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.


Difficulty with turning is a major contributor to mobility disability and falls in people with movement disorders, such as Parkinson's disease (PD). Turning often results in freezing and/or falling in patients with PD. However, asking a patient to execute a turn in the clinic often does not reveal their impairments. Continuous monitoring of turning with wearable sensors during spontaneous daily activities may help clinicians and patients determine who is at risk of falls and could benefit from preventative interventions. In this study, we show that continuous monitoring of natural turning with wearable sensors during daily activities inside and outside the home is feasible for people with PD and elderly people. We developed an algorithm to detect and characterize turns during gait, using wearable inertial sensors. First, we validate the turning algorithm in the laboratory against a Motion Analysis system and against a video analysis of 21 PD patients and 19 control (CT) subjects wearing an inertial sensor on the pelvis. Compared to Motion Analysis and video, the algorithm maintained a sensitivity of 0.90 and 0.76 and a specificity of 0.75 and 0.65, respectively. Second, we apply the turning algorithm to data collected in the home from 12 PD and 18 CT subjects. The algorithm successfully detects turn characteristics, and the results show that, compared to controls, PD subjects tend to take shorter turns with smaller turn angles and more steps. Furthermore, PD subjects show more variability in all turn metrics throughout the day and the week. Difficulty turning during gait is a major contributor to mobility disability, falls and reduced quality of life in older people and people with movement disorders. Studies have found that measurements of turning discriminate elderly fallers from non-fallers . Falls dCurrent clinical measures to assess mobility in PD and other movement disorders are coarse and insensitive. The most common objective method of tracking movement is based on optical Motion Analysis systems. However, these systems are costly, time consuming and can only be used in large laboratories. The need to overcome these limitations has led to an upsurge in research on the use of wearable inertial sensors for the study of human movement . SensorsGait research has focused primarily on straight-ahead walking ,23, and The assessment of gait and balance in the clinic does not adequately reflect mobility function during daily life, and little is known about turning characteristics in the home or community settings. Neurologists currently rely upon patient interviews and unreliable diaries for medical decision-making regarding fluctuations in motor function. As PD advances, patients experience frequent fluctuations in their motor function. Movement disorder specialists spend much of their effort on reducing these fluctuations by adjusting medication schedules and dosages, changing medications and referring to deep brain stimulation surgery. A brief medical examination of PD patients misses these diurnal fluctuations.et al. used activity monitors  to quantify changes in ambulatory activity following deep brain stimulation in advanced PD over a seven-day period. They found a significant increase in the length and variability of walking bouts, but the total number of steps per day did not change [Clinicians and patients would benefit from a system they can easily use to measure daily mobility and assess its fluctuations throughout the day, evaluate their risk of falling and measure the effects of treatment and exercise. However, no current system actually characterizes the quality of gait or turning or mobility fluctuations across days and weeks, because of the lack of sophisticated analysis and adequate technology. A few earlier studies to measure movement for long periods of time utilized activity monitors (Actigraphs) ,31. Theyt change . Human mIn this study, we use wearable inertial sensors to detect and analyze prescribed and spontaneous turns during gait in the laboratory and home. In addition to turning onset, the turn detection algorithm estimates other turn metrics, including duration, peak and mean velocity, number of steps to complete a turn and body jerk during a turn. We demonstrate the validity of our inertial algorithm in both the laboratory and home environment. In the laboratory, the sensitivity and specificity of the inertial algorithm is assessed using a Motion Analysis system and video data from a waist-mounted video camera aimed at the feet. We also evaluate the performance of the inertial algorithm during seven days of continuous data collected in subjects' homes. To the best of our knowledge, our study is the first to characterize spontaneous walking and turning in the home for an extended period of one week.2.In order to develop and validate the accuracy and reliability of the turn detection algorithm, we collected two sets of data. The first set was collected in the Balance Disorders Laboratory at the Oregon Health and Science University (OHSU). A second set of continuous monitoring data was collected in subjects' homes throughout a period of seven days. The following section describes the subjects, data collection protocol, and the algorithm for detecting turns and corresponding metrics.2.1.We examined 21 PD subjects  version III 24.5 ± 7.5) and 19 control subjects (67 ± 9 years) wearing an Opal inertial sensor  on the lumbar spine, as shown in 2.2.q in quaternion form and can be used directly to transform body frame sensor measurements into the inertial frame. We transform the angular velocity measurements from the body frame, bω, to the inertial frame, iω, using the following quaternion multiplication:iω to extract the z component, Angular rotational rate of the pelvis, measured by the gyroscope about the vertical axis, is an ideal signal to detect turns. The direction of gravity, measured by the accelerometer during a stationary period, can be used to project the gyroscope measurements on to the vertical axis throughout the trial, as described in . In our It is difficult for humans to make more than a very slight turn with a duration <0.5 s or to complete an extremely slow turn with a duration >10 s during gait. Therefore, only turns with durations between 0.5 and 10 s and turn angles over 45° were considered. We combine any turns in the same direction separated by a brief pause <50 ms. Relative turn angles are obtained by integrating Algorithm 1 Turn detection algorithm. 1:iωRotate angular velocity to Earth frame,  2:ωziExtract vertical component,  3:ωzi with cf = 1.5 HzLow pass filter  4:|ωzi|≥15°/sFind maxima of absolute value,  5:for each maximum do 6: find 5°/ s crossings preceding and following maximum 7: turn duration = final zero crossing - initial zero crossing 8:end for 9:for each turn do 10:if (intra-turn duration < 0.05 s) and (previous turn is same direction) then  11:  combine with previous turn 12:end if  13:end for 14:for each remaining turn do 15:if (turn duration > 10 s) or (turn duration < 0.5 s) then  16:  eliminate turn 17:end if  18:zθ = Σzω × sT turn angle  19:if turn angle zθ < 45° then  20:  eliminate turn 21:end if  22:end for2.3.We enrolled 12 subjects with PD  and 18 older control subjects (86 ± 7.5 years) in the home for seven days. On the morning of the first day, a study coordinator met to wear them and charge them at the end of each day. Three Opal sensors were worn: one on the pelvis at the lumbar vertebral level and one on each foot. The Opal's on-board data storage can hold 720 h worth of data. Opal sensors have sufficient battery life to continuously record data over 16 h throughout the day. Participants wore the Opal sensors all day for the rest of the seven days, recharging them each night. We expanded the algorithm to capture walking and turning events during spontaneous activities using the data collected in the home. The algorithm identifies periods of walking activities during normal daily activities in the community and calculates the hourly frequency of turning, the duration of each turn, the number of steps needed to complete a turn, the peak and average rotational turning rate and jerk, as well as the variability of these measures throughout the day and week.tω, of the lumbar sensor:tω exceeded a threshold of 15 °/ s for 10 s or longer. Consecutive bouts that were less than 10 s apart were merged together and considered one bout.Periods of walking (bouts) were detected from the total rotational rate, The number and duration of each step during detected walking bouts and turns were calculated using the rotational rate data collected from the Opal sensors attached to the subject's feet. For each step, the midpoint of the foot swinging motion (midswing) was first detected by the peak of the pitch angular velocity. Initial and terminal contact of the foot with the ground, marked by the zero-crossing of the pitch angular rate around the midswing, were used to detect each step and its duration.σ, to the mean, μ.We us the coefficient of variation (CV) to analyze the variability of the turn metrics throughout the day and week among the PD and control subjects. The CV summarizes the amount of variation as the ratio of the standard deviation, 3.First, we present the results related to the validation of the inertial sensor algorithms based on optical and video data collected in the laboratory. Then, we compare turning and characteristics between the subjects with and without PD obtained from the continuous inertial data collected in the home.3.1.The video data was reviewed by two independent raters who annotated the beginning and end time of each turn based on the rotation of the feet over the ground. The time indices of turns detected by the two video raters were upsampled from 30 to 128 Hz to match the sampling rate of the inertial data. Similarly, all turn metrics from Motion Analysis were resampled at 128 Hz. Rather than compare the inertial algorithm to video and Motion Analysis based on turn-by-turn detection accuracy, we validate the performance and agreement of the systems based on sample-by-sample accuracy. Although this might degrade the performance results, the timing accuracy of turn detection is important for characterizing turn duration. Therefore, a true positive turn sample was declared when detected by the inertial algorithm and the other gold-standard system. A false negative was declared when a turn sample was detected by the gold-standard system, but not detached by the inertial algorithm.A true negative was declared when no turn sample was detected by either the inertial algorithm or the gold-standard. A false positive was declared when a turn sample was detached by the inertial algorithm, but not by the gold-standard. The specificity of the inertial algorithm is 0.75, 0.69 and 0.60 with Motion Analysis and the two video raters.Motion Analysis has a better specificity than the inertial algorithm compared to the video raters; see Despite the good agreement between the inertial algorithm and the other gold-standard systems, some disagreement could be attributed to the different definition of a turn used with each system. For example, the video raters were instructed to annotate the time of the heel strike prior to and following the end of each turn. The heel strikes were chosen because the onset and end of each turn starting at the subject's waist is often ambiguous from the video recording. This, however, differs from the way that humans tend to turn by first rotating the pelvis around the vertical axis before turning their feet. This is consistent with the turn definition used for the inertial algorithm, which is based on the angular velocity of the lumbar. The optical marker-based algorithm is qualitatively similar, in that it first estimates a continuously varying yaw angle and then detects turns based on the turn angle. This might explain the better agreement between the Motion Analysis and inertial detected turns.The inertial sensor-based algorithm can be used to detect turns during walking at least as well as the Motion Analysis system. The number of detected turns for the inertial algorithm averaged 10.9 ± 1.6 across all subjects and trials. Similarly, the algorithm based on optical markers detected an average of 10.4 ± 3.4 turns. Subjects were inconsistent with one particular turn, when they were asked to pick up a light basket and turn. Some subjects avoided the turn altogether by picking up the basket without any turning at the waist. In this close pattern with the rapid series of turns, some subjects blended two turns together as they transitioned fluidly from one to the next rather than following the line precisely. Both of these factors contribute to variability in the number of detected turns, especially in the fast trials.Analysis of the pelvic rotation shows that PD subjects had a slower velocity compared to controls while turning at their self-determined slow, preferred and faster gait speeds. Even when matched for the same gait speed, patients with PD show impaired turning because of the significant challenge to their dynamic balance. However, turn duration was not significantly different between PD and control subjects.It has been shown that PD patients appear to walk better when they are examined in an outpatient clinic or in a research laboratory than caregivers report about their daily lives . This is3.2.Using the inertial algorithm validated with the laboratory data, we analyze the continuous data collected in the home. We expanded the algorithm to capture walking and turning events during spontaneous activities. The algorithm identifies periods of walking activities inside and outside the home and calculates the number of turns and corresponding turn metrics during each hour of the day. p-value for a t-test at a 5% level of significance to determine if PD and CT bout and turn metrics significantly differ from each other. Active rate is defined as the percentage of time when the subject is walking or turning compared to the full time of monitoring during the day. Bout duration and active rate are statistically significantly different between PD and control subjects. Active rate and number of bouts per hour show the largest variation among all subjects, with a clear trend of subjects being more active during the first three hours of the day. Activity rate declines for an average of three hours, then increases again during the 7–8th h. PD and control subjects generally complied with wearing the inertial sensors as instructed for the entire week, for an average of ten hours per day.p-value for the t-test.Subjects exhibit a great variability of the number of turns throughout the day, with an average of 65.2 and 67.3 turns/h for control and PD subjects, respectively. PD subjects tend to complete shorter turns with smaller turn angles, compared to the control group. Data also shows that PD subjects complete a turn with a larger number of steps. Consistent with the results obtained from the laboratory data, PD subjects show more variability than controls in all turn metrics throughout the day and the week.Compared to turn duration during the prescribed tasks in the laboratory (1.4 s), both control and PD subjects completed a turn with a longer average duration of 2.2 s and 2.0 s, respectively. Similarly, analysis of the pelvic rotational rate during turning shows that subjects significantly decreased their rotational rate in the home, compared to in the laboratory. In a previous study, it was demonstrated that even an identical instrumented test of mobility conducted in the home environment resulted in slower turns and walking than when conducted in the laboratory .4.In this study, we developed a wearable inertial system for continuous monitoring to detect and distinguish turning characteristics in healthy subjects and individuals with Parkinson's disease (PD). Assessment of the system in the laboratory showed that the inertial system can be used to detect turns with a comparable accuracy to that of the gold-standard Motion Analysis. We demonstrated that we could use the inertial system to measure locomotor activities and to characterize turns in the home throughout the day and week, using three wearable sensors. The algorithm successfully quantifies turns, and results show significant differences between turn characteristics of PD and control subjects. Compared to controls, PD subjects tend to take shorter turns with smaller turn angles and more steps. Furthermore, PD subjects show more variability in all turn metrics throughout the day and the week. Precise measures of turning mobility will improve intervention for balance disorders and fall prevention in the elderly and in patients with neurological diseases.


Exon recognition occurs through base-pairing interactions between two guide sequences on the ribozyme domain dI known as EBS1 and EBS2 and two stretches of sequence known as IBS1 and IBS2 on the 5' exon, whereas the 3' exon is recognized through interaction with the sequence immediately upstream from EBS1 [(δ-δ' interaction (subgroup IIA)] or with a nucleotide [(EBS3-IBS3 interaction (subgroup IIB and IIC))] located in the coordination-loop of dI. The δ nucleotide is involved in base pairing with another intron residue (δ') in subgroup IIB introns and this interaction facilitates base pairing between the 5' exon and the intron.Group II intron splicing proceeds through two sequential transesterification reactions in which the 5' and 3'-exons are joined together and the lariat intron is released. The intron-encoded protein (IEP) assists the splicing of the intron in vivo. We found that the EBS1-IBS1 interaction was required and sufficient for RmInt1 excision. In addition, we provide evidence for the occurrence of canonical δ-δ' pairing and its importance for the intron excision in vivo.In this study, we investigated nucleotide requirements in the distal 5'- and 3' exon regions, EBS-IBS interactions and δ-δ' pairing for excision of the group IIB intron RmInt1 in vivo of the RmInt1 intron is a favored process, with very few constraints for sequence recognition in both the 5' and 3'-exons. Our results contribute to understand how group II introns spread in nature, and might facilitate the use of RmInt1 in gene targeting.The excision  By contrast, most IIB and IIC introns display no statistical evidence of pairing of this δ position with the 3' exon. These group II ribozymes recognize the first nucleotide in the 3' exon, referred to as IBS3, rather than δ', by canonical base-pairing with an intron nucleotide, denoted EBS3 rather than δ, located in the "coordination loop" of dI [Group II introns are ribozymes and mobile retroelements consisting of a highly structured RNA organized into six distinct domains (dI to dVI). They have a multifunctional intron-encoded protein (IEP) ORF in dIV, encoding a reverse transcriptase-maturase that is required tructure . Group Itructure . In the tructure . In the p" of dI . The δ pSinorhizobium meliloti, a group IIB intron, is a mobile intron that self-splices in vitro in the absence of the IEP [in vivo and observed a low efficiency; only 0.07% of exons were joined [The RmInt1 intron of  the IEP ,6. Recene joined , but is in vivo. We found that exon recognition by the intron is subject to few constraints, the most critical of which is the EBS1-IBS1 interaction and the δ-δ' pairing. Our findings might have practical implications for the development of RmInt1 as a highly efficient gene targeting vector and support the extraordinary ability of RmInt1 for its dispersal in nature.In this study, we analyzed the nucleotide requirements in the distal 5' and 3' exon regions, EBS-IBS interactions and δ-δ' pairing for excision of the IIB intron RmInt1 in vitro trans-splicing depends on exon-intron binding sequences, the EBS1-IBS1 and EBS2-IBS2 pairings, whereas normal cis-splicing requires only the EBS1-IBS1 pairings [in vivo, by introducing single and multiple nucleotide substitutions into the IBS1 and IBS2 elements , primer extension or cRT-PCR (data not shown). Moreover, we showed previously that the lariat form accumulates in the splicing process and that this product is free of lariat-3' exon reaction intermediates Likewise, the RmInt1 splicing process was inefficient, shown by a low detection of ligated exons, more likely to be susceptible to cell degradation [The excision reaction was characterized by primer extension; with a primer P complementary to a sequence located 80-97 nt from the 5' end of the intron. In these assays, the excised intron RNA was detected as a 97 nt extension product Figure . In addiThe IBS2 stretch encompasses 5 nt (positions -13 to -9) in the 5' exon of RmInt1 that form Watson-Crick pairs with the EBS2 intron RNA sequence . The extension of the linker nucleotide between IBS2 and IBS1 by the addition of one nucleotide  led to a moderate decrease in excision efficiency (~87% of wild-type levels). Thus, in contrast to the critical positions of the EBS-IBS interactions in the DNA Target [By contrast, more heterogeneous results were obtained for nucleotide substitutions within the IBS1 element. The IBS1 stretch encompasses 7 nt, positions -7 to -1 with respect for the intron insertion site, that base-pair with the EBS1 sequence in the intron RNA  that rendered excision undetectable, and simultaneous changes of the -4, -3 and -2 nucleotides , which strongly reduced intron excision (~13% of wild-type levels). These results identified the IBS1 element in the 5' exon as the most critical for the intron excision reaction. A combined mutant (M2.3/-5G) lacking the canonical IBS2 sequence plus the substitution of the U-5 nucleotides by a G residue in IBS1 rendered intron excision barely detectable (lane 9), which reveals the additional contribution of the IBS2-EBS2 pairing to the efficiency of the intron excision reaction.(iv)[in vivo.In class IIB introns, position +1 at the 3' exon, known as IBS3 (C+1 residue in the RmInt1 3' exon), base-pairs with the EBS3 element in the intron RNA (position 329G), which is located in the "coordination loop" at dI and displaced only one nucleotide from the 3' branch of helix ID(iv). Interes(iv), resultein vivo is a highly favored process, with IBS1-EBS1 pairing the most critical step. As the intron lariat released from the 3'exon remains associated with the IEP, forming a ribonucleoprotein particle complex (RNP) that mediates intron mobility, we investigated whether the observed decrease in excision was correlated with reduced intron mobility. We carried out a homing assay with intron donor and recipient plasmids, in which intron mobility was assessed by DNA hybridization. Once RmInt1 was excised in vivo from the exon-mutant donor constructs, the RNPs produced were wild-type. Following introduction of either the wild-type intron (pKG2.5) or mutant constructs into a S. meliloti RMO17 strain harboring the target-recipient plasmid pJB0.6as, a broad correspondence was observed between the excision level detected by primer extension and homing efficiency  and reverse (intron retrohoming) splicing reactions. Our results contribute to understand how group II introns spread in nature, and might facilitate the use of RmInt1 in gene targeting.We show here that the intron-exon interaction IBS1-EBS1 and δ-δ' interaction in the domain dI of the rybozime, play an important role in the RmInt1 excision in vivo is subject to few constraints, the most critical of which is the EBS1-IBS1 interaction. Thus, the spread of group II introns into bacterial genomes from ectopic sites to homing sites may occur efficiently, increasing their ability to spread in nature.Group II introns can move site-specifically to homologous intronless genes, in a process known as retrohoming or, at much lower frequencies, to novel ectopic sites, in a process known as retrotransposition. It is believed that the latter process has led to the wide dispersal of mobile group II introns among bacterial species and also may have been used to invade eukaryotic genomes, where mobile group II intron are thought to have evolved into spliceosomal introns and non-long-terminal-repeat retrotransposons . Intron Gene targeting technology based on group II introns requires the modification of IBS2, IBS1 and IBS3, to render these sites complementary to the retargeted EBS2, EBS1 and EBS3 sequences for efficient RNA splicing ,14. We hS. meliloti RMO17, lacking the RmInt1 intron, was grown at 28°C on TY or defined minimal medium [E. coli DH5α was used for cloning and the maintenance of recombinant plasmids; it was cultured at 37°C in Luria-Bertani medium [S. meliloti and 50 μg/ml for E. coli; ampicillin, 200 μg/ml and tetracycline, 10 μg/ml.Rhizobial strain l medium . E. coliAltered Sites II in vitro Mutagenesis System , using pAL2.5 as a template. This plasmid was generated by inserting a 2.5 kb SphI fragment containing the entire RmInt1 sequence and flanking exons (-174/+466) from pGEmex2.5 [BamHI/SpeI fragment and inserted into pKG0 (pKG2.5 and mutant derivatives), which constitutively expresses RmInt1 and its derivatives under control of the promoter of the Km resistance gene [RmInt1 donor derivatives containing point or accumulative substitutions in the flanking regions of intron insertion sites and ribozyme domain dI, including the δ and δ' sequences, were generated with the GEmex2.5  into thence gene .600 nm of 0.6 to 0.9).RNPs were isolated essentially as previously described ,20. RNPsThese experiments were carried out on RNP preparations, since the lariat intron seems to be less susceptible to degradation than ligated exons, due to its association with the IEP forming ribonucleoprotein particles (RNPs). Primer extension reactions were essentially carried out as previously described . SamplesS.meliloti specific primers Sino 452 (5'-CGTTGTTCGGAATTACTGG) and Sino 952 (5'-TGTCTCCGATCCAGC). Primer specificity was determined by melting-curve analysis and gel electrophoresis , with the final volume made up to 23 μl with DEPC water. The PCR cycling conditions were as follows: hot start, with heating at 94°C for 3 min, followed by 40 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s. Controls without a template were also included. Two independent RNA preparations from each strain were tested. Relative quantification was carried out for splicing, based on the 2-ΔΔCT method [-ΔΔCT where ΔΔCT = ΔCT (DV* or +1A) - ΔCT (WT). ΔCT (DV* or +1A) = CT (DV or +1A mRNA amplified by 5-6 primers) - CT (DV or +1A mRNA amplified by S. meliloti specific 16S primers Sino 452 and Sino952). ΔCT (WT) = CT (WT mRNA amplified by 5-6 primers) - CT (WT mRNA amplified by S. meliloti specific 16S rRNA primers Sino 452 and Sino952). The result is expressed as a percentage related to WT construction. The error is calculated applying the formula: 2-ΔΔCT · ln2 · ΣSE.For the quantification of ligated RmInt1 flanking exons, a 283 bp fragment was amplified with 3'RTSp (5'-ATTCAACGGGAAACGTCTTG) and +9 (5'-AGCGGCGCCATGTTCGTCTT) primers. To normalize the loading amount of the starting material, a 500 bp fragment of the 16S ribosomal subunit RNA was amplified with T method . BrieflyS. meliloti RmInt1-less strain RMO17. We studied homing between compatible intron donor (pKG2.5 and mutant derivatives) and target-site recipient plasmids (pJB0.6as (-174/+466) or pJB-20/+5) as previously described [E. coli DH5α to rhizobial strains by tri-parental mating, using pRK2013 as a helper plasmid [Rm2011-2 element .Click here for file