TrkB allele (fB) mouse designed for deletion of TrkB to determine its role in the adult striatum.The high affinity tyrosine kinase receptor, TrkB, is the primary receptor for brain derived neurotrophic factor (BDNF) and plays an important role in development, maintenance and plasticity of the striatal output medium size spiny neuron. The striatal BDNF/TrkB system is thereby implicated in many physiologic and pathophysiologic processes, the latter including mood disorders, addiction, and Huntington’s disease. We crossed a mouse harboring a transgene directing cre-recombinase expression primarily to postnatal, dorsal striatal medium spiny neurons, to a mouse containing a floxed We found that there were sexually dimorphic alterations in behaviors in response to stressful situations and drugs of abuse. Significant sex and/or genotype differences were found in the forced swim test of depression-like behaviors, anxiety-like behaviors on the elevated plus maze, and cocaine conditioned reward. Microarray analysis of dorsal striatum revealed significant dysregulation in individual and groups of genes that may contribute to the observed behavioral responses and in some cases, represent previously unidentified downstream targets of TrkB.The data point to a set of behaviors and changes in gene expression following postnatal deletion of TrkB in the dorsal striatum distinct from those in other brain regions. Ntrk2). There are two major TrkB isoforms, including the full-length TrkB.FL containing the tyrosine kinase domain, i.e. tk+, and a C-terminal truncated form, TrkB.T1, lacking the catalytic tyrosine kinase domain. TrkB.T1 is a dominant-negative receptor that inhibits TrkB.FL signaling, but has many other functions, including as a signal transducing molecule -dATP. The probes had the following sequence: for Tacr1/substance P, complementary to bases 20–67, GenBank accession no. M68909; Penk/enkephalin, bases 304–351, M13227; Pdyn/dynorphin, bases 807–854, AF026537; Ntrk2/TrkB, bases 733–780, NM 008745; Ppp1r1b/DARPP-32, bases 1253–1300, BC031129. Striatal gene expression was determined in the nucleus accumbens . In the case of comparison between genotypes, unpaired two-tailed Student t-test was used. One-way ANOVA or two- way ANOVA with sex and genotype or drug and genotype factors were used to analyze the behavioral data. Bonferroni post-hoc comparisons were used with significant ANOVAs. Unpaired t-test was used to analyze western blot, Total RNA was extracted from the striatum of fB/fB and D9ΔTrkB transgenic mice using the Nucleospin RNA kit . RNA quantity was assessed with Nanodrop (Nanodrop Technologies) and quality with the Agilent Bioanalyzer (Agilent Technologies). Total RNA (200 ng) was amplified, biotinylated and hybridized on Illumina Mouse_8_V2 Beadchips, querying the expression of ~22,000 Refseq transcripts, as per manufacturer’s protocol. Slides were scanned using Illumina BeadStation and signal extracted using Illumina BeadStudio software . Raw data was analyzed as previously described using Bioconductor packages. Quality assessment was performed looking at the inter-array Pearson correlation and clustering based on top variant genes was used to assess overall data coherence. Contrast analysis of differential expression was performed using the ‘limma’ package . After lhttp://david.abcc.ncifcrf.gov/) to understand the potential biological relevance of differentially expressed genes. Canonical pathway analysis was performed using Ingenuity Systems Pathway Analysis (http://www.ingenuity.com).Gene Ontology (GO) terms were identified using the Database for Annotation, Visualization and Integrated Discovery (DAVID) (The authors declare that there is no conflict of interest.in situ hybridization or molecular genetic experiments, interpreted primary data and approved the final manuscript. All authors read and approved the final manuscript.EMU, MEP, TBB, EAT, HS and MEE conceived and designed the experiments, performed experiments, reviewed and interpreted primary data, and were involved in drafting and editing the manuscript. BX and LFR contributed reagents and edited the manuscript. JSM, KAP, JB, MR,and BT designed and performed behavior, Genes differentially expressed in striatum of D9ΔTrkB mice at P < 0.05.Click here for file


Long interspersed nuclear element 1 (L1) retrotransposons are the only autonomously mobile human transposable elements. L1 retrotransposition has shaped our genome via insertional mutagenesis, sequence transduction, pseudogene formation, and ectopic recombination. However, L1 germline retrotransposition dynamics are poorly understood because de novo insertions occur very rarely: the frequency of disease-causing retrotransposon insertions suggests that one insertion event occurs in roughly 18–180 gametes. The method described here recovers full-length L1 insertions by using hybridization enrichment to capture L1 sequences from multiplex PCR-amplified DNA. Enrichment is achieved by hybridizing L1-specific biotinylated oligonucleotides to complementary molecules, followed by capture on streptavidin-coated paramagnetic beads. We show that multiplex, long-range PCR can amplify single molecules containing full-length L1 insertions for recovery by hybridization enrichment. We screened 600 µg of sperm DNA from one donor, but no bone fide de novo L1 insertions were found, suggesting a L1 retrotransposition frequency of <1 insertion in 400 haploid genomes. This lies below the lower bound of previous estimates, and indicates that L1 insertion, at least into the loci studied, is very rare in the male germline. It is a paradox that L1 replication is ongoing in the face of such apparently low activity. Hum Mutat 32:1–11, 2011. © 2011 Wiley-Liss, Inc. Transposable elements are the most common class of repetitive DNA in the human genome, accounting for ∼45% of our DNA ) that can apparently insert anywhere within a large (3 Gigabase [Gb]) genome. Third, the frequency of insertion is likely to be extremely low, with the current estimates of de novo L1 activity predicting that a single insertion will occur in 1 in 9 to 1 in 186 humans, corresponding to a single de novo L1 insertion in 54 pg to 1.12 ng of germline DNA [Kazazian, Here we present the development of an L1 hybridization enrichment method capable of physically recovering complete L1 insertions into genomic targets devoid of L1 sequences. We illustrate the method's ability to recover full-length L1 insertions at the single DNA molecule level and present a study that enabled us to estimate an upper bound of the frequency of L1 retrotransposition in the human male germline at our selected loci.Sperm DNA was prepared as described previously [Jeffreys et al., http://www.repeatmasker.org), to locate nonrepetitive DNA suitable for primary and secondary polymerase chain reaction (PCR) primer design, as shown in Eight target loci known to have harbored disease-causing L1 insertions were selected for investigation, along with two additional loci (HoxD and MHC Class II) . None of2, 6.7 mM 2-mercaptoethanol, 113 µg/ml BSA, 1.1 mM dNTPs), 0.05 µM of each primer and 0.025 U/µl 20:1 Taq/Pfu polymerases . PCR cycling was performed in an MJ Tetrad PTC250 thermal cycler  at 96°C for 1 min, then at 96°C for 20 sec, 62°C for 1 min/kb of target amplicon plus 1 min for 30 cycles, followed by 61°C for 30 min. HPLC purified primers were supplied by Thermo Electron, and handled under PCR-clean conditions.A total of 20 µl PCR reactions contained 20 to 50 ng of genomic DNA (gDNA), PCR buffer  to an estimated concentration of 1 haploid genome/µl. PCRs were carried out in eight replicates of 1 µl input per dilution, then diluted 10-fold in 5 mM Tris HCl (pH 7.5) and 2 µl of the dilution used to seed nested secondary PCRs. Secondary PCR products were fractionated by agarose gel electrophoresis in the presence of 0.5 µg/ml ethidium bromide. The frequency of positive and negative reactions was used to Poisson estimate the maximum likelihood number of amplifiable molecules and its 95% confidence intervals [Jeffreys et al., Genomic DNA samples were serially diluted in 10-fold steps using single molecule diluent  to remove oligonucleotide primers and DNA polymerase that could interfere with hybridization enrichment. The aqueous phase was reextracted with chloroform and the purified DNA collected by ethanol precipitation. DNA was redissolved in 33 µl 5 mM Tris-HCl (pH 7.5) prior to hybridization enrichment.The principal stages of L1 hybridization enrichment are illustrated in 2, 6.7 mM 2-mercaptoethanol, 4.4 µM EDTA, 2 µg/ml single-stranded (heat denatured) high molecular weight herring sperm DNA). The washed beads were resuspended in a volume of 1 × DHB 1/12th that of the original volume. This working stock of beads was kept in the dark, on ice.M-280 streptavidin-coated super-paramagnetic Dynabeads  were captured with a Dynal MPC-S magnetic particle concentrator and washed three times at room temperature (resuspending each time) with 100 µl 1 × denaturing/hybridizing/binding buffer  prior to transfer to a fresh siliconized Eppendorf tube. The Dynabeads were finally captured and resuspended in 50 µl ED prior to thermal elution.Binding of the bio-oligo/DNA hybrids to Dynabeads , step 3 Single-stranded DNA was recovered from bead-bound bio–oligos , step 4 The unbound fraction collected at the first cycle of enrichment was reextracted, by adding more bio-oligos and following the annealing/binding/recovery procedure as above, to maximize DNA recovery. In total, one extraction and two reextractions were carried out per sample. The eluates from the extraction and the reextractions were pooled. A total of 33 µl of each pooled eluate was then subjected to a second round of hybridization enrichment as above, and again eluates from the secondary extraction and the reextractions were pooled. All eluates and washes were stored in the dark at 4°C.32P-labeled L1-specific oligonucleotide probe (PFLR5999). L1-sequence containing PCR products were detected by autoradiography. The remaining PCR products were fractionated by gel electrophoresis and stained with ethidium bromide (0.5 µg/ml). L1 sequence-containing bands identified by autoradiography were visualized using a Dark Reader trans-illuminator  and excised from the gel. DNA was extracted using the QIAquick gel extraction kit , cloned and sequenced (see below).The eluted DNA was subjected to nested PCR amplification using secondary target site primers Supp.  as shownE. coli cells. Plasmid DNA was recovered using the QIAprep Spin miniprep kit . A total of 20–30 ng/kb of plasmid DNA was sequenced using the Big Dye Terminator v3.1 ReadyReaction system  with M13F or M13R sequencing primers. Excess reaction components were removed using PERFORMA DTR Gel Filtration Cartridges  and samples were analysed on an ABI3730XL capillary sequencer (Applied Biosystems).Purified amplicons were ligated into the pGEM®-T Easy plasmid vector , following the manufacturer's protocol, and transformed into ultra competent DH5α http://www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cgi) and the GCG package . Assembled sequences were aligned with the human L1 element L1.3 (accession L19088) and the appropriate target site sequence  [Kazazian, 6 sperm, or ∼100 insertions per ejaculate (∼108 sperm).Ten target loci  were selBssSI allowed identification of DNA fragments derived from each target (32P-labeled target site-specific oligonucleotide probes (data not shown).Thermal cycling conditions were optimized to ensure efficient amplification of all 10 targets in a single 50-µl multiplex PCR seeded with 0.5 µg of sperm DNA, the maximum DNA input compatible with efficient PCR. Digestion of the 10-plex secondary PCR products with h target ; these fh target , indicatTo test whether multiplex PCR could also efficiently amplify molecules carrying a full-length L1 insertion, we added an extra primer pair specific for a locus containing the polymorphic AL121819 L1 insertion [Badge et al., Filled-site targets (12 kb) are much less efficiently amplified than empty sites (6 kb), as evident in 11 molecules over all 10 targets). It was therefore essential to recover de novo insertion PCR products by hybridization enrichment.To quantify the effect of this low PCR efficiency on de novo insert amplification and recovery, we analyzed the PCR product yield of an 11-plex PCR seeded with 0.5 µg of donor A gDNA, effectively containing ∼80,000 amplifiable molecules of the empty AL121819 site and ∼40,000 molecules of the filled site. This analysis indicated a gain of PCR products per cycle, from the empty and filled sites, respectively, of ∼1.8 and ∼1.6 over 20 cycles of PCR. This differential efficiency means that amplification of 0.5 µg gDNA containing a single amplifiable insertion molecule would produce 12,000 filled site molecules present in a huge excess of empty site molecules  [Jeffreys and May, −6% of empty target site molecules. This indicates a >500,000 fold enrichment of L1-containing amplicons. After multiplex PCR, a pool of DNA would contain ∼12,000 L1-containing molecules derived from each de novo insertion, plus ∼1011 empty target site molecules. Following a single round of enrichment, this ratio of filled to empty site molecules would increase from 1/8,000,000 to >1/16, allowing a single molecule of a de novo insertion to be readily detected.Following two rounds of optimized hybridization enrichment, we routinely recovered at least 2% of PCR-amplified L1-containing molecules, compared with <4.5 × 106 fold) of insert-free genomic DNA, and that hybridization enrichment was essential for their detection.To test whether DEASH could recover full-length L1 insertions at the single DNA molecule level, we mixed 24 pg sperm gDNA from donor A containing ∼2 amplifiable molecules of the AL121819 insertion (see above) with 0.5 µg of sperm gDNA from donor B, who lacks the AL121819 insertion. This DNA mixture, along with 95 additional reactions each containing 0.5 µg of sperm gDNA from donor B alone, was subjected to 11-plex PCR to amplify all ten targets plus the AL121819 locus. Amplified DNA from all 96 reactions was pooled and purified, and one-third of this DNA subjected to L1 hybridization enrichment. PCR amplification of the AL121819 target, either alone or as part of an 11-plex PCR for all targets, was followed by reamplification using primers designed to separately amplify the 5′ and 3′ junctions of the AL121819 insert . These PHaving established that hybridization enrichment could recover insertions at the single DNA molecule level, we screened 576 µg sperm gDNA from donor B for de novo L1 insertions. DNA was amplified by 10-plex PCR in 0.5 µg aliquots distributed over 12 96-well plates, and the PCR products from each plate pooled and enriched as above. Each of the 12 batches of enriched DNA was then screened for L1–containing target molecules using 10-plex (or duplex) primary PCR followed by nested PCRs as shown in A genuine de novo L1 insertion into one of the target loci should contain an L1 sequence and a poly A tail, flanked 5′ and 3′ by target site sequences. All of the 11 putative insertion amplicons showed sequence similarity to one of the target loci  most often associated with Alu elements (10/11). In 7 of the 11 cases the L1 and flanking sequence were >99% identical to regions of the genome harboring known L1 elements. Although no genuine insertions were identified, these chimaeric artifacts do provide further validation of the MP-HE approach, showing that L1 hybrid molecules generated during PCR amplification can be recovered by our strategy, but are easily identified as artifacts by sequencing.We used MP-HE to survey 576 µg of sperm DNA from a single donor for de novo L1 insertions. Eleven putative L1 insertions were identified, all containing L18 sperm, or 9.6 × 107 amplifiable molecules of each target under the assumption that single molecule PCR is 50% efficient when amplifying a 12-kb amplicon, as established for control target molecules carrying full-length L1 insertions. The 10 loci surveyed together cover 51 kb of target DNA per sperm, within which a de novo insertion could be detected. As half of the loci are on the X chromosome, and so only present in 50% of sperm, this is effectively reduced to 38 kb of DNA per sperm. We have therefore screened 38 kb × 9.6 × 107 = 3.7 × 109 kb genomic DNA for insertions. The lack of insertions places an upper bound on the L1 insertion frequency of three insertions in 3.7 × 109 kb (P = 0.05), or <1 event per 400 haploid genomes, lower than estimates of L1 retrotransposition frequency derived from the incidence of pathogenic L1 insertions in humans  [Brouha et al., This major survey of sperm DNA from a single donor failed to yield any genuine insertions. These data can be used to estimate an upper bound of the frequency of L1 insertion in this man's germline, with the caveat that this estimate only applies to the selected target loci. Indeed, because most of the target loci have accommodated pathogenic insertions in the past we may have ascertainment bias in favor of insertion-prone loci. This bias is likely to cause overestimation of the insertion rate, making even more significant the lack of insertions detected here. The DNA analysed was derived from 1.9 × 10The reason for this very low estimated frequency of de novo insertion of L1 elements in an individual male germline is unclear. It is unlikely that the chosen target loci are refractory to insertion because L1 insertion into the genome appears to be largely random [Feng et al., It is possible that donor B has an unusually low frequency of germline L1 retrotransposition due to the absence of active L1s in his genome. He does indeed lack the most active L1 identified to date, AC002980 [Brouha et al., Other factors could contribute to the very low rate of L1 retrotransposition observed here. Previously it was thought that L1 was primarily active during meiosis [Brouha et al.,


LINE-1 (L1) is the dominant category of transposable elements in placental mammals. L1 has significantly affected the size and structure of all mammalian genomes and understanding the nature of the interactions between L1 and its mammalian host remains a question of crucial importance in comparative genomics. For this reason, much attention has been dedicated to the evolution of L1. Among the most studied elements is the mouse L1 which has been the subject of a number of studies in the 1980s and 1990s. These seminal studies, performed in the pre-genomic era when only a limited number of L1 sequences were available, have significantly improved our understanding of L1 evolution. Yet, no comprehensive study on the evolution of L1 in mouse has been performed since the completion of this genome sequence.Using the Genome Parsing Suite we performed the first evolutionary analysis of mouse L1 over the entire length of the element. This analysis indicates that the mouse L1 has recruited novel 5’UTR sequences more frequently than previously thought and that the simultaneous activity of non-homologous promoters seems to be one of the conditions for the co-existence of multiple L1 families or lineages. In addition the exchange of genetic information between L1 families is not limited to the 5’UTR as evidence of inter-family recombination was observed in ORF1, ORF2, and the 3’UTR. In contrast to the human L1, there was little evidence of rapid amino-acid replacement in the coiled-coil of ORF1, although this region is structurally unstable. We propose that the structural instability of the coiled-coil domain might be adaptive and that structural changes in this region are selectively equivalent to the rapid evolution at the amino-acid level reported in the human lineage.The pattern of evolution of L1 in mouse shows some similarity with human suggesting that the nature of the interactions between L1 and its host might be similar in these two species. Yet, some notable differences, particularly in the evolution of ORF1, suggest that the molecular mechanisms involved in host-L1 interactions might be different in these two species. Long interspersed nuclear element-1 (LINE-1 or L1) constitutes the dominant category of transposable elements in mammalian genomes. L1s have accumulated in the genomes of their mammalian hosts in extremely large numbers and contribute to more than 20% of genome size in human and mouse -6. HowevThe pattern of L1 element evolution in mammals is very unusual. In most species analyzed so far, L1 evolves as a single lineage: a family of elements emerges, amplifies to hundreds or thousands of copies and then becomes extinct, being replaced by a more recently evolved family ,33-35. TMus musculus) have demonstrated the presence of multiple concurrently active L1 families with non-homologous promoters [The patterns described above result mostly from the analysis of the human genome and it is unclear how patterns of evolution in human recapitulate L1 evolution in other species. It is thus important to examine in greater detail the evolution of L1 lineages in other mammals. Pre-genomics studies in the house mouse ; (2) a 5’UTR that was not characterized before, named Mus (because it is absent from the rat genome); and (3) a 5’UTR that shows no similarity with any others, named N (for novel).A total of 20,459 L1 inserts with complete reverse transcriptase (RT) domains were identified using the Genome Parsing Suite (GPS). L1 elements were first grouped based on their 5’UTR. This was done by comparing the 5’ end of all elements with a library of previously described mouse 5’UTR using the Repeatmasker program . The A, Once elements were sorted based on their 5’UTRs, they were further categorized into families using a phylogenetic analysis of the 3’ terminus. A family is defined as a collection of elements that result from the activity of a highly homogenous group of progenitors, which are characterized by a unique combination of characters. In the first step of the phylogenetic analysis, neighbor joining trees  of elemeUsing this approach we identified 29 families and consensus sequences were derived for each of them (Table anc promoter (L1MdFanc_II). At the same time two families branched independently from the main lineage, one carrying a Mus promoter (L1MdMus_I) the other one the Fanc promoter (L1MdFanc_I). Preceding the L1MdFanc_II family a lineage made of four families with an Lx promoter was active. At two points in time the Lx promoter was replaced by the V promoter (yielding L1MdV_II and III) but these families did not persist or produce novel lineages.As L1 families have extensively recombined with each other (see below), various regions of L1 yield different evolutionary histories and it is impossible to build a single phylogenetic tree based on the entire length of the element. Figure anc promoter (L1MdFanc_II) and by the V promoter twice (L1MdV_II and III). The Fanc promoter was replaced independently twice by the Mus promoter as L1MdMus_I and L1MdMus_II do not form a monophyletic group. The Mus promoter was eventually replaced by the V promoter (L1MdV_I) and went extinct. The F promoter was then resuscitated approximately 6.4 MY ago and gave rise to families L1MdF_I to V. Approximately 4.6 MY ago the A promoter was recruited yielding the modern A lineage which extend from families L1MdA_VII to I. Within this lineage, an additional recruitment occurred resulting in the L1MdN_I family. Finally the F promoter was recently recruited twice, approximately 2.2 MY by the L1MdGf_II family and approximately 1.2 MY by the Tf/Gf_I lineage. Thus we estimate that L1 in mouse has experienced 11 replacements of 5’UTR.One of the most striking features visible on the tree is that families with similar 5’UTRs do not form monophyletic groups indicating that L1 families have frequently recruited novel 5’UTR, either from unknown sources or from ancient families. The oldest families in our study carried an Lx promoter, which was replaced three times: once by the FThe topology of the ORF2 tree indicates that mouse L1 families evolved mostly as a single lineage. This does not mean that a single family or single lineage was active at a time. In fact, the co-existence of multiple active families characterizes the evolution of L1 for the last 13MY of mouse evolution. For instance between 1 and 2.5 MY ago, six families  were active in the mouse genome as attested by the overlap in their average pairwise divergence  and L1MdF_II, near the 3’ end of the element Figure D. A 666 It should be noted that our criteria for identifying recombination events were stringent, as we only considered the recombination of large segments to be significant. Thus it is plausible that exchanges of sequences of shorter length have occurred between L1 families but were not detected due to the small number of defining characters in some conserved regions of L1, such as ORF2. The number of recombination events reported here suggests that recombination has played a significant role in the evolution of novel L1 families in mouse and can occur across the entire length of L1.The exchange of genetic information between families constitutes a significant challenge for evolutionary analyses as most phylogenetic algorithms do not allow for recombination. Thus we performed phylogenetic analyses using regions of L1 delimited by recombination breakpoints to fully assess the impact of recombination on the evolutionary history of FL L1 elements Figure . Trees AWe then examined the evolution of the protein coding sequences encoded in L1, ORF1, and ORF2. ORF2 is the most conserved region of L1. There are very few amino acid changes, in particular in the endonuclease and reverse transcriptase domains which are functionally indispensable ,58. All We examined the level of conservation of domains of ORF1 that are known to be functionally important ,59,60. TMore surprising is the degree of conservation at the amino acid level of the CC domain. Previous studies have shown that the CC domain of ORF1 has evolved under positive selection in primates ,39. In tanc_II and a 24 bp deletion in the second repeat found in L1MdFanc_I and L1MdF_IV. The 66–42 bp motif was followed by a duplication of the 42 bp unit resulting in a 66-42-42 bp structure which is found in families L1MdA_V to II, L1MdN_I, L1MdTf_III to I, and L1MdF_I, II, and V. This motif further evolved by the loss of the second 42 bp repeats in L1MdA_I and L1MdF_IV and by the addition of a third 42 bp unit in family L1MdGf_I. The ancestral 66–66 bp motif was recruited by recombination in families L1MdF_III, L1MdA_VI, and VII, and acquired a third 66 bp unit in family L1MdGf_II. These structural changes in the LPR resulted in changes in the length and structure of the CC. Coiled coils are formed from two or more α-helical peptide chains that contain a distinct arrangement of non-polar side chains. Domains that can form CC consist of heptads (or seven residue repeats) with non-polar or hydrophobic residues in the first and fourth positions [Although the CC domain is relatively conserved at the amino acid level, it shows a high level of structural variation. Previous studies have identified a region called length polymorphism region (LPR) ,61. Usinositions . The CC ositions . The shoMus and it has been proposed that some L1 families in the house mouse genome were acquired by hybridization [Finally, we examined the possibility of lateral transfer in the evolution of murine L1. In mammals, L1 is transmitted vertically and there is no evidence of lateral transfer , except dization ,64,65. IWe performed the first comprehensive analysis of L1 evolution since the completion of the mouse genome . The anaThe evolution of L1 in mouse fits the single lineage mode of evolution described previously in other mammals and particularly in human ,35,63,69anc_I (active 6.80 MY ago) to the ancestor of L1MdF_V (at 6.43 MY) and subsequently transferred from L1MdF_I (active 2.12 MY ago) to the recently active L1MdTf and L1MdGf families.The analysis of FL elements has revealed the extraordinary ability of L1 families to acquire novel motifs and to exchange sequences Figures  and 3. TThe first ORF is arguably the least understood region of L1, although it has been the subject of much attention in the past few years ,60,74-78We performed a comprehensive analysis of L1 evolution in mouse. This analysis covered the last 13 MY of mouse evolution, since the split between mouse and rat. The mouse L1 has evolved as a single lineage for most of its evolution, although co-existence between families carrying different promoter sequences was observed. L1 families have frequently acquired novel 5’UTR and have exchanged sequences over the entire length of the element. No evidence of rapid amino acid replacement in the ORF1 was detected, although it is likely that the structural instability of the CC domain is adaptive. The general pattern of evolution of mouse L1 is similar to the one in human suggesting that the nature of the interactions between L1 and its host might be similar in these two species. There are however some intriguing differences between mouse and human, particularly in the evolution of ORF1. These differences suggest that the molecular mechanisms involved in host-L1 interactions might be different in these two species.Mus musculus 2006 (mm8) genome built using the GPS [anc, V, Lx, Mus, or N; see result) followed by a roman number. The smaller the roman number, the younger the family is. For instance families L1MdA_I, L1MdA_II, and L1MdA_III are subsets of the previously described L1MdA family; family L1MdA_I is younger than family L1MdA_II and family L1MdA_III is the oldest of the three. We kept the Gf [Full-length (FL) elements were collected from the  the GPS . GPS con the GPS ,33. Thus the GPS  of eleme the GPS  based on the GPS . The modt the Gf  and Tf [t the Gf  names fohttp://darwin.uvigo.es/rdp/rdp.html) was used to search for evidence of recombination among families. RDP allows for the use of several recombination detection methods including substitution and phylogeny-based methods. Two substitution-based methods, MaxChi [NJ, maximum parsimony (MP), and maximum likelihood (ML) trees were calculated for each region of L1. Phylogenetic trees were reconstructed using the MEGA 5.01 package . The RDP, MaxChi  and Chim, MaxChi , as well, MaxChi , were us, MaxChi  which ishttp://www.datamonkey.com[a priori specification of branches in a phylogeny that may have evolved under different values of dN/dS. The dN/dS test is however not very sensitive, particularly if selection acts on a few codons. For this reason we used three methods designed to detect the action of positive or negative selection at specific sites in an alignment: Single Likelihood Ancestor Counting (SLAC), a Random Effects Likelihood (REL), and Fixed Effects Likelihood (FEL) [To test for evidence of selection in the evolution of L1 several methods implemented in the web server onkey.com of the Honkey.com were useonkey.com. A ratioonkey.com can deteod (FEL) . For eacod (FEL) ,39. CC sod (FEL) . Domainsod (FEL)  was usedMus/Rattus at 13MY [The age of each subfamily was estimated by calculating the average pairwise divergence based on the 3’UTR. CpG dinucleotides and the highly mutable polypurine tract located in the 3’UTR were removed from alignment. The average divergence between copies as well as the standard error was calculated using the maximum likelihood parameter distance (using the MEGA 5.01 software). Divergences were converted to time assuming a neutral rodent genomic substitution rate of 1.1%/MY .http://www.girinst.org/repbase/).The consensus sequences are available in Repbase .Click here for fileAlignments showing recombination break-points among L1 families. Only the parsimony-informative sites are shown.Click here for file


Continued improvements in Next-Generation DNA/RNA sequencing coupled with advances in gene annotation have provided researchers access to a plethora of annotated genomes. Subsequent analyses of orthologous gene structures have identified numerous intron gain and loss events that have occurred both recently and in the very distant past. This research has afforded exceptional insight into the temporal and lineage-specific rates of intron gain and loss among various species throughout evolution. Numerous studies have also attempted to identify the molecular mechanisms of intron gain and loss. However, even after considerable effort, very little is known about these processes. In particular, the mechanism(s) of intron gain have proven exceptionally enigmatic and remain topics of considerable debate. Currently, there exists no definitive consensus as to what mechanism(s) may generate introns. Because many introns are known to affect gene expression, it is necessary to understand the molecular process(es) by which introns may be gained. Here we review the seven most commonly purported mechanisms of intron gain and, when possible, summarize molecular evidence for or against the occurrence of each of these mechanisms. Furthermore, we catalogue indirect evidence that supports the occurrence of each mechanism. Finally, because these proposed mechanisms fail to explain the mechanistic origin of many recently gained introns, we also look at trends that may aid researchers in identifying other potential mechanism(s) of intron gain.This article was reviewed by Eugene Koonin, Scott Roy (nominated by W. Ford Doolittle), and John Logsdon. Although spliceosomal intron density varies widely among eukaryotes, no true eukaryote has ever been identified without a spliceosomal intron and some remnant of the spliceosome. Conversely, units of the spliceosome and/or spliceosomal introns have never been identified in any prokaryote ,2. Splicelements ,8.ReverseTranscriptase-MediatedIntronLoss (RTMIL) and genomic deletions, have been identified and are widely accepted [Intron Transposition[Transposon Insertion[Tandem Genomic Duplication[Intron Transfer[Intron Gain during Double-Strand Break Repair (DSBR)[Insertion of a Group II Intron[Intronization[.As researchers began to compare the structure of orthologous genes, it quickly became apparent that introns may be gained or lost throughout evolution . Subsequaccepted ,37. The sposition, TranspoInsertion, Tandem plication, Intron  Transfer, Intron ir (DSBR), InsertiII Intron and Intronization,42.Although seven possible mechanisms of intron gain have been proposed, researchers have identified thousands of novel introns whose mechanistic origins defy these explanations ,27,28,43The most commonly purported mechanism of intron gain is intron transposition . Intron Intron transposition has frequently been favored among the seven proposed mechanisms of intron gain by researchers ,37, likeEven if intron transposition does occur, it has been suggested that any mechanism of intron gain that relies upon reverse transcriptase (RT) may not be a prevalent mechanism of intron gain. One potential problem is known as the “rate paradox” . This maOther proposed molecular mechanisms of intron gain that mimic intron transposition, such as spliceosomal retrohoming or reverse transcription template switching , may occTransposon insertions into genic regions are generally viewed as deleterious mutations. However, one underappreciated outcome of this process may be intron creation. Such an insertion may completely intronize the transposon without disrupting the coding sequence when a transposon inserts into the sequence AGGT, resulting in the duplication of this sequence on each side of the transposon  are genomic lesions in which nearby phosphodiester bonds are severed on both strands of the DNA double helix. Such lesions may be caused by ionizing radiation, reactive oxygen species, or cellular processes. Because the repair of these lesions is paramount to cell viability, organisms have evolved two ubiquitous, genetically distinct, well conserved processes to repair these breaks: homologous recombination and non-homologous end joining (NHEJ) . The forDaphnia. These rDaphnia  are also causing intron gain. Therefore, we must look at trends in intron gain that may lead researchers towards a different mechanistic explanation.Micromonas isolate CCMP1545 [Micromonas, another recent study in fungi identified elements that create introns, dubbed introner-like elements. These elements were also only found co-linear to transcribed DNA and also did not appear to be canonical transposable elements [Perhaps the most tantalizing trend in intron gain is the role that transcription may play in intron gain. Studies in organisms with a dedicated germline have shown that genes that have experienced intron gain events are enriched for germline expression ,24. OtheCCMP1545 . These relements . Howeverelements  and, whielements . Thus, telements . If trueelements . Most imelements . Taken telements ,35, and elements , it is mFrequently a positional bias has been used to support possible mechanisms of intron gain or loss. A bias for intron gains or losses in the 3’ end of genes suggests that RT may have played a role in these events because RT transcribes from 3’ to 5’ and is known to frequently dissociate from templates before reaching their 5’ ends ,103. Ovein vivo[in vivo[in vivo, in particular intron gain during DSBR, intron transfer and intronization, is feasible. Demonstration of these mechanisms in vivo is essential to solidify them as genuine mechanisms of intron gain. Further genomic analyses, especially those performed at the population level, may then quantify the relative contribution of each mechanism, potentially identifying species-specific biases that may help account for the varied rates of intron gain among species [Out of seven proposed mechanisms of intron gain, six have indirect evidence to support their occurrence Table . The onlin vivo, suggesto[in vivo. Further species ,29.in vivo assays and genomic analyses performed at the population level, which will likely identify intron gains before extensive sequence divergence obscures their mechanistic origin, will likely prove the most fruitful avenues towards identifying and understanding the molecular processes underlying intron gain.Significant progress has been made in identifying the mechanisms of intron gain. However, this field still lies in its infancy. Even with one definitive and five likely mechanisms of intron gain, the vast majority of gained introns lack a known mechanistic origin. Therefore, it is essential that other mechanisms are envisioned and tested, as it is likely that undiscovered mechanism(s) of intron gain exist. Germline expressed and highly expressed genes tend to accumulate introns -25,98; yDSBR: Double-strand break repair; NHEJ: Non-homologous end joining; RT: Reverse transcriptase; RTMIL: Reverse Transcriptase-Mediated Intron Loss; DSB: Double-strand break.The authors declare that they have no competing interests.PY drafted, illustrated and revised the manuscript. LZ revised the manuscript. All authors read and approved of the final version of the manuscript.Reviewer #1: Dr. Scott Roy (nominated by Dr. W. Ford Doolittle)Reviewer’s comments: The authors provide a very useful and timely review on the mechanisms of intron gain, a field in which a tremendous amount of progress has been made in the past few years. I have very few criticisms, and even on those points where I might have a difference of opinion in regards to emphasis or interpretation, I think the authors' perspective is well supported and balanced, and so overall I do not think any changes need to be made.Authors’ response: We thank you for your review and comments. Because most of these mechanisms lack definitive evidence to support how/if they occur, we agree that there exist various, and at times conflicting, opinions and interpretations upon how/if these proposed mechanisms occur. Thus, we are delighted that you feel our presentation was balanced and supported by primary literature.Reviewer #2: Dr. Eugene KooninReviewer’s comments: This is a timely review of the important and fascinating problem of intron gain routes and mechanisms. The article is very well structured around the 7 distinct (proposed) routes of intron gain. My mild disappointments have to do with the excessive brevity of some of the sections. In particular, the section on Group II intron insertion is succinct to the point of being potentially misleading. I agree with the authors that there is no indication of recent intron gain via Group II intron insertion. However, this does not put into doubt the ultimate origin of spliceosomal introns from Group II introns whereas from the current version, the impression is that the authors refute that scenario. The situation can be easily remedied with a brief recapitulation of the evidence in support of the evolutionary links between self-splicing and spliceosomal introns.Authors’ response: We intentionally kept our review brief in order to meet the word limit of reviews in this journal (~3000 words). We apologize if, in our desire to write succinctly, we may have accidently expressed our beliefs and prior findings incorrectly, especially in regards to the origin of spliceosomal introns. As you mentioned, a significant amount of evidence exists to support the theory that group II introns, originating from an α-proteobacterium that would later go onto serve as the mitochondria, invaded the genome of their archaeal host, and in response to this invasion, selective pressures eventually gave rise to the nucleus, nonsense-mediated decay, and spliceosomal introns. We do note that in our original manuscript we directly stated that “group II introns are widely regarded as the progenitors of spliceosomal introns”. However, the wording of this section may have been misleading. Thus, we have rewritten the section “Insertion of Group II Intron” to better exemplify this point and have also added a brief primer on the origin of spliceosomal introns.Reviewer’s comments: In addition, the authors do not explicitly address the connection between alternative splicing and intron gain mechanisms. Emergence of a new alternative splice form often involves ("part-time") intronization, so I think this belongs in the paper.Authors’ response: Alternative splicing and, in particular, nonsense-mediated decay likely play key roles in the evolution of gained introns by alleviating negative fitness costs imposed upon organisms following the initial intron gain event . However, due to space limitations, we chose to focus only on the mechanisms that may initially cause the intron gain . The forces at play following this mutation that fixate this mutation in a population/species deserve a more in-depth treatment than we could provide with the space allotted. Regardless, we have added a brief discussion of these topics to the “Intronization” section, as this mechanism may rely most heavily upon evolutionary processes to yield efficient introns.Reviewer’s comments: Again, a very timely and useful review but with a little more attention to details, it could become even better.Authors’ response: We thank you for your review and apologize if we may have neglected some topics in an effort to be succinct.Reviewer #3: Dr. John Logsdon, University of IowaThis reviewer provided no comments for publication.


The diagnosis of sarcoidosis is still a significant challenge in China because of the need to exclude other diseases including granulomatous infections and malignancies that may be clinically and radiographically similar. The specific aim of the study is to search for serum protein biomarkers of sarcoidosis and to validate their clinical usefulness in differential diagnosis.Serum samples were collected from patients with sarcoidosis (n = 37), and compared to those from patients with tuberculosis (n = 20), other pulmonary diseases (n = 20), and healthy volunteers (n = 20) for determination of sarcoidosis-specific or -associated protein expression profiles. The first part of this study focused on proteomic analysis of serum from patients with sarcoidosis to identify a pattern of peptides capable of differentiating the studied populations using the ClinProt profiling technology based on mass spectrometry. Enzyme Linked Immunosorbent Assay (ELISA) was then used to verify corresponding elevation of the serum protein concentration of the potential biomarkers in the same patients sets. Receiver operating characteristic curve (ROC) analyses was performed to determine the optimal cutoff value for diagnosis. Immunohistochemistry was carried out to further confirm the protein expression patterns of the biomarkers in lung tissue.p < 0.05). The cutoff for serum SAA concentration determined by ROC analysis was 101.98 ng/ml, with the sensitivity and specificity of 96.3% and 52.5%, respectively. Immunohistochemical staining showed that the SAA depositions in lung tissue of the sarcoidosis patients were also significantly more intense than in non-sarcoid lung tissue (p < 0.05).An unique protein peak of M/Z 3,210 Daltons (Da) was found to be differentially expressed between the sarcoidosis and control groups and was identified as the N-terminal peptide of 29 amino acids (94-122) of serum amyloid A (SAA). ELISA confirmed that the serum SAA level was significantly higher in the sarcoidosis group than that of the other 3 control groups (This is the first study to investigate serum protein markers in Chinese subjects with sarcoidosis. This study shows that the serum SAA expression profiles were different between the sarcoidosis and non-sarcoidosis groups. SAA may be a potential serum biomarker for ruling-out the diagnosis of sarcoidosis in Chinese subjects. Sarcoidosis is a systemic disease of unknown etiology, with the pathological feature of non-caseous epithelioid granulomas. Many organs could be involved, and especially the lung and intrathoracic lymph nodes,2. SarcoAlthough great efforts have been devoted to establish methodologies that can differentiate sarcoidosis from other lung diseases-7, the dDifferential proteomics is the systematic investigation of changes in patterns of serum proteins in diseases. New tecIn the first part of this study, we used ClinProt profiling technology to discovery serum protein biomarkers associated with sarcoidosis. Serum amyloid A (SAA) was increased in the serum of patients with sarcoidosis, compared with non-sarcoidosis groups including healthy controls.http://inflammation against the human International Protein Index (IPI) database. The searching parameters were set up as follows: NoEnzyme, the variable modification was oxidation of methionine, the peptide mass tolerance was 5 ppm, and the fragment ions tolerance was 1 Da. Positive protein identification was accepted for a peptide with Xcorr of greater than or equal to 3.00 for triply and 2.50 for doubly charged ions, and all with ΔCn ≥ 0.1, peptide probability ≤ 0.001.To follow up the finding by mass spectrometry, human SAA ELISA assay  was used to measure the SAA level in 64 serum samples from patients with sarcoidosis. 99 serum samples were obtained from the comparison groups . Twenty (20) of 64 sarcoidosis patients were classified as the “treated” group, who had been successfully treated by corticosteroid and are showing improvement to complete cure. The remaining 44 patients were determined as “untreated” sarcoidosis, who had clinical symptoms to different extents but had not receive corticosteroid.The receiver operating characteristic (ROC) curve was plotted according to the SAA contents, as determined by the ELISA with the values of SAA in the sarcoidosis and non-sarcoidosis subjects. The optimal cut-off threshold is determined by ROC as the maximal value of Youden index (Youden index = sensitivity + specificity-1) for the quantitative analysis.The streptavidin-perosidase (SP) immunohistochemical assay was performed to detect the expression of SAA by a SAA antibody  in tissue biopsies of a subset of our test subjects: 10 patients with sarcoidosis  and lung tissues of 10 patients with other lung diseases .All experiments were carried out in triplicates, and the data were presented as mean ± standard deviation (SD). The statistical software SPSS 11.0 was used for data analysis. Independent-samples t test assuming equal variance and ANOVA were performed with p < 0.05 indicated statistically significant difference.Two samples from patients with sarcoidosis and one sample from a healthy control were used to verify reproducibility of the ClinProt system. Proteins were detected in these samples in a parallel experiment. The results showed that under the above-mentioned experimental conditions, M/Z bias was in a statistically acceptable range of errors (coefficient of variation or CV < 30%). FigureIn the range of M/Z 800-10,000, there were 8, 8 and 13 different protein peaks when the sarcoidosis subjects were compared with healthy controls, tuberculosis subjects and other lung disease subjects respectively. The protein peaks were then analyzed using the ClinProTools machine learning algorithms  to determine whether the identification and predictive ability of classification models in distinguishing sarcoidosis from the other disease subjects and from healthy controls Table.Specifically, of the total 8 differential peaks between the sarcoidosis group and tuberculosis group, 5 protein peaks  were overexpressed in sarcoidosis versus the other 3  that were more elevated and overexpressed in the tuberculosis group. In our study, the SNN method achieved the best results in identification and predictive capability.p < 0.05), but showed no significant differences between the non-sarcoidosis groups , which indicated that this is a disease-associated or specific protein for saroidosis.In addition, as shown in FigureThe LC-MS/MS trapped a molecular species of M/Z 3,211.57 (M + H), and the tandem MS analysis in the second stage obtained the parent ion of +4 charged 804.15. After LC-MS/MS data acquisition, the SEQUEST of IPI Human (3.45) database retrieved the consistent peptide identity of "SLADQAANEWGRSGKDPNHFRPAGLPEKY", which is the N-terminal 29 peptide of SAA   detected by semi- quantitative ELISA is plotted to generate the ROC curve using the cutoff point of 101.98 μg/L. The optimal cut-off yields the best sensitivity of 96.3%, and specificity of 52.5%, respectively; and the AUC of 0.763 . This is the first study demonstrating potential effectiveness of protein profiling in serum with ClinProt as a diagnostic tool for sarcoidosis in ethnic Han Chinese. We confirm that the SAA serum level and tissue expression of sarcoidosis are higher than in other lung diseases.Over the last decade, accumulated evidences in the research of protein biomarkers of sarcoidosis have shown that it is possible to find sarcoidosis-specific or associated proteins through analysis of body fluids. Researchers used to compare protein profiles from sarcoidosis patients with healthy controls in specimens of bronchoalveolar lavage fluid (BALF)-26. HoweThis study is the first effort using the ClinProt system of serum protein profiling in Chinese subjects to discover new diagnostic biomarkers in sarcoidosis. The ClinProt system is considered as more advanced technologies than SELDI-TOF-MS. We are SAA is an acute phase protein, which belongs to a group of heterogeneous proteins of the apolipoprotein family. It is possible that SAA could activate the NF-kB signaling pathway through interactions with Toll-like receptor 2 (TLR2), leading to the development of chronic granuloma as has been recently reported by Chen et al. AlthougA literature survey indicates that besides sarcoidosis, SAA is also over-expressed in several diseases such as COPD, lung caThe optimal cutoff of the serum SAA concentration obtained by the ROC analysis was 101.98 ng/ml yielding a sensitivity of 96.3% and specificity of 52.5% for diagnosis, respectively. By including not only healthy controls but by expanding the comparator groups to include patients with other inflammatory lung disease, our results demonstrated that SAA might be an important player in the development of sarcoidosis. However, although SAA is sensitive, it was less specific for the diseases tested in the study. This means that it is not for confirmative or for ruling- in a diagnosis of sarcoidosis.p < 0.001). In this study, we found that SAA is similarly increased in both “treated” and “untreated” sarcoidosis, which implies that SAA is not affected by the corticosteroid treatment.A follow up question is whether the SAA is a marker of disease activity or it was a sequel of the treatments? As early as 1989, Rubinstein et al had shown that SAA concentration in 25 sarcoidosis patients (13 cases with “active sarcoidosis” and 12 cases with “inactive sarcoidosis”) and 94 healthy volunteers. He showed that SAA concentrations in both types of sarcoidosis were significantly higher than that of healthy controls. However, SAA was of limited usefulness as a marker for disease activity[Since the usefulness of multiple markers for diagnosis is now widely recognized. ClinProIn summary, our results provide evidences that the SAA expression profiles by ClinProt-MALDI-TOF technique were different between the sarcoidosis and controls in Chinese subjects. The finding that the elevation of the SAA N-terminal peptide is associated with sarcodosis, is confirmed by ELISA in serum and further verified by immunohistochemical staining in lung tissue. Moreover experimental data obtained with current study suggested that multiple markers for diagnosis could be used in differential diagnosis of sarcoidosis despite its limitation in of its specificity.SAA: Serum amyloid A;Sarc: Sarcoidosis;TB: Tuberculosis;COPD: Chronic obstructive pulmonary disease;Da: Daltons;M/Z: The mass-to-charge ratio;ELISA: Enzyme Immunosorbent Assay;CV: Coefficient of variation;MALDI-TOF-MS: Matrix-Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry;SELDI-TOF-MS: Surface-enhanced Laser Desorption/ Ionization Time of Flight Mass Spectrometry;SNN: Supervised Neural Network;GA: Genetic Algorithm;QC: Quick Classifier;ROC: Receiver operating characteristic curveThe authors declare that they have no competing interests.HL and YZ were responsible for experimental design and finalized the manuscript. XC and YH participated in the clinical sample collection and conducted the ClinProt study. SD participated in the data analysis and manuscript preparation. LS, YH, YZ, XZ were involved in data collection and clinical follow-up. RCY was responsible for editing and finalizing the manuscript. All authors read and approved the final manuscript.


Caenorhabditis elegans strains that express full-length, untagged human TDP-43 and FUS in the worm's GABAergic motor neurons. Transgenic worms expressing mutant TDP-43 and FUS display adult-onset, age-dependent loss of motility, progressive paralysis and neuronal degeneration that is distinct from wild type alleles. Additionally, mutant TDP-43 and FUS proteins are highly insoluble while wild type proteins remain soluble suggesting that protein misfolding may contribute to toxicity. Populations of mutant TDP-43 and FUS transgenics grown on solid media become paralyzed over 7 to 12 days. We have developed a liquid culture assay where the paralysis phenotype evolves over several hours. We introduce C. elegans transgenics for mutant TDP-43 and FUS motor neuron toxicity that may be used for rapid genetic and pharmacological suppressor screening.Mutations in the DNA/RNA binding proteins TDP-43 and FUS are associated with Amyotrophic Lateral Sclerosis and Frontotemporal Lobar Degeneration. Intracellular accumulations of wild type TDP-43 and FUS are observed in a growing number of late-onset diseases suggesting that TDP-43 and FUS proteinopathies may contribute to multiple neurodegenerative diseases. To better understand the mechanisms of TDP-43 and FUS toxicity we have created transgenic  Amyotrophic Lateral Sclerosis (ALS) is a late-onset progressive disease affecting motor neurons ultimately causing fatal paralysis TDP-43 and FUS are evolutionarily conserved DNA/RNA binding proteins that shuttle between the nucleus and cytoplasm having multiple roles including DNA transcription and RNA processing Caenorhabditis elegans to investigate mutant TDP-43 and FUS age-dependent neurodegeneration. We created transgenic nematodes that express full-length wild type or mutant TDP-43 and FUS in the worm's GABAergic motor neurons. Transgenic TDP-43 and FUS worms recapitulate a salient feature of ALS; they display adult-onset, age-dependent, progressive paralysis and degeneration of motor neurons. Importantly, mTDP-43 and mFUS, but not wtTDP-43 and wtFUS, strains show the presence of insoluble proteins in extracts from whole animals suggesting that protein misfolding may be a primary cause of toxicity. We introduce a genetically tractable platform to investigate motor neuron toxicity caused by mutant TDP-43 and FUS that can be used for suppressor screening.Since TDP-43 and FUS are evolutionarily conserved we used the nematode unc-47) promoter  and inhibitory (GABA) inputs to coordinate muscle contraction/relaxation and facilitate movement unc-47 are hypersensitive to aldicarb-induced paralysis unc-47 mutants, mTDP-43 and mFUS animals were hypersensitive to aldicarb-induced paralysis, while wtTDP-43 and wtFUS transgenics showed a rate paralysis identical to non-transgenic N2 worms  that expresses GFP in the same GABAergic motor neurons C. elegans TDP-43 toxicity model unc-47p::GFP alone  and include: N2, oxIs12[unc-47p::GFP+lin-15], unc-47(e307), unc-47(gk192) and unc-119(ed3).Standard methods of culturing and handling worms were used unc-47 promoter , the unc-54 3′UTR plasmid pCM5.37  and the destination vector pCFJ150 to create unc-47::TDP-43 and unc-47::FUS expression vectors. Transgenic lines were created by microinjection of unc-119(ed3) worms, multiple lines were generated and strains behaving similarly were kept for further analysis. Transgenes were integrated by UV irradiation and lines were outcrossed to wild type N2 worms 5 times before use. The main strains used in this study include: xqIs132, xqIs133[unc-47::TDP-43[A315T];unc-119(+)], xqIs173, and xqIs98[unc-47::FUS[S57Δ];unc-119(+)].Human cDNAs for wild type and mutant TDP-43[A315T], and wild type and mutant FUS-TLS[S57Δ] were obtained from Dr. Guy Rouleau . The cDNAs were amplified by PCR and cloned into the Gateway vector pDONR221 following the manufacturer's protocol (Invitrogen). Multisite Gateway recombination was performed with the pDONR TDP-43 and FUS clones along with clones containing the For worms expressing TDP-43 or FUS, 20–30 adult day 1 animals were picked to NGM plates and scored daily for movement. Animals were counted as paralyzed if they failed to move upon prodding with a worm pick. Worms were scored as dead if they failed to move their head after being prodded in the nose and showed no pharyngeal pumping. All experiments were conducted at 20°C.Worms were grown on NGM-FUDR plates to prevent progeny from hatching. 20 animals/plate by triplicates were tested at 20°C from adult day 1 until death. Worms were declared dead if they did not respond to tactile or heat stimulus. Survival curves were produced and compared using the Log-rank (Mantel-Cox) test.To evaluate synaptic transmission, worms were grown on NGM and transferred to NGM plates +1 mM aldicarb at adult day 1. Paralysis was scored after 1 and 2 hours on aldicarb plates. Animals were counted as paralyzed if they failed to move upon prodding with a worm pick. All tests were performed at 20°C.Synchronized populations of worms were obtained by hypochlorite extraction. Young adult worms were distributed in 96-wells plate , containing DMSO or test compounds and incubated for up to 6 h at 20°C on a shaker. The motility test was assessed by stereomicroscopy. Videos of worms were taken with on an Olympus S7x7 stereomicroscope equipped with a Grasshopper GRAS-03K2M camera using Flycap software (Point Grey Research) at a rate of 300 frames per second.Age synchronized, adult day 1, whole worms were fixed and stained as described in WormBook in vivo. Animals were immobilized in M9 with 5 mM levamisole and mounted on slides with 2% agarose pads. Motor neurons were visualized with a Leica 6000 microscope and a Leica DFC 480 camera. A minimum of 100 animals was scored per treatment over 4–6 trials. The mean and SEM were calculated for each trial and two-tailed t-tests were used for statistical analysis.For scoring gaps/breaks from TDP-43 and FUS transgenics, synchronized animals were selected at days 1, 5 and 9 of adulthood for visualization of motor neurons 1/2 G syringe 10 times, sonicated and centrifuged at 16000g. Supernatants were collected.Worms were collected in M9 buffer, washed 3 times with M9 and pellets were placed at −80°C overnight. Pellets were lysed in RIPA buffer +0.1% protease inhibitors . Pellets were passed through a 27All supernatants were quantified with the BCA Protein Assay Kit (Thermo Scientific) following the manufacturer instructions.1/2 G syringe 10 times, sonicated and centrifuged at 100000g for 5 min. The soluble supernatant was saved and the remaining pellet was resuspended in extraction buffer, sonicated and centrifuged at 100000g for 5 min. The remaining pellet was resuspended into 100 µl of RIPA buffer, sonicated until the pellet was resuspended in solution and saved.For TDP-43 and FUS transgenics soluble/insoluble fractions, worms were lysed in Extraction Buffer ). Pellets were passed through a 27Worm RIPA samples (175 µg/well) were resuspended directly in 1× Laemmli sample buffer, migrated in 12.5% or 10% polyacrylamide gels, transferred to nitrocellulose membranes (BioRad) and immunoblotted. Antibodies used: rabbit anti-human-TDP-43 , rabbit anti-human-FUS/TLS , and mouse anti-actin . Blots were visualized with peroxidase-conjugated secondary antibodies and ECL Western Blotting Substrate (Thermo Scientific). Densitometry was performed with Photoshop (Adobe).For paralysis and stress-resistance tests, survival curves were generated and compared using the Log-rank (Mantel-Cox) test, and 60–100 animals were tested per genotype and repeated at least three times. For image analysis statistical significance was determined by Student's t-test and the results shown as mean ± standard error. Prism 5 (GraphPad Software) was used for all statistical analyses.Table S1Lifespan analysis for all experiments.(PDF)Click here for additional data file.Video S1mTDP-43 worms in liquid culture at time 0.(MOV)Click here for additional data file.Video S2mTDP-43 worms after 6 hours in liquid culture.(MOV)Click here for additional data file.Video S3mFUS worms in liquid culture at time 0.(MOV)Click here for additional data file.Video S4mFUS worms after 6 hours in liquid culture.(MOV)Click here for additional data file.Video S5wtTDP-43 worms in liquid culture at time 0.(MOV)Click here for additional data file.Video S6wtTDP-43 worms after 6 hours in liquid culture.(MOV)Click here for additional data file.Video S7wtFUS worms in liquid culture at time 0.(MOV)Click here for additional data file.Video S8wtFUS worms after 6 hours in liquid culture.(MOV)Click here for additional data file.Video S9N2 worms in liquid culture at time 0.(MOV)Click here for additional data file.Video S10N2 worms after 6 hours in liquid culture.(MOV)Click here for additional data file.Video S11unc-47p::GFP worms in liquid culture at time 0.(MOV)Click here for additional data file.Video S12unc-47p::GFP worms after 6 hours in liquid culture.(MOV)Click here for additional data file.


The films turn to be hydrophilic after exposed to AO. The contact angle increases with AO exposure time. This phenomenon is attributed to the lower surface roughness induced by collision and/or edge erosion of energetic ions to the film surface and is further explained by the Wenzel model. The demonstrated strategy can overcome limitations of Hummers method, provide possibility to gain functionalization and wettability transition in liquid-phase exfoliated basal-defect- and oxide-free graphene in the dry environment, and may extend the study and application of this material in spacecraft in low earth orbit.Though chemical modification of graphene based on Hummers method has been most widely used to tailor its properties and interfacial characteristics, a method which could achieve definitive and controllable groups and properties is still highly required. Here, we demonstrate a high-vacuum oxidation strategy by atomic oxygen (AO) and investigate the AO induced functionalization and wettability transition in films made from basal-defect- and oxide-free graphene dispersions. These graphene-based films are neither graphene nor graphite, but graphene blocks constituted by numerous randomly stacked graphene flakes. It is found that AO induced functionalization of these films through the formation of epoxy groups, sp Graphene, constituted by the two-dimensional honeycomb C–C network, has attracted a great deal of attention due to its exceptional and intriguing properties  by an L-600 centrifuge  in order to remove any largish flakes, eventually resulting in homogeneous colloidal suspension of graphene flakes in DMF. Graphene concentration after centrifugation, CG, was determined from Lambert–Beer law, A/l = αCG, where A is the absorbance measured at 660 nm by a 721E spectrophotometer (Shanghai Spectrum), l is the cuvette length, and α is absorption coefficient at 660 nm which equals to 2,460 mL/mg/m  by deionized water and then vacuum filtered to form a homogeneous film (Φ 40 mm) on the nylon membrane (pore size ~220 nm). The film was dried for 48 h in the vacuum oven at 100 °C. Subsequently, a 30 × 20 mm rectangular small film was cut from the film central region. Atomic force microscopy (AFM) was used to scan 96 points on the surface of the small film to determine the roughness. Average of the surface roughness (root-mean-square roughness) corresponding to each scan was used as the surface roughness of the small film. The small film was again cut into 6 bitty films with dimension of 10 × 10 mm. One bitty film was chosen to measure the contact angle with water. The other 5 bitty films were exposed into high-vacuum AO atmosphere with time of 3, 5, 10, 20, and 25 h. After AO exposure, the surface roughness and contact angle of these films were measured, and X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FTIR) spectroscopy analyses were performed. When the roughness of the 10 × 10 mm films was measured by AFM, 16 points were scanned in each sample and the average surface roughness was adopted.AFM images were captured by a CSPM5500 AFM (Being Nano-Instruments) with a scanning range of 13.56 μm in the tapping mode. Bright-field transmission microscope (TEM) and high resolution TEM (HRTEM) images were taken with a JEOL 2100FEF operating at 200 kV. When AFM and TEM were used to characterize the morphology and structure of the isolated or individual graphene flakes, AFM samples were prepared by spraying several microliters of dispersions onto the mica substrate and dried in vacuum oven, while TEM samples were prepared by pipetting several drops of dispersions onto holey carbon mesh grids (300 meshes). The Raman measurements were made on these films with a Renishaw Rm2000 using a 514 nm laser. FTIR spectra of the films were measured by a Nicolet iS10 spectrometer in the diffuse reflection mode. XPS investigation was performed on the films by an ESCALAB-250 photoelectron spectrometer (Thermo Fisher Scientific) with the monochromatic Al Kα X-rays source . For each spectrum, the binding energy position was calibrated by measuring the Fermi level position of an Au reference sample in contact with graphene-based film. Static contact angle was measured by placing a droplet of deionized water on the surface of the various films. An Easydrop DSA20 instrument was used to measure the contact angle at room temperature (~25 °C). The axisymmetric-drop-shape analysis profile method was used for estimating the contact angle of water on the film surface.2, O2+, O, O+, e, etc., as shown in Fig. 2+ could be as high as 15 eV  in which F is the effective flow rate of AO onto the sample surface and ΔM, ρ, A, t, Ey are mass loss, density, surface area, exposure time, and erosion yield, respectively. Ft is the accumulative AO flux exerting on the sample surface. For Kapton, Ey is equal to ~3.0 × 10−24 cm3/atom   component widens with increasing exposure time. These indicate that AO functionalization can disturb sp2 hybridization and induce sp3 hybridization in graphene. C3, C4, and C5 are attributed to single C–O–C bonds , double C=O bond, and triple O–C=O bond. In the case of O1s spectra, C=O groups often appear between the binding energy of 531 and 532 eV and C–O bonds in ether and hydroxyl appear between 533.8 and 534.6 eV , respectively.In order to ascertain the formation process of oxygen-containing groups during AO oxidation, we performed XPS analysis, as shown in Fig. 2 carbon in graphene and induce sp3 hybridization in the C atoms neighboring epoxy. So C2 components related to sp3 C also appear. The formation of ether groups may be attributed to three reasons: AO reacts with point defects in the basal plane of graphene; AO reacts with edge carbons; AO penetrates from edge and intercalates between randomly stacked graphene flakes to react with adjacent two carbon atoms from different flakes. The above Raman, FTIR, and XPS analyses of the pristine film have confirmed that the prepared graphene is largely free of basal defects and the film is constituted by numerous randomly stacked graphene flakes. Large quantities of edges and disorder stacks exist in the film. So the possibility of the first reason for ether formation is extremely low, and the formation of ether may be mainly attributed to the second and third reasons. Recently, some researchers investigated the oxidation of single-layer graphene prepared by chemical vapor deposition. They also found that AO can penetrate under the graphene from the graphene grain boundary and intercalate between the metal substrate and graphene to form ether-like groups . Figure 2+ and their energy is about 15 eV . Therefore, as the AO exposure time increases, the decreasing surface roughness would lead to a decreasing r and thus an increasing contact angle θw, as shown in Fig. Furthermore, we measured the contact angles of these films, as shown in Fig. θ holds constant and r decreases, rcosθ will decrease and thus θw will increase. If r holds constant and θ decreases, rcosθ will increase and thus θw will decrease. So r and θ have opposite effects on θw. The competition between r and θ will determine whether θw is decreased or not. The results on experimentally measured contact angle  will be predominately determined by the Wenzel model in the scope of surface physics (surface roughness) not surface chemistry .For a more detailed analysis, examining the correlation among surface content of the O-carrying groups, morphology roughness, and wettability (contact angle), we can find that as AO exposure time increases, the O-containing groups content increases Fig. c, d, indgle Fig.  show tha3 configuration, ether, and double and triple C–O groups. AO induced functionalization turns these films from hydrophobic to hydrophilic. The collision and/or edge erosion effects of energetic ions in the AO atmosphere can lower the film surface roughness, and thus result in larger contact angles which can be explained by the Wenzel model. The demonstrated high-vacuum oxidation strategy shows many advantages over Hummers method in controlling and determining functional groups. This strategy provides possibility to gain functionalization and wettability transition in liquid-phase exfoliated basal-defect- and oxide-free graphene in the dry environment. The study is also very valuable for graphene-based electronics and sensors to be used in spacecraft in low earth orbit where AO and energetic ions abound.In conclusion, we have demonstrated a high-vacuum oxidation strategy by AO and investigated the AO induced controllable functionalization and wettability transition in graphene-based films which are made from defect- and oxide-free graphene dispersions. These films whose surface roughness can be tailored are neither graphene nor graphite, but a randomly stacked graphene blocks which are constituted by a large number of graphene flakes in disordered arrays. High-vacuum oxidation conditions facilitate the determination and control of oxygen-containing groups. It is found that AO induced functionalization of these films through the formation of epoxy groups, sp


Wefound that intragenic L1 sequences have been conserved across evolutionary timewith respect to transcriptional activity and CpG dinucleotide sites formammalian DNA methylation. Then, we compared regulated mRNA levels of cells fromtwo different experiments available from Gene Expression Omnibus (GEO), adatabase repository of high throughput gene expression data, (http://www.ncbi.nlm.nih.gov/geo) by chi-square. The odds ratioof down-regulated genes between demethylated normal bronchial epithelium andlung cancer was high , suggesting cancer genome widehypomethylation down-regulating gene expression. Comprehensive analysis betweenL1 locations and gene expression showed that expression of genes containing L1shad a significantly higher likelihood to be repressed in cancer andhypomethylated normal cells. In contrast, many mRNAs derived from genescontaining L1s are elevated in Argonaute 2 (AGO2 or EIF2C2)-depleted cells.Hypomethylated L1s increase L1 mRNA levels. Finally, we found that AGO2 targetsintronic L1 pre-mRNA complexes and represses cancer genes. These findingsrepresent one of the mechanisms of cancer genome wide hypomethylation alteringgene expression. Hypomethylated intragenic L1s are a nuclear siRNA mediatedcis-regulatory element that can repress genes. Thisepigenetic regulation of retrotransposons likely influences many aspects ofgenomic biology.In human cancers, the methylation of long interspersed nuclear element -1 (LINE-1or L1) retrotransposons is reduced. This occurs within the context of genomewide hypomethylation, and although it is common, its role is poorly understood.L1s are widely distributed both inside and outside of genes, intragenic andintergenic, respectively. Interestingly, the insertion of active full-length L1sequences into host gene introns disrupts gene expression. Here, we evaluated ifintragenic L1 hypomethylation influences their host gene expression in cancer.First, we extracted data from L1base ( In cancer, DNA methylation in the rest of the genome, particularly at a longinterspersed nuclear element-1 (LINE-1 or L1) retrotransposon, is generally depletedand this event occurs within the context of genome wide or global hypomethylationDNA methylation is a fundamental molecular characteristic of the human genome, andalteration of this epigenetic regulation is associated with cancers L1s are also widely distributed in the genome Recently, we evaluated the methylation patterns of the 5′ UTRs of L1 sequenceshttp://l1base.molgen.mpg.de)http://www.ncbi.nlm.nih.gov/geo) Here, we extracted data from L1base  andhuman mesenchymal stem cells (hMSCs) after genome wide demethylation by5-aza-2′-deoxycytidine (Aza-dC) treatment demonstrated a greaterprevalence of intragenic L1s in down-regulated genes, interescis-regulatory element.Interestingly, hypomethylation down regulates both groups of genes, with L1 of analyzed samples. L1s are still active retrotransposons and new insertioncan be identified as LIDs Many studies have reported the methylation of tumor suppressor gene promoters incancer cells; this epigenetic regulation has become a potential candidate forbiomarker and therapeutic target development. To our knowledge, this is the firststudy to demonstrate that global hypomethylation down-regulates genes in cancer.Moreover, there may be several hypomethylation-mediated cis-suppressor elements,including intragenic L1s. Genome wide hypomethylation is common to many cancer typesNevertheless, not all intragenic L1s can repress gene expression in cancer, and L1smay regulate genes through several distinct mechanisms. Even though L1 sequenceanalysis showed that intragenic L1s have been conserved throughout human evolution,their sequences and distributions do vary considerably. Moreover, the methylationlevels of some L1 loci are independent of genome wide L1 hypomethylation in cancerHere, we identified one mechanism by which L1s can repress genes in cancer. First, L1hypomethylation increases L1 RNA levels. Then, the AGO2 protein regulates genespossessing L1. Finally, mRNA processing of genes harboring hypomethylated L1s isdisrupted. AGO2 was reported to commonly target L1 RNA This study proved that intragenic L1 hypomethylation represses genes via apost-transcriptionally mechanism, based on siRNA and AGO2. However, it is possiblethat there are other mechanisms that should be further explored such as theintereference with the elongating RNA Pol2 transcribing their host genes orformation of chromatin complex in relation with L1 methylation level.cis and in trans, such as L1 sequencevariations, boundary sequences, methylation, chromatin configuration, location,transcription factors that are correlated with the degree of L1 methylation,molecules involved in the AGO2-intronic L1 RNA processing mechanism and factors thatguide or prevent AGO2 recognition. In addition to their modifications in cancer, L1and other IRS methylation are altered by many biological processes including thedisease-related ones In conclusion, intragenic L1 produces L1 RNA upon hypomethylation in cancer tissues,and the host gene is consequently down-regulated when AGO2 is present. Furtherstudies should reveal additional factors that influence this process both int-test for categorical and non-categorical functionallyimportant features, respectively. For categorical features, the frequency ofintragenic L1 features was counted both according to the number of genes and thenumber of L1 sequences that contained the tested features.L1s reported in L1base t-test was performed on all probes. Some probesrepresented more than one gene (homologous probes). A gene was counted asdifferentially expressed (up- or down-regulated) by expression level of at leastone unique probe. If a gene contained only homologous probes, there must be atleast two homologous probes representing the same gene. Up- or down-regulatedgenes were counted when representing probes were significantly different betweentest and control groups at p<0.01. P<0.05 was used when the number oftests or controls was two or mRNA was prepared by immunoprecipitation.mRNAs from GEO To evaluate if intragenic L1s can influence host gene expression, up- ordown-regulated genes and genes without significantly increased- or decreased-expression were divided into two categories whether containing intragenic L1s ornot. Genes hosting L1s were listed in Supporting CU-DREAM is a method for analyzing databases of two expression arrays withdifferent conditions to determine if the two conditions share genome wide generegulation pathway. First, mRNAs from two different expression microarrays wereclassified as up- or down-regulated and not up- or not down-regulated dependingon the statistical significance determined by student's t-test. From dataof two arrays, each mRNA presented in both experiments were classified into 4groups: 1) regulated in both array experiments, 2) not regulated only in thefirst experiment, 3) not regulated only in the second experiment, and 4) notregulated in both array experiments. The numbers of genes in each subset werecompared using a chi-square test . Non ran2. To inhibit DNA methyltransferase (DNMT) activity,WSU-HN cells were supplemented with 4 µM 5-aza-2-deoxycytidine (Cat.No.A3656 Sigma-Aldrich) every 24 hours for up to 16 days for genomic demethylation.WSU-HN17 cells were transiently transfected with siRNA against eIF2C2 mRNA (AGO2siRNA), which was designed by and purchased from Santa Cruz Biotechnology, Inc.(eIF2C2 siRNA (h): sc-44409). A non-silencing siRNA with no homology to anyknown mammalian genes  was transiently transfected as a negative control siRNA for eachexperiment.Eleven HNSCC cell lines (WSU-HNs), including WSU-HN 4, 6, 8, 12, 13, 17, 19, 22,26, 30 and 31, were provided by Dr. Silvio Gutkind . Cells werecultured in Dulbecco's modified Eagle's medium  supplemented with 10% heat-inactivated fetalbovine serum . Cells were incubated at 37°Cin 5% COTaqI or 8U of TasIin 1xTaqI buffer (MBI Fermentas) at 65°C overnight and thenelectrophoresed in 8% non-denaturing polyacrylamide gels. The intensitiesof DNA fragments were measured with a PhosphorImager and analyzed usingImageQuant software (Molecular Dynamics). The methylated amplicons(TaqI positive) yielded 80 bp DNA fragments whileunmethylated amplicons (TasI positive) yielded 97 bp fragments.The L1 methylation level was calculated as a percentage (the intensity ofmethylated L1 digested by TaqI divided by the sum of theunmethylated L1 digested by TasI-and theTaqI-positive amplicons). The same set of DNAs was applied as apositive control in each set of COBRA experiments.The methods of genome wide L1 and specific loci L1 methylation measurements wereextensively validated ACAAATACCATATCCTTCAAGACAAATCG, were usedfor the RT step. The PCR oligonucleotides used were: L1-RNA, CAGGAAGGGGAATATCACACTC andTGCGCTGCACCCACTAACTC; and5′L1-RNA, GGCCAGTGTGTGTGCGCACCG and CCAGGTGTGGGATATAGTCTCGTGG; AGO2,CACAAGTTGGTTCTGCGCTA andTGAAACTTGCACTTCGCATC;GAPDH, TTCGCTCTCTGCTCCTCCTGTTC and CTGGTGACCAGGCGCCCAA; L1-EPHA3 RNA,CTAACCTGCACAATGTGCACATGTACCC and L1-EPHA3-IVS15F. For RNAimmunoprecipitation experiment control primers were, TRIM38, GCAAAAACCACAATTACTTTTGCAC andAAGAGAGAAAATTGGTAATCAGCTTG; negative control, PSENEN,GGCACCCCAGCCGGAGGA andCGGGTCGTCCCAAGGGTCTG; andBCKDK, CCCACCATGATGCTCTACGCTGG and CCTTGATGCGGTGAGCAATCCTC. Real-timeRT-PCR was performed for 40 cycles with an annealing temperature of 60°C.Real-time RT-PCR was performed in a Light Cycler machine  using QuantiTect SYBR Green I  according to the manufacturer's instructions. Toquantitate gene expression, each PCR product was cloned into the pGEM-T easyvector  and used as controls. All mRNA, L1 RNA andL1-EPHA3 RNA levels were normalized with GAPDH, total RNA and band intensity,respectively. The highest RT-PCR levels observed in each experiment wereadjusted to 1.Total RNA was extracted from cell lines using the Trizol reagent  according to the manufacturer's instructions. The RNAwas treated with RNase-free DNaseI(Fermentas) to remove contaminating genomicDNA and with RiboLock™ Ribonuclease Inhibitor (Fermentas) to preventdegradation. To synthesize cDNA, 5 µg DNA-free RNA was dissolved in 12µl of DEPC-treated water containing 0.5 µg oligo(dT)18 primer(Fermentas). The RNA was incubated for 5 min at 70°C and chilled on ice for5 min. Each sample was then incubated with 200U RevertAid™ M-MuLV ReverseTranscriptase (Fermentas), 20 U Ribolock™ Ribonuclease inhibitor(Fermentas) and 20 mM dNTPs for 1 hr at 42°C, followed by 10 min at 70°Cand subsequent chilling on ice. cDNA was amplified using the exon primers listedin the Appendix. RNA that had not been reverse transcribed was included asnegative control to evaluate the amount of LINE-1 DNA contamination.Oligo(dT)18, random Hexamer and L1-EPHA3-IVS15-P1, http://www.epigenome-noe.net/researchtools/protocol.php?protid=28)2 flask at 80% confluence, washed withPBS and trypsinized. Approximately, 1×108 cells were added to a15 ml conical tube, pelleted, and resuspended in 10 ml 1% formaldehyde inPBS. This crosslinking reaction was performed for 30 minutes at room temperatureand stopped by the addition of glycine at a final concentration of 125 mM. Thepellet was washed twice with ice-cold PBS containing 1× protease inhibitorcocktail. The cell pellet was resuspended in 200 µl Buffer A (5 mM PIPES(pH 8.0), 85 mM KCl, 0.5% NP40, 1× Roche protease inhibitorscocktail, SUPERase•in (50 U/ml)) and placed on ice for 10 minutes. Thecrude nuclei fraction was pelleted by microcentrifugation at 5000 rpm for 5minutes at 4°C. The pellet was washed once in Buffer A without NP-40, thenresuspended in 500 µl Buffer B , 1× Roche protease inhibitors cocktail, SUPERase in (50 U/ml))and incubated on ice for 10 minutes. Lysates were sonicated three times at4°C using a Branson Sonifier at constant power,output = 70%, and continuous sonication for 20seconds. After sonication, insoluble elements were cleared bymicrocentrifugation at 14,000 rpm for 10 minutes at 4°C. The sonicate wasdiluted 10-fold with IP Buffer to a final volume of 1 ml per immunoprecipitationreaction. A 1% aliquot was preserved as an input sample and frozen at−80°C until the reverse crosslinking step. For the precipitation step,5 µg of primary antibody or a normal IgG control was added to each tube.Immune complexes were allowed to form by slow mixing on a rotating platform at4°C overnight. To collect immune complexes, 50 µl Protein A/GAgarose-PLUS (Santa Cruz) was added to each tube and slow mixing rotation wascontinued for 2 hours. Immune complexes were pulled down by gentlecentrifugation at 1000 rpm for 2 minutes at 4°C. Each immune complex waswashed five times . After each wash , complexes were pelletedby gentle centrifugation  and the wash buffer was aspiratedusing a clean pipette tip. Immune complexes were eluted by the addition of 250µl Elution Buffer and collected by centrifugation .NaCl was added to a final concentration of 200 mM (including the input samples)and placed at 65°C for at least 2 hours to reverse crosslinking. Sampleswere subjected to Trizol LS reagent extraction and resuspended in 20 µlDEPC-treated water. DNA was removed from the samples by treatment withRNase-free DNaseI (Fermentas Inc.). TRIM38 was predicted to be positive. BCKDKand PSENEN were predicted as negative controls. TRIM38 was up-regulated inAGO2si experiment and possess a intronic miRNA binding site AGO2 antibody (sc-32659) and goat IgG (sc-2028) (Santa Cruz) were used toimmunoprecipitate RNA as described Click here for additional data file.Figure S2the distributions of genes commonly down-regulated in the independentexperiments compared between genes containing L1 and genes without L1including the list of L1-containing genesfound to be down-regulated in atleast one experiment.(PDF)Click here for additional data file.Table S1Genes containing L1 sequences.(PDF)Click here for additional data file.Table S2Analysis of L1 characteristics.(PDF)Click here for additional data file.Table S3GSE records, GSM samples, type of t-test, and 2×2 contingency tables ofchi-square tests corresponding to the expression analysis of genespossessing internal L1s.(PDF)Click here for additional data file.Table S4The 2×2 contingency tables corresponding to CU-DREAM chi-squaretests.(PDF)Click here for additional data file.Table S5The 2×2 contingency tables corresponding to genes possessing internalL1s which were down-regulated in both cancer and demethylated normalcells.(PDF)Click here for additional data file.Table S6List of GSE records and GSM samples, type of t-test, and 2×2contingency tables of chi-square tests for the analysis of up-regulation ingenes possessing internal L1s. The data were displayed for expression indemethylated lung cancer cells (Table S6.1 and S6.2), DICER1sh (Table S6.3and S6.4), AGO1sh (Table S6.5), AGO3sh (Table S6.6), and AGO4sh (TableS6.7).(PDF)Click here for additional data file.Table S7Odds ratios, p values and 2×2 contingency tables of chi-square testscomparing between expression of genes possessing nearby intergenic L1s andthe rest of genes in bladder carcinoma situ. The GSE and GSM records werelisted in supporting table (PDF)Click here for additional data file.


Carrier doping of graphene is one of the most challenging issues that needs to be solved to enable its use in various applications. We developed a carrier doping method using radical-assisted conjugated organic molecules in the liquid phase and demonstrated all-wet fabrication process of doped graphene films without any vacuum process. Charge transfer interaction between graphene and dopant molecules was directly investigated by spectroscopic studies. The resistivity of the doped graphene films was drastically decreased by two orders of magnitude. The resistivity was improved by not only carrier doping but the improvement in adhesion of doped graphene flakes. First-principles calculation supported the model of our doping mechanism. AlthoV-1 · s-1. The ratV-1 · s-1,8. Ease V-1 · s-1,10. FurtV-1 · s-1-14.2[Carrier doping is a common approach to tailoring the electronic properties of semiconductor materials. Carrier doping can also dramatically alter the electrical properties of graphene. Although several techniques aimed at the carrier doping of graphene have been demonstrated, including boron- or nitrogen-substitutional doping ,16, the 2, these d2,20 or po2,22, is a2,24. Conj2. EfficieWe demonstrate herein a novel carrier doping method for chemically derived graphene using radical-assisted conjugated organic molecules in the liquid phase. It is expected that liquid-phase chemical interactions between graphene and conjugated organic molecules induce high doping efficiency. Absorbance measurements provide direct evidence for charge-transfer (CT) interactions between graphene and radicalized TCNQ molecules in an organic solvent. Raman spectroscopy and ultraviolet photoelectron spectroscopy (UPS) have also been used to elucidate the effects of doping on doped graphene films, which showed improvements in resistivity of two orders of magnitude with highly stable doping effect. Previous attempts at carrier doping for chemically derived graphene have never decreased the resistivity by more than one order of magnitude . The dop4 and H2SO4. After centrifugation, the resulting graphite oxide was exfoliated into graphene oxide (GO) by ultra-sonication . Then, a GO aqueous dispersion was produced by centrifugation and dialysis to neutralize a pH.Chemically derived graphene was synthesized using a modified version of Hummer's method, a well-known approach to producing monolayered graphene via the liquid-phase exfoliation of graphite oxide, as described previously in the literature . Natural2H4, a strong reductant, with NH3 to adjust pH. The mixed solution was reacted in a water bath at 95°C for 1 h, and the color of dispersion changed from brownish color to gray. Finally, the solvent of reduced graphene oxide (RGO) dispersion was replaced by N,N-dimethylformamide (DMF) using an evaporator. RGO can be dispersed well in many kinds of organic solvents including DMF, while it is easily aggregated in aqueous solution because of its low electrostatic repulsion force.A reduction step of GO into graphene plays an essential role to determine the electrical properties of the resulting graphene films. GO was reduced as follows: GO was dispersed in aqueous solution containing NDoping graphene via charge transfer by TCNQ molecules was carried out as follows. First, 0.01 g of TCNQ powder  was dissolved into 5 ml of DMF solvent. Then, 5 ml of RGO dispersion and radicalized TCNQ in DMF were mixed and stirred for 1 week at room temperature. The color of mixture solution changed from yellow-green to orange. Our graphene films were deposited on glass substrates (Corning7059) by a spray coat method at a substrate temperature of 200°C in an atmosphere containing the solvent vapor. The thickness of the films was controlled by varying the spray amounts.-1, referred to as the 2D band. The size of the flakes is up to 50 × 50 μm2. After liquid phase reduction by N2H4 and NH3, the solvent of the RGO aqueous solution was replaced by DMF using an evaporator. RGO can be dispersed well in many kinds of organic solvents including DMF, while it is easy to aggregate in aqueous solution due to its low electrostatic repulsion force. The conductivity and the Hall carrier mobility of individual monolayered RGO flakes were as high as 308 S · cm-1 and 121 cm2 · V-1 · s-1, respectively. Hall measurements were conducted in air at room temperature using Hall-cross geometry and Au/Ti electrodes.The Raman spectroscopy was measured with a Jasco NRS-1000 . Absorbance and transmittance spectra were obtained with Shimadzu SolidSpec3700 UV–vis by using a quartz cell for absorbance measurements. The sheet resistance was measured by van der Pauw method at room temperature in air. The presence of monolayered GO flakes in our synthesized GO aqueous solution was verified by atomic force microscope images by Raman peak shifts and by the peak shape of the second-order two-phonons process peak at 2,700 cmk-points using the Monkhorst-Pack scheme for electronic properties calculations. It is necessary to ensure that the z axis of the periodic supercell  is large enough so that there is negligible interaction between the two graphene sheets. A distance of 170 Å along the z axis is found to be sufficient to ensure the energy convergence for configurations.The electronic structural analysis is carried out using the SIESTA3.1 code, which performs fully self-consistent calculations solving the Kohn-Sham equations . The KohDoping of graphene via CT by using TCNQ molecules was carried out as follows: first, TCNQ powder was dissolved into DMF solvent. It is expected that TCNQ molecules in DMF will be radicalized . Then, t-1), C = Cring , and C = Cwing , and a bending vibration mode of C-H . We observed all of the Raman peaks originating from TCNQ molecules in the spectrum of the RGO + TCNQ complex. However, these peaks shifted from those of the TCNQ single crystal relative to each other. The C = Cring stretching and C-H bending vibrations that appeared at 1,580 cm-1 and at 1,179 cm-1 respectively were similar to the vibration modes in a benzene ring. This indicated that the quinoid ring of the TCNQ molecules transformed to a benzene ring after CT, as in the case of adsorbed TCNQ on single-wall carbon nanohorns [-1 in the RGO + TCNQ complex sample. The degree of charge transfer, Z, was estimated at 0.39 from the C ≡ N vibration frequency, which should be a linear function of Z[Z value (degree of CT) was estimated at 0.27 from the C ≡ N vibration frequency in the Raman spectra. Doping effects from the surface adsorption were limited by the amount of adsorbed molecules, due to the strong intermolecular repulsive interaction [Z = 0.39).We made an attempt to conduct a Raman spectroscopic study of RGO + TCNQ films fabricated by spray coating and of TCNQ single crystals in order to elaborate the CT interaction. The obtained Raman spectra are summarized in Figure anohorns . Meanwhition of Z. Moreovetion of Z. The shering 1,60 cm-1, aneraction ,36. On teraction ,21,26. Ieraction ,37. We phν = 21.2 eV). We measured the UPS spectra of doped and non-doped RGO films under an applied sample bias voltage of -9 eV. The work function (Φ) increased by 0.4 eV from pristine RGO films relative to the RGO + TCNQ films as shown in Figure EF) shifting towards the Dirac point (ED) due to hole doping from TCNQ via CT, and the interface dipole effect for the TCNQ + RGO films might be smaller than that induced at a deposited F4-TCNQ/graphene interface [Additional evidence for the CT interaction was obtained via UPS using He1 radiation (nterface ,38.The extent of charge transfer between TCNQ and graphene was estimated using the Mülliken population analysis based on first-principles calculation. The amounts of charge transfer and adsorption energy  for the Rs, as a function of transmittance at 550 nm is summarized in Figure 10 cm-2 to 1.17 × 1012 cm-2 estimated from Hall measurement. Doping stability with time evolution at room temperature under ambient atmosphere was monitored. Rs increased by less than 10% after 1 year, whereas it increased by up to 40 % after 20 days in the case of AuCl3 which showed one of the highest doping effect [We verified the effects of doping on the electrical properties of graphene, and demonstrated the huge potential of transparent conductive graphene films. No dominant changes were observed in the optical transmittance spectra after doping, except for the appearance of a slight adsorption around 500 nm by TCNQ molecules . The sheg effect . Thermalg effect . Those sWe developed a novel method for the carrier doping of graphene using radical-assisted conjugated organic molecules in the liquid phase. The absorbance data and the Raman spectra results indicated strong charge transfer interactions between RGO and TCNQ. The high doping efficiency of our method was demonstrated as an improvement in sheet resistance by two orders of magnitude, without degradation of the optical transparency. First-principles calculation predicted the model of our doping mechanism and the origin of high doping efficiency. Furthermore, the doping effect was quite chemically stable. The doped chemically derived graphene films fabricated by all-wet process have huge potential as an alternative material for transparent conductive films in low-cost and low-temperature processes.The authors declare that they have no competing interests.RI designed and conducted all experiments and characterization and drafted the manuscript. PK, MB, and YK helped in technical support for experiments and drafting the manuscript. Both AS and MK have read and approved the final manuscript. All authors read and approved the final manuscript.