Daphnia pulex is an especially attractive system for interrogating the gain and loss of introns in natural populations. In this study, we used a comparative population-genomic approach to identify and characterize 90 recently gained introns in this species. Molecular clock analyses indicate that these introns arose between 3.9 × 105 and 1.45 × 104 years ago, with a spike in intron proliferation approximately 5.2 × 104 to 1.22 × 105 years ago. Parallel gains at homologous positions contribute to 47.8% (43/90) of discovered new introns. A disproportionally large number of new introns were found in historically isolated populations in Oregon. Nonetheless, derived, intron-bearing alleles were also identified in a wide range of geographic locations, suggesting intron gain and, to a lesser degree, intron loss are important sources of genetic variation in natural populations of Daphnia. A majority (55/90 or 61.1%) of the identified neointrons have associated internal direct repeats with lengths and compositions that are unlikely to occur by chance, suggesting repeated bouts of staggered double-strand breaks (DSBs) during their evolution. Accordingly, internal, staggered DSBs may contribute to a passive trend toward increased length and sequence diversity in nascent introns.As one of the few known species in an active phase of intron proliferation, the microcrustacean  Giardia lamblia  by the spliceosomal machinery after transcription . Among e lamblia  to an av lamblia . Althoug lamblia .Because introns are often found at the same positions in numerous orthologous genes of widely diverged eukaryotic species , it is lMost attempts to estimate rates of intron gain and loss rely on matrices of intron presence or absence in alignments of highly conserved regions of homologous genes , 2007b. The restrictions of such studies to a small subset of highly conserved genes sampled from distantly related species, to unambiguously aligned regions, and usually to just one sequence per species raise concerns with respect to the generality of the conclusions drawn from such studies. In addition, deep phylogenetic comparisons are of limited use for testing hypotheses regarding the molecular mechanisms of intron gain and loss because the molecular evidence of such events has generally been erased by accumulated mutations. Furthermore, broad phylogenetic comparisons of shared intron positions usually rely on assumptions of character-state irreversibility  repaired by nonhomologous end joining (NHEJ)   and gaing (NHEJ) . Other cg (NHEJ) . Additiog (NHEJ) , reinserg (NHEJ) , or prolg (NHEJ) . Moreovee common .D. pulex, expanding on our previous work (D. pulex clones and a subsequent polymerase chain reaction (PCR)-based survey of 84 additional D. pulex clones sampled from across North America. Because D. pulex harbors substantial nucleotide diversity, it was further possible to use gene phylogenies to assess the polarity of intron gains and losses. New insights regarding the age of derived, intron-bearing alleles and likely molecular events in the early evolution of introns after their initial formation are discussed in the context of our results.Although natural populations harboring intron presence/absence polymorphisms are ideal systems for identifying the molecular footprints of recent intron gains, to date, only a few studies have reported such polymorphisms . Here, wous work . These nD. pulex clones were sequenced and analyzed for intron presence/absence polymorphisms .Complete genomes of nine orphisms . Each of project . AnotherDNA used for high-throughput sequencing was extracted from >200 individuals of each clone using a Qiagen DNeasy tissue kit  following manufacturer’s instruction. DNA libraries were prepared and sequenced by the Beijing Genome Institute. Each clone was shotgun sequenced using an Illumina genome analyzer, yielding ≥20× coverage per site. Raw paired-end reads, each ∼75 bp in length, were de novo assembled using SOAP . More thA), a Perl script was used to generate 100-bp hybrid sequences comprising 50-bp portions of the exons flanking each intron in the TCO genome. These hybrid sequences were used to BLAST against contigs of sequenced genomes of the other eight clones of D. pulex. This strategy allowed us to find intron absence alleles in non-TCO clones of D. pulex. The same strategy was used for intron presence/absence comparisons between TCO and the D. magna Finnish clone.Two main strategies were used to search for intron presence/absence polymorphisms among newly assembled genomes. In Strategy I A, a PerlB), each exon in TCO was used to BLAST against newly assembled contigs of the other eight D. pulex clones to search for exonic insertions that are present in non-TCO clones but absent in TCO. Sequences of top hits were aligned using ClustalX  from conserved flanking protein-coding regions around the gain–loss polymorphisms to generate an ∼1 kb amplicon that contains at least two upstream or downstream introns. Including other nonpolymorphic introns in our targeted PCR amplifications facilitated detection of linked presence–absence polymorphisms, as linked intron loss might simply reflect the presence of processed pseudogenes. In such cases, these sites were excluded from further analysis. The same set of primers was also used to screen cDNA prepared from surveyed clones (see below).Primer pairs were designed using Primer 3 . The QIAprep Spin Miniprep Kit (Qiagen) was used for plasmid purification, and a T7 primer was used to sequence cloned inserts. To avoid PCR and cloning errors, 10–14 cloned products were sequenced per individual for these loci. Additionally, to ensure that cloning-induced errors were not the source of observed polymorphisms, sequences from cloned PCR products were compared with the directly sequenced PCR products .Daphnia individuals. mRNA was extracted using the Qiagen RNeasy Mini Kit (Qiagen). Extracted mRNA was quantified using a NanoDrop 3300 . A total of 1,000 µg of mRNA from each sample was used for cDNA synthesis using the SuperScript III first-strand synthesis kit  following the manufacturer’s instruction. Primers used in genomic DNA-based PCR were applied to synthesize cDNAs using the above-described amplification conditions. Obtained sequences of cDNA fragments were aligned with genomic sequences using ClustalX  part of the alignment of the gained intron sequences is completely conserved, but the remainder is highly divergent. Because introns that fall into these two patterns are homologous for at least part of their lengths, we scored them as a single gain event. To define parallel gains, we apply the following criteria: 1) If insertion sites for two functional introns are within 10 bases of one another, but not exactly the same insertion site, we regard these insertions as parallel gains  A gap in the alignment of gained introns from diverse t-tests were used to assess whether differences in the lengths of exons or the frequencies of their repeats were significant and chi-square tests were used to assess whether differences in base composition were significant and implemented using StatPlus:mac LE.2009 . An additional Python script (RepCheck), described below, was used to compare repetitive DNA content in the above three categories of exonic regions.To assess whether exonic regions surrounding the sites of established introns, singly gained or parallelly gained introns, differed substantively, an original Python script was used to compare their lengths, base composition, repeat content, and repeat frequency. πs) was calculated using DnaSP , and exon regions that are downstream of a derived intron-bearing exon. Sequences from outgroup species were excluded from our calculation. For each exon region, average pairwise sequence diversity at silent sites  Repetitive motifs (excluding microsatellites) within each new intron; 2) repetitive motifs shared by new introns at distinct loci; 3) repetitive motifs shared by exons at distinct loci hosting newly gained introns; and 4) repetitive motifs common to a neointron and one of its adjacent exons.P value of 0.0001 was used to reduce the chance of recovering coincidentally similar sequence segments. However, in the cases of a handful of examined neointrons, these search criteria revealed no significant motifs. For these introns only, an additional lower stringency screen (P ≤ 0.001) was used to recover repetitive elements of potential importance.Short sequence repeats in newly gained introns or adjacent exons were identified using REPFIND . A cut-oSpace_for_2nd_Repeat (stored in the program as Prob_of_2nd_Occurence). The program then compares each repeat identified by REPFIND with each intron in which it occurs. Repeats that have a probability of 0.05 or less of occurring a second time in a given intron are written to a tab-delimited output table. The source code for RepCheck is available from the authors upon request.For the repetitive motifs identified in category one, an addition Python script, called RepCheck (developed for this study), was used to evaluate the probability that any repeat identified by REPFIND would occur twice, by chance, in a particular intron. To evaluate this probability the following quantities were gathered: 1) The frequency of each nucleotide in an intron of interest; 2) the length of the intron of interest ( = Intron_Length); 3) the base composition of the repeat of interest; and 4) the length of the repeat of interest. The quantities are used to assess the probability of a given sequence occurring a second time in a given intron as follows: 1) Multiply together the frequencies of each nucleotide of a repeat in the intron where it is found (the result is stored in the program as Simple_Prob); and 2) determine the available space for a second occurrence of a given repeat in a given intron by taking the length of the intron and subtracting twice the length of the repeat less 1 (the result is stored in the program as Space_for_2nd_Repeat). The probability of the repeat occurring a second time is: 1 − (1 − Simple_Prob)For recovered direct repeats in classes two, three, and four, an additional Python script was used to determine the length, frequency, and base composition of each detected direct repeat. These metrics were subsequently evaluated using StatPlus:mac LE.2009  to assess the following: 1) Their distributions; 2) correlations among them; and 3) significant differences between them. The same methods of analysis were used to evaluate the repetitive content of exonic regions surrounding the sites of single and parallel gains, to assess whether there was a substantive difference between exons in these two categories.D. pulex alleles surveyed for a certain locus following the method in Daphnia species available for each locus to determine the derived state of SNPs with a congruent method. An allele phylogeny for each locus was used to find the most closely related Daphnia species of D. pulex. Once the derived SNPs were identified by this method, the derived allele frequencies of SNPs at silent sites were calculated by dividing the number of derived single nucleotides by the total number of single nucleotides of D. pulex at sampled positions.To compare the allele frequency spectra of gained introns and derived single nucleotide polymorphisms (SNPs), derived allele states of both types were inferred from an outgroup to polarize polymorphisms. Original Python scripts were used to calculate the frequencies for intron-bearing alleles and derived SNPs at silent sites. The frequencies of new introns were calculated by dividing the number of new intron-containing alleles by the total number of µ, for D. pulex is 6 × 10−9 per base per generation at silent sites  (D. pulex clones is 3.4 × 105 years (standard deviation = 8.02 × 104 years). This number is used as a calibration point for molecular clock dating using BEAST.To estimate the approximate age of the most recent common ancestor (MRCA) of th MEGA5  using th. pulex) . The appDaphnia  for a prior distribution, ucld.mean  was set to uniform, and an estimated silent site mutation rate of 6 × 10−8 per base per year was provided; and 6) for Markov chain Monte Carlo, the length of the Markov chain is 1,000,000 generations, echoing state to screen every 10,000 generations, and logging parameters every 200. Thus, the final sample files contained 5,000 trees.BEAST v1.7.2 and associated software applications, BEAUTi v1.7.2, TreeAnnotator v1.7.2, and FigTree v1.3.1, were used to facilitate molecular clock dating to estimate the age of new introns in Daphnia . To prepTreeAnnotator v1.7.2 was used to process the resultant trees. Parameters for this step were set as follows: 1) For burnin, we chose 50 to specify a 1% burnin; 2) for posterior probability limit, we chose 0.5, which summarizes the information for nodes with this posterior probability; and 3) for node heights, we used mean heights. FigTree was used to display the features of summary trees generated by TreeAnnotator. In FigTree, posterior was selected for display of branch labels and node ages for display of node labels.D. pulex populations were calculated in three categories: 1) An average age estimate of all derived intron-bearing alleles that are polymorphic in D. pulex; 2) an average age estimate of new introns that are endemic to Oregon populations only; and 3) an average age estimate of multiple unique introns gained at nearly the same location. Clades used for these calculations met two criteria: 1) They comprise only GIB alleles and 2) they have a posterior probability greater than 0.65 in the final summary tree generated by TreeAnnotator. A histogram of allele ages was generated in R 3.0.1.The results for gained intron-bearing (GIB) alleles that are still segregating in http://blast.ncbi.nlm.nih.gov/Blast.cgi, last accessed June 20, 2014). The full nonredundant/nucleotide (nr/nt) database in GenBank was searched for significant matches. BLAST algorithm parameters were set for “somewhat similar sequences” (BlastN), with other parameters left at default settings. Only hits with an e-value < 0.01 and sequence identify > 75% were deemed to be potential homologs of inserted sequences in Daphnia.BLAST searches were conducted using the sequences of newly gained introns as queries with the nucleotide BLAST program .Through a series of BLAST searches, we obtained orthologous sequences from the genomes of nine recently sequenced 85 genes   AT content in contrast to 55.0 ± 0.2 in adjacent exons of the same gene . Of these, 25 contain in-frame stop codons, an amount that is not significantly different from the expected value, given the overall rate of stop codon-bearing neointrons in our data set. Among the introns that did not contain stop codons, a majority (71%) would still result in frameshift mutations in downstream exons, if unspliced. In total, 88 of the 90 neointrons identified through this study would likely eliminate gene function if they were not spliced from pre-mRNA transcripts .The 90 ame gene . Approxibsupplementary fig. S63, Supplementary Material online), while the other has GG … AG . Rare, alternative splice sites have been observed in other species. For example, in the human genome, 99.1% of the introns have GT … AG boundaries, 0.9% of them have GC … AG boundaries, and 0.1% of them have AT … AC splice site junctions . For example, at locus Dpul_328763 , all outgroup species, all D. pulex clones from Oregon, and 14 non-Oregon clones have a plesiomorphic allele, Dpul_328763(+), that has an intron separating exons 8 and 9. In contrast, a set of geographically diverse populations in a nested clade have an alternative allele, Dpul_328763(−), that lacks this intron and, instead, has a single exon that is homologous to both exons 8 and 9 in Dpul_328763(+) alleles.Combining results of this survey of 84 us study  reveals µ) for D. pulex of 6 × 10−8 mutations per base per year  years, is approximately the same as the MRCA of all D. pulex.Based on the silent site mutation rate  is 1.40 ± 0.09 × 105 (mean ± SE) years. D. pulex populations in Oregon were thought to have experienced one or more historical bottleneck events (5 (mean ± SE) years.Distributions of derived intron-bearing alleles that are still segregating in el gains . Our estk events . Thus, tD. pulex resulted from parallel gain events. The estimate for the average age of allele clades encompassing multiple parallel intron gains at nearly the same location  is 1.13 ± 0.12 × 105 years. Overall, the distribution of ages of the 90 neointrons identified in this study is positively skewed and ranges from 1.45 × 104 to 3.9 × 105 years ago. A pronounced spike in intron accumulation occurred between 5.2 × 104 and 1.22 × 105 years ago .Based on sequence characteristics of these newly gained introns and adjacent exons, observed events of intron gain are associated with initial DSBs. Some appear to have been formed through staggered DSBs, evidenced by direct repeats at opposite ends. Others show evidence of a combination of blunt and staggered DSBs at the same site, although the order of these two events is sometimes unclear (D. pulex. Identified repeats were short segments (ranging from 4 to 17 bases) that occurred more than once (excluding microsatellites) and were discovered through either a high stringency (P ≤ 0.0001) or low stringency (P ≤ 0.001) REPFIND . Repair of staggered DSBs creates direct repeats, leaving a distinctive molecular footprint. However, the presence of some direct repeats is expected to be obscured by subsequent mutations on the timescale of 1/µ (µ being the mutation rate per base per generation).We discovered that a majority of neointrons contain internal repeats caused by staggered DSBs. Probability assessment by RepCheck indicates that 61.1% (55/90) of these internal repeats were unlikely to occur by chance . If internal staggered DSBs were common, recurrent events in introns, we would expect similar percentages of new and old introns to have internal direct repeats. However, a much higher percentage of neointrons have significant internal repeats (61.1%). A possible explanation for these observed differences is that internal DSBs occurred at precisely the time of new intron formation, by complex process that is yet to be explained. Over time, the initial short sequence repeats generated by staggered DSBs will have differentiated from one another through the accumulation of mutations as introns age.The same search strategy was applied to other established introns in the same gene for each surveyed locus. Among 617 examined introns, only 100 (16.2%) have internal repeats that pass the cutoff A; also see examples in supplementary figs. S5 and S11, Supplementary Material online); 2) single staggered DSB followed by sequence insertion ; 3) blunt and staggered DSBs that occurred side-by-side at the same locus, although the order is unclear ; 4) blunt-then-staggered  ; 5) blunt-then-blunt  ; 6) staggered-then-staggered ; and 7) staggered-then-blunt  . The category into which each new intron can most plausibly be assigned is listed in supplementary table S3, Supplementary Material online.New introns identified in this study can be categorized into seven categories, based on the apparent mechanism of their formation : 1) Singfig. 3D. pulex fall into three categories, based on phylogenetic patterns in inferred trees: 1) All D. pulex: an intron-bearing allele is present in all sampled D. pulex clones and no outgroup species; 2) Nested clade: an intron-bearing allele clade is nested within D. pulex, but is absent from other D. pulex clones and all outgroups; and 3) Parallel gain: independently gained, distinct neointrons are present at homologous sites in alleles of separate D. pulex clones, but are not present in any outgroup .1.D. pulex Intron GainsAll Cases of intron gain in Dpul_228023 locus . Here, all outgroup species have an uninterrupted exon 2, while the homologous protein-coding region is interrupted by a single orthologous intron in all sampled D. pulex clones.2.Nested-Clade Intron GainsEight gained introns fall into this category. An example is the D. pulex clones. In 19 of these cases, derived clones form a monophyletic group or groups nested within the larger D. pulex clade. For example, the derived allele Dpul_341016(+) at the Dpul_341016 locus bears a single insertion in a coding region that is homologous to exon 2 at this locus in all other D. pulex clones and outgroup species. Dpul_341016(+) alleles form a clade in the inferred tree and are present in all populations outside of Oregon .3.Parallel Intron Gains In the case of 61 introns in 57 genes, derived introns are present in only a subset of sampled D. pulex exhibit a pattern of parallel intron gain. In total, parallel gains account for 51 derived intron-bearing alleles in sampled populations . All outgroup species and a sizeable number of D. pulex clones have alleles with an uninterrupted exon 11. In three independent events, distinct introns were inserted into this exon in geographically diverse clones of D. pulex. We detected 19 additional genes in D. pulex with patterns similar to that observed at Dpul_308086, although the number of alleles per locus and affected exons varied. In one extreme case, locus Dpul_327924, six different, derived introns were identified in close proximity in alignments of one exon .Twenty genes in ulations . An illuπs, using the Jukes–Cantor correction and averaged across all sampled clones)  results reveal that values of πs of exons targeted by derived introns are significantly higher . These findings indicate that exons that bear derived introns are less conserved than their upstream or downstream neointron-free exons.Average pairwise sequence diversity at silent sites ( clones)  of exonsD. pulex, given that none of the outgroup species have introns at homologous locations. The rest of the neointrons have a frequency ranging from 1.4% to 98.3%. The frequency spectrum of derived intron-bearing alleles is skewed toward lower values  similar to that of derived SNPs at silent sites . One gained intron shares significant sequence identify with a D. pulex ribosomal RNA gene. Three additional significant hits were between Dpul_310811_TCO, Dpul_325592_TCO, and Dpul_210427_TCO, and genomic segments from Dania rerio, Enterocytozoon bieneusi, and Schistosoma mansoni, respectively. Possible explanation of these results would be horizontal transfer events from D. rerio, E. bieneusi, or S. mansoni, or from one of their close relatives for which genome sequences are not available. However, because these neointrons are rich with A and T mononucleotide repeats and e-values ranged from 0.01 to 3.0 × 10−5, microsatellite proliferation could also account for the observed similarity. No other neointrons yielded significant BLAST hits.In our previous study, one neointron shared significant sequence similarity to a sequence . BLAST aDpul_47827 locus reveals that when this intron is spliced out, it includes an 8 bp segment that was previously part of the upstream exonic sequence on its 3′ end . The second mechanism that can cause a length disparity between an inserted segment and the fragment excised during splicing is when an inserted segment has an internal splice site and its unspliced terminus extends the length of its adjacent exon.In a majority of cases, the newly inserted introns have canonical GT … AG splice sites at their termini. Comparison of cDNA sequences and other orthologous exons that do not have recently inserted introns reveals that the entire segment inserted into an interrupted exon was cleanly excised during RNA processing. In contrast, we identified a handful of cases in which the length of an inserted segment and the length of the RNA fragment removed during splicing are not identical. This length disparity may result from either of the two mechanisms. The first entails recruitment of a portion of an adjacent exon to become part of the excised fragment. For example, analysis of cDNA sequences at the Dpul_300453 locus is spliced out, one bp at the 5′ end of the inserted segment is recognized as an extension of the adjacent exon and 4 bp at the 5′ end of the downstream exon are included in the intron that is excised during splicing , resulting in a loss of one amino acid in the encoded protein. Similarly, at Dpul_341016 locus, 6 bp at the 5′ end of the inserted segment serve to extend the adjacent exon and 12 bp at the 5′ end of the downstream exon are excised along with the intron during splicing , resulting in a net loss of two amino acids in the encoded protein. These results indicate that exonic sequence deletion and new intron insertion co-occur at a subset of the surveyed loci.In other cases, both the mechanisms mentioned above may apply. For example, when the derived intron at the P ≤ 0.0001) screen of all neointron sequences of D. pulex using RepFind . There was a strong, positive correlation (P = 1.82 × 10−8) between AT content and the frequency of repeats that are present among several neointrons. High frequency repeats  had an average AT content of 93% and just over two-thirds of these, including the 23 most frequent repeats, had 100% AT content. Individually, high frequency repeats make up between 1.5% and 11.5% of the length of neointrons in which they reside. The abundance and AT richness of high frequency repeats help to explain the high AT content of new introns relative to adjacent exonic regions, which average only 54.7% AT content.This analysis allowed us to investigate if there are any shared motifs among the newly gained introns. A high stringency ( RepFind  identifiP ≤ 0.0001 in RepFind) of exons, which are the host of neointrons, revealed 130 repetitive motifs ranging in length from 4 to 15 bp and in frequency from 2 to 172 occurrences in the collective set of all such exon sequences . AT content of exon-specific repeats (54.6%) is generally similar to the AT content of the exons in which they reside (53.9%). Second, to search for potential sequence motifs that may facilitate single or parallel intron gain, we conducted two separate analyses: a) Exon sequences that exhibit parallel gains were compared with those having a single gain only and b) exons that contain established introns were compared with those bearing neointrons. Comparisons of sequence length, base composition, repeat content, and repeat frequency indicated no significant differences. Hence, we detected no distinguishing features in repeats found in exons that served as sites of neointron colonization once only or more than once. Additionally, none of the surveyed features appear to be unique to exons that have recent insertion of introns when compared with exons with established introns.This analysis allowed us to assess whether: 1) There are any shared motifs among the exons hosting new introns; 2) if so, do any shared motifs facilitate parallel intron gain, and 3) if there is any distinctive sequence features or motifs that distinguish exons colonized by neointrons from exons hosting established introns. Specific motifs in exons might encourage intron colonization or facilitate intron maintenance  . To scresupplementary table S6, Supplementary Material online).In cases where intron formation entailed a staggered DSB, a direct repeat of the terminal base pairs in an adjacent exon is produced at the opposite end of the neointron. Thirty-three direct repeats of this kind were identified in our data set. These repeats range in length from 4 to 12 bp. In the pool of associated intron and exon sequences, these repeats occur between 2 and 87 times and are marginally AT rich (57.3%), but no correlation between repeat AT content and frequency was detected . In sum, although it has been posited that 1) intron gains are rare, 2) intron gains are much less common than intron losses, and 3) intron gains are unlikely to occur more than once at homologous positions, our results show that these notions do not apply equally well to all lineages.Our findings indicate that several longstanding notions concerning the origin of introns differ from the results obtained by this study. First, numerous authors have suggested that intron gains are rare events . In cont Daphnia , we idenlineages , leadinglineages . In contlineages , perhapslineages . Our resDaphnia and previous comparative studies of highly diverged phylogenetic lineages may be attributable, at least in part, to the ages of the lineages investigated. Although the MRCA of sampled D. pulex is estimated to be merely 3.4 × 105 years old, prior studies compared intron presence and absence across widely diverse eukaryotes that may have shared a common ancestor no less than 2.2 × 109 years ago. Several of the neointron-bearing alleles detected in the current analysis are still actively segregating in D. pulex and may not persist in the long run. Although it remains unclear whether patterns that emerge through our analyses apply to lineages of far greater age, at present there is no formal basis for ruling this out. Notably, results similar to those of this study have been observed in other species with actively segregating neointron-bearing alleles  were discovered in these populations of Daphnia. A plausible explanation for this pattern is that the Oregon populations have smaller effective population sizes than their non-Oregon counterparts, and hence experience less effective selection, allowing a greater proportion of mildly deleterious intron-bearing alleles to become fixed . However, in several other cases, the Oregon-allele clade is nested well inside a larger clade comprising all other D. pulex populations . This latter pattern suggests that alleles at these loci originated in populations of D. pulex outside Oregon and were subsequently introduced to Oregon populations of D. pulex. In contrast, none of our inferred allele trees reveal non-Oregon clades nested within a larger Oregon clade. Hence, our results suggest that historical gene flow between Oregon and non-Oregon populations may have been largely or entirely unidirectional and that novel intron-bearing alleles received by Oregon populations are likely to be fixed or lost rapidly, due to drift, resulting in reduced overall genetic variation.In phylogenetic trees of alleles at numerous examined loci, clades comprising all Oregon alleles are sister to all other non-Oregon lineages, suggesting pronounced genetic differentiation between Oregon and non-Oregon populations  (mean ± SE), compared with that from derived SNPs at silent sites  years. In contrast, the average estimated age of derived intron-bearing alleles segregating in D. pulex populations is 1.40 ± 0.09 × 105 (mean ± SE) years, and 1.20 ± 0.9 × 105 (mean ± SE) years for derived intron-bearing alleles in Oregon populations. The frequencies of these alleles at polymorphic loci range from 1.2% to 88.9%. These results suggest that a majority of the derived intron-bearing alleles detected in this study are long lived, having persisted in natural populations of Daphnia for between 50,000 and 300,000 years, but only 6.7% of them appeared to be fixed during that time. We did not detect evidence of a selective advantage associated with new introns, although those that did become fixed may have done so as a result of hitchhiking effects caused by linked, beneficial loci. The distribution of estimated ages of new introns is positively skewed with a spike in the accumulation of recent introns gains in D. pulex between 52,000 and 122,000 years ago (A and B).Eight of the newly gained introns appear to be fixed in ears ago A and B.Arabidopsis reveal a tendency for initial length mutations to engender additional nearby length mutations that may serve to offset deleterious effects of the first indel  might help explain a standing mystery of the origin of intron gains—where do introns come from? Despite a few cases in which the sources of neointrons have been identified  , the souBased on our observation of frequent occurrence of internal repeats, we propose a new perspective to explain the mystery of the early evolution of novel introns. Regardless of the mechanisms of their initial creation, introns may subsequently expand or contract in length, possibly facilitated by repair of ongoing DSBs through NHEJ . AdditioDaphnia genome   or will cause frameshifts in downstream exons if unspliced. Our observation that there is no significant difference in the presence of PTCs between 3n and non-3n introns may reflect that neointrons surveyed in this study are robustly spliced, thus obviating a need for NMD to remove deleterious transcripts. However, a larger number of 3n introns would need to be examined to draw any firm conclusions. In the event that they are not spliced out, retained PTC-free introns might appreciably alter the length and composition of the encoded proteins, which would very likely be strongly deleterious.It has been hypothesized that the permissiveness of spliceosomes to recognize introns with weak splicing site opens the door for intron gain, and that NMD functions as a failsafe mechanism for weak splicing of new introns, especially in 3 introns . This hyintrons) . In contDaphnia genomes. For the various sequences inserted into exons, we propose that there are four potential fates: 1) Newly inserted sequences in exons that do not contain proper splicing signals are unlikely to be spliced and will likely affect gene function adversely, and can be rapidly removed by selection; 2) precise splicing of the entire insert as a neointron; 3) partial splicing that leaves a portion of the inserted sequence in the mRNA transcript; 4) activation of latent splice sites in adjacent exon regions, leading to the splicing of the inserted segment and portions of the adjacent exons. Partial splicing of inserted sequences or recruitment of adjacent exon sequences will cause the truncation or extension of the existing coding regions. If intron gain is a transient phase in the population, such events will accordingly cause elevated sequence diversity in alleles that had gained introns but lost them during the course of intron turnover relative to ancestral intron-free alleles. This might explain the higher sequence diversity (πs) of exons targeted by derived introns compared with adjacent exons. But, it is also possible that coding regions with higher than usual mutation rates may be more vulnerable to DSBs, which could potentially pave the way for new intron formation.Our results suggest that sequence insertions in coding regions are quite common in Supplementary figures S1–S77 and tables S1–S6 are available at Genome Biology and Evolution online .


The development of materials and strategies that can influence stem cell attachment, proliferation, and differentiation towards osteoblasts is of high interest to promote faster healing and reconstructions of large bone defects. Graphene and its derivatives (graphene oxide and reduced graphene oxide) have received increasing attention for biomedical applications as they present remarkable properties such as high surface area, high mechanical strength, and ease of functionalization. These biocompatible carbon-based materials can induce and sustain stem cell growth and differentiation into various lineages. Furthermore, graphene has the ability to promote and enhance osteogenic differentiation making it an interesting material for bone regeneration research. This paper will review the important advances in the ability of graphene and its related forms to induce stem cells differentiation into osteogenic lineages. Bone tissue regeneration is of high interest to promote faster healing and reconstruction of large bone defects created by tumor resection, skeletal abnormalities, fractures, and infection. The development of this field requires the use of substrates that enable cell attachment, proliferation, and differentiation –3. Sever2 carbon atoms and is the thinnest, lightest, and maybe the strongest material known [Graphene is a single atomic sheet of conjugated spal known , 11. Itsal known  making gal known –17. As gal known , 18–23.Since its discovery in 2004 by Nobel laureates Geim and Novoselov , graphenGraphene Physical, Chemical, and Mechanical PropertiesThinnest, strongest, and stiffest imaginable material .Almost transparent , 26.Most stretchable crystal (20% elasticity) .Recording thermal conductivity .Highest current density at room temperature .Completely impermeable .Highest intrinsic mobility (100 times more than in Si) .Conducting electricity in the limit of no electrons .2 g−1) [Large surface area (~2600 m2 g−1) .Longest mean free path at room temperature (micron range) .2 g−1, makes it an attractive platform for anchorage of large amounts of molecules [Due to its unique structure and a pure aromatic carbon system , it has olecules , 33.Graphene-related materials can be classified based on either number of layers  or their chemical modification such as graphene oxide (GO) or reduced GO (rGO) . GO is aGraphene in different forms can be obtained using “top-down” and “bottom-up” methods. The “top-down” approaches include mechanical and/or chemical exfoliation of graphite. The mechanical method, also known as the “Scotch tape” or peel-off technique, allows the detachment of micrometer-sized graphene flakes from a graphite crystal using adhesive tape , 32, 38.4 and H2) are pumped into a reaction chamber  at high temperatures (~1000°C). The high temperature leads to the pyrolysis of precursors and dissociates carbon atoms which then react with the substrate to produce the thin film of graphene [Graphene can also be obtained using “bottom-up” approaches like epitaxial growth and chemical vapor deposition (CVD) , 41. Thegraphene . CVD-grographene , 43–47.−1) arises from the stretching of the C–C bond in graphitic materials whereas the D band (~1340 cm−1) is only activated if disorder or defects are present. The 2D band (2500–2800 cm−1) is present in all types of sp2 carbon materials and is used to determine the number of layers of graphene [The different methods to produce graphene result in materials with different number of layers and/or chemical groups. Raman fingerprints for different groups and number of layers reflect changes in the electron bands and allow unmistakable identification and characterization of graphene by the analysis of three peaks: G, 2D, and D . The G agraphene .Graphene and its derivatives are interesting materials for biomedical applications since carbon is the basis of organic chemistry , 34. HowAnchorage-dependent cells need to adhere to substrates in order to spread, proliferate, and perform their functions , 50, 51.μg/mL) as it accumulates on the cell membrane leading to high levels of oxidative stress [μm can enter mammalian cells initiated by spontaneous penetration of lipid bilayers in a dominant edge-first or corner-first mode. Nonetheless, the uptake of microsheets larger than 5 μm in lateral dimensions is often incomplete [μg/mL), there is an increase in reactive oxygen species production and decrease in metabolic activity. However, at concentrations of 0.01 and 0.1 μg/mL, there is no increase in lactate dehydrogenase, an enzyme released upon membrane damage [Although substrates coated with graphene-based materials are not cytotoxic, the use of the material in solutions might pose hazards to cells and tissues. Cell viability may decrease significantly in solutions with high concentrations of pristine graphene  to minimize oxidation. There was no considerable toxicity after injecting mice with 20 mg/kg with PEGylated graphene as evidenced by histological and hematological analysis after 90 days. In fact, the graphene sheets levels in most organs were very low after three days from the injection. The relatively slow but persistent decrease of the material concentration in the liver and spleen suggests that the clearance of graphene nanosheets from the mouse body happens through both renal and fecal excretions .As graphene-based materials can be functionalized, there is an increased interest in using them for biomedical applications. In fact, the surface functionalization may be an important step for pacifying its strong hydrophobicity that may be associated with toxic effects. Nonetheless, the potential long-term adverse effects of functionalized graphene cannot be neglected . FurtherMajor bone reconstruction represents a major challenge and is a global health problem. Stem cell-based therapy might be a promising solution but it requires the constant development of biocompatible platforms that can promote and enhance cell viability, attachment, migration, and differentiation , 3. SeveGraphene has emerged as a promising material for stem cell research due to its unique mechanical, physical, and chemical properties  20, 24,,24, 61. 2) after 48 hours of incubation. Furthermore, MSCs on graphene assume a spindle shaped morphology whereas those cultured on SiO2 substrates form separate islands of polygonal cells [2 substrate [2, graphene oxide, and polydimethylsiloxane (PDMS) substrates [Cell adhesion, viability, and proliferation rate are directly related to the biocompatibility of the substrate , 6, 51. al cells . As cellal cells  the spinubstrate . Furtherbstrates , 46 but bstrates .2 were coated with CVD graphene, MSCs present high expression of osteocalcin (OCN) as compared to the uncoated materials [Although cell proliferation is not improved by pristine graphene, the material enhances stem cell differentiation towards osteoblastic lineage , 47, 52.aterials . These iaterials . It is katerials . Other faterials , 45, 47.aterials .It is known that the transport phenomena of cytokines, chemokines, and growth factors are drastically different between two- and three-dimensional 3D) microenvironments interfering in signaling transduction, cell-cell communications, and tissue development D microen, 70. Gra2 substrates treated with BMP-2 [β-glycerophosphate [π-π stacking between the aromatic rings in the molecules and the graphene basal plane [Although graphene holds the potential to induce spontaneous osteogenic differentiation of stem cells, this property is significantly enhanced by the use of chemical inductors for osteogenic differentiation , 47, 52.th BMP-2 . These hhosphate , 45. Dexal plane . GO is pal plane , 45. HenAlthough graphene has great benefits for osteogenic differentiations due its excellent physical properties, it can also be chemically modified  or combiPolymers have also been modified with graphene to provide better environments for cell survival and differentiation. The addition of 1 wt% of GO to gelatin-based composite improves significantly the tensile strength, Young's modulus, and energy at break by 84, 65, and 158%, respectively . The addPoly-L-lactide (PLLA) scaffold was modified with CNT and graphene of 1–3% wt by thermal-induced phase separation technique. The scaffold containing 3% wt of graphene enhances the differentiation of BMSC and increases the calcium deposition and formation of collagen type I. This can be attributed to the increase in specific surface area of the scaffold and the surface roughness that can increase adsorption of proteins. Furthermore, graphene provides more contacting surface to cells as compared to the same content of CNT .Graphene can form self-supporting graphene hydrogel (SGH) by the principal of colloidal chemistry due to intrinsic corrugation of graphene and solvation repulsion between neighboring graphene sheets, resulting in a large amount of separated graphene sheets in a collective manner inside SGH , 81. MulDue to the large surface area and delocalized electrons, GO and rGO have the potential to bind and solubilize molecules acting as drug delivery vehicles , 83, 84. in vitro and in vivo [ in vitro osteogenic differentiation of BMSC was higher on Ti/GO combined with BMP-2 than on Ti with BMP-2. Further in vivo experiment demonstrated the efficacy of BMP-2 delivered by Ti or Ti/GO substrate after implantation into mouse calvaria defects. After 8 weeks, Ti/GO implants conjugated with BMP-2 showed extensive bone formation revealed by microcomputed tomography and histological analysis as compared to Ti/BMP-2 substrate [Given that the induction of osteogenic differentiation of stem cells can take several weeks, the sustained release of inducing proteins, such as BMP-2, can accelerate this process. Recently the potential of titanium coated with GO (Ti/GO) has been explored for sustained release of BMP-2 to increase osteogenic differentiation in vivo . Ti subsubstrate .These findings corroborate graphene as a promising material that can increment bioactivity and differentiative potential of candidate materials for bone tissue regeneration. in vivo animal studies are needed to assess biodistribution and their metabolic pathways in tissues and organs to permit future clinical applications.The characteristics of graphene such as large surface area, excellent mechanical properties, and feasibility to be transferred to different substrates among others make it a unique material for stem cell research. Graphene-modified substrates and materials are biocompatible, allow cell adhesion and proliferation, and increase differentiation of stem cells into osteogenic lineage. In addition, it can be easily functionalized to bind biomolecules or elements of choice to induce and control stem cell behavior. Although some challenges remain, the advances obtained by using graphene to induce osteogenesis are exciting. One of these challenges is the lack of thorough and profound understanding of the mechanism and signaling pathways involved in stem cell differentiation stimulated by graphene. Further studies at cellular and subcellular level beyond proof of concept and focusing on underlying mechanism are necessary. Moreover, comparisons of graphene with current known and successful biomaterial and implants must be performed to conclude the benefits of the material. Furthermore,Due to the unique structures and remarkable properties, graphene and its derivatives hold great potential for biomedical applications. Although the research with graphene for bone tissue regeneration is still in early stages of development, the material may have bright future in clinical scenarios.


Idiopathic Pulmonary Fibrosis (IPF) is a progressive, incurable fibrotic interstitial lung disease with a prognosis worse than many cancers. Its pathogenesis is poorly understood. Activated platelets can release pro-fibrotic mediators that have the potential to contribute to lung fibrosis. We determine platelet reactivity in subjects with IPF compared to age-matched controls.Whole blood flow cytometry was used to measure platelet-monocyte aggregate formation, platelet P-selectin expression and platelet fibrinogen binding at basal levels and following stimulation with platelet agonists. A plasma swap approach was used to assess the effect of IPF plasma on control platelets.Subjects with IPF showed greater platelet reactivity than controls. Platelet P-selectin expression was significantly greater in IPF patients than controls following stimulation with 0.1 µM ADP (1.9% positive ±0.5 (mean ± SEM) versus 0.7%±0.1; p = 0.03), 1 µM ADP  and 10 µM ADP . Platelet fibrinogen binding was also increased, and platelet activation resulted in increased platelet-monocyte aggregate formation in IPF patients. Re-suspension of control platelets in plasma taken from subjects with IPF resulted in increased platelet activation compared to control plasma.IPF patients exhibit increased platelet reactivity compared with controls. This hyperactivity may result from the plasma environment since control platelets exhibit increased activation when exposed to IPF plasma. Idiopathic pulmonary fibrosis (IPF) is the most common fibrotic interstitial lung disease and has a prognosis worse than many cancers Epidemiological studies have demonstrated an association between IPF and vascular diseases including cardiovascular disease and venous thromboembolism Blood platelets play a central role in thrombosis through rapid activation and aggregation at sites of vascular injury. Transient activation of platelets also induces pro-inflammatory and pro-fibrotic effects In the present study we measured markers of platelet activation in IPF patients under basal conditions and following stimulation with platelet agonists and investigated the effect of plasma from IPF patients on platelet activation. Our data demonstrate that IPF is associated with platelet hyperactivity that may be caused by the plasma environment.Patients with a diagnosis of idiopathic pulmonary fibrosis according to ATS/ERS criteria Written informed consent was obtained from all participants and the study was approved by the Hull and East Riding Local Research Ethics Committee (LREC ref 08/H1304/54).Venous blood was collected using a 21G butterfly needle and vacutainer system . The first 4.5 mls of blood collected was discarded because artefactual platelet activation may occur during venepuncture. Citrated whole blood was added to tubes containing a saturating concentration of one or more of the following antibodies: fluorescein isothiocyanate (FITC)-conjugated monoclonal mouse anti-human CD42b ; phycoerythrin (PE)-conjugated monoclonal mouse anti-human CD14 ; PE-conjugated monoclonal mouse anti-human CD62P ; or FITC-conjugated polyclonal rabbit anti-human fibrinogen . To study the sensitivity of platelets to activation, some tubes were supplemented with adenosine diphosphate (ADP)  or the protease activated receptor - 1 (PAR1) agonist TFLLR . Single colour flow cytometry was used to assess platelet P-selectin expression and fibrinogen binding. Two-colour flow cytometry was used to identify the proportion of monocytes forming aggregates with one or more platelets  based on established methods In order to assess the effect of IPF plasma on control platelets a plasma swap approach was used. Blood from IPF patients and controls was used for the preparation of platelet-poor plasma. Washed platelets from control subjects were suspended in phosphate buffered saline (PBS) and divided into three aliquots. Plasma from the same donor was added to the first aliquot to form an autologous control. Plasma collected from a different control patient was added to the second aliquot to form an allogeneic control. Plasma from a patient with IPF was added to the third aliquot. The ratio of platelet suspension to plasma was 1∶1 (v/v). Samples were incubated with PE-conjugated anti-human CD62P antibody  under basal conditions and in the presence of ADP  for 20 minutes prior to fixation and flow cytometric analysis. Additional detail on the methods for assessing the effects of IPF plasma on control platelets is provided in data supplement Flow cytometry was performed using a FACS Calibur flow cytometer  and the data were analysed using CellQuest Pro software . Statistical comparisons of platelet-monocyte aggregate formation, platelet P-selectin expression and platelet fibrinogen binding between IPF and controls were performed using unpaired t-tests. A paired t-test was used to assess the difference in P-selectin expression between the groups in the plasma swap experiment. A p value of <0.05 was considered significant.Patients with IPF and controls were matched for age and gender. When whole blood was stimulated with ADP (1–10 µM) or TFLLR (1–10 µM), subjects with IPF demonstrated significantly greater platelet-monocyte aggregate formation compared with controls . The perHaving established that platelet-monocyte aggregates were increased in IPF, we next examined other markers of platelet activation. Consistent with increased platelet-monocyte aggregates we found that platelet P-selectin expression was elevated in whole blood from IPF patients compared with controls. Stimulation with all concentrations of ADP led to greater increases in platelet P-selectin expression in IPF patients compared with controls . Similar findings were observed following stimulation with TFLLR . No diffThe percentage of platelets binding fibrinogen was significantly higher in IPF patients compared with controls following stimulation with 0.1 µM and 1 µM ADP . There was no significant difference in fibrinogen binding under basal (unstimulated) conditions . The increase in fibrinogen binding in IPF subjects following stimulation with TFLLR did not reach statistical significance .We next examined whether the platelet hyperactivity observed in IPF was caused by a primary platelet defect or the plasma environment. Re-suspension of washed control platelets in plasma from IPF subjects led to elevated P-selectin expression when compared with platelets suspended in autologous control or allogeneic control plasma. IPF plasma increased P-selectin expression under both basal conditions (p<0.05) and following stimulation with ADP at concentrations of 0.1 and 1 µM (P≤0.01) . FurtherWe have demonstrated platelet hyperactivity in patients with IPF using two mechanistically different agonists (ADP and TFLLR) and three different markers of platelet activation, namely platelet-monocyte aggregate formation, platelet P-selectin expression, and platelet fibrinogen binding. Increased platelet-monocyte aggregate formation is a sensitive marker of platelet activation To explore whether the hyperactivity phenotype in IPF was the result of an alteration of the platelets or their environment we used the plasma swap approach, which has been used successfully in previous studies to examine the role of plasma The two patient groups in this study were well matched in terms of age and gender. At the time of undertaking the platelet assays there was no evidence-based disease modifying treatment for IPF Although not statistically significant, there was a higher prevalence of cerebrovascular disease in the IPF group. There is a recognised association between cerebrovascular disease and platelet activation It has recently been demonstrated that patients with COPD have increased levels of circulating platelet-monocyte aggregates compared to age and smoking status matched controls IIbβ3It is recognised that the choice of ex-vivo anticoagulant can impact platelet-monocyte association. Citrate results in lower levels of platelet-monocyte aggregates secondary to calcium chelation The present study does not establish a temporal relationship between platelet activation and development of IPF, and in isolation we cannot conclude whether the observed increased platelet reactivity is an important phenomenon at the onset of the disease or represents a secondary response to pulmonary fibrosis. Although epidemiological studies have reported that the increased incidence of cardiovascular events and venous thromboembolism in IPF patients predates the presentation of lung disease Therefore, in this small study of platelet activation in IPF we present clear evidence of increased platelet reactivity in IPF patients compared to controls and demonstrate that this effect can be reproduced in control platelets incubated in IPF plasma. This provides insight into the link between IPF and vascular diseases and we have described a mechanism by which platelet activation may perpetuate fibrosis. Activation of platelets represents an early stage in thrombosis and is unaffected by treatment with the vitamin K antagonist warfarin. The lack of benefit from anticoagulant therapies in IPF may be because the wrong component of the thrombotic process has been targeted We have demonstrated increased platelet reactivity in IPF patients that is reproducible with diverse agonists and different markers of platelet activation. The increased activation and reactivity of control platelets when incubated in IPF plasma suggests that the abnormal platelet response in IPF is due to the plasma environment. This is the first report of increased platelet reactivity in IPF patients providing insight into the relationship between IPF and vascular disease and highlighting the need for further investigation of the role of platelets in IPF pathogenesis.We thank Dr Laura Sadofsky and Chris Crow for helpful comments and technical support and Dr Jakob Dudziak for his ICT expertise.File S1Tables S1-S6.(DOCX)Click here for additional data file.


We demonstrated a method to construct high efficiency saturable absorbers based on the evanescent light field interaction of CVD monolayer graphene deposited on side-polished D-shaped optical fiber. A set of samples was fabricated with two different core-graphene distances (0 and 1 μm), covered with graphene ranging between 10 and 25 mm length. The mode-locking was achieved and the best pulse duration was 256 fs, the shortest pulse reported in the literature with CVD monolayer graphene in EDFL. As result, we find a criterion between the polarization relative extinction ratio in the samples and the pulse duration, which relates the better mode-locking performance with the higher polarization extinction ratio of the samples. This criterion also provides a better understanding of the graphene distributed saturable absorbers and their reproducible performance as optoelectronic devices for optical applications. The graphene is a two-dimensional carbon allotrope nanomaterial consisting of one atom thickness and that has important optical and electronic properties123456789101112131415161719234510111213141516171011121314151617et al. using exfoliated graphene dispersed in a polymer at all-fiber connector tip configuration, which pulses as short as ~800 fs with 3.2 nm bandwidthet al. demonstrated the generation of 756 fs pulses with 5 nm spectrum bandwidth1011121314151617Ultrashort pulses are suitable for numerous applications, which include telecommunications and the study of ultrafast processes3456234567891011121314151617In this work, we present an efficient and reproducible mode-locking performance criterion with seven D-shaped side-polished fiber samples based on CVD monolayer graphene saturable absorbers for EDFL, obtaining pulses duration of 256 fs, which is the shortest pulse reported in the literature with CVD graphene monolayer, for our knowledge. We carried out the characterization process in three steps after the fabrication of the sample by the transference of the graphene to the D-shaped optical fiber: the first step was to verify the quality of the monolayer graphene by Raman spectroscopy. Thereafter, the performance of the monolayer graphene sample as a polarizer was evaluated by the polarization relative extinction ratio between the modes of the light with polarization parallel (TE radiation) and orthogonal (TM radiation) to the graphene’s plane. Finally, we characterized the graphene saturable absorber mode-locking performance relating it to the polarization relative extinction ratio of each sample.−1), G (1592 cm−1) and 2D (2685 cm−1) characteristic bands of the graphene−1, full-width at half-maximun (FWHM), corresponds a monolayer while around 40 cm−1 indicates bilayer grapheneThe samples were analyzed with a Raman Microscopy System  using a 532 nm wavelength laser with an incident power of 2 mW. In TE/PTM), where PTE/PTM is the ratio between the power of the modes TE and TM relative to the graphene’s plane. Without graphene, the insertion loss of the D-shaped optical fiber was 0.60 dB, whereas with graphene the TE and TM losses for this sample were 20 and 6.1 dB respectively, resulting in an extinction ratio of 96%. The The polarization dependent loss of the monolayer graphene D-shaped fiber sample was performed by rotating input power of 754 μW with linear polarization from 0 to 360° degrees. The result is shown in the μm and PMMA thickness variation between 300 and 400 nm. An approaching to analyze this influence and give a support to the relative extinction ratio polarization measurements, were done by simulation, using the COMSOL Multiphysics software,version 5.1. The attenuation calculated by simulation for each orthogonal mode in each sample is summarized in As it can see in The EDFL continuous wave (CW) lasing threshold without the saturable absorber was achieved at a pump power of 15.7 mW. Incorporating the monolayer graphene saturable absorber on the D-shaped fiber, the lasing threshold increased to 20 mW. Non-self-starting mode-locking regime was obtained from 50 to 110 mW pump power, generating soliton-like pulses highly dependent of polarization state. In fact, because of symmetry breaking in the fundamental propagation mode caused by D-shaped optical fiber/graphene configuration and the unequal property of graphene as polarizer and saturable absorber, the mode-locking was activated for high absorption of the TE mode and different polarization states can generate distinct performances in the laser. In The mode-locking performance can be directly related to the sample’s polarization relative extinction ratio as shown in In this work, we present a simple and high performance criterion for D-shaped optical fiber based on monolayer graphene saturable absorber for the generation of ultrashort pulses in EDFL. With this method, it is possible to associate the polarization relative extinction ratio between the TE and TM radiation with the passively mode-locking optimization. In the EDFL, the samples with polarization relative extinction ratio higher than 85% resulted in pulse duration shorter than 300 fs. The shortest pulse duration of 256 fs was obtained at higher polarization relative extinction ratio of 96%, which is the shortest pulse reported in the literature using CVD monolayer graphene. This criterion can be used to evaluate the quality and performance of several optical devices based on transferred graphene in optical waveguides.The polarization relative extinction ratio is an important parameter because it is associated with the saturable absorption mechanism which generate ultrashort pulses. The saturable absorption mechanism is associated with electronic transitions from the valence band to the conduction band due to the interaction of light with graphene at high intensities. Due these transitions, the conduction band levels become fully occupied (Pauli blocking), so the graphene becomes transparent for high intensities. In the experiment and simulation results, high relative polarization extinction ratio implies a high absorption at TE mode, responsible for both saturable absorption in graphene, and ultrashort pulses generation.h) of 0 μm with transferred graphene length (l) of 10, 18.5 and 24 mm; and four samples with core-surface distance of 1 μm and 13, 14, 19 and 25 mm graphene length. The monolayer graphene was transferred from the cooper to the side-polished of the D-shaped optical fiber by using the wet transfer methodThe monolayer graphene was produced by chemical vapor deposition (CVD) grown on cooper and the polished D-shaped optical fibers (Phoenix Photonics) samples were provided, with distances from the fiber core to the surface of 0 and 1 μm and polishing lengths of 17, 25 and 30 mm. A total of seven samples were fabricated, three samples with core-surface distance μS. h = 1 μm; PMMA refractive index 1.49 and 300 nm thickness; fiber core and cladding refractive index 1.4492 and 1.4440 respectively and fiber core diameter 8.2 μm.The graphene was modeled using the boundary condition for the tangential magnetic field How to cite this article: Zapata, J. D. et al. Efficient graphene saturable absorbers on D-shaped optical fiber for ultrashort pulse generation. Sci. Rep.6, 20644; doi: 10.1038/srep20644 (2016).


Graphene/ZnSe film and graphene/CdSe film hybrid photodetectors fabricated by this method also show a high and reproducible performance. The general method is compatible with the conventional planar process, and would be easily standardized and thus pay a way for the photodetector applications.High- and reproducible-performance photodetectors are critical to the development of many technologies, which mainly include one-dimensional (1D) nanostructure based and film based photodetectors. The former suffer from a huge performance variation because the performance is quite sensitive to the synthesis microenvironment of 1D nanostructure. Herein, we show that the graphene/semiconductor film hybrid photodetectors not only possess a high performance but also have a reproducible performance. As a demo, the as-produced graphene/ZnS film hybrid photodetector shows a high responsivity of 1.7 × 10 High- and reproducible-performance photodetectors are critical to the development of many technologies, including communications, sensing, environmental protection, and imaging, etc1235678910121315162 bonded carbon atoms with ultrahigh carrier mobility192021222324252627et al. demonstrated a responsivity of 107 A/W in a hybrid photodetector that consists of graphene covered with a thin film of PbS quantum dotsWe now consider the film based photodetectors. They possess a low performance due to low carrier mobility of the film consisting of nanocrystals. The performance has been greatly enhanced by hybridizing semiconductor films with graphene, a two-dimensional single-layer of sp2/Si substrate by high vacuum method and are supposed to have a fixed amount of photo-generated carriers at different areas. The CVD grown graphene films on Cu foil were subsequently transferred onto semiconductor film29302 V−1 s−1, 7 A/W and a fast response speed of 50 ms, and shows a highly reproducible performance, in terms of narrow distribution of photocurrent (38–65 μA) and response speed (40–60 ms) for 20 devices. Graphene/ZnSe film and graphene/CdSe film hybrid photodetectors fabricated by this method also show a high and reproducible performance. Remarkably, our general method is compatible with the conventional planar process and will be easily standardized.Herein, we show that the graphene/semiconductor film hybrid photodetectors not only possess a high performance but also have a reproducible performance, taking ZnS, ZnSe and CdSe as examples. Semiconductor films were deposited onto SiO2/Si substrate shows that the thickness of ZnS film is observed to be 60 nm, in agreement with the setting value during e-beam evaporation process  obviously increased regardless of the gate bias (Vg) (Rλ) is a key parameter to evaluate the photodetector performanceRλ = ∆I/PSI is the difference between the current under illumination and dark current, namely photocurrent, P is the light power density irradiated on the channel, and S is the area of channel. Vsd) under different light power, with the back gate of Vg = 0 V. The photocurrent increases with the increase of Vsd and light power, while the calculated responsivity increases with the increase of Vsd yet decreases with the increase of the light power. It is consistent with the photodetector behavior reported before21Rλ value of our fabricated graphene/ZnS film photodetector is up to 1.7 × 107 A/W at an incident power of about 10 μW/cm2, indicating a high responsivity for our graphene/ZnS film hybrid photodetector. The responsivity can be expected to be further enhanced under light illumination with a lower power121516To fabricate graphene/ZnS film based field effect transistors, 10/50 nm Cr/Au electrodes were deposited by thermal evaporation method with standard photolithography and lift-off processes. After 365 nm light was focused on the channel with size of 5 × 5 μm inset in , the pho2126ias (Vg) . The spe2 V−1 s−1  and response speed (40–60 ms) for 20 devices. The method universality is confirmed by the realization of graphene/ZnSe film and graphene/CdSe film hybrid photodetectors with a high and reproducible performance. Our general method developed in this work is compatible with the conventional planar process, and would be easily standardized and pay a way for the detector applications.In summary, the combination of graphene films possessing an ultrahigh mobility and uniform conductivity with semiconductor films having a fixed amount of photo-generated carriers allows us to achieve graphene/semiconductor film hybrid photodetectors with a high and reproducible performance, based on that film based detector has a reproducible performance because it is collective one of the nanocrystals in film. As a demo, the produced graphene/ZnS film hybrid photodetectors shows a high responsivity of 1.7 × 102 was utilized as substrate for the semiconductor film deposition. The vacuum remained 2 × 10−4 Pa during the whole deposition process. Before graphene transfer, the semiconductor film on Si/SiO2 substrate was annealed for 30 min at 300 °C under low pressure with 10 sccm H2 and 20 sccm Ar, enabling the film adhered to the substrate as it was placed in water to pick up the graphene films.ZnS and ZnSe film with the thickness of 60 nm was deposited by e-beam evaporation, and 60 nm thick CdSe film was produced by thermal evaporation. ZnS, ZnSe and CdSe crystals (99.99% purity) with size of mm scale were used as targets. After the standard cleaning, the Si wafer with 300 nm SiO2 and 20 sccm Ar. This method enables the crackles transfer of graphene.The details of graphene growth and transfer can be found in our previous work312/Si, photolithography was used to fabricate transistors with channel lengths of 5 μm and widths 5 μm. For the performance reproducibility investigation of the graphene/ZnS, graphene/ZnSe and graphene/CdSe hybrids, 20 devices with channel length of 20 μm and width of 500 μm were fabricated with shadow mask. The doped Si substrate served as a global back gate. 10/50 nm Cr/Au used as the electrical contact metal for the source and drain electrodes were deposited by a high-vacuum thermal evaporation system.After graphene was transferred onto ZnS, ZnSe and CdSe film on 300 nm SiOSEM images were taken with a Hitachi SU8020 scanning electron microscope operated at 1 kV. XRD patterns of the samples were recorded with an X-ray diffractometer (X’ Pert Pro MPD) with λ = 1.54056 Å. Raman spectroscopy was performed on the samples with a confocal microprobe Raman spectrometer (Renishaw). UV-vis absorption (UV-3600) was conducted on the samples after transfer onto quartz substrate. The electrical and photoconductive properties of the graphene/ZnS film, graphene/ZnSe film and graphene/CdSe film hybrid devices were measured by a semiconductor parameter analyzer system (Keithley 2636B) at room temperature, using light emitting diodes with the corresponding wavelength of 365 nm, 460 nm and 620 nm as the incident light.How to cite this article: Huang, F. et al. High- and Reproducible-Performance Graphene/II-VI Semiconductor Film Hybrid Photodetectors. Sci. Rep.6, 28943; doi: 10.1038/srep28943 (2016).


Auditory feedback is required to maintain fluent speech. At present, it is unclear how attention modulates auditory feedback processing during ongoing speech. In this event-related potential (ERP) study, participants vocalized/a/, while they heard their vocal pitch suddenly shifted downward a ½ semitone in both single and dual-task conditions. During the single-task condition participants passively viewed a visual stream for cues to start and stop vocalizing. In the dual-task condition, participants vocalized while they identified target stimuli in a visual stream of letters. The presentation rate of the visual stimuli was manipulated in the dual-task condition in order to produce a low, intermediate, and high attentional load. Visual target identification accuracy was lowest in the high attentional load condition, indicating that attentional load was successfully manipulated. Results further showed that participants who were exposed to the single-task condition, prior to the dual-task condition, produced larger vocal compensations during the single-task condition. Thus, when participants’ attention was divided, less attention was available for the monitoring of their auditory feedback, resulting in smaller compensatory vocal responses. However, P1-N1-P2 ERP responses were not affected by divided attention, suggesting that the effect of attentional load was not on the auditory processing of pitch altered feedback, but instead it interfered with the integration of auditory and motor information, or motor control itself. Proficient motor control is achieved by using sensory feedback to plan, execute, and regulate motor movements The multiple resource theory of divided attention states that when performing two tasks simultaneously, the degree to which performance will decline on each task, compared to when each task is completed in isolation depends on: the resource demands of each of the two tasks, the similarity between the two tasks, and the allocation of resources between the two tasks The importance of auditory feedback for maintaining fluent speech has been demonstrated by individuals who have been deafened post-lingually, and experienced a gradual deterioration in the quality of their speech In addition to investigating vocal responses, auditory cortical responses to FAF recorded using electroencephalography (EEG) can provide information regarding the underlying neural mechanisms of speech motor control. The P1, N1, and P2 event-related potentials (ERPs) are reliably elicited by FAF Although it is unclear how FAF modulates these ERPs under divided attention, when elicited by other forms of auditory stimuli, auditory ERPs have shown sensitivity to divided attention. In particular, the N1 component is often enhanced when participants attend to an auditory stimulus, relative to passive listening of the same stimulus For the current experiment, we used a dual-task paradigm to investigate whether divided attention impacts the compensatory vocal responses and ERPs elicited by FAF. In order to reduce the allocation of attentional resources to auditory feedback during the FAF task, participants simultaneously monitored a rapid serial visual presentation (RSVP) of letters. The RSVP contained target letters, which participants identified and later reported. Attentional load was manipulated by varying the rate of the RSVP. Increasing the rate of the RSVP decreased the inter-stimulus interval (ISI) between letters, which modulated the perceptual load by increasing the number of stimuli. As a result of the increased number of stimuli, participants had to process more irrelevant information, which directly impacted the perceptual selection of relevant information, and thus increased the overall attentional demand of the task  participated. Vocal and behavioural responses were recorded from all 65 participants, while ERP responses were also recorded from a subset of 33 right-handed participants . All participants were Canadian-English speakers who did not speak a tonal language, with the exception of one participant who spoke a tonal language, but identified English as their primary language. This tonal language speaker did not show any differences in vocal compensations nor ERP responses to the FAF perturbations compared to the other non-tonal language-speaking participants. All participants also had normal or corrected to normal vision, had not been diagnosed with attention deficit (hyperactivity) disorder , epilepsy (or had a family history of seizures), visual deficits that could not be amended by corrective lenses, and did not have a speech or language disorder.Sixty-five participants between the ages of 16 and 38 years ; and 2. an “X,” that occurred pseudorandomly before or after the white letter. All letters in the stream were capitalized and in black font with the exception of the white target letter. Each letter was displayed at the same location in the center of a grey field, where the varied ISI was seen as a uniform grey field. For the single-task trials, each letter stream ended with a blank grey field.th and the 37th letter, and the “X” randomly occurred between the 1st and 15th letter before or after the white letter . Intermediate attentional load trials were 5.4 s in duration and consisted of 16 letters in the visual stream, with an ISI of 300 ms. The white letter appeared at random between the 4th and 16th place in the letter stream, while the “X” occurred at random between the 1st and 12th letter before or after the white letter (minimum 2nd letter and maximum 16th letter). Low attentional load trials were 5.5 s in duration, with 11 letters in the stream and an ISI of 500 ms. The white letter occurred randomly between the 4th and 11th place in the visual stream, and the “X” occurred at random between the 1st and 15th letters from the white letter . The presentation rate of the RSVP was manipulated to impose a high, intermediate, and low attentional load. The RSVP started with a green fixation cross that lasted for 750 ms, followed by a stream of successively presented letters. Each trial was approximately 5.5 s in duration. Each letter appeared for 50 ms, and each trial occurred with an ISI of either 100, 300, or 500 ms . High attentional load trials were 5.4 s in duration and consisted of 37 letters, with an ISI of 100 ms. The white letter appeared at random between the 4During the single-task condition, participants were instructed to attend to the RSVP, as the start of the RSVP was their cue to start vocalizing, and the termination of the RSVP was their cue to stop vocalizing. During the dual-task condition, participants were instructed to attend to the RSVP and monitor the letter stream for their cue to start and stop vocalizing, but also so they could identify two targets: a white letter, and an “X.” At the end of the letter stream, participants were required to answer two questions about the target letters. The first question appeared on the screen at the end of the trial and stated, “Identify the WHITE letter. If you are unsure, please guess.” The second question, which appeared immediately after the participant’s response to the first question stated, “indicate when the “X” appeared with reference to the white letter.” The participant was required to press a key labelled “YES” if they believed that the “X” appeared before the white letter, and a key labelled “NO” if the believed that the “X” appeared after the white letter. During each vocalization, the FAF perturbation occurred either 250–1000 ms before the “X,” or 100–1000 ms after the “X.” Emphasis was placed on both maintaining a steady vocalization and on responding accurately to the two questions at the end of the trial. Since participants could take as much time as they needed to respond to the two questions, participants moved on to subsequent trials at their own pace during the dual-task trials, whereas single-task trials occurred with an inter-trial interval of 3000 ms. The total duration of the experiment ranged from 40 to 60 minutes, and depended on the participant’s reaction time to the questions during the dual-task blocks, and the duration of breaks between blocks.Participants were seated 76 cm from a 15-inch LCD monitor in an electrically shielded booth  and were fitted with a HydroCel GSN 64 1.0 Cap , Etymotic ER-3 insert earphones , and an earset microphone . The presentation of visual stimuli and shift onsets and offsets were controlled by Max/MSP 6 . Keyed behavioral responses to questions were also recorded using Max/MSP 6, using a standard keyboard with labeled keys. During the experiment, voice signals were sent to a mixer , then to a digital signal processor , which altered the F0 of the voice signal. This process introduced a ∼10 ms delay in the feedback signal, that was then presented back to the participant through headphones as auditory feedback. The unaltered voice signal was digitally recorded  at a sampling rate of 44.1 kHz for later analysis.For each trial, records were kept of the trial’s attentional load condition, the white letter in the stream, whether the “X” appeared before or after the white letter, the participants’ keyed responses and reaction time to question one, as well as the participants’ keyed responses and reaction time to question two. Participants who did not answer either of the questions according to the instructions were excluded from the analysis. One participant’s answers were excluded from the white letter identification analyses for this reason. Accuracy and reaction time for questions one and two were averaged for each of the three categories of attentional load in the dual-task condition . Accuracy for high, intermediate, and low attentional load trials were then averaged across all participants for each question. Only accuracy data were examined since response accuracy was emphasized during the participant instructions.Each participant’s unaltered voice recording was segmented into separate vocalizations. The swipe algorithm Missing and incomplete vocalizations were excluded from the statistical analysis. Participants with more than 67%  rejected trials for any of the four attentional load conditions  were removed from further analysis. A total of 11 participants were excluded from the experiment due to F0 tracking issues. For the 54 remaining participants, an averaged F0 trace for retained trials was constructed for each of the four attentional load conditions. The average number of trials in each condition was 42 for the dual task-high, 40 for the dual task-intermediate, 41 for the dual task-low, and 39 for the single task attentional load trials. Traces were averaged across all participants, for each condition. For each participant, the magnitude of the compensatory response and the latency of the response were assessed. The amplitude of the compensatory response was determined by finding the maximum point at which the participants’ average F0 trace deviated from the baseline mean. Latency was determined as the time at which the maximum peak in the compensatory response occurred.EEG signals were recorded from 64-electrodes on the scalp, and referenced online to the vertex (Cz). Signals were bandpass filtered (1–30 Hz) and digitized (12-bit precision) at 1000 samples per second. Impedances were maintained below 50 kΩ for the experiment Six electrodes were included in the analysis: Fz, Cz, F3, C3, C4, and F4. These six electrodes were selected for analysis through visual inspection, having demonstrated the most robust P1-N1-P2 components, and based on previous research, which suggests that front-medial and centro-frontal regions display the most robust responses to pitch shifts Two separate repeated-measures analysis of variances (RM-ANOVAs) were conducted to look at the influence of attentional load  with block-order  as a between-subjects factor, on response accuracy for the identification of the white letter and the placement of the “X”. The Greenhouse-Geisser Two separate RM-ANOVAs were conducted to assess the influence of attentional load  and block-order  on vocal response magnitudes and response latencies. Follow-up RM-ANOVAs were run to investigate significant interactions. The Greenhouse-Geisser Separate two-way RM-ANOVAs were conducted to look at the impact of attentional load  and electrode site  with the between subject factor of block-order  on P1-, N1-, and P2- amplitudes, and P1-, N1-, P2- latencies. The Greenhouse-Geisser F = 78.662, p<0.001, η2 = 0.607, where white-letter identification was significantly more accurate for the dual-task low attentional load condition, relative to the dual-task high attentional load condition, p<0.001. While the dual-task intermediate attentional load condition also elicited higher response accuracy for the white-letter identification, relative to the dual-task high attentional load condition, p<0.001  = 0.686, p = 0.411, η2 = 0.013, and the interaction between attentional load and block-order were not significant, F = 0.107, p = 0.819, η2 = 0.002.A RM-ANOVA was conducted to look at the effect of attentional load on white-letter identification accuracy with block-order as a between subjects factor. There was a main effect of attentional load, .001 see . The maiF = 106.443, p<0.001, η2 = 0.672, where accuracy was much higher for the dual-task low attentional load condition, compared to the dual-task intermediate, and dual-task high attentional load conditions, p<0.01. In addition, “X”-placement accuracy was also greater for the dual-task intermediate attentional load condition, compared to the dual-task high attentional load condition, p<0.001  = 0.159, p = 0.691, η2 = 0.003, and the interaction between attentional load and block-order were not significant, F = 2.454, p = 0.107, η2 = 0.045.A RM-ANOVA was conducted to look at the effect of attentional load on “X”-placement accuracy, with block-order as a between subjects factor. There was a main effect of attentional load, .001 see . The maiPearson-product moment correlations were conducted to look for potential relationships between the magnitude and latency of vocal responses, and the accuracy with which the white-letter and “X”-placement were identified, for the dual-task high, dual-task intermediate, dual-task low attentional load conditions. The correlations between white letter identification accuracy and vocal response magnitude, and white letter identification accuracy and vocal response latency, were not significant, both p>.05. Similarly, the correlations between accuracy for “X”-placement and vocal response magnitude, and accuracy for “X”-placement and vocal response latency, were also not significant, both p>.05.F = 1.175, p = 0.321, η2 = 0.022  = 0.573, p = 0.453, η2 = 0.011. However, a significant interaction was found between attentional load and block-order, F = 5.782, p = 0.003, η2 = 0.1  = 7.303, p = 0.001, η2 = 0.207, where the vocal response magnitudes were significantly larger in the single-task condition, compared to all attentional load conditions, p<0.02. However, the effect of attentional load on vocal response magnitudes in the dual-task first condition was not significant, F = 1.083, p = 0.362, η2 = 0.043.A RM-ANOVA was conducted looking at the effect of attentional load on vocal response magnitudes, with block-order as a between subjects factor. The main effect of attentional load on vocal response magnitudes was not significant, .022 see , nor was 0.1 see . Follow-p = 0.173, η2 = 0.031, nor was the main effect of block-order, F = 0.542, p = 0.465, η2 = 0.01, or the attentional load by block-order interaction F = 0.739, p = 0.53, η2 = 0.014.A RM-ANOVA was conducted to look at the effect of attentional load on vocal response latencies, with block-order as a between subjects factor. The main effect of attentional load was not significant, F = 1.684, See F = 1.438, p = 0.238, η2 = 0.054, no main effect of electrode site, F = 1.375, p = 0.261, η2 = 0.052, and no main effect of block-order, F = 0.009, p = 0.926, η2<0.001. The attentional load by block-order interaction was also not significant, F = 0.979, p = 0.407, η2 = 0.038, as was the electrode site by block-order interaction, F = 2.067, p = 0.15, η2 = 0.076, the attentional load by electrode site interaction, F = 0.281, p = 0.945, η2 = 0.011, and the three-way interaction between attentional load, electrode site, and block-order, F = 0.853, p = 0.531, η2 = 0.033.A RM-ANOVA was conducted to look at the effect of attentional load, electrode site, and block-order on P100 amplitudes. There was no main effect of attentional load, F = 1.349, p = 0.265, η2 = 0.051, no main effect of electrode site, F = 0.299, p = 0.695, η2 = 0.012, and no main effect of block-order, F = 0.885, p = 0.356, η2 = 0.034. The attentional load by block-order interaction was not significant, F = 0.034, p = 0.992, η2 = 0.001, as was the electrode site by block-order interaction, F = 0.753, p = 0.45, η2 = 0.029, the attentional load by electrode site interaction, F = 0.338, p = 0.916, η2 = 0.013, and the three-way interaction between attentional load, electrode site, and block-order, F = 1.246, p = 0.286, η2 = 0.047.A RM-ANOVA was conducted to look at the effect of attentional load, electrode site, and block-order on P100 latencies. There was no main effect of attentional load, F = 2.05, p = 0.114, η2 = 0.076, nor a significant main effect of block-order, F = 1.065, p = 0.312, η2 = 0.041. However, the main effect of electrode site was significant, F = 4.057, p = 0.023, η2 = 0.14, such that the right electrode site recorded smaller N1 amplitudes  than the medial electrode site, p<0.01  = 1.22, p = 0.308, η2 = 0.047, as was the electrode site by block-order interaction, F = 0.747, p = 0.479, η2 = 0.029, the attentional load by electrode site interaction, F = 0.795, p = 0.523, η2 = 0.031, and the interaction between attentional load, electrode site, and block-order, F = 0.989, p = 0.413, η2 = 0.038.A RM-ANOVA was performed to look at the effect of attentional load, electrode site, and block-order on N100 amplitudes. There was no significant main effect of attentional load, 0.01 see . FurtherF = 0.588, p = 0.625, η2 = 0.023, no main effect of electrode site, F = 1.367, p = 0.265, η2 = 0.052, and no main effect of block-order, F = 2.225, p = 0.148, η2 = 0.082. Furthermore, the attentional load by block-order interaction was not significant, F = 1.061, p = 0.371, η2 = 0.41, nor was the electrode site by block-order interaction, F = 2.644, p = 0.081, η2 = 0.096, the attentional load by electrode site interaction, F = 1.534, p = 0.195, η2 = 0.058, and the interaction between attentional load, electrode site, and block-order, F = 1.649, p = 0.164, η2 = 0.062.A RM-ANOVA was performed to look at the effect of attentional load, electrode site, and block-order on N100 latencies. There was no main effect of attentional load, F = 0.603, p = 0.615, η2 = 0.024, no main effect of electrode site, F = 2.013, p = 0.144, η2 = 0.075, and no main effect of block-order, F = 0.016, p = 0.9, η2 = 0.001. The attentional load by block-order interaction was nonsignificant, F = 0.055, p = 0.983, η2 = 0.002, as was the electrode site by block-order interaction, F = 2.259, p = 0.115, η2 = 0.083, the attentional load by electrode site interaction, F = 0.311, p = 0.93, η2 = 0.012, and the three-way interaction between attentional load, electrode site, and block-order, F = 1.354, p = 0.237, η2 = 0.051.A RM-ANOVA was conducted to look at the effect of attentional load, electrode site, and block-order on P200 amplitudes. There was no main effect of attentional load, F = 2.066, p = 0.112, η2 = 0.076, no main effect of electrode site, F = 0.991, p = 0.378, η2 = 0.038, and no main effect of block-order F = 1.181, p = 0.288, η2 = 0.045. Furthermore, the attentional load by block-order interaction was not significant, F = 1.294, p = 0.283, η2 = 0.049, as was the electrode site by block-order interaction, F = 0.204, p = 0.816, η2 = 0.008, the attentional load by electrode site interaction, F = 0.369, p = 0.898, η2 = 0.015, and the interaction between attentional load, electrode site, and block-order, F = 0.802, p = 0.57, η2 = 0.031.Similar results were obtained from the RM-ANOVA performed to determine whether an effect of attentional load and electrode-site, with block-order as a between-subjects factor, existed for the latency of the P200. There was no main effect of attentional load, The aim of this study was to investigate whether increases in attentional load modulate vocal and neural responses to FAF perturbations. Participants produced vocalizations while exposed to FAF in both single and dual-task conditions. To manipulate participants’ attentional load, participants produced vocalizations while they either passively viewed a RSVP of letters, or while they attended to a RSVP of letters that was either presented at a low, intermediate, or high rate, in order to later identify target stimuli. A main effect of attentional load on both white letter identification accuracy, and “X”-placement accuracy was found, as target identification was better following the dual-task low and dual-task intermediate attentional load trials, relative to the dual-task high attentional load trials. These results suggest that the participants’ attentional load was successfully manipulated by increasing the stimulus presentation rate. Despite the differences in accuracy found across the different presentation rates, even in the highest attentional load condition participants accurately identified the white letter 87.5% of the time, while the “X”-placement was correctly identified 78.2% of the time. The high level of accuracy found even in the most attentionally demanding condition, suggests that while the attentional load manipulation was successful, participants were still able to maintain a high level of performance.Examination of speakers’ compensatory responses to the brief FAF perturbations revealed that vocal response magnitudes were modulated by an attentional load by block order interaction. Participants who were exposed to the single-task condition, prior to the dual-task condition, produced larger vocal responses in the single-task condition, relative to the dual-task condition. However, participants who were exposed to the dual-task condition, prior to the single-task condition, produced similar vocal responses in both conditions. This interaction suggests that when participants completed the single-task prior to the dual-task, they were able to passively view the visual stream during the single-task, with sufficient attentional resources remaining to monitor their auditory feedback and correct for production errors. In contrast, when attentional resources were then split between the vocalization task and the target identification task, fewer attentional resources were available for participants to monitor their auditory feedback, resulting in smaller vocal responses. Although divided attention modulated responses to FAF when the single-task condition occurred prior to the dual-task condition, this was not the case when the dual-task condition occurred prior to the single-task condition. When the dual-task condition was completed prior to the single-task condition, vocal responses in the single-task condition were no different than those in the dual-task condition. We suggest that when participants were exposed to the dual-task, prior to the single-task, the single-task demanded more attentional resources as participants were unable to passively view the visual stream. Previous research has shown that extensive and consistent training on a task can lead to automatic processing of stimuli The results of this study suggest that when attention is divided, smaller vocal responses to FAF are produced. One possible explanation for these smaller responses is that under divided attention auditory feedback is less salient. Previous research has shown that under visual attentional load, the loudness of tones is attenuated by 7 dB Although the size of the vocal responses to FAF were modulated by divided attention, vocal response latencies were not. We hypothesized that increasing attentional load would increase the processing demands of the visual task, and reduce the amount of attentional resources allocated to the processing of auditory feedback, resulting in later vocal responses. Although it is possible that vocal response latency is not affected by divided attention, it is also possible that the attentional load manipulation was not strong enough to affect the vocal response latencies recorded in this study. As previously mentioned, response accuracy was quite high, even for the highest level of attention load. This being said, while it appears as though attention can be divided without influencing the timing of vocal responses, this may not be the case in situations where attentional resources are more heavily taxed.Divided attention resulted in the modulation of compensatory vocal responses to FAF; however, divided attention did not modulate the amplitude or latency of the P1-N1-P2 ERP components elicited by the FAF in this study. Although vocal responses were smaller under divided attention, they were still produced. This suggests that the FAF perturbations were detected by the auditory system in both the single- and dual-task conditions. Based on these results, we suggest that the effect of attentional load was not on the auditory processing of FAF, but instead it interfered with the integration of auditory and motor information, or motor control itself. It has previously been suggested that speech motor control may be disrupted under divided attention. For example, when participants were required to read sentences while also completing a secondary motor task, divided attention resulted in decreased displacement and velocity of labial movements Even though divided attention was not found to modulate ERP responses to FAF, the amplitude of the N1 component was found to vary as a function of electrode site. N1 amplitudes were smaller  at the right electrodes sites, relative to medial electrode sites. This result is unsurprising, as N1 responses elicited by FAF are generally largest at medial sites Much like the size of the ERPs elicited by the FAF perturbations, and the vocal response latencies, the latencies of the ERP components were not found to differ when attention was divided. We hypothesized that divided attention may result in later ERP latencies as a result of increased processing demands. Although this hypothesis was not confirmed, it is possible that the dual-task condition was not demanding enough to increase processing demands to the extent that ERP latencies were affected. As mentioned previously, even during the most demanding attentional load condition, accuracy was still quite high, thus a more demanding task may be required to produce changes in ERP latencies as a function of attentional load.Fluent speech production relies on auditory feedback for the regulation of ongoing and future speech motor commands. The aim of the current study was to investigate how attention modulates the processing of auditory feedback during ongoing speech. The results of this study suggest that divided attention can reduce the size of compensatory motor responses to FAF. However, the results of this study also suggest that the P1-N1-P2 ERP components elicited by FAF are less sensitive to divided attention. While the attentional load manipulation utilized in this study was successful at reducing target identification accuracy as the attentional load increased, accuracy was still quite high, even at the highest level of attentional load. With this being said, it is possible that auditory-cortical responses are less sensitive to increases in attention load, but may show attentional modulation if a secondary task is utilized that imposes a higher attentional load. Alternatively, sensorimotor integration, or motor control itself, may be more susceptible to increases in attentional load.Throughout a typical day, we often encounter situations where we must speak while also performing other tasks. Recently, there has been a focus on assessing the impact of conversation on the performance of secondary tasks such as driving. We instead assessed the impact of divided attention on low level control of speech and found that our ability to use auditory feedback to monitor and regulate our speech may be compromised during many of our daily encounters. While typically developed adults have internal models that can execute fluent speech in a feedforward manner, previous research has suggested that children rely more on auditory feedback