was set in the test chamber, and its resistance variation under different humidity was measured by the LCR meter. The humidity sensor with readout circuit was set in the test chamber. The test chamber provided different humidity levels to the humidity sensor. When the humidity in the test chamber rose or dropped, the resistance of the humidity sensor produced a change. The readout circuit converted the resistance variation of the sensor into the output voltage, and the oscilloscope recorded the output voltage. To characterize the influence of temperature, the humidity sensor was measured under different temperatures. The output voltage of the humidity sensor was detected at different temperatures. 6.A humidity sensor equipped with a readout circuit was successfully manufactured using the commercial 0.18 μm CMOS process and a post-process. The post-process was compatible with the commercial CMOS process. The humidity sensor consisted of a sensitive film and interdigitated electrodes. The sensitive film of the humidity sensor was titanium dioxide that was prepared by the sol-gel method. The titanium dioxide was deposited on the interdigitated electrodes. The humidity sensor generated a change in resistance as the sensitive film absorbed or desorbed water vapor. The resistance variation of the sensor was converted by the readout circuit into the output voltage. The sensor integrated with readout circuit on-a-chip would reduce noise and interference. The experiments showed that the output voltage of the humidity sensor decreased from 2.27 to 2.05 V as the humidity changed from 30 to 90 RH% at 25 °C. The humidity sensor had a sensitivity of 4.5 mV/RH% at room temperature.


We fabricate a nonlinear optical device based on a fiber pigtail cross-section coated with a single-layer graphene grown by chemical vapor deposition (CVD) method. Using the fabricated graphene-assisted nonlinear optical device and employing Nyquist 16-ary quadrature amplitude modulation (16-QAM) signal, we experimentally demonstrate phase conjugated wavelength conversion by degenerate four-wave mixing (FWM) and transparent wavelength conversion by non-degenerate FWM in graphene. We study the conversion efficiency as functions of the pump power and pump wavelength and evaluate the bit-error rate (BER) performance. We also compare the time-varying symbol sequence for graphene-assisted phase conjugated and transparent wavelength conversions of Nyquist 16-QAM signal. Recently, a variety of graphene-based photonic devices have been reported, including wide bandwidth polarizers7912131415(3) nonlinearities20Graphene234It is well known that the increase of spectral efficiency has become an effective way when scaling up the data rate2327In this paper, considering the combination of advanced optical modulation format signal  and the optical nonlinearity of a single-layer graphene coated fiber device, we show an experimental observation of degenerate/non-degenerate FWM based wavelength conversion of a 5 Gbaud Nyquist 16-QAM signal. We study the wavelength tuning properties and the conversion efficiency as functions of the pump wavelength and pump power. Moreover, we characterize the performance of Nyquist 16-QAM wavelength conversion by measuring the BER as a function of the received optical signal-to-noise ratio (OSNR). Time-varying symbol sequence for phase conjugated wavelength conversion by degenerate FWM and transparent wavelength conversion by non-degenerate FWM are also measured for comparison.The line-width of ECL1 laser is 100 KHz. At the transmitter, the information data is firstly mapped into 16-QAM format and then packed into data frames. In each frame, 38400 data symbols are transmitted after the preamble. We insert 1 pilot in every 63 data symbols for phase recovery. The preamble includes two 63-symbol M-sequences as synchronization sequences and four 127-symbol M-sequences as training sequences. After 2 samples per symbol up-sampling, the signals are digitally shaped with a root-raised-cosine (RRC) filter. The roll-off factor of the RRC is 0.01. After digital to analog convertors (DAC), electrical low-pass filters with the bandwidth of 4.4 GHz are used as anti-aliasing filters to remove out-of-band radiation. At the receiver, a matched RRC filter is adopted. After synchronization, the signals are re-sampled to 2 samples per symbol and the training sequences are picked up for time-domain channel estimation and equalization. After equalization, the phase is corrected with pilots and further estimated with the blind phase search (BPS) algorithm. Finally, the bit error ratio (BER) is measured by error counting. The RF spectrums of Nyquist pulse waveform before RRC filter and after RRC filter is shown in We first demonstrate the wavelength conversion of Nyquist 16-QAM signal based on degenerate FWM process in the single-layer graphene coated fiber device. In the experiment, the signal wavelength is fixed at 1552.52 nm. pump = 1550.12 nm, λsignal = 1552.52 nm). The conversion efficiency increases with the pump power. The conversion efficiency η of FWM can be approximately expressed as η = (γ∙Pp∙L)γ is the effective nonlinear coefficient, Pp is the pump power, and L is the length. In fact, the conversion efficiency curve in pump and a data signal at wavelength λsignal are combined together and sent into a graphene-assisted nonlinear device with high third order nonlinearity (χ(3)). Due to the energy conservation in the degenerate FWM process, the wavelength of the newly converted idler wave can be written by 1/λconv = 2/λpump−1/λsignal. Hence, one can achieve tunable wavelength simply by adjusting the pump wavelength or the signal wavelength, i.e. variable λpump or λsignal results in changeable λconv. So the converted idler wavelength can be tuned simply by changing the pump wavelength, i.e. tunable wavelength conversion even for a fixed input signal wavelength. We define the conversion efficiency as the power ratio of converted idler to signal. pump1 = 1549.32, λpump2 = 1552.52 nm, λsignal = 1551.32 nm).We then demonstrate the wavelength conversion of Nyquist 16-QAM signal based on non-degenerate FWM process in the single-layer graphene coated fiber device. idler ∝ E2pumpE*signal, where Eidler, Epump and Esignal represent the complex electric fields of newly converted idler, input pump and input signal, respectively. “∗” denotes the complex conjugate of the electric field. Hence, the newly converted idler does not take the same data information carried by the original signal but its “phase conjugated” copy.For the wavelength conversion based on degenerate FWM process as shown in idler ∝ Epump1EsignalE*pump2, where Epump1 and Epump2 represent the complex electric fields of two input pumps. Therefore, the newly converted idler copies exactly the same data information carried by the original signal, i.e. fully transparent wavelength conversion.For the wavelength conversion based on non-degenerate FWM process as shown in The converted idler is “phase conjugated” copy of the original data signal in the process of graphene-based degenerate FWM−3 (7% forward error correction (FEC) threshold) for Nyquist 16-QAM wavelength conversion with the converted idler at 1547.71 nm. The received OSNR penalties of ~1.4 dB at a BER of 1 × 10−3 are observed for converted idlers at 1546.12 and 1544.52 nm. The increased OSNR penalty is mainly due to the reduced conversion efficiency for converted idlers at 1546.12 and 1544.52 nm. −3. The right insets of To characterize the performance of Nyquist 16-QAM wavelength conversion, we further measure the BER curve as a function of the received OSNR for back to back (B-to-B) signal and newly converted idler. (3) nonlinearities. Therefore, the nonlinear optical device based on a fiber pigtail cross-section coated with a single-layer graphene grown by CVD might show favorable performance in practical applications.Luo and co-authors(3) nonlinearities−20 m2/W, ~10−18 m2/W and ~10−11 m2/W, respectively313217Due to possessing linear band structure that allows interband optical transitions at ultrabroadband range (across the infrared and visible range), graphene has been suggested as a material that might have large χ(3) of graphene could also give rise to nonlinear Kerr-effects such as FWM, TPA, stimulated Raman scattering (SRS), stimulated Brillouin scattering (SBS), SPM, and XPM. So, on may wonder whether the graphene based phase conjugated and transparent wavelength conversions still can occur, especially for advanced optical modulation formats signal, e.g., Nyquist 16-QAM. One may also wonder what is the influence of the XPM, SPM on the signal quality  in the process of graphene-assisted FWM based wavelength conversion of Nyquist 16-QAM signal. Previous experiments on enhanced FWM in graphene-assisted nonlinear optical devices have been demonstrated with impressive performance, such as the works by C. W. Wong and Y. J. Rao18202137(3) nonlinearities, and compatibility with existing optical fiber transmission systems might enable novel architectures for optical signal processing applications.The phenomena of degenerate and non-degenerate FWM have been demonstrated in semiconductor optical amplifiers−3 with the converted idler generated at 1547.71, 1546.12 and 1544.52 nm, respectively. For transparent wavelength Nyquist 16-QAM conversion based on non-degenerate FWM, the observed OSNR penalty is around 1.6 dB at a BER of 1 × 10−3 with the converted idler generated at 1548.11 nm. It is expected that graphene-assisted nonlinear optical devices might find more interesting optical signal processing applications.Firstly, FWM in graphene which is mechanically transferred on the end face of optical fiber has been experimentally observed. The degenerate/non-degenerate FWM based wavelength conversion of advanced modulation format signal (e.g. Nyquist 16-QAM) is further demonstrated in the experiment. Secondly, we compare in detail the measured conversion efficiency with and without graphene. The power of converted idler without graphene is observed to be ~5.8 dB lower than the one with graphene. We also clarify that the enhanced conversion efficiency mainly comes from the high nonlinearity of graphene. Additionally, the converted idler wavelength can be flexibly tuned by changing the pump wavelength and the conversion efficiency varies slightly. Lastly, for phase conjugated Nyquist 16-QAM wavelength conversion based on degenerate FWM, the observed OSNR penalties are around 1, 1.4 and 1.4 dB at a BER of 1 × 103 solution. Then, the floating PMMA/graphene sheet is mechanically transferred onto the fiber pigtail cross-section and dried in a cabinet. After drying at room temperature for about 24 hours, the carbon atoms could be self-assembled onto the fiber end-facet thanks to the strong viscosity of graphene. The PMMA layer is finally removed by boiling acetone. By connecting this graphene-on-fiber component with another clean and dry FC/PC fiber connector, the nonlinear optical device is thereby constructed for degenerate/non-degenerate FWM based wavelength conversion applications. Here, the fiber is standard single mode fiber and serves as the brace. The grown graphene sheet is transferred on silicon-on-insulter (SOI) for SEM characterization as show in −1, respectively. The measured I2D /IG ratio of 1.65 confirms the formation of monolayer graphene2/Si substrate is almost defect-freeTo fabricate the nonlinear optical device based on a single-layer graphene, as shown in How to cite this article: Hu, X. et al. Phase Conjugated and Transparent Wavelength Conversions of Nyquist 16-QAM Signals Employing a Single-Layer Graphene Coated Fiber Device. Sci. Rep.6, 22379; doi: 10.1038/srep22379 (2016).


The aim of this opinion article is to understand to what extent idiopathic pulmonary fibrosis (IPF) can be considered, in its clinical and pathogenic features, similar to cancer. Indeed, IPF has common risk factors with cancer, a low survival, and, most importantly, epigenetic and genetic alterations, abnormal expression of microRNAs, cellular and molecular aberrances, and the activation of similar signalling pathways.The pathogenic link between the two diseases may have a number of practical consequences. It may improve our understanding of IPF drawing on cancer biology knowledge. In addition, the recognition of similar pathogenic pathways may also encourage the use of cancer drugs for the treatment of IPF. Nintedanib, an inhibitor of tyrosine kinase receptors initially developed for cancer, has been recently approved for the treatment of IPF thanks to the observation that these receptors are also abnormally activated in IPF.The vision of IPF as a cancer-like disease may improve our understanding of the pathogenesis of this disease also opening new scenarios for repositioning cancer drugs for IPF. In addition, it may increase the level of awareness towards this dreadful disease at the public, political, and healthcare level. According to the current pathogenic hypothesis, idiopathic pulmonary fibrosis (IPF) is the result of chronic damage of the alveolar epithelium leading to a series of events responsible for abnormal tissue repair and the profound changes of the alveolar structure that characterize this disease . This alHowever, the study of the association between IPF and lung cancer with the relative clinical and therapeutic implications is far beyond the objectives of this opinion article. The aim of this article is instead to go through the scientific reasons that may support the intriguing vision of IPF as a disease with a cancer-like nature and to understand if this point of view may have positive consequences on the overall management of IPF.Aging, cigarette smoke, diet, and chemical and physical factors, through the methylation of suppressor genes or the hypomethylation of oncogenes, may affect the activity of some genes without changing their DNA sequences. It is recognized that these “epigenetic” changes are involved in the initiation and progression of cancer. For instance, the reduced expression of the Thy-1 glycoprotein, due to the hypermethylation of the promoter region of the gene coding for this glycoprotein, is associated with a more invasive behaviour of cancer. Similarly, the loss of expression of the same glycoprotein in IPF is related to the differentiation of fibroblasts into myofibroblasts within “fibroblast foci” , 9. MoreTissue homeostasis is maintained by cell-to-cell communication through junctional channels named connexins that facilitate the interaction and coordination of cell functions. Cancer cells are instead characterized by an impairment of intercellular communication suggesting that, in order to proliferate, these cells need to isolate themselves from the influence of surrounding normal cells. Lower levels of connexin 43 (Cx43) have been described in many cancers, including lung and gastric cancer. Similarly, IPF myofibroblasts, have a reduced ability to express connexin 43, suggesting that the loss of proliferative control in IPF cells could be caused by an altered fibroblast-to-fibroblast communication caused by a reduced expression of connexin 43 –27. The ex vivo fibroblast cell lines. Furthermore, both p110γ pharmacological inhibition and gene silencing were able to significantly inhibit proliferation rate as well as a-SMA expression in IPF fibroblasts. This suggests that PI3K p110γ isoform may have an important role in the pathogenesis of IPF and can be a specific pharmacological target. More recently, researchers have focused on another signal transduction pathway frequently altered in many cancers: the JAK-STAT pathway [in vitro and in vivo in a rat model of bleomycin-induced fibrosis [A large variety of signal transduction pathways are involved in the pathogenesis of cancer and IPF. The Wnt/β-catenin signalling pathway regulates the expression of molecules involved in cancer progression and tissue infiltration such as matrilysin, laminin, and cyclin-D1. This pathway is also strongly activated in IPF lung tissue as evidenced by extensive nuclear accumulation of β-catenin at different involved sites such as bronchiolar lesions, damaged alveolar structures, and fibroblast foci , 39. The pathway . This pa pathway , a lower pathway , 46. Thefibrosis , 48. Thefibrosis . NintedaAccording to the long list of pathogenic analogies between cancer and IPF, we might easily conclude that these two diseases are indeed similar. However, there are at least three arguments, apparently against this concept, that still need to be clarified: (1) myofibroblasts within fibroblast foci are polyclonal whereas cancer cells are thought to be monoclonal; (2) IPF is limited to the lungs, cancer metastasizes; and (3) IPF is by definition bilateral, cancer is always unilateral. These arguments, in our opinion, are largely based on general beliefs and do not weaken in any way the concept of the cancer-like character of IPF. Effectively, a number of studies have unequivocally shown that only some cancers are monoclonal whereas many others are instead marked by the same cytogenetic heterogeneity observed in IPF cells. Similarly to IPF, myofibroblastic cancers, such as desmoid tumours, are locally aggressive but unable to metastasize and ultimately 2–6 % of cancers have a bilateral and synchronous involvement –58.Having removed any conceptual obstacle to the view of IPF as a disease with a close link to cancer biology, this commentary will briefly underline the main reasons explaining why this vision may be relevant not just conceptually but also clinically. The awareness of cancer as a severe and potentially deadly disease is widespread in public opinion, leading to fund-raising campaigns and gatherings of private and governmental funds, resulting in the continuous progression of basic and clinical research. The development of dedicated diagnostic tools, the realization of large screening programs, the launch of a variety of new anti-cancer drugs, as well as the creation of personalized treatments have been achieved due to the fight against cancer during the last few decades. On the contrary, the awareness of IPF as a fatal disease, with a prognosis worse than the majority of cancers, is still limited to few experts and to those patients and families who are directly involved with the disease. The concept of IPF as a cancer-like disorder of the lung may help in meeting the urgent need for a better understanding of the disease and may increase the attention given to IPF at a public, political, and healthcare level. In addition, the existence of pathogenic links between IPF and cancer may improve our understanding of the pathogenesis of IPF, taking advantage of the large knowledge that already exists of cancer biology or even “borrowing” drugs, as in the case of nintedanib, which have been developed or used for cancer. Finally, the recognition of new common pathogenic mechanisms between IPF and cancer may also encourage new IPF clinical trials with other common oncologic drugs, alone or in combination with other drugs, or even through personalized treatments as largely experimented in cancer.