By merging bottom-up and top-down strategies we tailor graphene's electronic properties within nanometer accuracy, which opens up the possibility to design optical and plasmonic circuitries at will. In a first step, graphene electronic properties are macroscopically modified exploiting the periodic potential generated by the self assembly of metal cluster superlattices on a graphene/Ir(111) surface. We then demonstrate that individual metal clusters can be selectively removed by a STM tip with perfect reproducibility and that the structures so created are stable even at room temperature. This enables one to nanopattern circuits down to the 2.5 nm only limited by the periodicity of the Moiré-pattern, i.e., by the distance between neighbouring clusters, and different electronic and optical properties should prevail in the covered and uncovered regions. The method can be carried out on micro-meter-sized regions with clusters of different materials permitting to tune the strength of the periodic potential. Two main routes are usually followed to modify graphene's electronic and optical properties. On the one hand, bottom up approaches have proven to be efficient to change the overall electronic structure of graphene, enabling for example, the gap opening at the Fermi energy124578911121315131719π-bands with the characteristic linear dispersion and Fermi velocity of free standing graphene are only modified by the appearance of a small gap less than 100 meV227Let us first outline the bottom-up approach12452112325Additionally, this Moiré pattern formed by the graphene monolayer and the Ir(111) substrate can be used as a template for networks of monodisperse clusters of transition metals25The experimental bottom-up procedure is the following. We first grow a graphene monolayer on an Ir(111) substrate by chemical vapor deposition (CVD) of ethylene in UHV environments with the Ir(111) substrate held at 1050°C. Then, by evaporationg W or Ir from high purity filaments, we subsequently cover it with a hexagonal array of metal clusters with 2.5 nm periodicity (see methods for details on the sample preparation).‘graphene’ regions, i.e., ones covered with clusters and uncovered ones, with supposedly different electronic properties. It is noteworthy that our method can be used with clusters of different metallic elements, see We will now turn to the above mentioned top-down approach where STM appears as an ideal technique to tackle local manipulation on such samples due to its ability to modify and pattern 2D samples with ultimate resolution3031The procedure we have developed to engineer graphene nanostuctures consists in selectively removing single metallic clusters on top of graphene by gently approaching the STM tip towards them, as schematized in During the patterning process, the tip resolution is very robust and we usually observe almost no changes in our resolution after each cluster removal see . It furt25The possibility of picking up or manipulating individual clusters formed on the Moiré-pattern was previously mentioned25To this end, we have performed a careful study of the probability of removing a cluster as a function of both the tip-sample approaching distance and the bias voltage applied to the sample during the whole process, see 0G = 2e2/h, e: electron charge; h: Planck's constant), indicating that the extraction of a metal cluster involves the formation of an atomic size contact. Similar G ≈ 0G values have been reported for contacts between an atomically sharp Au tip and graphene regions strongly bonded with a metal substrate2 to sp3, in a similar way as reported for graphene regions underneath metal clusters on the graphene/Ir(111) systemTo get more insight into the physical processes involved in the cluster extraction, we recorded the current during the vertical displacement of the STM tip (I-Z curves), see for example inset of 35363736372 STM image of a graphene region uniformly covered by metallic clusters is shown in Let us now address several key points to infer the actual potential of our method to architecture functional graphene nanostructures, in particular, size limits, stability and quality. The range of applicability is obviously limited by the size of the nanostructures that can be created. We can build nanostructures from the 2.5 nm limit given by the Moiré pattern distance to the few micrometers one which is given by the typical STM scanning range; the possibility of growing single Moiré patterns domains extends over several micrometersFinally, we want to comment on the state of the graphene layer after the removal of the clusters. We aim to use pristine graphene on Ir(111) domains as one of our building blocks, thus, we need our cluster extraction method to produce perfectly clean graphene regions. To this end, we show in The full potential and applicability of our nanostructures is realized if the covered and uncovered regions display different electronic properties which has only been demonstrated for the homogeneous systemsπ→π* transitions between the valence and conduction band, so-called interband plasmonsE ≤ 0.4 eV should be forbidden in the covered, but allowed in the uncovered regions and can thus be localized to small graphene areas by removing the upper Ir-clusters, see L posses normal modes corresponding to the wave number q = nπ/L of the interband plasmon (q ≈ 0.03(0.05)Å−1 and energy E ≈ 0.25(0.375) eVL ≈ 10.5(6.2) nm for n = 1 or L ≈ 21(12.5) nm for n = 2. These length scales are well within the reach of our technique  system, plasmonic excitations have been measured by electron energy loss spectroscopyR can be obtained, see R ≤ 7 nm. In this regime, the uncovered area could resemble a quantum bit with qubit states “zero exciton” or “one exciton”. The excitonic states can further arbitrarily be connected by conventional wave function overlap or via Förster energy transfer which is mediated by the Coulomb interaction between the excitonic states, see A second and exciting new feature is given by the possibility to confine charged carriers, i.e., electrons as well as holes, within arbitrary geometrical regions due to locally modifying the electronic gap. One could hence design graphene quantum dots or nanowires of arbitrary size and form limited only by the cluster size of 2.5 nm which has to be contrasted with graphene nanostructures obtained by electron beam lithography and subsequent etching which have typical dimensions L = 20–100 nm. Using the effective-mass-approximation and thus the standard Dirac Hamiltonian with a variable mass profile, the discrete spectrum of a circular quantum dot as function of the radius sp2- to sp3-bonding on the covered regionsThe feasibility of the above proposals crucially depends on the impact that the Iridium substrate and the clusters on top have on the electronic properties of the graphene layer. Even though from ARPES experiments the band structure hardly seems to be affected beyond the gap of Δ ≈ 0.1 eV and ≈ 0.4 eV, respectively, the graphene Dirac cone has been reported to hybridize near the Fermi level with the S1 surface state of Ir(111)To conclude, we have presented a perfectly reproducible nanopatterning technique for graphene that combines bottom-up with top-down approaches. The precision is related to the periodicity of the Moiré-pattern that is formed by the graphene layer with the underlying substrate. Presupposing locally distinct electronic gaps in the covered and uncovered regions, new devices could be tailored with nano precision and we propose a novel platform for plasmonics relying on inter- rather than on intraband transitions. Also single graphene quantum dots/wires could be designed at will and arranged to arbitrary circuitries. Determining the optical gap and relaxation properties of mass-confined Dirac electrons via optical near-field scanning spectroscopy, emission spectroscopy or even transmission spectroscopy by chemically reduce the thickness of the sample would provide new insight on the role of the Ir(111) substrate on the excitonic decay rate. Finally, we note that the electronic spectrum drastically changes in the presence of a magnetic field due to the appearance of the zeroth Landau level not present in conventional semiconductor quantum dots which could be observable via Terahertz magneto-Raman spectroscopy.The STM experiments were performed with a home-built variable temperature instrument44+ sputtering at 850°C. The growth of graphene on the clean Ir(111) surface was performed by chemical vapor deposition (CVD) of ethylene (3 × 10−7 Torr during 1 min) in UHV environments with the Ir(111) substrate held at 1050°C. Under such conditions, small areas of the Ir(111) substrate remained intentionally uncovered by graphene, which allowed us to estimate the coverage of W or Ir used for the cluster formation. W and Ir were evaporated from high purity filaments composed of each corresponding material. An accurate calibration of the deposition rate as a function of the filament temperature, measured by an infrared pyrometer, was performed by means of STM images acquired on areas of bare -uncovered by graphene- Ir(111).Ir(111) surfaces were cleaned by 1 keV ArThe experimental data (plasmonic excitations in graphene on Ir(111) and Pt(111)) was obtained from the original publications and fitted to the theoretical predictions using the least-square method. The electronic properties of graphene were modeled using the standard effective-mass-approximation. Exciton lifetimes and hopping amplitudes were estimated via Fermi's Golden Rule.A.J.M.G. carried out the experiments supported by I.B. and J.M.G.R. A.J.M.G. and J.M.G.R. designed the research helped by I.B. A.J.M.G., I.B and J.M.G.R. analyzed the data. T.S. performed the main calculations with the collaboration of A.G.R. I.B. wrote the manuscript with the help of T.S. All authors contributed to the scientific discussion and revised the manuscript.Supplementary Material for the article Towards scalable nano-engineering of grapheneNano-lithographing Graphene with 2.5nm precisionNano-lithographing Ir on graphene with 2.5nm precisionNano-lithographing W on graphene with 2.5nm precision


When Hubel  referred Our interest in layer 1 (L1) of cortex sprang from the knowledge that it was the final destination of basal ganglia output. In spite of the fact that the whole cortex is represented in striatum and movement is certainly not its only function, here we review thalamic output to motor cortex because of our long-standing interest in movement and the basal ganglia. Similarly L1 is not only present in motor cortex but invests the complete cortex. In theorizing about basal ganglia and their influence on the “crowning enigma” it is as important to remember that movement is only the most obvious output and the easiest outcome to measure from both the dark basements of the brain and its crowing glory. With its plastic spines and input from so many parts of the environment L1 might be the best place to provide the necessary context for movements, or for perceptions, both of which, like all decisions of the cortex, need information from many sources including historical experience that can be coded in the pattern of spines.L1 is recognized throughout the entire cerebral cortex. Cajal  describeFrom work performed in mice and rat neocortex  three types of neurons in L1 are described as non-pyramidal GABAergic: Cajal-Retzius, elongated neurogliaform and single bouquet. Cajal-Retzius are the earliest born  containing axons reach any given square millimeter of rat L1  and midline rhomboid nucleus  is considered the area where afferents from globus pallidus , substantia nigra reticulata (SNR) and deep cerebellar nuclei form terminal fields in separate nuclei of the ventral thalamus: ventrolateral (VL), ventral anterior (VA) and ventromedial (VM). According to Scheibel and Scheibel  the bestTerminal sites of afferent axons to MT are conserved across species  in relation to basal ganglia output indicated that increases in GPi(EP) and SNR activity resulted in decreased VM activity  and inhibitory GABAThe presence of a cooperative integration between the distal dendritic tuft and the axosomatic compartment of pyramidal cells has been studied following co-activation of both compartments. It has been observed for example, that axosomatic action potentials can be generated by the coincidence of back propagation of action potentials and the effect of distal dendritic excitatory potentials  or the hyperpolarization-activated current (Ih) plus the effects of specific activation of neurotransmitter receptors are important mechanisms for microcircuit control. Kv channels in the distal apical dendrite of layer 5 pyramidal neurons control the interaction between subthreshold tuft excitatory input and trunk spike generation  and at low density in the somatic region with two different effects: an inhibitory action in the tuft that controls initiation and propagation of dendritic spikes, and an excitatory action in the soma that decreases the threshold for action potentials  on L1 is also important in the control of tuft currents and local interneurons. For example, α-adrenergic agents decrease Other neurotransmitters acting on L1 are also relevant for cortical function. For example, ascending ACh axons extend preferential terminations in L1 ) and caudolateral VA/VL receives GLU terminals from cerebellum. VM and rostromedial VA/VL project to L1 and caudolateral VA/VL to layer 2/3.Apart from cerebellar and basal ganglia influences relayed to motor cortex via MT, indirect cerebellar inputs reach basal ganglia through midline/intralaminar outputs.Plastic related changes in spines of pyramidal dendritic tufts make learning-related structural changes a likely substrate for learning in L1.What is the role of the large synaptic terminals from SNR to VM described as “detonator drives” that typically favor neurotransmitter spillover and volume transmission?What is the function of the synaptic contacts made in basal ganglia from MT and midline/intralaminar axons as they travel to motor cortex? Which neurons are being contacted?What is the function of the electrically coupled elongated neurogliaform interneurons?Is 5HT released in L1 involved in learning-induced dendritic changes as are other neurotransmitters released in the area?The excitatory input of L1 as studied in many cortical areas regulates the active regenerative electrical activity of dendrites of pyramidal cells of layer 2/3 and 5. This excitatory top down control on the dendritic tuft is crucial for integration and further generation of axosomatic discharge.Recent combined electrophysiological and behavioral observations indicate that L1 can be considered as a driver of pyramidal neurons with important behavioral consequences such as attention, expectation, anticipation, and action command.We would like to consider projections from MT and midline/intralaminar nuclei to L1 as a regulatory entity of pyramidal cell excitability in motor cortex. These projections can provide the necessary binding input to trigger or suppress final patterns of activity that would ultimately generate appropriate behavioral responses. Motor thalamus has already been labeled a “super integrator” (Bosch-Bouju et al., A source of concern when dealing with MT and midline/intralaminar afferents to L1 is the extent of their projection as observed in other neocortical areas. It is hard to conceive the point-to-point modulation suggested by some electrophysiological results e.g., Jiang et al. . PerhapsChanges in dendritic spines induced by motor activity and learning observed in dendritic tufts in L1 underline the functional rewiring observed as changes in spine morphology and neuronal dynamics (Yuste and Bonhoeffer, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


By conducting BLAST searches of the databases, we screened 22 introns (37–65 nt) with conserved lengths and sequences among closely related species. We then provide experimental and bioinformatic evidence for the splicing of 15 introns, of which 12 introns were remarkably G-rich and 9 introns contained completely inefficient splice sites and/or branch sites. These unorthodox characteristics of ultra-short introns suggest that there are unknown splicing mechanisms that differ from the well-established mechanism.According to the length distribution of human introns, there is a large population of short introns with a threshold of 65 nucleotides (nt) and a peak at 85 nt. Using human genome and transcriptome databases, we investigated the introns shorter than 66 nt, termed ultra-short introns, the identities of which are scarcely known. Here, we provide for the first time a list of  Pre-mRNA splicing is an essential process in eukaryotic gene expression. In higher vertebrates, however, the length of the target introns that need to be recognized vary markedly, from <50 nucleotides (nt) to >500,000 nt with degenerated splicing signals, or 5′ and 3′ splice sites at the ends of the introns. Interestingly, there are two statistical modes in the length distribution of eukaryotic introns; in humans, there is a narrow distribution of short introns with a peak at ~90 nt and a broad distribution of long introns with a peak at ~2000 nt ,2,3.in vivo and in vitro. According to this system, the essential splicing sequences in pre-mRNA, namely the 5′ splice site, the branch-site sequence, and the 3′ splice site, are simultaneously bound by the splicing factors U1 snRNP, U2 snRNP, and U2AF65/U2AF35, respectively, leading to early ATP-dependent formation of the spliceosomal A complex , which are very efficiently spliced iewed in ,5). Eleciewed in ). Nevertin vitro . Therefobona fide tiny human introns that are actually spliced. There are currently no objective- or evidence-based rules to determine the minimum intron length in the gene annotation procedure. We could list 22 evolutionarily conserved introns that are shorter than the 66-nt threshold, which we termed ultra-short introns. These ultra-short introns have not been characterized elsewhere. We then confirmed 15 introns with mRNA-Seq data, database annotations, and experimental evidence obtained by reverse transcription–polymerase chain reaction (RT–PCR) analysis. We demonstrated that nine ultra-short introns are actually spliced out, a process that is often required to escape from nonsense-mediated mRNA decay (NMD), suggesting that splicing of these ultra-short introns is a prerequisite for the expression of the host genes.To address this question, it is essential to identify and verify Using sequence data deposited in a human annotated transcriptome database (H-InvDB), we plotted the distribution of intron lengths . All of The authenticity of introns in the ultra-short range has never been investigated. Here, we validated the candidate introns in the ultra-short range to make a list of genuine ultra-short introns.To select unequivocal introns in the ultra-short range, we performed three screening steps in 4528 tentative intronic sequences retrieved from H-InvDB. Using a human mRNA/cDNA sequence database, we first eliminated the transcripts that were erroneously mapped in H-InvDB with insertions/deletions to the genomic sequence. From the resulting 4027 candidate introns, conserved introns were selected by two kinds of BLAST searches: one targeted a genomic database using flanking exons of the candidate introns as queries, and the other targeted a transcriptome database using concatenated flanking exons as queries. Only candidate introns for which the results of both BLAST searches contained the same species with high similarity scores were selected. As a result, we obtained 1253 short introns (37–85 nt), of which 23 were in the ultra-short range (37–65 nt). By removing one ultra-short intron, which was a partial transcript, 22 candidate introns were finally determined as conserved ultra-short introns .The authenticities of the 22 candidate ultra-short introns were further validated based on three criteria: (i) The presence of individually sequenced data in the International Nucleotide Sequence Database Collaboration (INSDC) databases (DDBJ/EMBL/GenBank); (ii) Verification with read sequences in the published mRNA-Seq data; and (iii) Our experimental verification by RT–PCR analysis of endogenous transcripts. Finally, we identified 15 ultra-short introns that fulfilled at least one of these three criteria (“SN” in (i) We inspected the original transcript data submitted to the INSDC database to judge whether the transcripts were cloned and sequenced individually at the specific interests of the investigators, or whether they were automatically sequenced in a high-throughput sequencing project. We found that eight introns were individually sequenced  We confirmed whether the 22 ultra-short introns are spliced out by searching the publicly available RNA-Seq database of healthy human tissues—A total of 20 samples from 18 tissues; 16 tissues from BodyMap 2.0 project see  and an aSpliced” in in vitro [(iii) We performed RT–PCR experiments to examine whether the selected ultra-short introns are actually spliced out by endogenous splicing in human tissues. These experiments demonstrated that nine ultra-short introns were spliced out from the endogenous transcripts  were located within the untranslated region (UTR) while the other 20 ultra-short introns were located within the coding sequences CDS; . Of thes and SN16GGGGCTGGG) in 43-nt  and 49-nt  introns that functions as an intronic splicing enhancer (ISE) [We found that the G-content in conserved human introns gradually increases from the short range 85 nt) to the ultra-short range ≤65 nt; , which i5 nt to tThe essential core splicing signals  in the listed ultra-short introns were evaluated using the SROOGLE Web tool see . AlthougUsing the SROOGLE tool, we also searched for various exonic splicing enhancer (ESE) sequences in upstream and downstream of the ultra-short introns, which would potentially underpin nearby suboptimal, or weak, splice sites . TheThe evident steric hindrance of the ultra-short introns together with the unorthodox sequence features suggests the presence of multiple splicing mechanisms that are distinct from the authentic spliceosome-dependent mechanism of action . Herevia nonfunctional splice sites and branch site indicates that the splicing reaction occurs without either U1 or U2 snRNP. U1 snRNP-independent splicing was reported to occur under conditions of enriched SR proteins [Drosophila spliceosome with a shorter intron (62 vs. 147 nt as the control) have a smaller head domain containing the early “A” complex, suggesting that the complex with a shorter intron consists of fewer splicing factors [(i) Splicing of ultra-short intron proteins ,23 or inproteins ,25. This factors .trans-acting factor that recognizes a particular ISE. The 5′ splice site activation mechanism in which the G-triplet interacts with U1 snRNP via non-canonical base pairing with the U1 snRNA [in vivo splicing studies in HeLa cells showed partial splicing depression of G-rich 49-nt  and 43-nt  introns by disruption of U1 snRNA (unpublished data). The non-canonical U1 snRNA binding in these two introns (SN5 and SN3) remains to be analyzed.(ii) The G-rich ISEs, instead of containing authentic splice site and branch-site sequences, could be critical for the recognition of ultra-short introns for splicing. We have demonstrated that a specific ISE, CAGGGGCTGGG, is essential to splice out two ultra-short introns (SN3 and SN5) , and we U1 snRNA  might bein vitro [(iii) A curious but important question is whether the G-rich ultra-short intron, which lacks an authentic branch site, is excised as a canonical lariat structure or not. We previously showed that a 56-nt intron  containing effective 5′ and 3′ splice sites and a branch site  was exciin vitro . Howeverhttp://hinv.jp/) [http://www.insdc.org/). Therefore, we used all of the human mRNA/cDNA sequences in the INSDC, except for computationally generated transcripts (pHITs and eHITs).An integrated database of human genes and transcripts, H-InvDB Web server  , was useftp://ftp.ncbi.nih.gov/genomes/H_sapiens/ARCHIVE/BUILD.36.3/Assembled_chromosomes/). Candidate intron sequences lacking a terminal GT and AG  were eliminated.The sequences of the candidate introns were extracted from the human genome reference sequence , we performed three screening steps using 4528 tentative introns retrieved from H-InvDB. The first BLAST searches against the human transcriptome were queried by exonic sequences derived from genome coordinates of H-InvDB annotation. We could accordingly eliminate the H-InvDB transcripts that were erroneously mapped to genomic sequences with insertion/deletion generated by over-fitting to the GT–AG rule in the automated H-InvDB annotation process. This process yielded 4027 candidate introns, which were then screened by two kinds of BLAST searches: (i) NCBI Transcript Reference Sequences were queried for concatenation of both flanking exons, and (ii) Genomic Reference Sequences were queried for introns with both flanking exon sequences. These procedures were applied to each intron with the following thresholds: Length of obtained sequence >55; Bit score >99; E-value ≤1×10−19; Identity ≥96%.To select unequivocal introns in the short range  were first checked against the source of the transcript sequences described in the INSDC database.http://www.ensembl. info/blog/2011/05/24/human-bodymap-2-0-data-from-illumina/). This dataset contains mRNA-Seq data from 16 human tissues, including adrenal, adipose, brain, breast, colon, heart, kidney, liver, lung, lymph, ovary, prostate, skeletal muscle, testes, thyroid, and white blood cells. We also investigated the sequence read archive databases (http://www.insdc.org/), which contain mRNA sequences derived from healthy human tissues: Brain (SRP002274), pancreatic islet (SRP010483), gastric tissue (SRP012016), and white blood cells (ERP000177). We generated index FASTA files for exonic fragments with defined lengths across each splice junction of the 22 ultra-short introns using ERANGE (getsplicefa.py program) [To gather evidence for the splicing of the 22 selected introns, we examined the following mRNA-Seq datasets. The BodyMap (2.0) project was searched via the Ensembl browser (program) , and theprogram) .We experimentally confirmed the splicing of these 22 introns using RT–PCR assays targeting endogenous gene transcripts. Total RNA extracted from human tissues  was reverse transcribed with PrimeScript reverse transcriptase (Takara Bio) and oligo-dT primers. To detect the spliced products, the reverse-transcribed cDNAs were amplified by PCR with specific primers , and collected annotated information for each intron and the hosted gene  through H-InvDB (http://www.ensembl.org/index.html). We finally identified the encoded proteins of the host genes using the curated results of H-Inv proteins, which were categorized based on the similarity between the predicted amino-acid sequence and the known sequences (http://hinv.jp/hinv/help/help_proteins.html).The frequency of the HITs spliced at the exact splice junctions corresponding to the ultra-short introns is represented as the ratio of the number of HITs spliced at the ultra-short introns to the number of all aligned HITs across the ultra-short intron region. We also checked for the presence of ultra-short introns in the Ensembl transcript database (http://sroogle.tau.ac.il) [The essential core splicing signals  of the 22 selected ultra-short introns were analyzed using SROOGLE Web server (u.ac.il) . This inu.ac.il) .


Vascular smooth muscular cell (VSMC) involve in vessel tone regulation and endothelial cell function. It is known and critical player in pathological conditions including atherosclerosis. VSMC is dynamic structure and response to different stimuli. MiRNAs molecules are important regulatory mechanism to promote/suppress VSMC proliferation and phenotype switch.Aim: We hypothesized that patients with acute myocardial infarction have altered VSMC transcriptome affecting cell phenotype and proliferation.® Human Genome U133 Plus 2.0 Array. The differently expressed mRNA/miRNA data were validated on independent cohort of patients with RT-PCR method assay.Methods: Aortic wall tissue obtained during coronary artery bypass surgery underwent laser dissection of VSMC. Frozen aortic sections were stained with Arcturus LCM kit to identify cells of interest follow by cell collection and tRNA extraction. The transcriptome profile will be performed using Affymetrix GeneChipResult: We identify nine miRNA differentially expressed in patients with acute myocardial infarction, with four of them related to VSMC proliferation and differentiation. The most prominent down regulated miRNA was known regulator of VSMC phenotype miR-143. Among up regulated miRNA, miR-486-5p and miR-29a-3p reported before as a potent regulator of VSMC function. Pairwise analysis with mRNA data from the same patients reveal reciprocal and statistically significant changes in the phenotype and proliferation related mRNA.Conclusion: In patients with acute myocardial infarction we observe remote phenotype and proliferation shift in the aortic wall VSMC. Switch of contractile phenotype and proliferation alteration might play compensatory role in patient with unstable plaque and myocardial infarction.


Gas permeability data are presented for mixed matrix membranes (MMMs) of few-layer graphene in the polymer of intrinsic microporosity PIM-1, and the results compared with previously reported data for two other nanofillers in PIM-1: multiwalled carbon nanotubes functionalized with poly(ethylene glycol) (f-MWCNTs) and fused silica. For few-layer graphene, a significant enhancement in permeability is observed at very low graphene content (0.05 vol.%), which may be attributed to the effect of the nanofiller on the packing of the polymer chains. At higher graphene content permeability decreases, as expected for the addition of an impermeable filler. Other nanofillers, reported in the literature, also give rise to enhancements in permeability, but at substantially higher loadings, the highest measured permeabilities being at 1 vol.% for f-MWCNTs and 24 vol.% for fused silica. These results are consistent with the hypothesis that packing of the polymer chains is influenced by the curvature of the nanofiller surface at the nanoscale, with an increasingly pronounced effect on moving from a more-or-less spherical nanoparticle morphology (fused silica) to a cylindrical morphology (f-MWCNT) to a planar morphology (graphene). While the permeability of a high-free-volume polymer such as PIM-1 decreases over time through physical ageing, for the PIM-1/graphene MMMs a significant permeability enhancement was retained after eight months storage. High-free-volume polymers, such as polymers of intrinsic microporosity (PIMs), have attracted attention for their high gas permeabilities [a) [et al. [et al. [Membranes are currently used for a variety of industrial gas separations, including nitrogen generation from air and the removal of COural gas . Howeverbilities . Howeverbilities ,5. The abilities , can givties [a) –9, the a [et al. ,11 for m [et al.  for a fuPMMM of a MMM containing a volume fraction ϕF of impermeable, spherical filler particles within a polymer of permeability PP, is given by equation (??).2 [Simple models of MMMs predict that the addition of an impermeable nanofiller to a permeable matrix will reduce permeability compared with the unfilled material. For example, the Maxwell model  predictsnau & He  reportednau & He , MgO [15 & He [2 , in varib). The randomly twisted shape makes it difficult for molecules to pack, and because there are no single bonds in the backbone about which rotation can occur, they cannot undergo the types of conformational change that enable conventional polymers to rearrange and fill space.The permeability of a glassy polymer is linked to the amount and distribution of free volume within the material. Glasses are in a non-equilibrium state, and many factors may influence the way the molecules pack together and, thus, the free-volume distribution. High-free-volume glassy polymers have high permeabilities as a result of frustrated packing of the constituent macromolecules. In a PIM, this is a consequence of the molecular structure of the polymer chain, which is composed of fused-ring sequences interrupted by sites of contortion such as spiro-centres b. The rac–e).The presence of a nanofiller surface restricts the conformational freedom of polymer chains in its vicinity, which may further frustrate the ability of the chains to pack together, thus increasing free volume and enhancing permeability. It is a reasonable hypothesis that the effect of a nanofiller will depend not only on its available surface area but also on the curvature of the surface at the nanoscale. Here we compare an essentially planar filler (graphene), with a cylindrical nanofiller (f-MWCNTs), and a filler composed of more-or-less spherical nanoparticles (fumed silica) c–e.ca 10 regularly stacked layers is generally regarded as graphitic. For the present work, few-layer graphene was considered a suitable planar nanofiller for comparison with multiwalled carbon nanotubes.The term ‘graphene’ may be applied to a wide range of materials. A classification framework for graphene-based materials has been suggested  and a re2.(a)′,6,6′-Tetrahydroxy-3,3,3′,3′-tetramethyl-1,1′-spirobisindane  was dissolved in methanol and re-precipitated from dichloromethane before use. Anhydrous K2CO3  was dried in an oven at 110°C overnight before use. Anhydrous dimethylacetamide (DMAc), toluene and methanol (MeOH), were purchased from Sigma-Aldrich and used as received. Natural graphite was purchased from NGS Naturgraphite GmbH.Tetrafluoroterephthalonitrile  was purified by sublimation; it was heated to around 150°C and the pure product collected without vacuum. 5,5(b)3 min−1 and the injection volume was 100 μl. Polystyrene standards of known molar mass were used for calibration.Gel permeation chromatography (GPC) measurements were carried out using a Viscotek GPC max VE 2001 instrument with two PL mixed B columns and a refractive index detector. Tetrahydrofuran was used as solvent at a flow rate of 1 cm1H nuclear magnetic resonance (NMR) spectroscopy was carried out using a Bruker 400 MHz spectrometer. For NMR sample preparation, PIM-1 (≈5 mg) was dissolved in deuterochloroform  and transferred into a 5 mm NMR tube.Ultraviolet–visible (UV–Vis) spectroscopy was carried out using a Cary 60 UV–Vis spectrophotometer at room temperature.(c)et al. [2CO3 (0.03 mol), DMAc (20 ml) and toluene (10 ml) were added to a round-bottomed flask equipped with a mechanical stirrer, nitrogen inlet and Dean–Stark trap. The mixture was refluxed at 160°C for 40 min, then the viscous solution was added to methanol to precipitate the product. The yellow product was dissolved in chloroform and reprecipitated from methanol. Further purification was carried out by refluxing the precipitate in deionized water overnight, then it was dried overnight in a vacuum oven at 110°C. GPC: Mn=43000, Mw=170000, Mw/Mn=4.0. 1H NMR : δ6.81 , 6.42 , 2.33 , 2.17 , 1.36 , 1.31 . IR : 3000–2800, 2238, 1443, 1262, 1107, 1009, 751. Elemental analysis, calculated for C29H20N2O4 (wt.%): C, 75.64; H, 4.38; N, 6.08. Found: C, 71.99; H, 4.17; N, 6.02.PIM-1 was synthesized by a method based on that of Du et al. . TFTPN ((d)Mn, and only a modest decrease in weight-average molar mass, Mw. To form the membrane with the highest graphene content (sample KA1-7-12(3)), a 5 ml portion of the stock solution was transferred into a 7.8 cm diameter flat-bottomed glass petri dish, and the solvent was allowed to evaporate over 3 days. For preparation of membranes with lower graphene contents, portions of the stock PIM-1/graphene solution were mixed with portions of a pure PIM-1 solution of concentration 35 mg ml−1. For samples KA-1-7(1), KA-1-7(2) and KA-1-7(3), 2 ml of stock solution were mixed with 2, 5 and 10 ml, respectively, of PIM-1 solution. For samples KA-1-7(5) and KA-1-7(6), 4 ml of stock solution were mixed with 20 and 40 ml, respectively, of PIM-1 solution. A 7.8 cm petri dish was used for samples KA-1-7(1) and KA-1-7(6), and a 6.8 cm dish for other samples.A stock PIM-1/graphene dispersion was prepared as follows: natural graphite (600 mg) and PIM-1 (1.4 g) were added to chloroform (100 ml). The solution was sonicated for 84 h  and then centrifuged twice at 6000 r.p.m. for 20 min to remove graphitic particles. Although prolonged sonication of a polymer solution might be expected to cause cleavage of polymer chains, control experiments with PIM-1 solutions showed little effect on number-average molar mass, (e)CG, was determined by UV–Vis spectroscopy. The absorption was measured at 660 nm and CG evaluated using a value of α=3620 ml mg−1 m−1 for the absorption coefficient, as determined by the Coleman group [−1 graphene, as well as 14 mg ml−1 PIM-1. The graphene contents of membranes used for permeation measurements were then calculated from the known dilutions. For calculation of the percentage graphene by volume, the graphene was assumed to have the same density as graphite (2.2 g cm−3).For determination of the graphene content, membrane KA-1-7(5) (0.23 g) was redissolved in 23 ml of chloroform. The graphene concentration of the solution, an group . This ga(f)Single gas permeation measurements were carried out in a fixed volume/pressure increase apparatus  at 25°C at a feed pressure of 1 bar, as described previously ,23.3.(a)Self-supported PIM-1/graphene MMMs were obtained by slow evaporation of the solvent from PIM-1/graphene dispersions in chloroform. A stock PIM-1/graphene dispersion was prepared by ultrasound-assisted exfoliation of natural graphite into a PIM-1 solution. Dilution of the stock dispersion with fresh PIM-1 solution enabled dispersions of various concentrations to be obtained, and hence MMMs with various graphene contents as indicated in 2 > H2 > He ≈ O2 > CH4 > N2. In the simplest model of membrane permeation, the solution-diffusion model, permeability coefficient P is the product of a diffusion coefficient D and a solubility or sorption coefficient S .3.1Theed PIM-1 .A significant enhancement in gas permeability is observed at low graphene content. This is discussed further and compared with the effect of other nanofillers below.(b)et al. [figure 2a. It can be clearly seen that while the Maxwell model predicts a decrease in gas permeability, the experimental results show a significant increase in the effective permeability of the MMM with increasing filler loading over the range 7–24 vol.%. Filler loadings above 24 vol.% were not investigated; it is difficult to achieve homogeneous dispersions at higher loadings. The nanoparticles used in that study were reported to have particle diameters in the range 11.1–13.3 nm, although there was evidence of larger aggregates in the MMMs. Filler density and Brunauer–Emmett–Teller (BET) surface area were reported as 2.2 g cm−3 and 205−245 m2 g−1, respectively.Data of Ahn et al.  for methet al. [figure 2b. The MWCNTs used for functionalization (FutureCarbon GmbH) were reported to have 8–12 walls and diameter in the range 12–15 nm. The BET surface area was 250 m2 g−1, similar to the fused silica described above. For the plot in figure 2b, a density of 2.1 g cm−3 was assumed in order to calculate vol.% filler; in practice, the density may vary depending on the diameter and number of walls [ca 1 vol.% filler, and then decrease.Permeability data from Khan et al.  for methof walls . It can b. For calculation of vol.% filler, the density of bulk graphite (2.2 g cm−3) was assumed. A similar pattern is seen with graphene as for f-MWCNTs, with an initial increase followed by a decrease with increasing filler content. While PIM-1 itself can exhibit substantial differences in permeability depending on sample history, it is noteworthy that broadly similar results were obtained by three different laboratories working with three different fillers, the CO2 permeability for methanol-treated PIM-1 being in the range 5000–6200 barrer, and the highest CO2 permeability achieved for these MMMs being about double, in the range 12 200–13 400 barrer. The significant difference between the fillers is the filler loading at which the greatest permeability enhancement is seen, which increases from ca 0.05 vol.% for few-layer graphene, to ca 1 vol.% for f-MWCNTs, to ca 24 vol.% for fused silica.Permeability data for methanol-treated PIM-1/graphene MMMs from the present work are included in figure 22 g−1 , but it(c)et al. [ca eight months storage under ambient conditions  to a cylindrical morphology (f-MWCNT) to a planar morphology (graphene).For this series of samples, the addition of just 0.05 vol.% few-layer graphene to PIM-1 gave a substantial enhancement in gas permeability; for CO


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.