C. elegans motor circuit dynamics. The Six/SO-family homeobox transcription factor UNC-39 governs lineage-specific neurogenesis to give rise to a neuron RID. RID bears the anatomic hallmarks of a specialized endocrine neuron: it harbors near-exclusive dense core vesicles that cluster periodically along the axon, and expresses multiple neuropeptides, including the FMRF-amide-related FLP-14. RID activity increases during forward movement. Ablating RID reduces the sustainability of forward movement, a phenotype partially recapitulated by removing FLP-14. Optogenetic depolarization of RID prolongs forward movement, an effect reduced in the absence of FLP-14. Together, these results establish the role of a neuroendocrine cell RID in sustaining a specific behavioral state in C. elegans.Neuromodulators shape neural circuit dynamics. Combining electron microscopy, genetics, transcriptome profiling, calcium imaging, and optogenetics, we discovered a peptidergic neuron that modulates DOI:http://dx.doi.org/10.7554/eLife.19887.001 While the hard-wiring of circuits through chemical and electrical synapses provides the anatomic basis for behaviors, their outputs are subjected to modulation and reconfiguration by neuromodulators such as biogenic amines and neuropeptides  to contain both clear and dense core vesicles. C. elegans modulators of known functions act through neurons that clearly participate in classical chemical synaptic transmission. These notions led to a speculation that all C. elegans neurons secrete neuromodulators  reconstruction of several C. elegans larvae , fixed by tannic acid and high-pressure freezing that allows for better preservation of intracellular structures in physiological states , a fascicle consisting of multiple motor neuron processes that innervate the dorsal body wall muscles. We reconstructed the entire length of a first stage larva (L1), and fragments of multiple young adults, by serial transmission EM (sTEM) .10.7554/en passant neuromuscular junctions (NMJs)  . The RIDs (NMJs) . These vs (NMJs) .The morphological features of these varicosities are distinct from those of their neighboring NMJs . In bothC. elegans neuropeptide marker   were expressed by muscles and multiple neurons during embryonic development and into the adulthood  for RID (and a few other neurons)  fate marker shortly after the second round of mitosis   exhibite alleles . Second,poptosis ; illustrpoptosis . By simu mitosis . In unc-poptosis . Blockinpoptosis , or globpoptosis  apoptoti mutants , furtherunc-39 mutants exhibits striking lineage-specificity. The CAN, ALA, RMED and RIA neurons are lineage-related to RID by sharing progenitors that give rise to ABalappaa  in adults. RID differentiation thus may be particularly sensitive to the perturbation of UNC-39 activity.The loss of RID in Balappaa . They arC. elegans larvae. Due to the absence of a strong RID-specific promoter that is required for cell sorting, we devised a subtractive transcriptome strategy, taking advantage of the complete and specific loss of RID in unc-39 larvae to detect RID-enriched transcripts.The morphology of RID suggests that it functions as a specialized peptidergic neuron. To identify neuropeptides that are expressed by RID, we performed mRNA sequencing on differentiated RID neurons isolated from Pceh-10-GFP-positive (GFP+) cells from wild-type and unc-39(hp701) second stage larvae (unc-39 GFP+ cells against their respective unsorted whole cell populations (All Cells) . The non-overlapping transcripts thus represent candidates that are either differentially expressed in RID, or differentially regulated by UNC-39 , hen-1  . By contrast, flp-14, ins-17, and nlp-34 showed more enrichment in the wild-type dataset, with flp-14 and ins-17 exhibiting the most significant difference and abundant expression, making them candidate genes highly-expressed in RID. Reassuringly, using transcriptional reporters , PDE, and PLN  , and pdfTo address the function of the RID neuron, we monitored its activity pattern in behaving animals by calcium imaging. A genetic calcium sensor GCaMP6, fused with RFP (mCherry), was expressed in RID to monitor relative neuronal activity change. Parameters for spontaneous locomotion  and neuronal activity (GCaMP/RFP ratio) were simultaneously acquired as described ; MateriaPflp-14-positive neurons exhibited obvious activity change that correlated with forward movement. During the transition from reversals to forward movement, we consistently observed an activity rise in RID, typically with a significant lag (~10 s) in reaching its peak when compared to the velocity rise , when animals transited between forward movement and reversals, respectively  over reversals and pauses (<5% combined). RID-ablated animals drastically reduced the propensity for forward movement , and increased the propensity for reversals (~20%) and pauses (~20%) (nd turns . Our spond turns . We quans (~20%) . RID-abls (~20%)  and velos (~20%)  of each s (~20%) . They als (~20%) , withouts (~20%) . Hence, unc-39 mutants, harboring an anatomic loss of RID among other defects, exhibited motor characteristics strikingly reminiscent to the RID-ablated animals – replacing long foraging with more frequent, shorter and slower forward runs, more pauses, and more reversals  in flp-14 mutants led to an increase in the duration of the forward runs and a decrease of the re-initiation frequency for both forward and reversal movements . Both manipulations led to reduced duration and velocity in forward runs, and increased bout initiation frequency , which inhibits a premotor interneuron (AVB) to halt forward movement, and simultaneously activates the GABAergic motor neurons (VDs) to induce deep bending  provide electrical and chemical synaptic inputs, respectively, to potentiate a cholinergic motor neuron group (the B motor neurons), which innervate and organize muscle contractions in a sequential order to propel the animal forward , and/or, FLP-14 released from additional neurons exert an opposing effect on the forward runs.In our behavioral assays, RID-ablated animals bear close resemblance to the characteristics and severity of the motor defects exhibited by our ins-17 mutants did not exhibit obvious motor defects. We also tested a possibility that defective GABA uptake may also contribute to the influence of RID on forward runs. RID expresses snf-11, a plasma membrane GABA transporter    or inhibes (ALA) . MoreoveC. elegans neurons likely co-express classical neurotransmitters and neuropeptides, and the latter may interact with multiple receptors, an incomplete phenotypic recapitulation upon the perturbation of neurons, neuropeptides that they produce, and receptors that they act upon, may not be uncommon.We propose that while RID secretes FLP-14 as a key effector in modulating motor circuit output, RID may also function through additional co-effectors, and FLP-14 may play opposing roles via different neurons. Because most C. elegans, while multiple cells are affected by unc-39 mutations, the RID lineage is particularly sensitive to the perturbation of UNC-39 activity. A key feature of the RID lineage defect was the random timing of the second round of mitosis in precursor cells. Such a phenotype implies a crucial role for UNC-39 in the onset of and exit from the cell cycle that is to be followed by RID terminal differentiation. Mechanisms that couple cell cycle and terminal differentiation have remained elusive  plates seeded with OP50, and maintained at 22°C. hp701 was isolated by EMS mutagenesis of a strain carrying an integrated Pceh-10-GFP array (hpIs202), identified through a combination of SNP mapping and whole genome sequencing  were backcrossed at least 4x against N2 prior to usage. See Supplemental Methods for a complete list of mutants, transgenic lines, and constructs.quencing , followehp701) were packed into 3 mm diameter aluminum carriers and frozen using a Leica EM HP100 high-pressure freezer. Subsequent fixation and freeze substitution was performed as described (L1 (N2) or young adult animals  were cultured and recorded at 25°C. Embryos were mounted at 1–4 cell stages, and recorded every 35 s with 25 z-levels. After ~350 z-scans (200 min) with DIC optics, the GFP fluorescence optics was automatically activated and exposed for every 20th z-scan till 320 min of development, and then every 30th z-scan till the three-fold stage. Recording was performed at 630x on a 4D microscope controlled by TimeToLive . RID and neighboring lineage cells were traced using SIMI°Biocell  until the late 1.5-fold stage (340 and 360 min) as described (Strains (escribed .hpIs202 (Pceh-10-GFP) and unc-39(hp701); hpIs202 larval stage 1 (L1) animals were grown on 150 mm 8P plates seeded with NA22 for approximately ~12 hr until they reached larval stage 2 (L2). Cells were extracted from L2s. See Supplemental Information for detailed methods.Synchronized hpIs202 (Pceh-10-GFP) L1 animals were immobilized and RID ablated using the MicroPoint laser as described  animals were placed on a drop of M9 buffer on top of a 2.5% agar pad, covered with a cover slip. Four ~1 mm × 1 mm 0.001 inch-thick polycarbonate pieces  between the agar pad and coverslip served as spacers to allow movement. Each recording lasted for 3 min. Images were captured using a 20x objective on a Zeiss Axioskop 2 Plus equipped with an ASI MS-40000 motorized stage, a dual-view beam splitter (Photometrics) and a CCD camera (Hamamatsu Orca-R2). The 4x-binned images were obtained at 10 frames per second. See Supplemental Information for detailed methods.The third stage (L3) flp-14(gk1055), unc-39(hp701) and npr-4(tm1782)npr-11(ok594) animals carrying the same transgene using a Diode red laser while animal locomotion was tracked under dark-field imaging by infrared light. The stimulation and tracking were performed using the Colbert  system  system . The loc) system . See Suphttp://www.blender.org).Images were stitched and aligned using TrakEM2 . In the Pceh-10-IDA-1::cherry) and neuropeptide fluorescent markers (Pceh-10-INS-22::GFP), expression patterns of UNC-39  and RID phenotypes in unc-39 mutants using RID fluorescent markers (Pceh-10::GFP) and RID cell fate markers , images were captured using a 63x objective on a Zeiss Axioplan two connected to a Hamamatsu ORCA-ER digital camera and processed using Improvision Open Lab software. Images were processed using minimal deconvolution levels to remove background fluorescence. Confocal images of transgenic strains carrying either Pflp-14-GFP, Pins17-GFP, unc-3fosmid::SL2::GFP, and unc-39fosmid::GFP were acquired on a Nikon Eclipse 90i confocal microscope. Confocal image processing was conducted using Adobe Photoshop. The primer pair to generate a 4.3 kb Pflp-14 fragment are tactgtcgaccgacaaacacccaaatatcc  and aactggatcctccttcggattgtgtggag .For imaging dense core vesicle , which softens the cuticle, was added for no longer than 4 min . Post-incubation with SDS-DTT, the sample was neutralized and washed 5x times with 1x Egg Buffer. To break apart animals, Pronase solution (15 mg/mL) was added, and the sample was pipetted ~40x for 30 min. After several washes, cells were resuspended in 1x Egg buffer. Propidium iodide (1 μg/mL) was added prior to cell sorting to identify damaged cells.hpIs202 control and the unc-39 (hp701); hpIs202 strains, respectively.GFP+ cells from L2 worms were sorted onto Trizol-LS (Invitrogen) using a BD FACSAria with a 70 micron nozzle (BD Biosciences) operated at the University of Toronto Flow Cytometry Facility. Profiles of GFP+ strains were compared to an N2 standard to identify and exclude autofluorescent cells. For each independent replicate, 20,000–50,000 events, most likely representing cells, were FACS-isolated from each strain. Three and four biological replicates with corresponding All Cells reference samples were collected for the For the all cells reference samples, flash-frozen pellets were pulverized using a mortar and pestle and dissolved in Trizol-LS (Invitrogen). RNA was extracted from these pulverized worm pellets (All and from sorted cells collected directly in Trizol-LS (GFP+ cells sample). DNA contamination was removed using the Zymo DNA-free RNA Kit  according to manufacturer’s instructions.RNA sample concentration and quality were determined using an Agilent Bioanalyzer at The Centre for Applied Genomics . RNA Integrity Numbers (RIN) scores of seven and above were used for subsequent RNA-sequencing analysis. RNA concentration was also verified using the Quibit RNA HS Kit (Thermo Fisher Scientific).5 ng of RNA was used as starting material for cDNA library preparation. cDNA was synthesized from RNA using the SMARTer Ultra Low Input RNA Kit for Sequencing (Clontech) and cDNA libraries prepared using the Low Input DNA Library Prep Kit (Clontech) according to manufacturer’s instructions. RNA-sequencing was performed on an Illumina Hiseq 2000 according to standard protocols, generating 100 base paired-end reads.C. elegans genome (WS235) using RNA STAR under default settings ;hpIs202 datasets. For hpIs202 and unc-39(hp701);hpIs202 datasets, transcripts were considered significantly enriched in GFP+ cells over sample matched All Cells by applying the following criteria or filters: (1) Differentially expressed transcripts with False Discovery Rate (FDR) adjusted p values<0.05, and, (2) Transcripts where the ratio of log2 transformed Mean Counts from GFP+ samples to log2 transformed Mean Counts from All Cells samples was greater than 1 (Mean GFP+ Counts / Mean All Cells Counts >1). Final datasets resulted from analyses of >3 experimental replica from All cells and GFP+ samples for wild-type and unc-39 mutant strains.Using DESeq, we identified differentially expressed transcripts between GFP+ cells and All Cells reference samples in the ttx-3, hen-1, glc-3, pdfr-1)   or neuronal subtypes not included in GFP+ Cells - the glutamatergic (eat-4) and GABAergic (unc-25) neurons  , and in d kal-1) ; Second, neurons  by repor neurons  and the  neurons .C. elegans typically spend most of the time moving forward, with brief interruptions of backward movement. As previously described with some modifications .Tracking and analysis were performed using Micromanager and ImageJ software plugins developed in-house (courtesy of Dr. Taizo Kawano). Image sequences were sampled at 100-msec exposure (10 frames per second). The directionality of movement (forward vs. backward) was determined by first identifying the anterior-posterior axis or the ‘head' and 'tail’ points, which were manually defined at the first two frames and verified throughout the recording. To calculate directionality of movement, the displacement of the midline point in relation to the head and tail for each worm was determined based on its position in the field-of-view and the stage coordinates. Image sequences where animals touched the edge of the recording field or crossed over on themselves were not processed.Analyses of the output data were carried out using an R-based code developed in-house (courtesy of Dr. Michelle Po). The following parameters were quantified by the program: (1) Initiation ; (2) Duration ; (3) Velocity . Frequency of initiations, durations, and velocities were calculated for forward and reversal locomotion separately.peak – Velocitytrough / # of Frames). The rate of Deceleration during transitions from forward to backward locomotion was calculated similarly (Velocitytrough – Velocitypeak / # of Frames). Rise and decay in calcium transients during transitions from backward to forward locomotion and vice versa, respectively, were calculated using the linear slope. Cross-correlation analyses were performed between rate of acceleration and calcium rise, as well as deceleration and calcium decay.Regions of interest (ROIs) containing the RID neuron was defined using a MATLAB script developed in-house. GCaMP and RFP fluorescence intensities from RID were then measured. To analyze overall RID activity, the ratio of GCaMP to RFP was calculated in order to control for possible motion artifacts detected. The velocity of each time point was measured using an Image J plug-in developed in-house . The ratPceh-10, in a subset of neurons , and the expression of ZIF-1::SL2::GFP by Pgpa-14, Pttx-3, and Parr-1, which overlap with Pceh-10 for ALA, AIY, and CAN, respectively. ZIF-1 targets chrimson::GFP::ZF for degradation. GFP and RFP were used to confirm the specificity of expression pattern of these constructions in targeted neurons.To restrict expression of the Chrimson protein to RID, we drove the expression of a Chrimson::GFP:ZF construct by hpIs626 from an Ex array made from co-injected Pceh-10-chrimson::GFP::ZF, Pgpa-14-ZIF::SL2::RFP, Pttx-3-ZIF::SL2::RFP and the lin-15 co-injection marker. This transgene exhibits restricted chrimson::GFP expression in RID and CAN. Parr-1-ZIF-1::SL2-RPF was then injected in hpIs626;; ~10% of the double transgenic animals that carries the Ex array  exhibited RID-specific chrimson::GFP expression. These array were then crossed into unc-39, flp-14, and npr-4 npr-11 backgrounds.We first generated an integrated transgene For optogenetics experiments, we first picked late larvae to young adult transgenic animals by selecting for those with RID-specific GFP expression onto the OP-50 retinal plates under the dissecting fluorescent microscope. After 24 hr incubation, each animal was individually recorded using the COLBERT stimulation system (see below). After recording, each animal was mounted on slides to be examined for GFP signals; data collected from animals that exhibited residual GFP signals in CAN were discarded.flp-14 and npr-4 npr-11 animals, and 4 s before and 4 s after the switch for unc-39 animals. To quantify the forward run length, all runs during the laser on phase and laser off phase were included.To make OP50-retinal plates, we seeded each 60 mm NGM plate with a mixture of 250 μL OP50 in LB with 1 μL of 100 mM retinal in ethanol. Animals were then individually washed in the NGM buffer before allowed to navigate on the surface of a 100 mm NGM plate without food. We used the Colbert system to stimulate Chrimson with a Diode red laser  and carry out dark-field imaging using infrared light and a dark field condenser (Nikon) using a 10X Plano Apo objective (NA = 0.45). A motorized stage and custom real-time computer vision software kept the animal in the center of the field of view. The locomotion of each animal was recorded for 30 min or until it reached the edge of the plate. The laser was set at a 3 min on/3 min off cycle during the recording. Data was collected at 30 f/s and analyzed using a customized particle-tracking and shape analysis algorithms. Each trajectory was segmented into periods of forward movement (runs) separated by sharp orientations (turns). Turns were automatically flagged when the heading change of the center of the mass trajectory was >60° over 1 s. To quantify the change of phase velocities before and after the laser was switched on , we quanFor locomotion , statistical significance was determined using Mann-Whitney or Kruskal-Wallis tests for comparing two and more than two variables, respectively, and subjected to post-hoc analysis. For calcium imaging , Pearson tests were used to calculate correlation coefficients and statistical significance. For optogenetic stimulation and locomotion analyses, Wilcoxon matched-pairs signed rank test was used to compare the velocity difference during periods of Lights On and Off for the same strain. The Kruskal-Wallis Test with post-hoc test (Dunn’s Multiple Comparison) was used to assess the significance of difference between groups. p<0.05 were considered to be statically significant. All statistics were performed using Prism software (GraphPad). 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.C. elegans Forward Motor State" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Eve Marder as the Senior Editor and Reviewing Editor. The following individual involved in review of your submission has agreed to reveal his identity: Akinao Nose (Reviewer #2).Thank you for submitting your article "Neuroendocrine Modulation Sustains the The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.In this manuscript the authors go to significant length to argue that a newly identified neuroendocrine neuron is important in behavior. All of the reviewers found the manuscript interesting, but have some specific issues that need addressing.Essential revisions:Most notably, please move I am leaving portions of the initial reviews for your information because I don't think they are in disagreement and might be informative to you.Reviewer #1:This manuscript takes the novel approach of identifying possible neuroendocrine cells by sTEM and determining their functions. The authors identify the RID neuron as a neuroendocrine cell because of its large number of dense core vesicles. In a screen for mutants that specifically disrupt RID differentiation, they identified the Six/SO homeodomain transcription factor UNC-39. Loss of unc-39 results in loss of several RID markers and its distinctive axonal morphology. Although the unc-39 results are solid, they were not well integrated into the overall theme of the paper (neuropeptide modulation of forward movement).During forward movement, calcium levels in RID increased, whereas forward to reverse transitions caused a decrease in calcium levels, suggesting that RID is involved in forward movement. The calcium imaging was supported by behavioral work that showed that unc-39 mutants and RID-ablated animals had shorter forward runs and more turns than wild type, although unc-39 mutants had a more severe phenotype than the RID-ablated animals , suggestThe authors performed a subtractive transcriptome analysis to identify three candidate neuropeptide genes that are enriched in RID: flp-14, ins-17, and nlp-34. flp-14 mutants show a decreased average forward run duration, an increased number forward initiations, and an increased number of reversal initiations compared to wild type. However, as the authors note, the defects in flp-14 are not as severe as in unc-39 mutants and RID-ablated animals, suggesting that other peptides may act in concert with FLP-14 to modulate forward movement. In addition, the behavioral assays were highly variable . For instance, the wild-type values for average forward duration were significantly different in The authors indicate that ins-17 mutants acted similarly as wild type and the double mutants acted similar to the flp-14 mutants alone. However, flp-14 ins-17 double mutants showed more severe phenotypes than flp-14 mutants alone , contrarAnalysis of the possible FLP-14 receptors was not very informative. Double knockout of the possible receptors, NPR-4 and NPR-11, showed phenotypes that were not significantly different than wild type, but showed the same general trend as flp-14 mutants. Despite this negative result, The paper lacks a model of how RID inputs into the motor circuit to sustain forward motion. RID synapses onto hypodermal cells, DD neurons, and muscle, but could also act humorally. Do the authors hypothesize that the effect of FLP-14 peptides released from RID is on DD neurons, muscle, or other targets? In Ascaris, application of FLP-14 on muscle causes relaxation followed by rhythmic contractions or sustained contractions (depending on which paper one reads). The authors should have a model for the neuromodulation.Reviewer #2:1) Specificity of the activation experiments with Chrimson. Please explain more in detail how the authors restricted the expression of Chrimson to RID neurons using the ubiquitin ligase system. Since the specificity of Chrimson expression is critical for the interpretation of the results, inclusion of a picture showing the specific expression as a supplementary figure would be desirable. I also wonder how one should interpret the results of the optogenetic activation in unc-39 mutants. Is the reversion of the phenotype due to lack of Chrimson expression in RID or defective function of the neuron?￼￼2+ or other signaling molecules  in DDs.2) It is not clear how RID regulates forward locomotion. I would expect more through discussion on putative target cells of RID in relation to known locomotory circuits. An obvious candidate would be DD MNs whose terminals reside in the immediate neighbor of RID varicosities. What happens to forward locomotion when DD MNs are inhibited or activated? If feasible, the authors may also examine if the activity manipulation of RID or application of FLP-14 affects the level of CaReviewer #3:1) There seems to be a caveat implied by If so, the optogenetic assay does not strongly support the conclusions of the manuscript although it doesn't contradict them either. It is perfectly reasonable for a particular assay of a complex phenomenon to fall short of providing conclusive evidence. It is even useful to present such results by way of transparency and completeness. Thus, the data is useful and should be presented in the main text without detracting from the interest of the manuscript or the validity of the conclusions.If I am mistaken, perhaps the caveat should be briefly mentioned and refuted for the sake of clarity. Otherwise, the caveat should be discussed and the conclusion of the relevant Results section should be revised to state that the optogenetic assays were consistent with the overall theme of the work but in and of themselves inconclusive.C. elegans researchers and of conceptual importance to any researcher that felt the need to tuck away a valid result in the name of a 'cleaner', more linear story.2)  Essential revisions:Most notably, please move I am leaving portions of the initial reviews for your information because I don't think they are in disagreement and might be informative to you.Response: Thank you. We gladly moved the figure and related texts to the Results .Response: Thank you. The unc-39 work, including the embryonic lineage analyses that denote its deficiency of RID neurogenesis, its EM analyses, its motor behavioral deficit, and RNA profiling of unc-39 neurons, could indeed be a stand-alone study. The focus of such a ‘paper’ would have been UNC-39’s requirement for RID’s development. However, the focus of our paper, representing the main interest of my group, is the RID neuron and its role in the motor circuit.unc-39 mutants, there is failure of RID neurogenesis. The absence of a neurite with neuroendocrine properties in the dorsal nerve cord of unc-39 mutants confirms that the one that we observed in the wild-type dorsal nerve cord was RID. 2) RID promotes forward run. The motor behaviors of unc-39 mutant reiterate those that we observed for RID-ablated animals; the absence of a behavioral response to optogenetic stimulation of the RID neuron serves as a control and confirms the cellular origin of the effect was RID. 3) FLP-14 and other neuropeptides are expressed by RID. Since we do not have a RID-specific promoter, we took advantage of unc-39’s absence of RID to perform subtractive RNA profiling to search for RID-enriched transcripts. Indeed UNC-39 transcriptional targets are likely present in this list, but this was not our focus. The sole reason to provide those data was to be useful to those who are interested in pursuing UNC-39’s targets, and we are confident of the reproducibility and quality of these datasets.In our study, UNC-39 is an essential tool for three key aspects of RID: 1) RID’s identity as a peptidergic neuron. In unc-39-relatedresult sections focused on evidence for RID’s absence in unc-39 mutants; the rest of the unc-39 work was distributed in our Results sections where they are integrated as controls. In the Discussion, we now further highlight the relevance of the unc-39 data to the overall theme of the paper: the absence of RID in unc-39 mutants has provided our work a genetic tool to investigate the property, function, and molecular mechanisms of RID. We further refer to published work on Drosophila Six; together they suggest that UNC-39/Six/SO transcription factors may have a shared role in endocrine development.This is why our During forward movement, calcium levels in RID increased, whereas forward to reverse transitions caused a decrease in calcium levels, suggesting that RID is involved in forward movement. The calcium imaging was supported by behavioral work that showed that unc-39 mutants and RID-ablated animals had shorter forward runs and more turns than wild type, although unc-39 mutants had a more severe phenotype than the RID-ablated animals , suggestThe authors performed a subtractive transcriptome analysis to identify three candidate neuropeptide genes that are enriched in RID: flp-14, ins-17, and nlp-34. flp-14 mutants show a decreased average forward run duration, an increased number forward initiations, and an increased number of reversal initiations compared to wild type. However, as the authors note, the defects in flp-14 are not as severe as in unc-39 mutants and RID-ablated animals, suggesting that other peptides may act in concert with FLP-14 to modulate forward movement.Response: These are our conclusions. We agree that behavioral differences summarized by bar graphs may not be sufficiently informative for readers without direct experience performing C. elegans motor behavior assays. To place these quantitative analyses in a more visual context, we now provide supplemental figures where we show the raw data that were used for quantification. They allow a qualitative, but very intuitive view of the data variability of the same genotype, and the degree of difference across groups. It should be evident from these raw data that the motor phenotypes of unc-39 and RID-ablated animals were quite similar, and flp-14’s defect was of the same trend but less severe compared to the unc-39 or RID-ablated animals. These points have been articulated in Results and Discussion.In addition, the behavioral assays were highly variable . For instance, the wild-type values for average forward duration were significantly different in Response: We appreciate the reviewer’s keen observations on the behavioral defects of flp-14 mutants.a) We now provide raw behavioral recording data  and 2 soflp-14 rescue in former flp-14 and unc-39 mutants were derived from. In Si(FLP-14), a transgenic line with a single copy integrant of the FLP-14 rescuing construct. In Si(FLP-14);hpIs202 – hpIs202 being a GFP marker for visualizing RID. These controls were not to be compared with each other. The genotype information is provided in figure legends. Second, even for strains of the identical genetic background, the absolute value of the motor behavioral index would exhibit variability when they are assayed on different days. In our behavioral assays, we only compare values of animals assayed on the same day and on the same plate, as we explained in the Materials and methods and Appendix 1. The variability we observed is standard for these motor behavioral assays that were performed by our group, so we are confident with conclusions drawn from these data.b) Regarding the concern about the variability, and Si(FLP-14) did not rescue flp-14 back to Si(FLP-14). There was indeed one motor index between flp-14;Si(FLP-14) and Si(FLP-14) that showed really small, but statistically significant difference. An exogenous or even an overexpression of neuropeptides could easily generate effects on motor behaviors, and it was difficult to assert cellular origin of secreted molecules by standard transgenic rescue experiments. We generated a single copy insert of flp-14 construct Si(FLP-14) under a portion of its own genomic regulatory elements to avoid these caveats as much as possible, but it is quite possible that flp-14;Si(FLP-14) did not behave identical to Si(FLP-14). With the raw data, the rescuing effect should be quite obvious. The key point of this experiment was to ablate the RID neurons in the Si(FLP-4) background to determine if its rescuing effect was reversed.c) Regarding the concern that We have reworded the statement to reflect these points.The authors indicate that ins-17 mutants acted similarly as wild typ￼e and the double mutants acted similar to the flp-14 mutants alone. However, flp-14 ins-17 double mutants showed more severe phenotypes than flp-14 mutants alone , contrarResponse: In the original flp-14 ins-17 double mutants were compared against wild-type and ins-17. During revision, we realized that we had forgotten to replace an older version of the figure, resulting in the absence of the flp-14 panel in the previous submission altogether.ins-17 did not significantly enhance flp-14’s motor defects. Double mutants did exhibit a trend of slightly decreased reversal duration, forward, and reversal velocity when compared to flp-14 alone, but the change is so small, excluding ins-17 being a main regulator of motor behaviors. We have reworded the text to state that ins-17 is not a main contributor. We thank the reviewer for bringing it to our attention.We have replaced it with the proper panel. We moved these results to the main figure and provided raw behavior data as a supplemental figure. As shown in Analysis of the possible FLP-14 receptors was not very informative. Double knockout of the possible receptors, NPR-4 and NPR-11, showed phenotypes that were not significantly different than wild type, but showed the same general trend as flp-14 mutants. Despite this negative result, Response: Agree. We have incorporated this data into the Results section . The authors should have a model for the neuromodulation.Response: Indeed. We do not have any positive data or conclusions on RID’s downstream targets. As we previously discussed, the Ascaris electrophysiology work alluded to a possibility of muscles and forward driving motor neurons being the potential targets. This notion would contradict the candidate neurons based on expression pattern of the predicted FLP-14 receptors (interneurons). Because of our negative data on these receptor mutants, we favor clues from the Ascaris work.To make a speculative model that does not mislead other researchers, we placed question marks on these candidate targets in the model sketch. In the Discussion, we further emphasize the necessity to identify FLP-14’s physiological receptors in order to determine RID target cells. Although DD and hypodermis were the postsynaptic partner of RID in the adult wiring diagram, this was based on their anatomic apposition to the few active zones observed along the RID axon. This approach may have its limitations because ~90% of RID axonal boutons lack active zones; for the ~10% with active zones, we never observed docked clear or dense core vesicles.Reviewer #2:1) Specificity of the activation experiments with Chrimson. Please explain more in detail how the authors restricted the expression of Chrimson to RID neurons using the ubiquitin ligase system. Since the specificity of Chrimson expression is critical for the interpretation of the results, inclusion of a picture showing the specific expression as a supplementary figure would be desirable.Response: We added the description in the Results, and expanded the methods (see Appendix 1) on obtaining these strains. A supplementary figure containing the requested image has been added  in DDs.2) It is not clear how RID regulates forward locomotion. I would expect more through discussion on putative target cells of RID in relation to known locomotory circuits. An obvious candidate would be DD MNs whose terminals reside in the immediate neighbor of RID varicosities. What happens to forward locomotion when DD MNs are inhibited or activated? If feasible, the authors may also examine if the activity manipulation of RID or application of FLP-14 affects the level of CaResponse: We thank the reviewer for this suggestion. We do not understand how RID regulates forward locomotion in the context of other circuit components. We provide more background and expand Discussion in the revised manuscript. DD could be a candidate due to its proximity, but other motor neuron neurites are also fairly close. A main issue is that the post-synaptic partner of peptidergic neurons could not be reliably predicted by anatomic apposition. We also suspect that the predicted FLP-14 receptors are incorrect: we did not find any supportive experimental evidence for the predicted FLP-14 receptor mutants (Reviewer #3:1) There seems to be a caveat implied by If so, the optogenetic assay does not strongly support the conclusions of the manuscript although it doesn't contradict them either. It is perfectly reasonable for a particular assay of a complex phenomenon to fall short of providing conclusive evidence. It is even useful to present such results by way of transparency and completeness. Thus, the data is useful and should be presented in the main text without detracting from the interest of the manuscript or the validity of the conclusions.If I am mistaken, perhaps the caveat should be briefly mentioned and refuted for the sake of clarity. Otherwise, the caveat should be discussed and the conclusion of the relevant Results section should be revised to state that the optogenetic assays were consistent with the overall theme of the work but in and of themselves inconclusive.Response: We thank the reviewer for bringing this observation to our attention. We have re-examined all data to see if there is a general trend of higher activity in flp-14 mutants before stimulation when compared to other animals, and whether the post-stimulation velocity represented the highest velocity in these recordings. This was not the case, and we take this opportunity to elaborate on these experiments and analyses done.flp-14, we refer to flp-14.In To reiterate the methodology used for the optogenetic stimulation experiment , we recorded one animal at a time, with a 3 minute light on/3 minute light off protocol for 30 minutes, or until we lost track of the animal before 30 minutes. Stimulation on and off events were pooled (N) from recordings of the same genotype, and we compared the motor behaviors with and without stimulation within the same recording using two indexes: a) The mean duration of all forward run events during the ON and OFF periods . b) The unc-39 mutants: these animals almost exclusively exhibited short runs; if we chose a longer time, N from the unc-39 dataset would be too small.The reviewers may have noticed that the N number for quantified velocity change before and after stimulation  is much unc-39 exhibited a non-ambiguous drastic reduction in velocity when compared to both wild-type and flp-14 animals , we picked as many candidate RID-specific old larvae to young adult animals as possible from three genotypes under low magnification fluorescent microscopes, cultured them on retinal plates, and recorded all animals on the following day. This amounted to 12-14hr recording, for three genotypes of ~6~10 animals/genotype). After recording, we mounted each animal to examine with a high magnification microscope, and discarded data from animals that showed residual chrimson signals in neurons other than RID. Three independent repeats were performed. The scarcity of samples prevented us from using animals at the identical developmental stage, and we had to pool recording data obtained on different dates. Together with the small number of Ns for velocity comparison, we felt that we should only use these datasets to compare the motor behavior within the same recording, hence the normalized difference at the ON and OFF period. In fact, we knew that the absolute velocity value in these panels did not represent the spontaneous velocity as observed under culturing conditions –  animals  at all.flp-14 and npr-4 npr-11 mutants (a more representative mean speed), while keeping unc-39’s cut off to 4 seconds. These data led to the same conclusion  Response: Agreed. We moved


First‐principles calculations show that the surface oxygen vacancies can facilitate the electrochemical charge transfer by creating midgap electronic states. The specific capacitance of the UCNG electrode reaches 978.1 F g−1 (135.8 mA h g−1) at the current densities of 1 A g−1 and retains a high capacitance retention of 916.5 F g−1 (127.3 mA h g−1) even at current density up to 10 A g−1, showing remarkable rate capability (more than 93.7% capacitance retention). Additionally, 99.3% of the initial capacitance is maintained after consecutive 20 000 cycles, demonstrating enhanced cycling stability. Moreover, this proposed laser‐assisted growth strategy is demonstrated to be universal for other metal oxide/graphene composites with tuned electrical conductivity and electrochemical activity.The metal oxides/graphene composites are one of the most promising supercapacitors (SCs) electrode materials. However, rational synthesis of such electrode materials with controllable conductivity and electrochemical activity is the topical challenge for high‐performance SCs. Here, the Co Many approaches are available for fabricating the Co3O4/graphene composites for the charge‐storage performance. Dong et al. and Liao et al. reported that the Co3O4/graphene hybrids were in situ synthesized by a simple hydrothermal procedure, which delivers a high‐specific capacitance and exhibits an excellent cycling stability.3O4 nanosheets on graphene foam through the electrodeposition and annealing process, in which the 3D hierarchical structure and the synergetic effect of Co3O4 and graphene enhance the rate of ion and electron transport in electrodes.3O4/graphene nanostructures by the microwave irradiation method, which is used as the electrode for lithium ion batteries and shows an excellent rate capability and highly reversible large capacity.3O4 nanoparticles on graphene,3O4) and a short transport length of electrolyte ions and electrons in the composites.3O4 nanoparticles, high conductive graphene, and tight coupling between those two remains challenging, and there are still somewhat limited . What's more, controlling the intrinsic conductivity of Co3O4 nanoparticles and their electrochemical activity is another great challenge in promoting the SCs performance.The CoVO) has been explored to improve the intrinsic conductivity and electrochemical activity of transition metal oxides, and thus to increase their SCs performance,VO) can change the geometric and electronic structures as well as the chemical properties of transition metal oxides. However, owing to the limitations of synthetic methods ,In recent years, intentional creation of oxygen vacancies (3O4 nanoparticles/graphene (UCNG) composites with high rate capability (93.7%) for the first time. Without introducing any reducing agents and organic additives, ultrafine Co3O4 nanoparticles (≈10 nm) anchoring on graphene were obtained. Under laser irradiation, the graphene oxide (GO) was reduced to high conductive graphene through photothermal effects, accompanied the fragmentation (size reduction) of the porous Co3O4 nanorods (P‐Co3O4). More importantly, abundant oxygen vacancies were created on the ultrafine Co3O4 nanoparticles surface and the Co3O4 nanoparticles tightly anchored on the graphene, which significantly modifies the electric conductivity and the electrochemical activity of Co3O4 nanoparticles and hence enables the UCNG electrode with fast and reversible Faradaic reactions. The ultrafine Co3O4 nanoparticles/graphene composites are the promising candidates as the supercapacitor electrode. The capacitance of the composites reached 978.1 F g−1 (135.8 mA h g−1) at the current densities of 1 A g−1. It shows high rate capability, with more than 93.7% capacitance retention at even 10 A g−1. The superior performance also benefits from the surface midgap electronic states, which are enhanced by oxygen vacancies (VO) and facilitate the electrochemical charge transfer, as shown by our first‐principles calculations. The discovery in our paper not only establish a new route to prepare the Co3O4/graphene composites and shed light on their formation mechanism, but also opens a new avenue for realizing high‐performance SCs electrodes, especially for rate capability.Herein, we proposed a novel one‐step laser irradiation route through simultaneous laser‐induced reduction and fragmentation to prepare oxygen‐vacancy abundant and ultrafine Co22.1Figure3O4 nanorods (P‐Co3O4) and GO as the mixed precursor, and ultrapure water as solvent. During krypton difluoride (KrF) excimer laser  irradiation process, the fragmentation (size reduction) of the P‐Co3O4 nanorods occurs in the liquid, which may be driven by the laser–nanorods interaction‐induced plasma3O4 can also absorb this laser. Meanwhile, GO was transformed into reduced graphene oxide  for clarity) after the laser irradiation, mainly because its large surface area with strong optical absorption can result in a rapid temperature rise in GO  and subsequently lead to a significant temperature rise of water, which has been demonstrated to be a facile reduction method for the synthesis of graphene by recent reports.3O4 nanoparticles. As a result, the coupling of the fragmentation of the P‐Co3O4 with the reduction of GO to LG through laser irradiation could provide a novel method for the synthesis of the UCNG composites. For the yield and production rate of the laser processing, it has been demonstrated that the laser fluence dominates nanomaterials throughput for many materials.The proposed laser‐assisted growth strategy for the UCNG composites is illustrated in 2.23O4 nanoparticles/graphene composites prepared under various laser parameters were surveyed to prove the above strategy and optimize the preparation condition associated with excellent electrochemical properties for SCs electrodes applications . The optimized product (UCNG composites) with ultrafine Co3O4 nanoparticles and high conductive graphene was selected and discussed below. The morphology and microstructure of the UCNG were examined by the scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The SEM images  and Fourier transform infrared (FTIR) spectroscopy .A number of Co3O4 and LP‐Co3O4, was characterized by X‐ray diffraction (XRD) in a scan range of 10°–80° , their diffraction lines are in line with the UCNG composites, indicating that the laser irradiation and the graphene introduction have no effect on the Co3O4 crystal structure.The crystal structure of the UCNG composites, P‐Co3O4 and the UCNG composites  can be assigned to the Co 2p3/2 and Co 2p1/2 spin‐orbital peaks of Co3O4, respectively.3O4, the Co 2p peaks of the UCNG composites show two more obvious satellite peaks centered at about 786.3 and 802.5 eV, which are attributed to the Co2+ oxidation state, indicating that a part of the Co3+ ions was transformed to Co2+ and formed oxygen vacancies. A similar phenomenon of the transformation from higher valence metal ions to lower valence metal ions under laser irradiation is also found in our previous work,3O4 in the UCNG composites is reduced by laser irradiation and plentiful oxygen vacancies on the surface are produced, while the main body of the nanoparticles is still the Co3O4 crystal. The peaks in the XPS spectrum of LP‐Co3O4 are similar to those of the UCNG composites , indicating the oxygen vacancies are induced through laser irradiation and are not affected by the introduction of GO.To further investigate the oxidation status of cobalt, XPS measurements were carried out to examine both the P‐Co3O4 among the UCNG composites . The prominent resonance line of paramagnetic phase in the EPR absorption spectrum for the UCNG composites demonstrates that there are more Co2+ ions in the Co3O4 crystal, which further manifest the presence of oxygen vacancies.3O4 nanoparticles, the Co3O4 phase has not been altered as indicated by XRD  was furthermore employed to investigate the oxygen vacancies in the Co3O4, GO, and LG were analyzed by Raman spectroscopy. As observed in Figure E2g vibrational mode within aromatic carbon rings and the degree of graphitization for the area of excitation, while the D peak is ascribed to the small and isolated domains of aromaticity and the disorder of graphene. The weak 2D band (≈2700 cm−1) confirms the few‐layer graphene structures obtained by laser irradiation.3O4, three characteristic peaks at 470.0, 512.1, and 676.8 cm−1 were showed, which corresponds to the Eg, F12g, and A1g modes of the crystalline Co3O4. It should be noted that the peaks at 480.6 and 603 cm−1 from Co3O4 can be observed in the UCNG composites. Compared to LP‐Co3O4, the obvious peak shift and disappearance for the Co3O4 particles in the UCNG composites could be attributed to the effective bonding between Co3O4 and graphene,3O4 nanoparticles on the graphene sheets. In addition, after the laser irradiation, the D/G ratio associated with both LG and UCNG composites is increased (from 0.93 for GO to 0.97 for LG and 0.95 for UCNG composites), indicating a high concentration of structure defects in the graphene lattice induced by laser irradiation.3O4 nanoparticles, which restricts their mobility and prevents the Ostwald ripening process, i.e., the formation of large Co3O4 particles and the aggregation of the nanoparticles during the synthesis processes. These results are consistent with the SEM and SEM results. In order to discover the effect of GO on the formation of the ultrafine Co3O4 nanoparticles, we conducted the control experiments with the same experimental conditions except in the absence of GO. It can be seen that the porous Co3O4 nanorods completely fragmented after laser irradiation, but most of ultrafine Co3O4 nanoparticles agglomerate and even fused into larger spherical particles , which confirms that GO is necessary for the formation of the ultrafine Co3O4 nanoparticles.The local structures of the UCNG composites, LP‐Co2.3m KOH aqueous solution. Figure−1 in a potential range of −0.05–0.45 V. Two obvious redox peaks are observed in every curve, indicating that the capacitances are mainly from pseudocapacitance characteristics due to Faradaic redox reactions. The anodic and cathodic peaks exhibit a symmetrical shape, demonstrating the high redox reversibility of the UCNG composites.3O4 and CoOOH (P1/P2) and between CoOOH and CoO2 (P3/P4). The surface redox reactions are presented as follows (Equations We carried out cyclic voltammetry (CV), galvanostatic charge/discharge (GCD), and electrochemical impedance spectroscopy (EIS) measurements in a three‐electrode electrochemical cell with a platinum foil (1 cm × 1 cm) counterelectrode and an Ag/AgCl reference electrode in 2 −1 are 978.1, 955.6, 941.3, 929.2, 921.8, and 916.5 F g−1 , respectively. Figure −1. It was observed that the specific capacitance of the UCNG composites is highest among all samples  and most of other reported Co3O4‐based electrodes and even superior to some advanced carbon materials for high‐rate supercapacitors . The reason can be explained by the high electrical conductivity of the graphene sheets and the ultrafine Co3O4 nanoparticles. More importantly, the oxygen vacancies on the ultrafine Co3O4 nanoparticles surface in the UCNG composites induced by laser irradiation may play a key role in enhancing the rate capability. This result is confirmed by the fact that LP‐Co3O4 also shows high capacitance retention (85.8%) with increasing the current density from 1 to 10 A g−1.Figure RS, RCT, CDL, CF, and Wo in the circuit represent solution resistance, charge‐transfer resistance, double‐layer capacitance, pseudocapacitance, and the finite‐length Warburg diffusion element, respectively. The impedance spectra can be classified into high and low frequency regions. The equivalent series resistance  of the UCNG composites electrode is obtained from high‐frequency region of Nyquist plots , indicating a decreased electric resistance of the UCNG composites and interface resistance due to the incorporation of graphene. The enhanced conductivity of UCNG composites can be attributed to the combination of the unique synthesis procedures without using any surface modifying agent and organic additives, the plentiful oxygen vacancies created on the ultrafine Co3O4 nanoparticles surface, and the tightly anchoring of Co3O4 nanoparticles on the graphene. In the lower frequency region, the straight line was determined by ion diffusion. Obviously, the near‐zero slope for the UCNG composites and LP‐Co3O4 electrodes shows a lower diffusion resistance and an ideal polarizable capacitance in comparison with the P‐Co3O4 electrode, indicating an enhanced ion transmission properties from the laser‐induced effect (laser‐induced reduction and fragmentation). On the basis of above discussion, we might conclude that the combination of higher electrical conductivity and faster ion diffusion is responsible for the excellent rate capability of the UCNG composites.The electrochemical performance of the UCNG composites was further investigated by using EIS Figure d. The im−1 for 20 000 cycles, which is a very significant requirement for the practical applications of supercapacitors. Figure3O4 and graphene . Besides, Figure S18 (Supporting Information) represents the Co 2p XPS spectrum for the UCNG composites after 20 000 cycles. Although the intensity of the satellite peaks related to the Co2+ oxidation state decreases, it still can be clearly observed, demonstrating that abundant oxygen vacancies are still retained on the ultrafine Co3O4 nanoparticles surface. These results further indicate the excellent cyclability of the UCNG composites. The cycle stability of the UCNG composites is remarkably superior to those of P‐Co3O4, and LP‐Co3O4 electrodes. After 20 000 cycles, the P‐Co3O4 and LP‐Co3O4 electrodes retain about 84.7% and 92.8% of its original capacitance, respectively. The enhanced cycle stability of the UCNG composites is mainly owing to the synergistic effect derived from ultrafine Co3O4 nanoparticles with surface oxygen vacancies and the graphene with high conductivity. As we know, a significant number of defect sites on the graphene surface induced by laser irradiation of GO can provide more active sites to anchor the Co3O4 nanoparticles and strengthen the binding energy between Co3O4 and graphene sheets, which leads to a more stable structure and a little loss of the capacitance during the cycling test. In addition, the UCNG exhibited high power density (4000 W kg−1) and energy density (43.1 Wh kg−1), as shown in Figure S19b (Supporting Information).The cycle stability of the UCNG composites is investigated by repeating the charge/discharge test between −0.05 and 0.45 V at a high current density of 10 A g2.4VO) enhanced performance, we perform spin‐polarized density functional theory (DFT) calculations with the local density approximation + Hubbard U correction (LDA+U) approach. The calculation details are shown in the Supporting Information. Co3O4 has a spinel structure, with Co2+ ions in tetrahedral interstices and Co3+ ions in the octahedral interstices of the cubic close‐packed lattice of oxide anions. We choose the (001) surface to study because it is the most common surface observed in experiments and has the lowest energy.FigureB. For comparison, the Co atoms in the bulk octahedral sites (Co3+) have μ = 0, while those in the tetrahedral sites (Co2+) has μ ≈ 2.6 μB. The surface contributes new electronic states inside the band gap of bulk Co3O4, as shown in Figure To understand the origin of the oxygen vacancies VO enhanceVO). The VO further reduces the coordination of some of the surface Oh Co atoms to from 5 to 4. This is accompanied by the change of μ to ≈2.6 μB, similar to the Co2+ atoms in the bulk tetrahedral sites. Hence, the valance state of those Oh atoms is reduced to 2+, in agreement with the experiments. The VO also reduces the coordination of some of the Co atoms in the subsurface octahedral sites from 6 to 5, enhancing their contribution to the band gap states  The oxygen vacancies by laser irradiation dramatically increase the active sites on the Co3O4 surface and the conductivity of Co3O4, which promotes the electrochemical activity and facilitates the electrochemical reaction. (iii) The defect sites created by the laser on graphene and the large surface area of graphene efficaciously prevent the aggregation of Co3O4 nanoparticles, thus enhancing the rate performance and cycling stability.From the above characterization results, it should be noted that UCNG composites would be highly suitable as a high‐rate electrode material for SCs, primarily due to the following factors. (i) Well‐dispersed ultrafine Co2O3 and MnO2. To demonstrate the generality of this laser synthesis method, MnO2/graphene and Fe2O3/graphene composites were also fabricated under laser irradiation . MnO2/graphene and Fe2O3/graphene composites fabricated under laser irradiation also exhibit excellent electrochemical performance, especially for high‐rate capability .Researches for the generation of appropriate oxygen vacancies in metal oxide and the formation of reduced graphene oxide have long focused on traditional reductive method by reducing agent, reducing gas, etc.,33O4/graphene composite as a typical example and have developed a universal in situ laser‐assisted strategy to grow ultrafine metal oxides/graphene composites via simple laser irradiation in solution at ambient conditions. Under laser irradiation, the GO is reduced to LG with a low transfer resistance for efficient charge storage and delivery, while the porous Co3O4 nanorods completely smash into ultrafine Co3O4 nanoparticles with short ionic diffusion distance for fast ion transport. The as‐obtained UCNG composites exhibit high‐specific capacitance and excellent cycling stability. In particular, when the current density is increased from 1 to 10 A g−1, the capacitance of UCNG composites still retains 93.7%, exhibiting outstanding rate capability. ESR measurements and DFT calculations also reveal that new oxygen vacancies defect states are generated after laser irradiation, which serve as electron reservoir during the electrochemical process. We demonstrate that the unique UCNG composite nanostructures with the abundant oxygen vacancies on the ultrafine Co3O4 nanoparticles surfaces and the tightly anchoring of Co3O4 nanoparticles onto the graphene sheets could provide good electrical conductivity, high electrochemical activity, and effective pathways for rapid ionic/electronic transport and fast reversible Faradaic reactions. Combining the remarkable rate capability, outstanding capacitance and excellent cycle stability, we conclude that the UCNG composites are good electrode materials for supercapacitor compared to previous samples in literature. In addition, this novel synthetic method can be extended to other metal oxide/graphene composites, which will have great potentials in not only the energy storage field, but also in numerous other frontiers.In summary, we take the Co43O4 Nanorods (P‐Co3O4)Synthesis of Porous Co: All chemicals were of analytical grade and used without further purification. In a typical synthesis, 0.996 g of cobalt acetate tetrahydrate (Co(CH3COO)2·4H2O) and 1.0 g of polyvinyl‐pyrrolidone (PVP K‐30) were added in 40 mL of polyethylene glycol (PEG‐400). The mixture was kept vigorous stirring at 60 °C until Co(CH3COO)2·4H2O and PVP K‐30 were completely dissolved. Then, 1.0 g of urea (CO(NH2)2) was added to the resultant homogeneous solution. After stirring for 15 min, the reaction mixture from the first step was transferred to a Teflon‐lined stainless autoclave for solvothermal reaction at 160 °C for 20 h. After that, the product was washed with deionized water and ethanol for several times, and then dried in a vacuum oven at 60 °C for 12 h. The as‐prepared powder was annealed at 300 °C for 2 h in air with a heating rate of 1 °C min−1 to acquire porous Co3O4 nanorods (denoted as P‐Co3O4).Synthesis of UCNG Composites: GO was synthesized from natural flake graphite  according to the modified Hummer's method.3O4 nanorods (0.25 g) were dispersed into 10 mL of GO solution (1.5 mg mL−1). Then the mixture solution was irradiated with the beam of a KrF excimer laser  under a continuous magnetic stirring. The pulsed laser beam was guided by a reflection mirror and focused by a quartz lens on the mixture solution. The laser fluence was measured by a laser FieldMaxII Meter (Coherent). The optimal laser parameters are selected as following: 400 mJ pulse−1 cm−2 of laser fluence and 30 min of irradiating time. After laser irradiation, the products were collected by freeze drying for further characterization. The yield of ultrafine Co3O4 nanoparticles/graphene (UCNG) composites is about 94.3%. The maximal experimental throughput is ≈0.5 g h−1 for this typical ns laser system. For comparison, the laser‐reduced graphene was synthesized under the same experimental conditions in the absence of Co3O4 nanorods. Also, LP‐Co3O4 were also obtained in the absence of GO for controlled SCs measurement under similar laser conditions as described above.Characterization: UV–vis absorption spectra were recorded on a Shimadzu UV‐3600 spectrophotometer in the 200–1000 nm wavelength range at room temperature. The powder XRD measurements were performed using a Bruker D8‐Advance diffractometer with Cu Kα radiation (λ = 1.5418 Å). The powder samples were mounted flat and scanned over the range 10°–80°. The microstructures of the samples were observed by SEM (FEI Quanta 250 FEG) and TEM (JEOL JEM‐2010F). Raman spectra were taken on a Thermo Scientific Raman Microscope DXR with a 532 nm laser excitation length. The XPS was measured on a Kratos AXIS Ultra DLD spectrometer with a monochromatic AlKa X‐ray source. The EPR spectra were obtained using a Bruker A300 EPR spectrometer at X‐band (≈9 GHz) at room temperatures. FTIR spectra were recorded using a Nicolet 6700 FTIR spectrometer.Electrochemical Measurements: All electrochemical measurements were performed on a Zahner/Zennium electrochemical workstation at room temperature. The working electrodes were fabricated as follows. Briefly, the as‐prepared composites, acetylene black, and polyvinylidene difluoride were mixed in a mass ratio of 85:10:5 and dispersed in N‐methyl pyrrolidone solvent. Then, the slurry was pasted on the nickel foam substrate (1 cm × 1 cm) with a spatula and dried at 60 °C for 24 h. Finally, the electrode plate was pressed under a pressure of 10 MPa. The mass of active materials of (ultrafine Co3O4 nanoparticles/graphene composites) on current collector (nickel foam) was 6.5 mg cm−2. The monolithic electrode was tested in three‐electrode configuration assembled with a platinum foil (1 cm × 1 cm) auxiliary electrode and the Ag/AgCl reference electrode by CV, GCD, and EIS in an aqueous KOH electrolyte (2.0 m). The specific capacitance of electrode material was calculated from the charge and discharge curves, according to C = IΔt/ΔVm, where C is the specific capacitance (F g−1), Δt is the discharge time (s), I is the discharge current (A), ΔV is the operating potential window (V) during the discharge, and m is the mass (g) of active materials (UCNG composites) in as‐prepared electrode. EIS measurements were conducted with an AC amplitude of 5 mV in the 100 mHz–100 kHz frequency range.Spin‐Polarized DFT Computation: Spin‐polarized DFT calculations were performed using the Vienna Ab‐initio Simulation Package (VASP)U = 3 eV for Co d‐electrons, which has been shown to reproduce well the experimental structural parameters, heat of formation, and the band gap.−1. 5 × 5 × 1 k‐points with Monkhorst–Pack samplingThe authors declare no conflict of interest.SupplementaryClick here for additional data file.


The ongoing mobilization of mammalian transposable elements (TEs) contributes to natural genetic variation. To survey the epigenetic control and expression of reporter genes inserted by L1 retrotransposition in diverse cellular and genomic contexts, we engineered highly sensitive, real-time L1 retrotransposon reporter constructs.de novo integrants retrotransposed by L1 in pluripotent mouse embryonic stem (ES) cells underwent rapid silencing by dense cytosine methylation. Similarly, de novo cytosine methylation also was identified at new integrants when studied in several distinct somatic tissues of adult founder mice. Pre-existing L1 elements in cultured human cancer cells were stably silenced by dense cytosine methylation, whereas their transcription modestly increased when cytosine methylation was experimentally reduced in cells lacking DNA methyltransferases DNMT1 and DNMT3b. As a control, reporter genes mobilized by piggyBac (PB), a DNA transposon, revealed relatively stable and robust expression without apparent silencing in both cultured cancer cells and ES cells.Here we describe different patterns of expression and epigenetic controls of newly inserted sequences retrotransposed by L1 in various somatic cells and tissues including cultured human cancer cells, mouse embryonic stem cells, and tissues of pseudofounder transgenic mice and their progeny. In cancer cell lines, the newly inserted sequences typically underwent rapid transcriptional gene silencing, but they lacked cytosine methylation even after many cell divisions. L1 reporter expression was reversible and oscillated frequently. Silenced or variegated reporter expression was strongly and uniformly reactivated by treatment with inhibitors of histone deacetylation, revealing the mechanism for their silencing. By contrast, de novo methylation marks at newly inserted sequences retrotransposed by L1 in early pre-implantation development are maintained or re-established in adult somatic tissues. By contrast, histone deacetylation reversibly silences L1 reporter insertions that had mobilized at later timepoints in somatic development and differentiation, e.g., in cancer cell lines. We conclude that the cellular contexts of L1 retrotransposition can determine expression or silencing of newly integrated sequences. We propose a model whereby reporter expression from somatic TE insertions reflects the timing, molecular mechanism, epigenetic controls and the genomic, cellular and developmental contexts of their integration.We hypothesize that the The online version of this article (doi:10.1186/s13100-017-0091-2) contains supplementary material, which is available to authorized users. Approximately half of the human and mouse genomes is comprised of various classes of transposable elements (TEs). These TE insertions have mobilized by distinct mechanisms and accumulated over evolutionary time –4. Untilde novo insertions causing diseases in mouse and man [This ongoing movement of endogenous TEs including L1 retrotransposons can result in diverse genetic consequences. These include insertional and deletional (indel) gains and losses of genomic fragments, exon shuffling, insertional mutagenesis of genes, probably chromosomal translocations and inversions, and expression of retrotransposon-initiated fusion transcripts (RIFTs), among others –22. Much and man –25. By cCytosine methylation is a key epigenetic regulatory mark localized predominantly within extant L1 retrotransposons and other TEs in mammalian genomes. It has been strongly associated with their transcriptional silencing and regulation, and may affect expression of adjacent genes , 27. Cytde novo transcriptional gene silencing (TGS) [de novo cytosine methylation. By contrast, more strongly repressive epigenetic marks including cytosine methylation have been identified at recently inserted L1 elements that were transmitted via meiotic cell division through the mouse germ line in a transgenic mouse model [A recent study of host epigenetic responses to L1 retrotransposition in various somatic cells including embryonal carcinoma (EC) cells showed that newly integrated L1 reporters were silenced by ng (TGS) . The epise model . Similarse model , 34. SucIn classic examples of variable epigenetic silencing at mammalian TEs, changes in epigenetic marks  at pre-existing, integrated endogenous retroviruses (ERVs) have resulted in highly variable expression of nearby genes, resulting in variable phenotypes in genetically identical siblings. Variable but heritable phenotypes in the classic pseudo-agouti mouse model illustrate this impact of epigenetic regulation of an existing TE on neighboring gene expression –37. The piggyBac (PB) insertions. We corroborated recently reported results about cytosine methylation established at new L1 integrants transmitted through the mouse germ line [To investigate how different cellular contexts and mechanisms of transposition may impact reporter expression and epigenetic silencing of newly mobilized TE insertions, we developed new and highly sensitive real-time reporters. We compared the reporter expression and silencing of newly integrated L1 insertions vs. of new erm line . We obsede novo L1 retrotransposition, several research groups have engineered L1 donor constructs to track mobilization [To define the genetic consequences of lization , 40–42. TEM1, encoding a beta-lactamase, to generate an exquisitely sensitive reporter assay in living cells. Its expression levels can be quantified over a very large dynamic range extending over four orders of magnitude , respectively. The AI would be spliced from L1 RNA transcripts at the time of retrotransposition, so a newly integrated reporter cDNA would lack the AI and therefore could be expressed as the intact gene [To assess expression of L1 reporters that are newly mobilized by retrotransposition, we constructed novel, real-time reporter assays. Their expression levels would not be influenced by positive or negative selective pressures imposed on the cells. We first chose act gene .TEM1-AI reporter cassette. The resulting construct was subcloned into a stably maintained, episomal vector, i.e., pCEP4, resulting in pDES46  AI junction [bona fide L1 retrotransposition events  HCT116 cells, which lack both the maintenance DNA methyltransferase gene e DNMT3b , cytosinBecause cytosine methylation is a strong and stable mediator of transcriptional gene silencing (TGS) , we compTEM1 reporter integrants. After their retrotransposition, inserted L1 reporter sequences were retained in the host cell genomes in the absence of imposed positive selection. As was the case with L1-NeoR integrants , and an L1 integrant including a portion of the SV40 promoter and TEM1 reporter (including 20 CpGs) were almost completely unmethylated , suggesde novo L1 insertions do not reflect spreading of the repressive marks already maintained at neighboring, extant repetitive elements.Several of the newly integrated sequences retrotransposed by L1 that were recovered from HeLa cells had inserted into repetitive elements pre-existing in the host genome  inhibitors including 100 nM trichostatin A (TsA), 10 mM sodium butyrate, 1 μM scriptaid, 1 nM apicin, and 5 mM nicotinamide  was performed to recover any new L1 ORFeus integrants, regardless of their expression of GFPuv. They were sequenced and mapped, and custom bisulfite sequencing primers were designed. Genomic DNA was modified with sodium bisulfite, and then PCR amplification was performed using primers internal to the reporter, or alternatively to target individual integrants. Amplicons were cloned and sequenced. The results showed heavy methylation of the retrotransposed reporter gene, assayed either in bulk or from individual L1 integrants . Minimal expression of integrated L1 reporters was observed, consistent with dense de novo cytosine methylation resulting in strong silencing. An alternative explanation for low beta-lactamase expression was that L1 ORFeus did not retrotranspose efficiently in mES cells. However, this same donor element mobilized very actively in various cultured cancer cell lines [To evaluate silencing of a second, independent reporter gene, we also transfected pJL5, a donor plasmid encoding L1 ORFeus marked by ll lines , 55. UpoTo study the epigenetic modifications established at new L1 integrants in vivo, we obtained several tissues from a transgenic mouse model, in which L1 ORFeus had retrotransposed initially in “pseudofounder” animals. Pseudofounder mice were defined as those mice in the first generation that harbored new, spliced L1 insertions but lacked the unspliced donor element. Their progeny also harbored some of the same, newly retrotransposed L1 ORFeus insertions as were present in the initial pseudofounders themselves, showing that these genomic L1 insertions could be transmitted through the germ line. The new L1 insertions likely had retrotransposed from the donor episome, immediately after its injection as a transgene and before its loss due to cell division during early embryogenesis .de novo cytosine methylation established at the newly mobilized L1 ORFeus integrants in the pseudofounders, as well as at some of the same integrants transmitted to offspring [de novo L1 integrants were methylated in two independent pseudofounder mice, F235 and F234  methylation was maintained through differentiation into a range of tissues in the developing organism; and c) methylation at such new L1 insertions was maintained and/or re-established upon their transmission through the germline.Taken together, these results demonstrated that: a) new L1 insertions occurring early in embryogenesis underwent dense, TEM1 and green fluorescent protein (GFP), were engineered to be mobilized as cargo by PB DNA transposons. A large majority of HeLa cells harboring newly transposed integrants displayed stable, robust expression of the reporters when mobilized by PB , distinct from that observed after L1 retrotransposition.In additional control experiments, we also launched ure Fig. . No varide novo L1 integrants has not been evaluated fully in the wide-ranging biological contexts in which retrotransposition can occur [Endogenous retrotransposons comprise a substantial portion of the mouse and human genomes. Several distinct TE families have modified the mammalian genome profoundly over evolutionary time , 58. Thean occur , 59.Here we investigated the expression and epigenetic silencing of newly integrated L1 reporters in cultured human cancer cells, mouse ES cells, and in several tissues of pseudofounder transgenic mice and their progeny. The results revealed distinctive patterns of L1 reporter expression and associated epigenetic marks, which are associated with the genomic, cellular and developmental contexts of mobilization.Newly retrotransposed and integrated L1 reporters in cultured human cancer cells frequently were silenced. This silencing was heritable, as daughter cells tended to display levels of reporter expression that were similar to their parents after mitotic cell divisions Fig. . New L1 de novo methyltransferases are expressed at low levels [We speculate that this variegated expression of reporter sequences retrotransposed by L1 in cultured cancer cells may reflect the timing of their integration, i.e., at later stages of somatic development Fig. , when cew levels . Retrotrw levels . Thus thw levels . The varw levels . An altede novo CpG methylation  cells, histone deacetylation was identified as the silencing mechanism . We noteine PA-1  represenine PA-1 . HoweverPB, a DNA transposon, to compare with L1 reporter silencing. These control vectors differ in their mechanisms of integration, genomic target sites, and the frequency of insertions generated per host genome. Each of these factors could play significant roles in shaping the downstream epigenetic silencing marks established at the new insertions.We also studied silencing of newly mobilized L1 insertions in vivo, both in differentiated tissues of pseudofounder mice and in their offspring. By contrast, the prior study did not include an analysis of silencing of new integrants in vivo . In addiPB donor elements using engineered, heterologous reporters including several different strong promoters and terminators. In comparison with native, unmarked elements, these reporter genes incorporated into donor TEs potentially could interfere with their mobilization. Moreover, upon integration they could trigger antisense transcripts [We acknowledge potential limitations in our study. First, to investigate the state of expression and epigenetic regulation of newly inserted sequences that were mobilized by TEs, we artificially marked the L1 and nscripts  or othernscripts .de novo cytosine methylation [de novo methylation in somatic tissues [de novo methylation.Second, we did not investigate L1 insertions that had newly integrated in germ line tissues. Extensive research has been conducted on the epigenetic control of pre-existing TEs in germ tissues during embryonic development. They appear to undergo a wave of demethylation followed by two distinct waves of hylation , 79. PIW tissues . We curr tissues , in targThird, new insertions were not identified immediately after their integration in single cells. This approach has become technically possible recently, so we could identify and characterize new insertions in individual cells or very small subclonal populations within a few cell divisions of integration. Their minimal allelic fractions would require use of ultra-deep sequencing, making further analysis technically difficult but feasible for the first time.Fourth, we did not perform chromatin immunoprecipitation (ChIP) experiments to assess enrichment of particular histone modfiications at the new L1 integrants, although we measured cytosine methylation in detail. However, in a recent paper describing L1 reporter silencing by histone deacetylation in hEC cells, confirmatory ChIP data  were strThe donor TE constructs used here did not include a means to terminate conditionally their capacity for ongoing retrotransposition. Future designs will incorporate this feature, to avoid the potential for ongoing genetic variability, e.g., after subcloning individual cells.In some cases, rather than assay for reporter expression, we monitored retrotransposition by using PCR-based assays to identify genomic insertions of newly transposed reporter genes. As was the case with reporter expression assays used here, we did not select positively or negatively on cells harboring such inserted sequences to preferentially include expressed reporters or exclude silenced reporters.PB donor elements have been developed, whereby we could compare their silencing during development and in diverse tissues, such strains were unavailable to us. However, published studies have indicated that PB insertions typically are not silenced even when selection is not imposed [And finally, although in vivo mouse models harboring control  imposed , 80.In summary, we showed here that the expression and silencing of newly integrated sequences mobilized by L1 retrotransposition appear to be associated with the cellular, developmental and genomic contexts of their integration. We hypothesize that the distinct epigenetic marks set up at new TE insertions that integrated in different cellular or developmental contexts may have various downstream consequences. For example, recent evidence suggests that most new somatic L1 insertions mobilized during human cancer development can mediate only minimal, if any, impacts on neighboring gene expression, unless they cause direct insertional mutagenesis of coding exons , 25. By We propose that these findings may have important practical implications for evaluation and understanding of new TE insertions in various biological contexts. For example, they may facilitate a novel experimental assay of transposition timing, i.e., to identify when the elements mobilized in vivo. Thus we would expect to find dense cytosine methylation strongly repressing newly integrated, polymorphic L1 insertions that were mobilized early in development or passed through the germ line Fig. . Such inde novo cytosine methylation at new integrants also was observed in several distinct somatic tissues of adult pseudofounder mice. We conclude that the host cellular and developmental contexts of retrotransposition are significant determinants of reporter expression and epigenetic silencing at newly integrated sequences mobilized by L1 retrotransposition. We have proposed a model whereby reporter expression of somatic TE integrants reflects the timing, molecular mechanism, epigenetic controls and the genomic, cellular and developmental contexts of their mobilization.Analysis of newly inserted genomic sequences retrotransposed by L1 in various somatic cells and tissues revealed distinct patterns of expression and epigenetic regulation. In cancer cell lines, the newly retrotransposed integrants typically underwent rapid transcriptional gene silencing, but they lacked cytosine methylation, and their reporter expression was reversible and oscillated frequently. Silenced or variegated reporter expression was strongly and uniformly reactivated by treatment with inhibitors of histone deacetylation. By contrast, newly inserted sequences retrotransposed by L1 in mouse embryonic stem (ES) cells underwent rapid silencing by dense cytosine methylation. Similarly, TEM1-AI beta-lactamase/artificial intron (AI) reporter cassette inserted into its 3′ UTR, we first deleted the BamHI site in ORF2 of human L1.3 as present in pJM101/L1.3  using site-directed mutagenesis. The L1.3/NeoR-AI promoter cassette was then moved into pBSII-KS using NotI and BamHI. We then introduced double-stranded oligonucleotides (containing Bst1107I -HSVTKpolyA-MluI) to replace the NeoR-AI and promoter fragments from pJM101/L1.3. BseRI sites were introduced to flank both TEM1 (sequences obtained from the vector pBLAK-b which were then codon optimized) and beta-globin AI, using fusion PCR. Both of these constructs were cut out using BseRI and ligated together seamlessly. The resulting L1.3/TEM1-AI construct was then excised from pBSII-KS using NotI and BamHI and ligated into pCEP4 (Invitrogen), yielding pDES46.To construct an L1 donor launched from an episomal plasmid, i.e., pDES46  cells, provided by Dr. John V. Moran (University of Michigan), were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with the same supplements as HCT116 cells. E14Tg2a.4 mouse embryonic stem (ES) cells, derived from 129P2 ES cells, were provided by Dr. Allan Bradley (Sanger Institute) and the BayGenomics resource. They were cultured without feeder cells in Glasgow’s Modified Eagle’s Medium (Sigma Aldrich) supplemented with 10% ES cell-qualified FBS, 1% non-essential amino acids, 1% L-glutamine, 1% sodium pyruvate, 0.1 mM 2-mercaptoethanol (2ME) and ESGRO (Millipore) at 1000U/mL in a 7% CO2 atmosphere.HCT116  cells, kindly provided by Drs. Ina Rhee, Christoph Lengauer and Bert Vogelstein (Johns Hopkins University), were cultured in McCoy’s 5A modified medium  supplemented with 10% heat inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin (P/S), at 37 °C and 5% COFugene 6  was used to transfect the cultured cancer cell lines. Cells were transfected with pDES46 and were selected on Hygromycin (Invitrogen) at 0.3 mg/ml for 2 weeks. Resulting HygroR cells were cloned by limiting dilution into 96-well plates and screened for single colonies. Colonies were assayed for TEM1p beta-lactamase expression using the CCF2-AM assay . Certain2. For feeder cells, Neo2-expressing mouse embryonic fibroblasts (MEFs) were cultured in DMEM supplemented with 10% FBS and 1% P/S, in 5% CO2, arrested using Mitomycin C or gamma irradiation, and seeded in dishes. The mES cells were added 2–3 days after feeder cell seeding.Mouse ES cells including Bruce4 cells , and thlacZ LSL cassette. Briefly, one million Truck_305 cells were incubated for 30 min with the adenoviral Cre vector  in a 7% CO2 incubator, at various multiplicities of infection (MOIs) ranging from 10 to 200 per cell, and then plated into a well of a 6-well dish that was pre-seeded with mouse feeder (PMEF) cells. Cells were evaluated for cell death by light microscopy after 18 h incubation. Based on the extent of cell death and colony morphology, MOIs 25 and 50 were found to be optimal. Cell clones exposed to these MOIs were expanded. After at least 12 d in culture, with periodic changes of culture medium, cells were stained with crystal violet and X-gal, to visualize the colonies and assay for presence of the lacZ LSL cassette. ES cell clones that did not stain blue with X-gal, which indicated activated smL1 expression and potential retrotransposition, were propagated.To activate L1 retrotransposition in these cells, an adenoviral vector encoding Cre recombinase was introduced to excise the To quantify beta-lactamase activity and protein expression, cells were stained with CCF2-AM substrate (Life Technologies)  by replade novo L1 integrants were transmitted in heterozygosity.Transgenic founder and pseudofounder mice were generated by pronuclear injection of linearized, marked smL1 donor cassette into fertilized eggs as described previously . The unsTo extract genomic DNA, we added 600 μL lysis buffer with 420 μg/mL of proteinase K to cells growing in a 48 well plate. Lysis buffer consisted of 50 mM Tris-Cl, pH 8.0, 100 mM EDTA, 100 mM NaCl and 1% SDS. Next, 200 uL saturated NaCl was added, followed by an equal volume of isopropanol. After centrifugation, DNA pellets were washed with 70% ethanol, dried and resuspended in TE buffer.TEM1 reporter using standard methods.Southern blotting was performed to identify lengths and amounts of genomic fragments harboring new TE insertions. Genomic DNA was electrophoresced, blotted, and probed for the de novo insertion sites, Sanger sequence reads were aligned against the reference human genome (hg19) using Blat.For recovery of TE integrants in cultured cancer cells using inverse PCR (iPCR), genomic DNA isolated from clonal populations of cells was digested with a restriction enzyme (RE) such as XbaI, EcoRI or HindIII. Upon heat inactivation of the RE, digested products were diluted to 1 ng/μL in a total of 500 μL, and incubated with T4 DNA ligase overnight at 16 °C for intra-molecular ligation. After ethanol precipitation, DNA was resuspended and used in iPCR reactions using primers DES682 and DES209 which annealed to the 3′ end of the retrotransposed cassette. PCR reactions consisted of 40 cycles at 94 °C for 30 s, 55 °C for 30 s and 72 °C for 2 m 20 s. Each of the several bands observed by gel electropheresis after PCR were cloned into pCR2.1 using TOPO cloning kit (Invitrogen) and transformed into bacteria. Colony PCR using M13 forward and reverse primers was used to identify bacterial colonies containing cloned insert. PCR products were cleaned up and sequenced. To map To map new L1 integrants in mES cells using linear amplification-mediated (LAM-) PCR, we chose three ES cell clones  in which the spliced  reporter gene had been identified by PCR assays. LAM-PCR reactions were set up with 50 ng gDNA from each ES cell clone, 2 nM dNTPs, 5 nM 5′-biotinylated primers DES3171 or DES3174, 1 uL of Advantage 2 enzyme in 1× buffer (Clontech), for 50 cycles . Streptavidin-coated magnetic beads (200 mcg) were washed twice in 100 μL of binding buffer  using a magnetic separation stand, resuspended in 50 μL of 2× binding buffer, and mixed with the linear PCR reaction. The suspension was incubated for 60 min at RT under constant agitation, and then washed three times in 200 μL of wash buffer . For second-strand synthesis, the matrix-bound DNA was resuspended in 20 μL of a reaction mixture containing 500 nM dNTPs, 100 ng/μL random hexamers, 5 U Klenow enzyme (New England Biolabs) and 1× NEB Buffer 2, and incubated at 37C for 60 min. After washing first with wash buffer and then twice in 1× reaction buffer, dsDNA was restricted using HaeIII or Sau3AI (NEB) at 37C for 2 hr, washed again in wash buffer followed by twice in 1× ligation buffer, and ligated with either HaeIII adapter (DES3177 and DES3178) or Sau3AI adapter (DES3177 and DES3179) using T4 DNA ligase at 16C overnight. To elute products, beads were resuspended in 5 μL of 0.1 N NaOH and incubated at RT for 10 min. The eluate was separated from the matrix using the magnetic stand, and was neutralized by adding 5 μL of Tris-Cl, pH 7.0.To perform nested PCR, 1 μL of the eluate or from a 1/100 dilution from the first round of PCR was added as template in a second (nested) PCR reaction in 50 μL. Primers for the adapter (DES3181) and nested adapter (DES3182) were paired with DES3172 and nested primer DES3173 (HaeIII), or DES3175 and nested primer (Sau3AI), respectively, in the donor plasmid. Products were cloned into the Topo-TA pCR2.1 backbone (Invitrogen). To map integration junctions, Sanger sequencing was performed.Bisulfite sequencing was performed using either the EZ DNA Methylation kit (Zymo Research) or the Qiagen Epitect kit, following the manufacturers’ instructions, respectively. Alternatively, we prepared fresh sodium hydroxide, sodium bisulfite and hydroquinone solutions to denature and treat DNA samples, which were then purified using Microcon-30 centrifugal spin columns  .The culture medium for cells growing at 50–70% confluence was replaced by medium containing one of HDAC inhibitors including TsA, scriptaid, apicidin, butyrate and nicotinamide  at the specified concentrations. After treatment for a specific time (12–24 h), the cells stained using the CCF2-AM assay and observed using a fluorescence microscope.