L1-encoded ORF1 and ORF2 proteins bind L1 RNAs, forming ribonucleoproteins (RNPs). These RNPs interact with diverse host proteins, some repressive and others required for the L1 lifecycle. Using differential affinity purifications, quantitative mass spectrometry, and next generation RNA sequencing, we have characterized the proteins and nucleic acids associated with distinctive, enzymatically active L1 macromolecular complexes. Among them, we describe a cytoplasmic intermediate that we hypothesize to be the canonical ORF1p/ORF2p/L1 Our genome consists of about two percent genes, while around 60 to 70 percent are made up of hundreds of thousands of copies of very similar DNA sequences. These repeats have accumulated over time due to specific genetic elements called transposons.Transposons are often referred to as ‘jumping genes’, as they can move within the genome and thereby create mutations that may lead to cancer or other genetic diseases. LINE-1 is the only remaining active transposon in humans, and it expands by copying and pasting itself to new locations. To do so, it is first transcribed into RNA – the molecules that help to make proteins – and then converted back into identical DNA sequences.In a never-ending battle, our cells have been fighting to keep LINE-1 and its ancestors from replicating, and so evolved various defense mechanisms. Yet, LINE-1 has learned to circumvent these barriers, and continues to replicate and cause disease. Our understanding of these defenses and of how LINE-1 evades them is limited.Previous research has shown that the LINE-1 RNA and its two encoded proteins, called ORF1p and ORF2p, interact with a series of other proteins, with which they can form different types of complexes. Now, Taylor, Altukhov, Molloy et al. used human embryonic kidney cells grown in the laboratory with different LINE-1 mutations to identify how they affect the bound proteins and RNAs. The results showed that LINE-1 can form at least two different sets of complexes with other proteins.The complex containing ORF1p and ORF2p and several other proteins was located in the cytoplasm, the fluid that fills the cells. However, the experiments also revealed a new complex in the cell nucleus, which contained ORF2p and proteins involved in DNA replication and repair, but not ORF1p. The results suggest ORF1p delivers RNPs to the nucleus around the time the cell divides. Another group of researchers has looked more closely at what happens during cell division.A next step will be to study how exactly LINE-1 contributes to cancer. In the future, overactive LINE-1 proteins could be targeted to kill cancer cells, to identify cancer early, or to see if the cancer has come back. LINE-1 may also provide clues on how the genome has evolved. ORF0, has been identified on the Crick strand of the L1 gene; this ORF encodes a 71 amino acid peptide and may generate insertion-site-dependent ORFs via splicing  to investigate the proteomic characteristics of endogenously assembled ectopic L1-derived macromolecules present in an assortment of affinity-enriched fractions. We revisited RNase treatment and split-tandem affinity capture approaches and complemented them with RNA sequencing, enzymatic analysis, and in-cell localization of ORF proteins by immunofluorescence microscopy , such that the proteomes are thoroughly labeled. Protein fractions derived from the differently labeled cell populations, obtained e.g. before and after experimental manipulations are applied, are mixed and the relative differences in proteins contributed by each fraction are precisely measured by mass spectrometry. In addition to the above cited studies, these approaches have been adapted to numerous biological questions using a variety of analytical frameworks e.g.  from a tetracycline inducible minimal-CMV promoter, harbored on a mammalian episome . All L1−4, see Materials and methods). These same proteins were previously observed as specific L1 interactors in I-DIRT experiments targeting ORF2p but not in those targeting ORF1p; in contrast, the proteins that demonstrated RNase-sensitivity: ORF1, MOV10, ZCCHC3, and UPF1 were observed in both ORF1p and ORF2p I-DIRT experiments (−11). This analysis also revealed a statistically significant overrepresentation of genes associated with the exon junction complex . Hence, the overlapping portion of the ORF1p- and ORF2p-associated interactomes appeared to depend upon intact L1 RNA. Host-encoded proteins segregated into groups that responded differentially to RNase treatment, with a substantial population of RNase-resistant interactors linked to both ORF2p and the nucleus. This observation led to the hypothesis that our ORF2p-3xFLAG affinity captured L1s constitute a composite purification of at least, but not limited to, (1) a population of L1-RNA-dependent, ORF1p/ORF2p-containing L1 RNPs, and (2) an ORF1p-independent nuclear population associated with ORF2p.eriments . Stated eriments . The ORFeriments , they toWhile effects of PABPC1, MOV10, and UPF1 on L1 activity have been described , effectsboth ORF2p and ORF1p, whereas the supernatant contained the proteins associated only with ORF2p . The results corroborated our previous observations that: (i) almost all of the ORF1p partitioned into the elution fractions, (ii) a quarter of the ORF2p (~26%) followed ORF1p during the α-ORF1p affinity capture, (iii) roughly half of the UPF1 (~55%) followed ORF1p, and (iv) most of the PCNA (~87%) remained in the ORF1p-depleted supernatant fraction . In contrast, all sixteen proteins exhibiting ORF1p co-partitioning approximately equal to or less than that of ORF2p were predominantly found in the supernatant fraction and were enriched for cell-compartment-specific association with the nucleus . The nodes representative of PURA, PURB, and PCNA appeared to exhibit a striking proximity to one another, suggesting highly similar co-fractionation behavior potentially indicative of direct physical interactions. In an effort to examine this possibility, we graphed the frequency distribution of the proximities of all three-node-clusters observed within −7 (see Appendix 1). We therefore concluded that PURA, PURB, PCNA, and (perhaps at a lower affinity) TOP1, likely constitute a physically associated functional module interacting with L1. In further support of this assertion, we noted that known functionally linked protein pairs PABPC1/PABPC4 (cytoplasmic)   and HSPAplasmic)  also exhTo validate our hypothesis that these proteins are associated with ORF2p in the nucleus, possibly engaged with host genomic DNA, we carried out ORF2p-3xFLAG affinity capture from chromatin-enriched sub-cellular fractions and found that the co-captured proteins we identified  overlappBecause the L1 RNA is an integral component of proliferating L1s, and because we observed that interactions between ORF2p, ORF1p, and some host proteins were sensitive to treatment with RNases, we sought to characterize the RNAs present in our samples. We extracted RNAs from each of the three fractions produced by split-tandem affinity capture  and carrcis preference’), presumably at the site of translation in the cytoplasm (ΔORF1 construct (pLD561) as a control  analyses did not reveal an apparent nuclear population, leading us to revisit IF studies. Previously, IF of ORF1p and ORF2p in HeLa and HEK-293T cells yielded two striking observations: (i) ORF2 expression was seemingly stochastic, with ORF2p observed in ~30% of cells; and (ii) while ORF1p and ORF2p co-localized in cells that exhibited both, we did not observe an apparent nuclear population of either protein . Subsequrd also exhibited co-localized cytoplasmic ORF2p  resulted in the majority of cells exhibiting cytoplasmic ORF2p, consistent with our previous work , and a D702Y mutation to inactivate the reverse transcriptase activity (RT-/pLD624)  immediately preceding DNA cleavage. Notably, there is a third class of proteins, including PURA/B, PCNA, TOP1, and PARP1, that all respond similarly to both EN- and RT- mutants compared to WT, exhibiting reduced associations with the mutant L1s; although, the RT- mutant showed a larger effect size on the PURA/B proteins. These data suggest that cleavage of the host genomic DNA by ORF2p fosters associations between L1 and this third class of proteins, but that interactions with PURA/B may be further enhanced by L1 cDNA production. Other nuclear L1 proteins: HSPA8, HAX1, HSPA1A, TUBB, and TUBB4B were increased in both mutants. To better visualize the range of behaviors exhibited by our proteins of interest, and the population at large, we cross-referenced the relative enrichments of each protein detected in both experiments, shown in - mutants and reduced in EN- mutants. We therefore speculate that the sum of observed interactomic changes include effects attributable directly to the catalytic mutations as well as potential indirect effects resulting in increased cytoplasmic RNPs (including ORF1p)in the RT- mutant.Based on the hypothesis that our composite purifications contain bona fide nuclear intermediates, we decided to explore the effects of catalytic point mutations within the ORF2p endonuclease and reverse transcriptase domains, respectively. We reasoned that such mutants may bottleneck L1 intermediates at the catalytic steps associated with host gDNA cleavage and L1 cDNA synthesis, potentially revealing protein associations that are important for these discrete aspects of target-primed reverse transcription (TPRT), the presumed mechanism of L1 transposition . For thi/pLD624) . Figure /pLD624) , our fin/pLD624) . Two claWe next decided to measure the in vitro dynamics of proteins copurifying with affinity captured L1s, reasoning that proteins with comparable profiles are likely candidates to be physically linked to one another or otherwise co-dependent for maintaining stable interactions with L1s. To achieve this, we first affinity captured heavy-labeled, affinity-tagged L1s and subsequently incubated them, while immobilized on the medium, with light-labeled, otherwise identically prepared cell extracts from cells expressing untagged L1s . In thisHaving observed coordinated and distinctive behaviors exhibited by groups of L1 interacting proteins across several distinctive biochemical assays, we then integrated the data and calculated the behavioral similarity of the I-DIRT-significant interactors, producing a dendrogram; Alu elements exhibit ORF2p-dependent mobilization that does not require ORF1p, but appears to be enhanced by ORF1p in some contexts  and ORF1p– (immuno-depleted ‘supernatant’) populations . To our - and RT- catalytic mutations, decreasing together in EN- mutants, and increasing together in RT- mutants  our L1 preparations exhibit RT activity . In contrast, the supernatant from the α-ORF1p affinity capture contained the proteins we speculate to be associated with ORF2p, but not ORF1p; moreover, fully intact RNA does not appear to be essential to the maintenance of these interactions. An exciting alternate interpretation to direct protein-protein linkage is that some of the L1 RNAs in this population may be at least partially hybridized to L1 cDNAs, which would render them RNase resistant: at the salt concentration used in our RNase assay 0.5 M; , RNase Autations , with a activity , with TOactivity , PCNA/TOactivity , and PURactivity . Notablyactivity . Given tactivity  and HSPAactivity , and becactivity , we propactivity . Furtheractivity . This isactivity , no suchactivity . These pactivity , but exhAs mentioned above, we previously speculated that an RNase-insensitive fraction of L1-associated UPF1 may support retrotransposition in conjunction with PCNA in the nucleus . In contAn obvious need is the continued validation of putative interactors by in vivo assays. Genetic knockdowns coupled with L1 insertion measurements by GFP fluorescence  provide -RNA-containing cytoplasmic L1 RNPs can be prepared at sufficiently high purity and yield (Throughout this and our prior study  we have nd yield  to enablnd yield ), providnd yield .LD cells  1L square glass bottles each containing 200 ml of SILAC suspension culture at ~2.5 × 106 cells/ml were transfected using 1 μg/ml DNA and 3 μg/ml polyethyleneimine ‘Max’ 40 kDa . A common transfection mixture was made by pre-mixing 800 μg DNA and 2.4 mL of 1 mg/ml PEI-Max in 40 ml Hybridoma SFM medium  and incubating for 20 min at room temperature (RT); 10 ml of the mixture was added to each bottle. On day 1, cells (200 ml) were split 1:2.5  without changing the medium. On day 3, the cells were induced with 1 µg/ml doxycycline, and on day four the cells were harvested and extruded into liquid nitrogen. Aliquots were tested by western blot and the per-cell expression of both ORFs was indistinguishable from puromycin-selected material described previously (Appendix 1); transfection efficiency was assessed at >95% by indirect immunofluorescence of expressed ORF proteins. The median lysine and arginine heavy isotope incorporation levels for cell lines presented in this study were >90%, determined as previously described . The gels were subsequently subjected to colloidal Coomassie blue staining (Four sets of 200 mg of light (L) and heavy (H) pLD401 transfected cell powders, respectively, were extracted 1:4 (w:v) with 20 mM HEPES-Na pH 7.4, 500 mM NaCl, 1% (v/v) Triton X-100 (extraction solution), supplemented with 1x protease inhibitors . After centrifugal clarification, all of the L and H supernatants were pooled, respectively, and then split, resulting in two sets of cleared L and H extracts equivalent to duplicate 400 mg samples from each SILAC cell powder. These four samples were each subjected to affinity capture upon 20 μl α-FLAG magnetic medium. After binding and washing, one set of L and H samples were treated with a control solution consisting of 2 μl of 2 mg/ml BSA  and 50 μl extraction solution, v:v (Ctrl); the other set of L and H samples was treated with a solution of 2 μl 2 mg/ml RNase A/5000 u/ml RNase T1 (Thermo Fisher Scientific #EN0551) and 50 μl extraction solution, v:v (RNase). Samples were then incubated 30 min at RT with agitation, the supernatant was removed, and the medium was washed three times with 1 ml of extraction solution. The retained captured material was eluted from the medium by incubation with 40 μl 1.1x LDS sample loading buffer (Life Technologies #NP0007). To enable quantitative comparisons of fractions, the samples were combined, respectively, as follows: 30 ul each of the staining  and the LSup with HElu, and 28 μl each of the LElu with HSup. These samples were then prepared as gel-plugs (as above) and processed for MS analyses, as described below.400 mg of light (L) and heavy (H) pLD401 transfected cell powders, respectively, were extracted and clarified as above. These extracts were subjected to affinity capture on 20 μl α-FLAG magnetic medium, 30 min at 4°C, followed by native elution with 50 μl 1 mg/ml 3xFLAG peptide . 45 μl of the elution were subjected to subsequent affinity capture upon 20 μl α-ORF1 magnetic medium, resulting in a 45 μl supernatant (Sup) fraction depleted of ORF1p. Finally, the material was eluted (Elu) from the α-ORF1p medium in 45 μl 2.2x LDS sample loading buffer by heating at 70°C for 5 min with agitation. To enable quantitative comparisons of fractions the samples were combined, respectively, as follows: 28 μl each of the ∗Gel ODS-A, YMC, Allentown, PA). Peptides were gradient-eluted  into an LTQ-Orbitrap-Velos or an LTQ-Orbitrap-XL mass spectrometer (Thermo Fisher Scientific) acquiring data-dependent CID fragmentation spectra. In vitro exchange samples were loaded onto an Easy-Spray column  and gradient-eluted  into an Q Exactive Plus mass spectrometer (Thermo Fisher Scientific) acquiring data-dependent HCD fragmentation spectra. In SILAC experiments comparing inactivated ORF2p catalytic mutants to WT  peptides were extracted from the gel in two 1 hr incubations with 1.7% v/v formic acid, 67% v/v acetonitrile at room temperature with agitation. Digests were partially evaporated by vacuum centrifugation to remove acetonitrile, and the aqueous component was desalted on Stage Tips. Peptides were loaded onto an Easy-Spray column  and gradient-eluted  into an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific) acquiring data-dependent fragmentation spectra .Gel plugs were excised, cut into 1 mm cubes, de-stained, and digested overnight with enough 3.1 ng/μl trypsin  in 25 mM ammonium bicarbonate to cover the pieces. In RNase-sensitivity and split-tandem SILAC analyses based on pLD401, as well as in vitro protein exchange experiments based on pMT302 and pMT289, an equal volume of 2.5 mg/ml POROS R2 20 µm beads (Life Technologies #1112906) in 5% v/v formic acid, 0.2% v/v TFA was added, and the mixture incubated on a shaker at 4°C for 24 hr. Digests were desalted on Stage Tips , eluted,http://www.coxdocs.org/; ‘contaminants’). Additionally, proteins with the ‘POTENTIAL CONTAMINANT’ column value ‘+' were filtered out. Proteins with at least two razor+unique peptides were retained for the analysis. H/(H + L) and L/(H + L) values were derived from unnormalized ‘ratio H/L’ values and were used for plotting label-swapped RNase-sensitivity and split-tandem data. Unnormalized ‘ratio H/L’ values were used to calculate H/(H + L) in ORF2p catalytic mutant comparisons and in vitro exchange experiments. These values have been referred to as ‘affinities’ within the Supplementary Materials. Normalization and clustering procedures applied to data presented in the figures (Raw files were submitted to MaxQuant  version  figures  are deta- and RT- mutant affinity capture results (- and RT-) was filtered to retain only proteins detected in at least two replicate experiments. The difference between the affinity value of ORF2p and 0.5 value was calculated for each experiment. The affinities of each protein were shifted by the calculated difference. To determine the statistical significance of differentially co-captured proteins between EN- or RT- and WT, respectively, we used a 1-sample t-test and applied Benjamini-Hochberg p-value correction. To determine the statistical significance of differentially co-captured proteins between EN- and RT- we used an unpaired t-test and applied Benjamini-Hochberg p-value correction. To plot in vitro dynamics  and reference FASTA and GTF files were created by combining the human genome and ORFeus-Hs from pLD401  using the following parameters: -runThreadN 8, --quantMode GeneCounts, --outSAMtype BAM SortedByCoordinate, --outFilterMatchNmin 30; (3) the results were output to one binary alignment map file for each sample matched to the reference; (4) genes with the coverage of 10 or more reads in at least three experiments were selected; and (5) data was normalized using the ‘DESeq2’ (Human genome hg19 GRCh37.87 (FASTA) and annotation (GTF file) were downloaded from ENSEMBL  as a di-peptide composed of repeats of the same sequence linked by a four-unit polyethylene glycol moiety; which was used to elute ORF1p-containing complexes from α-ORF1p medium at a concentration of approximately 2 mM  selecting x nuclei among all nuclei. The random distribution was used to calculate Bonferroni corrected p-values for the pairwise distances between ORF2p+ nuclei. The distribution of ORF2p+ inter nuclei distances was then compared to the distribution of random inter-nuclei distances using Welch’s t-test. The custom scripts used to select nuclei and calculate statistics, extracted data, calculated distances, p-values, and raw images are presented in the supplement . In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and James Manley as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Geoffrey Faulkner (Reviewer #2); Victoria Belancio (Reviewer #3).Thank you for submitting your article "Dissection of purified LINE-1 reveals distinct nuclear and cytoplasmic intermediates" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:This paper reports the protein content of LINE-1 complexes in the cytoplasm and nucleus of cells undergoing retrotransposition events, and uncovers an array of distinctive complexes. The reviewers uniformly found the story to be of much interest and to constitute a significant advance beyond published work. They are enthusiastic.We include all their reviews here for consideration. There were raised many shared issues of what were judged to be overinterpretations or overstatements of the data. I think most of the comments can be addressed by rewriting.A common thread is the point that we cannot know whether the complexes are on a direct pathway to retrotransposition, or whether they are even on a pathway at all. Some disclaimers here are needed.Essential revisions:Reviewer 1:The point about Orf2 traversing the RNA in the cytoplasm, given what we think we know about the process of RT here, seems cogent. Here it is probably correct that the data do not rule out the presence of Orf1 in the nucleus at the time of RT. This needs to be addressed.Reviewer 2:There are real issues about the cell localization data. We should not assume that the localization of cellular proteins is according to the dogma in non-transposing cells, outside of the Orf complexes. There are real issues about IF quantification that need to be addressed.Reviewer 3:Almost all the comments here are valid and readily addressed. Again, the issue of Orf1 in the nucleus is raised as a red flag that needs to be addressed, likely by softening of claims.We look forward to receiving a revised version with a point-by-point response to the reviews.Reviewer #1:This study provides a significant advance in our understanding of biochemical interactions of LINE-1 encoded proteins, with solid evidence for multiple, varied interactions with distinct host cell components in both the nucleus and the cytoplasm. The authors employ sophisticated, quantitative methods to interrogate protein interactions of (primarily) the LINE-1 ORF2 protein, thereby gaining new insight into the complexity of the host-parasite relationship and process of LINE-1 retrotransposition. It represents a significant step forward for the field, building on results of Taylor published in 2013 in Cell. There are however some issues with the wording of some conclusions overstepping the limits of what is documented by the data presented, as specifically noted below. With wording changes to more accurately describe/interpret the data the most serious limitation of this work , could be considered minor.In Abstract "our findings support the presence of multiple L1-derived retrotransposition intermediates in vivo"; oversteps the actual data because there is no direct evidence to address which of these various complexes are bona fide retrotransposition intermediates vs which reflect the various ways the cell interferes with/prevents retrotransposition.In numerous instances the term "purified" is used when the data support "enrichment" but not purification. This leads to confusion when the "purified" complex turns out to be, as is the point of the work, multiple distinct complexes.The conclusion "[…]ORF1p probably does not interact with ORF2p in the nucleus", is not supported by the data, which simply show that there are RNA- and ORF1p-independent interactions between ORF2p and non-L1 cellular proteins in the nucleus. Agreed that the data show a persistent complex of ORF2p that lacks ORF1p, but that is not the same as the above. There is likewise no data to support "ORF1p may be displaced from the L1 RNA during the nuclear portion of the lifecycle" – maybe there is also no remaining L1 RNA by the time the ORF2 complexes lacking ORF1p are formed. The authors should provide supporting data to support this claim or remove it.Subsection “4.4. The effects of retrotransposition-blocking point mutations on the interactomes of purified L1 RNPs”. TPRT is target primed reverse transcription, not "template".Subsection “4.4. The effects of retrotransposition-blocking point mutations on the interactomes of purified L1 RNPs”. What evidence suggests that ORF2 traverses L1 RNA in cytoplasm? Given that this RT gets its primer from the target DNA this comment seems unwarranted. This unnecessary speculation is not appropriate for the Results section. In fact the effects of the RT and EN mutations are actually so subtle (~2x) compared to the robust effects they have on retrotransposition (>>100X) it may speak more to the fact that neither of these most abundant L1-containing protein complexes (both nuclear and cytoplasmic) are on the productive retrotransposition pathway.Reviewer #2:Molloy et al. perform affinity purifications followed by quantitative mass spectrometry to identify proteins that interact with the L1-encoded proteins and mRNA in ribonucleoprotein particles (RNPs). Their results suggest the existence of multiple different L1 RNPs that contain the L1-encoded proteins ORF1p and ORF2p and various cellular factors at distinct stoichiometries and in discrete subcellular locations. The major advance of this manuscript over the work of Taylor et al.  is the resolution of the subcellular localisation of the different RNP intermediates. These data are really interesting and should be the starting point for a range of follow-up investigations. However, the strength with which some of the conclusions are presented is not sufficiently justified by the available data and this should be addressed prior to publication.1) Most of the conclusions about the subcellular localisation of ORF1p/ORF2p versus ORF2p-only RNP complexes are drawn from the known subcellular localisation and ontological classification of their interacting protein partners. While these data are interesting, the paper would be greatly improved by the direct demonstration of differential subcellular colocalisation of the L1 proteins with their interacting partners. Indeed, the experiments aimed at verifying these conclusions are not entirely convincing, and in some cases the results are overstated or overgeneralised (see below).2) The authors performed ORF2p-3XFLAG affinity capture from chromatin-enriched subcellular fractions to support the conclusion that ORF2p-only RNPs localise to the nucleus. The text states that proteins co-purifying with chromatin-associated ORF2p "largely overlapped" with those described as nuclear ORF2p-only RNP interactors. However, comparing the list of these proteins in 3) Immunofluorescence experiments have little quantitative information. Statements like "these cells accounted for <10% of the population" and "Expression of ORF2p in the absence of ORF1p resulted in >90% of cells exhibiting cytoplasmic ORF2p" and "they always occurred in proximal pairs" would be much more convincing with some statistical information about the experiment. How many cells were analysed? How many times were the results replicated? Clarity here would bolster the results.4) Immunofluorescence experiments could also be used to demonstrate colocalisation of ORF1p and ORF2p with their presumed interaction partners in the cytoplasm and nucleus, and would provide more direct support for the conclusions drawn regarding subcellular localisation of different L1 RNPs. Such experiments would understandably be limited by the availability of antibodies to the interacting factors, but would almost certainly be feasible in some cases.5) Analysis of host factors differentially associated with RT and EN mutant L1 RNPs is also intriguing, but the results are over-interpreted. As a whole, most of the conclusions drawn in the manuscript need to be backed up by more direct functional assays, or the language in which the conclusions are stated needs to be softened considerably.Reviewer #3:This is an experimentally sound, well-written manuscript demonstrating the existence of distinct L1 complexes with potentially different roles in L1 retrotransposition. The results presented in this manuscript are novel and provide significant insights into L1 RNPs and its role in the L1 replication cycle. In addition to identifying L1 interactors with a previously reported role in L1 retrotransposition, the authors provide experimental validation of additional and previously unknown interactors. Another important finding is the demonstration that the proportions of proteins within L1 complexes is affected by individual mutations of the L1 ORF2p catalytic activities. I am very enthusiastic about the findings described in this manuscript. I think they significantly advance the research related to transposable elements and viruses and would be helpful to the future progress of studies conducted by other labs.This reviewer did not have access to the co-submitted manuscript by Mita et al.• Indicate if the status of L1ORFeus mRNA in the different fractions shown in • Subsection “4.2. Split-tandem separation of compartment-specific L1 ORF-associated complexes”, the last paragraph provides experimental validation of the significant overlap between putative nuclear L1 complexes and complexes detected in chromatic enriched fractions. Please discuss this validation earlier in this study as it provides important experimental support for the references to these complexes as nuclear.• Comment on how these nuclear and cytoplasmic complexes align with the existing literature showing that L1 RT present in the cytoplasm generates cDNA when provided with a primer (LEAP protocol).• "We did not observe instances of nuclear ORF2p using the construct  the LINE-1 ORF2 protein, thereby gaining new insight into the complexity of the host-parasite relationship and process of LINE-1 retrotransposition. It represents a significant step forward for the field, building on results of Taylor published in 2013 in Cell. There are however some issues with the wording of some conclusions overstepping the limits of what is documented by the data presented, as specifically noted below. With wording changes to more accurately describe/interpret the data the most serious limitation of this work , could be considered minor.We added LEAP and RNA-seq experiments in order to account for the state of the L1 RNA  across various fractions – now presented in new In Abstract "our findings support the presence of multiple L1-derived retrotransposition intermediates in vivo"; oversteps the actual data because there is no direct evidence to address which of these various complexes are bona fide retrotransposition intermediates vs which reflect the various ways the cell interferes with/prevents retrotransposition.We agree that the diversity of putative assemblies are of unknown molecular physiology (and that their ultimate outcome is unknown), and accordingly, more nuance is required to avoid over-lumping and over-interpretation. We amended the text and title. In numerous places we substituted e.g. “L1 macromolecules” and “protein complexes” in place of “intermediates.” A few instances of “intermediate” were retained where it seemed appropriate for the concept being conveyed. We have also shown the preps to be enzymatically active by LEAP assay new .In numerous instances the term "purified" is used when the data support "enrichment" but not purification. This leads to confusion when the "purified" complex turns out to be, as is the point of the work, multiple distinct complexes.Agreed: we have modified numerous instances of “purified,” with alternative descriptors such as “enriched” and “affinity captured.”The conclusion "[…]ORF1p probably does not interact with ORF2p in the nucleus", is not supported by the data, which simply show that there are RNA- and ORF1p-independent interactions between ORF2p and non-L1 cellular proteins in the nucleus. Agreed that the data show a persistent complex of ORF2p that lacks ORF1p, but that is not the same as the above. There is likewise no data to support "ORF1p may be displaced from the L1 RNA during the nuclear portion of the lifecycle" – maybe there is also no remaining L1 RNA by the time the ORF2 complexes lacking ORF1p are formed. The authors should provide supporting data to support this claim or remove it.We removed the text “ORF1p probably does not interact with ORF2p in the nucleus" and “ORF1p may be displaced from the L1 RNA during the nuclear portion of the lifecycle.”Subsection “4.4. The effects of retrotransposition-blocking point mutations on the interactomes of purified L1 RNPs”. TPRT is target primed reverse transcription, not "template".Fixed.Subsection “4.4. The effects of retrotransposition-blocking point mutations on the interactomes of purified L1 RNPs”. What evidence suggests that ORF2 traverses L1 RNA in cytoplasm? Given that this RT gets its primer from the target DNA this comment seems unwarranted. This unnecessary speculation is not appropriate for the Results section. In fact the effects of the RT and EN mutations are actually so subtle (~2x) compared to the robust effects they have on retrotransposition (>>100X) it may speak more to the fact that neither of these most abundant L1-containing protein complexes (both nuclear and cytoplasmic) are on the productive retrotransposition pathway.We removed the speculative text pertaining to ORF2p traversing L1 RNA in cytoplasm.We agree that ORF1p may play a role in the nucleus, but the current data lack granularity that would allow us to define these complexes further. We note that data in the companion manuscript by Mita et al. suggest that any role of ORF1p in the nucleus is transient and show no nuclear co-localization between ORF1p and ORF2p. Accordingly we have modified the figure to show both RNA-linked and non-linked ORF1p in the nucleus, with a semi-transparent shade to imply our lack of knowledge regarding these interactions now .We now provide two lines of evidence supporting the presence of the L1 RNA in the ORF2p-containing nuclear complex lacking ORF1p: (1) high levels of LEAP activity and (2) RNA sequencing data. We respectfully disagree, therefore, that the ORF1p-lacking complex should also lack the L1 RNA. The remaining schematic accordingly shows ORF2p as the protein interaction hub, in keeping with our lack of evidence to support RNA as a key player in those interactions. We note additionally that the presumed mechanism of L1 insertion, TPRT, would require an ORF2p-L1 RNA interaction at the chromatin. Disruption of e.g. PCNA and UPF1 interactions with ORF2p have strong negative effects on retrotransposition; mild positive effects were observed when knocking down TOP1 and PURA/B; we also identified most of these proteins in ORF2p affinity captured from fractionated chromatin. We conclude that these ORF2p interactions modulate retrotransposition and very likely occur at the chromatin.Reviewer #2:[…] 1) Most of the conclusions about the subcellular localisation of ORF1p/ORF2p versus ORF2p-only RNP complexes are drawn from the known subcellular localisation and ontological classification of their interacting protein partners. While these data are interesting, the paper would be greatly improved by the direct demonstration of differential subcellular colocalisation of the L1 proteins with their interacting partners. Indeed, the experiments aimed at verifying these conclusions are not entirely convincing, and in some cases the results are overstated or overgeneralised (see below).2) The authors performed ORF2p-3XFLAG affinity capture from chromatin-enriched subcellular fractions to support the conclusion that ORF2p-only RNPs localise to the nucleus. The text states that proteins co-purifying with chromatin-associated ORF2p "largely overlapped" with those described as nuclear ORF2p-only RNP interactors. However, comparing the list of these proteins in Fixed. The overlapping proteins have been more explicitly stated in the main text, and the supplementary table has been amended to draw attention to the proteins in question. We purposefully restricted the stated overlap between the proteins identified in the two different experimental designs to those proteins meeting the conservative filter of “I-DIRT specific;” detection alone was not considered sufficient. The rationale is set out in the first two paragraphs of the Results. E.g. the proteins SSBP1, PRPF6, RBM14, U2SURP, SRSF5 (not described as overlapping) were detected in the chromatin fraction ORF2p IP as well as at least one other IP presented in this study, but we do not consider that a compelling enough case to present them as putative L1 interactors at this time. Moreover, we note that in the chromatin fraction ORF2p IP experiment presented in the supplement, of the proteins found to be enriched with ORF2p in the chromatin fraction, only PCNA was also detected after IP of ORF2p from unfractionated cell extracts – this is a general limitation in the sensitivity and dynamic range provided by shotgun proteomic experiments – such that it is not uncommon to observe limited overlap in different proteomic experimental regimes.3) Immunofluorescence experiments have little quantitative information. Statements like "these cells accounted for <10% of the population" and "Expression of ORF2p in the absence of ORF1p resulted in >90% of cells exhibiting cytoplasmic ORF2p" and "they always occurred in proximal pairs" would be much more convincing with some statistical information about the experiment. How many cells were analysed? How many times were the results replicated? Clarity here would bolster the results.eLife manuscript.These images were collected a couple years ago and at the time we did not have the foresight to plan a rigorous quantitative analysis. We counted many cells we observed and estimated frequencies of subpopulations, but did not record images from the population that were not “interesting” with respect to observed ORF1p/ORF2p expression – moreover, aside from the nuclear ORF2p cells, the results were comparable to those we previously published in Taylor et al. 2013. Unfortunately, we currently lack the personnel to revisit this experimental regime. We therefore softened our language regarding the precise proportions of the populations observed. However, to respond to the reviewer and strengthen the manuscript, we added a new companion analysis. We possess 11 such IF images displaying the described ORF2p staining phenomenon, among other non-staining cells . We analyzed the distributions of cell-cell proximities (nuclei) in these images to provide a quantitative validation of our claim regarding the proximity of cells displaying nuclear ORF2p – now presented in 4) Immunofluorescence experiments could also be used to demonstrate colocalisation of ORF1p and ORF2p with their presumed interaction partners in the cytoplasm and nucleus, and would provide more direct support for the conclusions drawn regarding subcellular localisation of different L1 RNPs. Such experiments would understandably be limited by the availability of antibodies to the interacting factors, but would almost certainly be feasible in some cases.We do certainly agree that in some cases these experiments would be feasible and informative. As stated above, there are some expected limitations related to identifying reagents, as well as expectations that some may not generate discrete/enriched co-localization foci. E.g. as annotated in Uniprot, many have broad, diffuse, or complex localizations – we also agree with the reviewer’s point that localization of these proteins in the context of L1 expression may differ from that defined by normal conditions, curated in databases. Hence, this experiment would require some time for trial and error on our part and, given IF is not the expertise of our core-team, was not an experiment we could implement within the post-review timeframe.We have instead softened our language throughout (in response to this and other reviewer points), in order to compensate for the reviewer’s degree of skepticism regarding putative L1 complexes, and we have added additional IF co-localization as a future experimental direction in the discussion. We also believe this point is at least partly addressed by the chromatin fractionation experiment  which showed compositional overlap with our proposed nuclear L1 intermediates inferred from alternative fractionation schemes .5) Analysis of host factors differentially associated with RT and EN mutant L1 RNPs is also intriguing, but the results are over-interpreted. As a whole, most of the conclusions drawn in the manuscript need to be backed up by more direct functional assays, or the language in which the conclusions are stated needs to be softened considerably.The manuscript as a whole has been modified to soften tone, including the changes made in response to reviewer #1; in this section we removed the text pertaining to ORF2p traversing L1 RNA in cytoplasm, as well as other modifications.Reviewer #3:[…] • Indicate if the status of L1ORFeus mRNA in the different fractions shown in RNA-seq and LEAP analysis of the fractions has now been provided, see new • Subsection “4.2. Split-tandem separation of compartment-specific L1 ORF-associated complexes”, the last paragraph provides experimental validation of the significant overlap between putative nuclear L1 complexes and complexes detected in chromatic enriched fractions. Please discuss this validation earlier in this study as it provides important experimental support for the references to these complexes as nuclear.We attempted to accommodate this critique, however, we cannot move this section to an early position in the manuscript because the section, as written, leverages the observation of TOP1 as part of the putative nuclear L1 complex. TOP1 was not detected in the RNase experiment, and therefore, moving this section requires some acrobatics to narrate the significance of the TOP1 observation and testing of TOP1 by over-expression, etc. If the reviewer is adamant on this request, we could accommodate it with a more involved re-write where we excise the text describing the chromatin fractionation and move it to the previous section, but leave the over-expression results where they are (following the observation of TOP1). We note that, as it stands, this information is provided in the Results section attached to • Comment on how these nuclear and cytoplasmic complexes align with the existing literature showing that L1 RT present in the cytoplasm generates cDNA when provided with a primer (LEAP protocol).We have done LEAP experiments on split-tandem affinity captured L1 fractions those of , and all• "We did not observe instances of nuclear ORF2p using the construct . Note that, for technical reasons related to quantitation using shotgun proteomics, zero-values for proteins  are not recorded in any sample, and small values instead are derived either from noise or imputed. ORF1p was shown to be ~100% in the final elution fraction by western blot  and by MS (this study). Other proteins generated much of their signal (as graphed) in the ORF1p containing fraction, but some proportion may have decayed away (remaining in the supernatant) or be partially associated with the ORF2p-only fraction. We cannot distinguish between these scenarios based on that data alone. The inverse is also true for proteins in the supernatant. UPF1 represents a protein that we hypothesize to be specifically present in both complexes because of its borderline behavior in numerous experiments. Being an RNP that evolves from one lifecycle stage to the next, proteins likely exchange on and off the complex dynamically along the way. These proteins may exhibit absolute or conditional dependencies, resulting in heterogeneous mixtures or continua of RNPs. • Statements that are supported by unpublished data that are not shown in this or the accompanying manuscript should be eliminated or restated.Done.• Discuss the possibility that the lack of detection of the L1 ORF1p in the nucleus by IHC could be due to technical problems rather than the lack of the protein?This section has been re-written; we do not make a point of not detecting ORF1p in the nucleus in our IF assays anymore, and instead refer to Mita et al. the co-submission that does demonstrate nuclear ORF1p. We understand that this reviewer did not have access to the co-submitted manuscript by Mita et al. – in its current state the text stands on its own and does not suggest a failure to detect nuclear ORF1p means a lack of ORF1p in the nucleus.


The G-doped/p-Si gas sensor was synthesized by a simple capillary force-assisted solution dropping method on p-Si substrates, whose porosity was generated through an electrochemical etching process. G-doped/p-Si was fabricated with various graphene concentrations and exploited as a H2 sensor that was operated at room temperature. The sensing mechanism of the sensor with/without graphene decoration on p-Si was proposed to elucidate the synergetic gas sensing effect that is generated from the interface between the graphene and p-type silicon.In this study, a graphene-doped porous silicon (G-doped/p-Si) substrate for low ppm H In general, gas sensors are of great interest because of their ability for real time analysis of gaseous chemicals over a wide range of applications. In the vast area of gas sensing, hydrogen sensors are based on metal oxide films that are configured in a chemiresistor mode [Hydrogen gas is widely used as a clean fuel in various industrial fields, and is expected to be the fuel to replace fossil fuels . Since Htor mode . Typicaltor mode ,6. When tor mode . p-Si istor mode . Varioustor mode  because tor mode .Gas sensing is one of the most promising applications for graphene, because the delocalized pi(p) bonds of graphene allow for charge carriers to have zero rest mass and high mobility ,12, and 2 gas detection based on graphene-doped porous silicon (G-doped/p-Si) substrate are investigated utilizing graphene as a catalyst material. Graphene doping was performed by a facile, inexpensive synthetic procedure, using solution dropping onto a p-Si substrate with a one-step method of decorating graphene on the high surface area porous media created via an electrochemical etching process. Electrochemical etching is typically a simple, inexpensive procedure for synthesizing p-Si layers [In this study, the sensor properties for low ppm Hi layers . The loaWater-dispersible graphene with oxygen functionalization at basal edges was developed by MExplorer Co., Ltd. , the thickness and lateral dimension of the as-received graphene was <5 nm and 2–3 μm, respectively See .2 was applied for 1h in a mixture solution of 30 wt % H2O2 and 10 wt % HF. After the chemical etching, the porous silicon substrate was dipped in 10 wt % HF to remove the formed oxide layer from the silicon surface for the electrochemical etching processes, and the samples were then rinsed copiously in DI water and dried at room temperature.The synthesis of the high porosity silicon substrate was performed by a technique combining both metal-assisted chemical etching (MacE) and electrochemical etching , utilizi2) was deposited (2) that was located in the middle part on the surface of substrate a × 100%, where Ra and Rg are the electrical resistance of the sensor under dry air and the concentration of H2 gas, respectively.Doping graphene on the synthesized p-Si substrate was performed by a facile solution drop method using a micro pipet. The 100 μL solution volume as a function of graphene concentration ranging from 0 to 10 mg/mL in an aqueous solution on the p-Si substrate size (5 × 10 mmeposited  and drieubstrate . The for chamber  and reco2). The estimated graphene volume is (a) 0 mg/mL, (b) 0.1 mg/mL (graphene volume: 0.01 mg), (c) 1 mg/mL (graphene volume: 0.1 mg), and (d) 10 mg/mL (graphene volume: 1 mg). The size of the graphene identified from the SEM view (the dotted domains) ranged from several to tens of microns. The external average pore size of the synthesized p-Si is 4.5 μm macroporosity, whose width narrows to meso- and micro-porosity toward a vertical hole depth of approximately 90 μm  due to operation at room temperature in comparison with micro hotplate sensors, which are known for low consumption sensors (250 mW at 400 °C) [The G-doped/p-Si does not have a sensing property because p-Si hinders the diffusion of H 400 °C) .2 + 2h+ → 2H+, resulting in a reduction in the carrier density [ density . The con2 gas detection by an inexpensive synthesis route was proposed, where the p-Si substrate with a large specific area was employed as a sensor matrix and graphene as a catalyst material. The H2 sensing properties as a function of graphene concentration on the p-Si substrate were analyzed at room temperature. The G-doped/p-Si has an enhanced sensing property in comparison to that of pristine p-Si. The catalytic effect of graphene on the surface of p-Si was elucidated by an enhancement in the carrier transfer to the adsorbed hydrogen gas molecules due to the doped graphene on the p-type silicon. The hierarchical hybrid structure of G-doping on porous silicone shows potential as an extended application of optical and medical sensors, and electronics for storage devices.In this study, a graphene decorated porous silicon (G-doped/p-Si) substrate for low ppm H


When encoding a real-life, continuous stimulus, the same neural circuits support processing and integration of prior as well as new incoming information. This ongoing interplay is modulated by attention, and is evident in regions such as the prefrontal cortex section of the task positive network (TPN), and in the posterior cingulate cortex (PCC), a hub of the default mode network (DMN). Yet the exact nature of such modulation is still unclear. To investigate this issue, we utilized an fMRI task that employed movies as the encoded stimuli and manipulated attentional load via an easy or hard secondary task that was performed simultaneously with encoding. Results showed increased intersubject correlation (inter-SC) levels when encoding movies in a condition of high, as compared to low attentional load. This was evident in bilateral ventrolateral and dorsomedial prefrontal cortices and the dorsal PCC (dPCC). These regions became more attuned to the combination of the movie and the secondary task as the attentional demand of the latter increased. Activation analyses revealed that at higher load the prefrontal TPN regions were more activated, whereas the dPCC was more deactivated. Attentional load also influenced connectivity within and between the networks. At high load the dPCC was anti-correlated to the prefrontal regions, which were more functionally coherent amongst themselves. Finally and critically, greater inter-SC in the dPCC at high load during encoding predicted lower memory strength when that information was retrieved. This association between inter-SC levels and memory strength suggest that as attentional demands increased, the dPCC was more attuned to the secondary task at the expense of the encoded stimulus, thus weakening memory for the encoded stimulus. Together, our findings show that attentional load modulated the function of core TPN and DMN regions. Furthermore, the observed relationship between memory strength and the modulation of the dPCC points to this region as a key area involved in the manipulation of attentional load on memory function. Traditional theories postulate that the information required for current task performance is actively maintained in a designated working memory component . This viThe framework of However, using active traces of past information to process current information does not happen in isolation, but rather is modulated by attention . NumerouAttention is assumed to influence regions that are attributed to distinct neural networks, namely the prefrontal sections of the task positive network TPN; , and theTwenty-four healthy adults completed the task . All participants were right-handed, except for two ambidextrous individuals with a tendency to use the right hand more, as indicated by the Hebrew translation of the Edinburgh Handedness Inventory . All parFigure 1). A short movie was played during the encoding phase. The distraction phase immediately followed and consisted of mathematical questions meant to diminish rehearsal and recency effects .The paradigm was divided into three runs of 582 s each. A run started with a fixation cross for 24 s and a movie that was not followed by distraction or recognition phases. The purpose of this single movie was to habituate the neural response to the visual stimuli and to accustom the participants to the task. This movie was discarded from the analyses. A run consisted of 8 cycles of encoding, distraction and recognition phases. Participants responded to the linguistic, mathematical, and mnemonic tasks by pressing either the right or left button on a hand-held response box. Prior to the experiment, each participant received an explanation about the paradigm and practiced until he or she indicated that the task was clear. The practice session presented different stimuli from those used in the actual experiment.During the encoding phase, a short, 21 s movie was presented. The movies in the task were unfamiliar, non-emotional, silent clips edited from feature length films. The original feature length films were not silent but sound was omitted during editing.Context was achieved during encoding by presenting pairs of related movie clips in successive cycles. Each pair of movies portrayed the same characters in a similar setting performing a similar action. The resemblance between the clips in a pair of related movies was strong. The resemblance between unrelated pairs of movies was weak. Overall, there were 12 pairs of short related movies. The order of the movie clips within a pair  was counter balanced across participants. Notably, each movie clip had its own recognition questions, independent of its paired movie clip. We remind the reader that for the purposes of this article, the context manipulation was irrelevant to the analyses and did not affect the attentional load manipulation (see below).n = 19; mean age 33.4; 14 females) who performed this task alone. Accuracy and reaction time (RT) of 213 words and 197 pseudo-words were evaluated. The 54 stimuli with the shortest RT and highest accuracy level were selected for the easy category. The 54 stimuli with the longest RT and lowest accuracy level were selected for the hard category. In each category, half of the stimuli were words and half were pseudo-words. The easy and hard categories differed in terms of accuracy level and RT, as indicated by two t-tests . Three words were presented during each movie: two words and one pseudo-word or vice versa. The words appeared one at a time, like subtitles below the movie, for 2 s each. Each word appeared 0.5 s before the frame that served as a target in the recognition questions. Consequently, for each movie the words were presented at the same exact time for all participants, regardless of the task type  and regardless of the order of the movie (first or second in a pair). The word disappeared when the participant pressed the response button or after the 2 s time frame elapsed. Overall, there were 12 movie clips  coupled with an easy linguistic task and 12 movie clips with a hard linguistic task . The coupling of movie pairs to easy or hard linguistic task was counter balanced across participants.Attentional load was changed during encoding by the simultaneous performance of an easy or a hard linguistic task. The task required participants to indicate whether a string of letters was a word or pseudo-word. Participants were requested to attentively watch the movie and perform the linguistic task as accurately as possible. Stimuli for the linguistic task were selected based on an independent behavioral pilot study conducted with a different group of participants  at the top of the screen, with the correct answer and a foil underneath it in two separate rectangles. If the response was given within the question’s time frame, the equation disappeared and the rectangles became blank.During the recognition phase, three recognition questions were presented for 5 s each. The recognition questions were designed analogously to the mathematical questions. Namely, the question “What did the last movie show?” was presented at the top of the screen with two picture choices presented below. The correct answer was a picture frame taken from the short movie that immediately preceded the question. The foil was a frame taken from the original feature length film presenting the same characters and setting, but from a segment that did not appear in either of the two short movies viewed. If the response was given within the question’s time frame, the question disappeared and the rectangles became blank.∗-weighted imaging: repetition time (TR) = 3000 ms; echo time (TE) = 35 ms; flip angle (FA) = 90°; field of view (FOV) = 200 mm; matrix size = 96 × 96; 39 axial slices of 3 mm thickness, 0 gap. A high-resolution anatomical T1-weighted fast spoiled gradient echo imaging was acquired: FOV = 256 mm; matrix = 256 × 256; TR = 9.2 ms; TE = 3.5 ms; axial slices of 1 mm thickness, no gap. This anatomical scan was used for surface reconstruction. To minimize head movements, participants’ heads were stabilized with foam padding. Stimuli were controlled using PsychoPy software  using an eight channel head coil. Blood-oxygen-dependent-level (BOLD) functional MRI was acquired with T2software , 2008 anThere were three behavioral tasks: linguistic (performed during the encoding phase), mathematical (performed during the distraction phase) and mnemonic (performed during the recognition phase). In each, accuracy level and mean RT were measured. The accuracy level was calculated as the proportion of correct responses. Questions with RT shorter than 0.1 s were discarded from analysis. Incorrect questions were discarded from the RT analysis.t-test, comparing low vs. high attentional loads. This was done once for accuracy level and once for RT.The analyses commenced with validation of the distracting and attentional load manipulations. Analysis of the distracting mathematical task aimed to verify that the participants performed the task above chance level. To this end, accuracy level across all the experimental conditions was evaluated. The linguistic task was analyzed in order to confirm that it had indeed two difficulty levels. This was done using a paired-sample n = 18; mean age 29.16; 9 females). These participants completed the same movies task , while sitting in front of a laptop in a quiet room. Accuracy levels for each question were calculated across all participants. Three of the 72 questions had accuracy levels lower than 0.5 overall  and in the easiest condition , and were discarded from further analyses.Before analyzing the recognition questions, the efficiency of each question was examined using the data of an independent behavioral study with a different group of participants . Therefore, the first and the second movies were collapsed together in all subsequent analyses.Following the aforementioned procedure, the recognition task was analyzed using a 2-way repeated measure analysis of variance (ANOVA) with context (first/second movie) and attentional load (low/high) as within-subject variables. There was neither effect for context nor any interaction between context and attentional load, for either accuracy level or RT . Head movements did not exceed 3 mm in any of the runs. Spatial smoothing was applied using a Gaussian spatial filter . The functional images were superimposed on 2D anatomical images and incorporated into the 3D datasets through trilinear interpolation. The complete functional dataset was transformed into Talairach space .k and all the other participants who saw the same movie as a low load first movie). The averaged BOLD signal was constructed across 4 to 5 participants, depending on the movie. The correlation coefficients were transformed using a Fisher transformation. Note that in the inter-SC analytic method, the average time course of the participants serves as a model for the time course of the “left out” participant, and therefore, that model should include only participants which are identical to the participant with regard to the specific experimental condition. However, once the correlation maps were constructed, they can be averaged across conditions to create the contrast of interest, namely low vs. high attentional load. Since participants watched 12 movies within each manipulation condition , every participant had 12 correlation maps per experimental condition, which were averaged together, yielding one correlation map per condition for each participant.Inter-SC analysis measures the progressive changes in neural signal that arise when encountering dynamic naturalistic stimuli , 2010. Ht-test was used cross all the 22 participants to determine whether the correlation was different from zero. This was done once for the low and once for the high attentional load conditions. Controlling for multiple comparisons across the whole brain was done using a false discovery rate (FDR) method  method , with a t-test. For this t-test, controlling for multiple comparisons was done using the aforementioned FDR method, with a threshold of p < 0.05 and minimum cluster size of 50 × 33 voxels. The results of the analysis are presented in a correlation map for each condition . Like in the inter-SC analysis, this step prevented comparison of two non-significant activations. Therefore, the next stage of analysis was conducted only in regions whose average activation was significantly larger than zero in at least one condition. Third, the difference between the low and high attentional load was determined using a paired-sample t-test. Regions were further sorted into TPN (%SC > baseline and high load > low load) or DMN (%SC < baseline and low load > high load). Only regions that fulfilled either of these criteria were regarded as regions of interest (ROI) and included in subsequent analyses.We predicted that high attentional load would be associated with a higher level of inter-SC. However, a higher inter-SC level indicates only that the neural signal of a region became more correlated across participants, but not if the region was more activated or deactivated. In order to further characterize the level of the signal and sort the regions into TPN and DMN, the inter-SC analysis was complemented by a standard activation analysis. Activity level was examined in regions which demonstrated significant changes in inter-SC patterns using the following steps in each region. First, the timecourse of the encoding phase, averaged across all the voxels in a region, was extracted, incorporating a hemodynamic delay of 2 TRs (6 s). The timecourse was used to calculate the region’s percent signal change (%SC) from baseline in the low and high attentional load conditions. Second, across all participants, a one-sample t-test was used to determine whether the correlation in either the low or the high attentional load condition was significantly different than zero. The fourth step was conducted only in cases where at least one condition was significantly different than zero and consisted of a paired-sample t-test to assess the difference between the low and high attentional load conditions. Controlling for multiple comparisons was done using the Bonferroni method, taking into account the total number of paired comparisons.We examined the hypothesis that higher attentional load would be associated with greater FC within each functional network  and larger negative correlation between the networks. The analysis was conducted as follows for every possible pair of ROIs. First, for a given participant, the time course of each movie was extracted from a pair of ROIs, and a Pearson correlation was calculated between the two time courses. This resulted with 24 correlation coefficients for each participant, one for each movie, and all of them were Fisher-transformed. Second, all the correlations corresponding to the low attentional load condition were averaged together, and the same was done for the high attentional load condition. Third, across all participants, a one-sample The final stage of analysis aimed to clarify the role of the identified inter-SC patterns in memory when dealing with increased attentional loading. To this end, a step-wise regression analysis was used. One model was built to predict accuracy level in the recognition task and another to predict RT. The predicting variables were the inter-SC patterns of all ROIs. Accuracy level, RT and inter-SC were modeled as the difference between high and low attentional load conditions . In order to verify that this brain-behavior relationship was specific to inter-SC, parallel models were calculated, once when the predicting variables were the ROIs’ activation indices and once when they were the FC indices.t(23) = 8.09, p < 0.001] and a shorter RT  for the easy compared to the hard linguistic questions.First, we validated the efficiency of the mathematical and linguistic tasks. In the former, the accuracy level was above chance [mean (SD): 88.7% (8.15)]. This indicates that participants were engaged in the task (distracted from the movies), and confirms that performance in the recognition task was not based on active working memory. Examination of the linguistic task verified a significant difference between the low and high attentional load conditions, thus validating the attentional load manipulation. The analysis revealed a significantly higher accuracy level , there was a significant effect in RT. Thus, participants recognized stimuli from movies encoded during the low load condition faster than the high load condition .Finally, the influence of the attentional load manipulation (which occurred during encoding) on the ability to recognize a stimulus from the encoded movies (which was assessed during recognition) was examined. Although the low and high attentional load conditions did not significantly differ in terms of accuracy level . This means that a larger increase in inter-SC level of the dPCC during the high attentional load predicted lower memory strength in that condition. In order to clarify whether this relationship was specific to inter-SC patterns, a parallel model was examined with the activation indices as the predicting variables and one with the FC indices as predicting variables. These models were non-significant, suggesting a specific relationship of memory to inter-SC. Finally, the model that predicted RT index of the mnemonic task was non-significant for either inter-SC, activation or FC indices.To clarify the functional significance of the inter-SC patterns, we examined their ability to predict memory strength. This was done using a step-wise multiple regression analysis with the inter-SC index of the TPN and DMN ROIs as the predicting variables. The model that predicted the accuracy level index of the mnemonic task contained only the inter-SC index of the dPCC, which explained 37.5% of the variance [The current study portrayed how attention modulates the interplay between processing and memory of dynamic stimuli by utilizing movies and analyzing inter-SC. We found that high attentional load was associated with both lower memory strength and higher synchronization of neural signal across participants in lateral and medial prefrontal regions and dPCC. The experimental design also allowed a unique opportunity to further characterize activity level in each region as well as their functional coupling, using activation and FC methods, respectively. We found that in the prefrontal TPN regions, both inter-SC and activation increased at higher attentional load, and FC within the network was higher. In contrast, in the dPCC, higher attentional load was accompanied by greater inter-SC, greater deactivation and greater anti-correlation with the TPN regions. Critically, only the inter-SC level of the dPCC predicted memory strength.In terms of behavior, responses to the easy linguistic task were faster and more accurate than the hard linguistic task, confirming two levels of attentional load during encoding. Importantly, encoding during low attentional load was associated with shorter response latency for the mnemonic questions that followed. Since this measure is considered an indication for memory strength , this reFigure 2), which are known to accumulate information across long timescales . Given the known involvement of the PCC in mnemonic operations  as anticipated from TPN regions. These activation results are in agreement with previous studies that utilized still stimuli and measured activity, which showed that dividing attention during encoding recruited lateral and medial prefrontal regions . This activation pattern is expected since the PCC is part of the DMN, which is known to be deactivated as load increases . This is in line with finding that the anti-correlation between the DMN and TPN increases at higher loading (Functional connectivity analysis revealed that as attentional load increased, the prefrontal section of the TPN network became coherent within itself and negatively correlated with the dPCC ( loading . This an loading . TherefoTogether, our results show that attentional load modulated the function of regions known to accumulate information over long timescales when facing continuous real-life stimuli . The modNO: Substantial contributions to the conception and design of the work, acquisition, analysis, and interpretation of data and writing the work. IS-L: substantial contributions to the conception and design of the work, analysis, and interpretation of data and revising the work critically. NO and IS-L share first authorship equally. YL and RT: substantial contributions to the analysis and interpretation of the data and revising the work critically. TH, NG, and ELA: substantial contributions to the conception of the work and revising the work critically. All authors: final approval of the version to be published and agreement to be accountable for all aspects of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In addition, X-ray photoelectron spectroscopy data revealed that the C1s peak of the air-assisted graphene film was higher than the one of a pristine graphene film, indicating that a cleaner graphene surface was obtained.The polymer residues still present on a chemical vapour-deposited graphene surface after its wet transfer by the poly(methyl methacrylate) method to the arbitrary substrates, tend to cause problems such as electrical degradation and unwanted intentional doping. In this study, by using an effective cleaning method for the graphene surface by air-assisted plasma, the graphene surface was cleaned significantly without damaging the graphene network, which resulted in the reduction (approx. 71.11%) of polymer residues on its surface. The analysis reveals that this approach reduced the D-band  formation while maintaining the π-bonding of the graphene, which affects conductivity. By characterizations of the optical microscope, Raman spectroscopy and atomic force microscopy, we obtained a significantly cleaner graphene surface (roughness of 4.1 nm) compared to pristine graphene (roughness of 1.2 nm) on a SiO For further investigation, the Raman spectra were carried out for the as-received Cu foil and the graphene grown on Cu (figure 2b). In the case of the as-received Cu foil, Raman data showed no signal, while the D, G and 2D peaks presented in the case of graphene/Cu as typical fingerprints of graphene identification.The OM images of CVD graphene grown on a Cu foil with graphene domains showed white points  which indicated impurities on it during CVD graphene growth. Besides a hexagonal shape, the graphene domains also were pentagonal with non-symmetric morphology, as shown in the AFM image in figure 22 wafer even packaged in the box from the company, however, still shows a lot of tiny impurities on its surface. Consequently, further cleaning is necessary by wet cleaning  water) and dry cleaning (air-assisted plasma) to obtain an ultra-clean SiO2 surface. Before transferring the graphene/Cu on to the SiO2 substrate, the SiO2 substrates were perfectly cleaned by air-assisted plasma for 5 min for removing impurities on its surface, which still existed after rinsing by acetone, ethanol and DI water for 20 min with each chemical, as shown in the OM images in figure 3a,b. After obtaining the ultra-clean SiO2 surface, graphene/Cu was transferred onto it by a kind of popular polymer named PMMA (figure 3c). Actually, the PMMA chemical still remained as polymer residues on graphene/SiO2 even after acetone rinsing (20 min) owing to weak van der Waal interactions and chemical bonding at the interface between the PMMA and graphene films. Therefore, it requires a cleaning process to remove as much polymer residues as possible. Consequently, a very clean graphene surface on SiO2 was obtained after 30 s of air-assisted plasma treatment (figure 3d).The SiO2 surface before and after air-assisted plasma cleaning at the same position . The XPS spectra were also studied in two cases of pristine graphene/Si and air-plasma cleaned-graphene/Si (figure 5b). The C1s peak of air-plasma-cleaned graphene/Si showed a very high peak compared with pristine graphene/Si, indicating the attainment of a cleaner graphene surface.A demonstration by Raman spectra was carried out for the cleaning effect. The Raman data  of polymer residues on its surface. Analysis reveals that this approach reduced the D-band  formation, which affects conductivity. By characterizations of OM and AFM images, and Raman spectroscopy, we obtained a significantly cleaner graphene surface (roughness of 1.2 nm) compared to pristine graphene (roughness of 4.1 nm) on the SiO


Ultra-thin graphene coating has been reported to provide considerable resistance against corrosion during short-term exposures, however, there is great variability in the corrosion resistance due to graphene coating in different studies. It may be possible to overcome the problem of hampered corrosion protection ability of graphene that is caused due to defective single layer graphene by applying multilayer graphene. Systematic electrochemical characterization showed that the multilayer graphene coating developed in the study provided significant corrosion resistance in a chloride solution and the corrosion resistance was sustained for long durations (~400 h), which is attributed to the multilayer graphene. Since corrosion is an age-old problem, several mitigation strategies have been investigated and adopted with different degrees of success. The use of graphene as an ultra-thin coating has emerged as a novel and exciting approach to the corrosion protection ,14,15,16diamond) , impermediamond) , and chediamond) . The spediamond) ,3. In fadiamond) . In a stdiamond) .In the light of the recent research findings on graphene for corrosion resistance ,5,28,30,2 for 2 min using Solarus Advanced Plasma System  and cut to the size of 1 × 1 cm and placed in the hot zone of quartz tube of a furnace for chemical vapour deposition (CVD) of graphene. The CVD set-up, shown in 2 mixture.The copper foils of thickness 0.05 mm were procured from Storm Copper Components, Decatur, TN, USA. The copper samples were plasma cleaned under Ar/O2 was introduced at a pressure of 6 to 9 Torr and flow rate of 400 sccm, to create a reducing atmosphere to avoid oxidation of copper samples at high temperatures. The furnace is heated to 1000 °C while maintaining a reducing atmosphere of Ar/H2 flow. The copper coupons were annealed for 50 min at 1000 °C under the reducing atmosphere, before Ar/H2 flow was replaced with n-hexane vapour that was passed at 2 sccm and 500 mTorr for 7–10 min. Then, the copper coupons were cooled under the flow of Ar/H2 at 400 sccm and at a pressure of 8 to 9 Torr.The quartz tube was first pumped down to a pressure of 70 mTorr, and then a gas mixture of 85% Ar and 15% HThe coating developed as discussed earlier was characterized using Raman spectroscopy, optical microscopy, and scanning electron microscopy. −1 with a laser spot size of 1 μm. Raman spectroscopy provides confirmatory information on whether the deposited layer is graphene. The deposited layer was characterized using Renishaw micro-Raman Spectrometer  HeNe (632.8 nm) laser operating at 100% power. Extended scans (10 s) were performed for a spectral range between the Raman shifts of 150 to 3200 cmThe optical micrographs of bare copper and graphene coated copper were obtained using Olympus BX 51  optical microscope. The scanning electron microscopy (SEM) images of graphene coated copper samples were obtained using JEOL 7001 with an accelerating voltage of 10 kV at 12 nA probe current and 10 mm working distance. 2 acted as the working electrode, platinum mesh as counter electrode and saturated calomel electrode as the reference electrode). Ecorr vs. time plots were generated after immersion in 0.1 M NaCl for 1 h, to ascertain the stabilized open circuit potential (OCP). Since the perturbation potential for the EIS test was 10 mV, any fluctuation in the open circuit potential of less than 10 mV for a period of 1000 s was considered as a stable potential for the purpose of EIS experiments. A small sinusoidal potential of 1 to 10 mV AC was applied which rendered the system pseudo-linear. EIS runs were carried out by applying a sinusoidal signal at Ecorr with perturbation potential of 10 mV. Impedance response was measured over frequencies between 1 MHz and 10 mHz, recording 10 points per decade of frequency using Powersuit software (version 2.58). These frequencies are chosen such that they reach asymptotic limits in which the imaginary impedance tends to zero at the lowest and highest frequencies of the employed frequency range. Electrochemical impedance spectroscopy (EIS) was performed on the graphene coated and bare copper samples in 0.1 M NaCl solution using a Princeton Applied Research (PAR) potentiostat  and an electrochemical cell with three electrodes , i.e., a measure of the number of graphene layers, is ~0.36 for a single layer, and >1 for multi-layer graphene [G/I2D ratio in a typical Raman spectrum in −1 signifies the extent of defects in graphene [The two Raman signature peaks are G peak at 1580 cmi et al. ). The ragraphene ,33. The graphene . The higgraphene .Graphene coated copper has features of thermally etched grain boundaries b which iScanning electron microscopy (SEM) of graphene coated Cu in the present study shows dark areas in The diameter of semicircular loop of a Nyquist plot and the magnitude of impedance at the lowest frequency in a Bode impedance plot are broad measures of corrosion resistance. The Bode and Nyquist plots of graphene coated and uncoated samples show the impedance of graphene coated copper to be 4–5 times higher than the uncoated copper . The impThough the corrosion resistance due to the multilayer graphene is not quite impressive (only 4–5 times), it is still of great interest to assess whether this corrosion resistance was durable, and hence, EIS study was undertaken after immersion in 0.1 M NaCl for different durations . The EIS data representing the improvement in corrosion resistance are reproducible as established in c) values, that are the measure of the corrosion resistance are 4.718 × 104 Ωcm2, 4.52 × 104 Ωcm2, 5.249 × 104 Ωcm2, 4.875 × 104 Ωcm2, and 6.260 × 104 Ωcm2 after the immersions for 1, 16, 32, 64, and 386 h respectively, whereas that of the uncoated/bare copper after 1 h immersion is 1.245 × 104 Ωcm2 . Therefore, the data infer the multilayer graphene coating to improve, corrosion resistance of copper by ~5 times, and more importantly sustains this improvement for 386 h, which is largely consistent with the data in f of multilayer graphene which suggests a lower number of conductive pathways in multilayer graphene film whereas the relatively low value of Cdl of multilayer graphene film indicates lower exposure of metal–electrolyte interface (see f and Cdl). The exponent of CPE is very close to 1, indicating a fully capacitative nature of coating. The low Chi squared value represents fair accuracy of the results. The data in It is noted that graphene in the study of Singh Raman et al.  had a smContrary to the observations of improvement in the corrosion resistance due to graphene coating in this study and those reported in the literature ,2,3, a vThe above discussion would suggest that graphene domain boundaries may be the major defect type that may impair corrosion/oxidation resistance. This assumption is consistent with the results and schematics for deterioration in corrosion resistance of graphene coated copper reported in the recent studies ,30. TherThe role of plugging/masking the graphene domain boundary locations in effectively improving corrosion resistance is strongly corroborated by the findings of Hseih et al. . In thisIn the light of the above discussion, the superior surface coverage of the multilayer graphene led to a durable corrosion resistance for the entire test duration (~400 h) in the present study. In the context of the beneficial effect of the multilayer graphene in corrosion resistance, the work of Prasai et al.  that comIt may be relevant to note that the multilayer CVD graphene on Ni may be more defect-free because the mechanisms of deposition of graphene on Ni and Cu are different. Graphene deposition on the metals having considerable solubilities of carbon  progresses by an intricate mechanism. In this mechanism, the carbon produced due to hydrocarbon decomposition at the metal surface diffuses into the bulk metal, and is subsequently rejected out during cooling since carbon solubility decreases considerably with temperature. On the other hand, metals having little/very limited solubility of carbon at reaction temperature—e.g., Cu—are more likely to develop a single layer graphene instead of multilayer graphene because of the catalytic effect is likely to cease after the first layer is deposited . HoweverIn conclusion, graphene coating was synthesized on copper using a chemical vapour deposition technique. Electrochemical results indicated that the graphene coating acted as a corrosion barrier for copper and provided a durable corrosion protection in 0.1 M NaCl. Contradictory findings as reported in the literature regarding corrosion protection ability of graphene are attributed to the quality of graphene. Corrosion protection ability of graphene can be retained for long exposure times by applying multilayer graphene instead of single layer graphene. Multilayer graphene is suggested to be effective in blocking the pathways of corrosive species.


Humidity sensors have been widely used in areas such as agriculture, environmental conservation, medicine, instrumentation and climatology. Hydrophobicity is one of the important factors in capacitive humidity sensors: recent research has shown that the inclusion of graphene (G) in polyvinylidene fluoride (PVDF) improves its hydrophobicity. In this context, a methodology to fabricate electrospun membranes of PVDF blended with G was developed in order to improve the PVDF properties allowing the use of PVDF/G membrane as a capacitive humidity sensor. Micrographs of membranes were obtained by scanning electron microscopy to analyze the morphology of the fabricated samples. Subsequently, the capacitive response of the membrane, which showed an almost linear and directly proportional response to humidity, was tested. Results showed that the response time of PVDF/G membrane was faster than that of a commercial DHT11 sensor. In summary, PVDF/G membranes exhibit interesting properties as humidity sensors. Currently, humidity sensors are widely used in diverse areas such as agriculture, environmental conservation and medicine. Specifically, in the medical field, these sensors are used in respiratory equipment, incubators, sterilizers and biological products, where humidity control is very important ,2,3,4. BOne of the most common ways to express this parameter is through relative humidity (RH). RH is defined as the ratio, expressed as a percentage, of the amount of humidity present in a gas, at a certain temperature, to the maximum humidity level that the gas can hold at that same temperature ,4.The humidity sensors research has generated a wide variety of devices composed of polymers, ceramics, blended materials, graphene (G)-based materials, and others ,12,13,14and 47 s ,16,17.These kinds of humidity sensors are mostly made of hydrophobic materials, avoiding the storage of water molecules in the sensing material and letting water vapor flow freely. The formation of clusters of absorbed water molecules leads to hysteresis; this is a serious disadvantage in capacitive humidity sensors ,4,18.Polymeric materials have been widely used as sensing materials due to their advantages of low cost, easy preparation, excellent stability and good mechanical properties . Nanofib2 carbon atoms arranged in a hexagonal lattice and it is the elementary structure of graphite. In recent years, there has been an increasing interest in G because of its excellent electrical conductivity, strong mechanical strength, thermal conductivity and ease of functionalization. Therefore, G-based materials are highly attractive [The incorporation of nanoparticles into the polymeric solution is another method for changing membrane properties . Graphentractive ,27. G hatractive . Recent tractive ,25,29.PVDF has received especial attention among a wide variety of polymers due to its thermal and chemical stability, mechanical and ferroelectric properties which can be easily conformed to complex surfaces ,33,34,35It has been shown that the RH of the surrounding atmosphere can modify the dielectric properties of electrospun PVDF . The aimThis paper focus on a mixture of PVDF with G in order to obtain a humidity sensor with different characteristics with regard to sensitivity, response time and hysteresis. Recently, studies have shown that mechanical and thermal properties of PVDF are improved by the addition of G ,29,37,38N,N-dimethylformamide (DMF) at room temperature. This kind of polymer solution was used to fabricate nanofibers by electrospinning. Rough and porous structures are attractive elements for humidity sensors because they allow water vapor to pass through and are hydrophobic [PVDF is dissolved in polar solvents such as rophobic ,20.PVDF powder and DMF were purchased from Sigma Aldrich . Ultra-high Concentration Dispersion of Graphene Nanoplatelets was purchased from Graphene Supermarket . Two solutions were prepared: PVDF/DMF and PVDF/G/DMF. The PVDF/DMF solution with a 20 wt % composition was prepared by dissolving PVDF powder in DMF at 60 °C, with the purpose of ensuring a homogeneous mixture. PVDF/G/DMF solution with 20 wt % composition was prepared as follows: PVDF powder and G were thoroughly weighted in order to obtain PVDF/G membranes with 0.5 wt % composition, which was chosen based on the investigation about hydrophobic PVDF/G membranes ,25,29. TPVDF/G/DMF and PVDF/DMF solutions were used to fabricate membranes by electrospinning as illustrated in In order to perform a morphological analysis, micrographs of membranes were acquired by a JSM-6360LV scanning electron microscope . Circular samples of 1 cm diameter were obtained from the PVDF and PVDF/G electrospun membranes for the purpose of testing the behavior of these materials. The average thickness of PVDF and PVDF/G membranes was around 124.4 µm and 127 µm, respectively. Finally, copper tape electrodes were placed on both faces of the PVDF (PVDF-Cu) and PVDF/G (PVDF/G-Cu) samples. The choice of the electrodes for the samples depends on the membrane morphology because the porosity can allow that the conductive material is deposited inside the membrane. Taking into consideration the morphology of PVDF/G membrane, other sample was tested (PVDF/G-Ag), in this case a layer of silver paint was used as the electrode. The samples with electrodes are illustrated in This paper will focus on a PVDF/G membrane as a humidity sensor; hence the humidity dependence of the dielectric constant was tested. If samples are used as the dielectric material in a parallel plate capacitor, the dielectric constant of electrospun samples shows a directly proportional behavior with respect to capacitance of the condenser ,33. TherConsidering that samples were fabricated with electrospinning, the sensing principle of the proposed sensor is based on the dielectric constant change of membranes (measured with a LCR bridge) due to water vapor inside fibrous structure of these samples as shown in 3 and capacitive impedance was measured with a LCR bridge  at 1 kHz for a varying humidity between 35 and 90% in concentration RH at room temperature (25 °C ± 1 °C).In order to measure the response of the PVDF-Cu, PVDF/G-Cu and PVDF/G-Ag membranes to relative humidity changes, the samples were placed inside an insulation chamber of volume 0.018472 mFour commercial DHT11 humidity sensors  with the specifications listed in Micrographs of membranes, acquired by the JEOL JSM-6360LV scanning electron microscope are shown in Two humidity characterization tests (Test 1 and Test 2 for each sample) were done to analyze the humidity responses of PVDF-Cu, PVDF/G-Cu and PVDF/G-Ag samples; a comparative test between each sample and the commercial DHT11 humidity sensor was also performed to examine the response time to humidity drop. Taking into account that the humidity changes for different points inside chamber, the humidity measurements were acquired by the DHT11 nearest to the membrane. Firstly, PVDF membrane with copper electrodes was tested twice (Test 1 and Test 2) to analyze the behavior of PVDF membrane without G to relative humidity changes from 40% to 90%. The capacitive response to relative humidity changes can be seen in Subsequently, comparative tests between PVDF membrane and DHT11 sensor were done. In this case, the response of the sensors was measured when humidity level drops rapidly inside transparent chamber . The graph of The next test was performed with PVDF/G-Cu membrane. The capacitive response to relative humidity changes from 35% to 85% can be seen in A comparative test between PVDF/G-Cu membrane and DHT11 sensor when humidity level drops rapidly inside the chamber  was also performed.The graph of One last test was performed using PVDF/G-Ag sample. Capacitive response to relative humidity changes can be seen in Finally, comparative response times, between PVDF/G-Ag membrane and DHT11 sensor, were determined when humidity level drops rapidly inside chamber . The graph of 2 = 0.9609) with a NRMSE of 5.25%, however, the Test 2 showed a poor linear fit  and low noise in the acquired signals, however, it was noticeable that there was a difference in the magnitude of the capacitance between the samples. The sensitivity of PVDF/G-Cu and PVDF/G-Ag were better than PVDF sample as can be seen in PVDF/G samples have a faster recovery time than the commercial sensors; this phenomenon can indicate that there no significant formation of clusters results from the adsorption of water molecules by the sensing material, which could cause hysteresis and low response time. A reliable hysteresis measurement should be done in future research to confirm that there was no water molecules absorbed by PVDF/G samples.Despite the similar behavior of the PVDF/G membranes, analyzing In this paper, it was possible to apply a methodology to fabricate PVDF/G membranes by electrospinning and use PVDF-Cu, PVDF/G-Cu and PVDF/G-Ag samples as capacitive humidity sensors. The sensing principle of the proposed sensor was based on the dielectric constant change of membranes due to water vapor inside fibrous structure of these samples. The morphology of membranes and the addition of G were used to modify the hydrophobicity of PVDF, which is an important factor for capacitive humidity sensors. The PVDF membrane can function as capacitive humidity sensor, however, the design of the PVDF capacitive humidity sensor must be improved in order to obtain good response time, better sensitivity, repeatability and low noise. PVDF is commonly used in different biomedical applications due to its biocompatibility, piezoelectric properties and the facility to be cast or molded by different techniques.On the other hand, based on results, the sensitivity and linearity of PVDF/G samples were interesting and the response time was better than that of the commercial DHT11 sensor and the PVDF sample. A good response of PVDF/G as a humidity sensor was thus obtained by using the electrospinning technique and adding graphene, nevertheless, although scientific research has proven that the incorporation of G into the PVDF enhances its hydrophobicity, other tests such as hydrophobicity tests, hysteresis measurements and repeatability should be done in future work to know the mechanisms involved in the response of PVDF/G samples and then design sensors with improved features. PVDF/G membranes not only work as humidity sensors, but can also be used for temperature, light and pressure sensing due to their ferroelectric properties. Other possible applications could be filtration, growth of living tissues and the manufacture of prosthesis.