Since the expression of let-7 is modulated by LINE-1 activity, we discuss possible mechanisms for this effect and how the silencing of LINE-1 activation could provide new therapeutic options for cancer treatment. Based on the deep sequencing of small RNAs in parallel with gene expression profiling in breast cancer cells, we have identified potential pathways linking L1 activity to let-7 processing and maturation and ultimately to the control of stemness in human cancer cells.Long interspersed nuclear element-1 (LINE-1 or L1) retrotransposons are insertional mutagens capable of altering the genomic landscape in many ways. Activation of the normally silent LINE-1 retrotransposon is associated with a high level of cancer-associated DNA damage and genomic instability. Studies of LINE-1 have so far focused mainly on changes in gene expression, and our knowledge of its impact on functional non-coding RNAs is in its infancy. However, current evidence suggests that a significant number of human miRNAs originate from retrotransposon sequences. Furthermore, LINE-1 is generally not expressed in normal tissues while its expression is widespread in epithelial cancers. Based on our recent studies, we demonstrate a functional link between aberrant LINE-1 expression and deregulation of  L1 is an insertional mutagen capable of copying itself and reinserting into the genome at multiple sites and is thereby capable of wreaking mutational havoc on the genome. The activation of L1 retrotransposons and ensuing L1 retrotransposition is also associated with a high frequency of DNA breaks and genomic instability  and seve mapping .c-MET, typically via alternative promoters, has been recognized (A recent survey of whole genome sequences from a variety of tumors included in the Cancer Genome Atlas (TCGA) project also identified a number of L1-mediated insertional mutations in colon, prostatic, colorectal, and ovarian cancers, suggesting that L1-induced mutations are common in cancer cells and tissues . Despitecognized , little cognized . StrikinSLC7A2, leads to a defect in the expression of the lncRNA and results in a lethal encephalopathy phenotype , which binds to polycomb group proteins and interacts with multiple target gene promoters during the process of atherosclerosis . In particular, let-7a miRNA was strongly upregulated from 149,428 normalized mapped reads to 1,855,633 reads in L1 silenced cells, accounting for 40% of the increase in the total normalized read counts in the L1-silenced cells compared to cancer cells in which L1 remained active. This massive increase in let-7a expression is intriguingly similar to the differential expression of let-7a seen in normal cells and a variety of cancer cells in which the expression of let-7 is repressed , which binds to corresponding sequences located in the 3′ UTRs of genes. Let-7a is also known to target many oncogenes including c-Myc, HMGA2, and Lin28, and its expression is a hallmark of cell differentiation. Notably, the loss of let-7a expression is often considered to have prognostic value since it indicates poor survival in many cancers. Studies performed in lung and renal cell carcinoma reveal that the overexpression of let-7a inhibits in vitro cancer cell proliferation and in vivo tumor regeneration by reducing the expression of c-Myc and c-Myc targeted genes  and negative factors . The p68/p72 RNA helicases, as components of the Drosha microprocessor complex, stimulate the processing of pri-let-7 miRNAs into mature RNAs by Dicer-mediated processing. The mature let-7a also binds to a complementary region in the pri-let-7 miRNA, recruiting Argonaute and promoting its own downregulation . In another study, inhibition of L1 activity by antiretroviral drugs was shown to reduce c-Myc expression in cancer cells , which is a component of Drosha/Dicer complexes and is known to positively regulate processing of miRNAs . Strikingly, except for pre-let-7i, all the members of the let-7 family interact with hnRNPA1. Thus, there is evidence of a relationship, at least in the case of let-7a miRNAs, between the L1 activity and miRNA expression.There is little or no direct evidence for a reciprocal relationship between the silencing of L1 and 7 miRNAs , it is c7 miRNAs . Regardlan cells . Severalan cells . Importalet-7 biogenesis. Intriguingly, our gene expression microarray profiling of L1-silenced cells showed significantly upregulated expression of the DEAH box-containing DHX9 protein, which shares high sequence similarity to the Drosophila maleless protein (MLE), a protein that regulates dosage compensation (let-7a miRNAs by recruiting a DHX9-like RNA helicase to promote its own translation (let-7a miRNAs. However, further research is needed to evaluate the precise function of L1 in the activation of the let-7 miRNAs. Given that let-7 is often viewed as a tumor suppressor miRNA, a strategy in which L1 activity is selectively inhibited pharmacologically could provide new therapeutic options for human cancer treatment.An alternative but not mutually exclusive possibility is that L1 silencing may activate miRNA-inducing proteins such as transcription factors and RNA helicases that activate ensation . DHX9 isensation  as an RIensation . Strikinnslation . Togethelet-7a. As we propose above, there is a clear link between let-7a expression and the silencing of L1 elements. Questions remain, however, as to how L1 elements, either directly or in combination with other host proteins contribute to the loss of let-7 expression in the various types of cancer. Further studies are required to thoroughly test the mechanisms proposed above by which L1 might affect miRNA expression. Regardless of the answers to these questions, the available data suggest that silencing of L1 expression holds great therapeutic potential for the treatment of various epithelial cancers.In summary, this study has explored the relationship between the expression of L1 elements and cancer onset and progression. In this study we have shown, first, that L1 activity is widespread in epithelial cancers. Second, the expression of non-coding RNAs including miRNAs and lncRNAs is closely associated with L1 activity. Third, we have demonstrated the interplay between the aberrant expression of L1 elements and miRNAs, and in particular, the tumor suppressor miRNA 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.


The effect of breast milk fatty acid (FA) composition, particularly levels of docosahexaenoic acid (DHA), on infant health outcomes is unclear. Part of the reason for this is difficulties in collecting, storing and shipping milk samples to the laboratory. Here we report the validation of a dried milk spot (DMS) system to measure FA composition to help overcome these obstacles.trans FA (iTFA). Experiments were carried out using pooled milk samples from US (n = 5) and Malawian women (n = 50). Experiments compared liquid vs. DMS samples (n = 55), assessed stability of FA composition under different storage conditions (n = 5), and compared the results from two different labs using the same methods (n = 5).Milk FA were measured by gas chromatography and reported as percent of total FA; the FA of primary interest in this study were DHA and industrially produced 2 = 0.99 and 0.99, respectively, P < 0.0001). The % DHA in DMS samples was stable for up to four weeks at room temperature and up to three years at -80 °C; only slight deviations from the acceptable range of variability (±15 %) occurred in the 4 °C and -20 °C conditions for % DHA. The % iTFA was stable under all conditions. All % DHA and % iTFA were within 15 % of the referent when analyzed in two laboratories.Both % DHA and % iTFA levels in liquid and DMS samples were strongly correlated  contains supplementary material, which is available to authorized users. After cooling, 40 parts of both hexane and distilled water were added. After briefly vortexing the samples were spun to separate layers, and an aliquot of the hexane (upper) layer which contained the FA methyl esters was taken for analysis by gas chromatography  as described previously [Fifty-five samples of milk from Malawian and US mothers were used to compare the FA composition of liquid vs. DMS. To make DMS samples, 1iquid milk samples were thawed and one drop of milk was placed on the collection cards. Then the DMS samples were stored at room temperature in the dark for one week before analysis . The liquid samples analyzed immediately after thawing.The five pooled milk samples from US mothers were spotted onto pretreated cards. Several DMS cards from each pool were stored in the dark in reclosable plastic bags under each of the following conditions: room temperature (23 °C), refrigeration (4 °C), standard freezer (-20 °C), and research freezer (-80 °C). At Days 4, 7, 14, 21, and 28, DMS from the first three storage temperatures were analyzed for FA composition; the Day 0 sample was analyzed immediately after spotting and served as a reference. Additionally, DMS stored at -20 °C and -80 °C were tested at months 3, 6, 9, and 12, and then every 6 months for up to 3 years.Pooled milk samples from five US mothers were placed on DMS cards and sent by express mail at ambient temperature to an affiliated lab in Seoul, South Korea (OmegaQuant Asia). Identical DMS samples were saved at OmegaQuant Analytics in Sioux Falls, SD. Sample analysis was coordinated to be done on the same day (about 1 week after shipping). Identical methods were used in both laboratories.t plus C18:2 t). If necessary, the FA were log-transformed to meet normality requirements. Spearman correlations, linear regression, and paired t-tests were used to compare the liquid and DMS samples. Correlations between liquid and dry milk samples were calculated across the full range of DHA values, and for only those samples with DHA < 1.0 %. The latter is the range in which the vast majority of milk DHA levels lies worldwide [All statistics and graphs were analyzed and created with MiniTab  and GraphPad Prism 6 . The primary FA of interest were DHA C22:6n-3) and iTFA . The linear regression equation to estimate the liquid milk % DHA using DMS data was: Liquid % DHA = 0.8974 (DMS % DHA) – 0.0015 . For the < 1 % DHA subset, the regression equation was: Liquid % DHA = 0.9116 (DMS % DHA) – 0.0054 .The % DHA of the liquid and DMS samples were highly correlated for both the whole set . The regression equation to estimate liquid % iTFA from DMS was: Liquid % iTFA = 0.9605 (DMS % iTFA) - 0.0146 . There was a greater than 15 % difference between the liquid and DMS samples for six FA  condition for up to 4 weeks or in the -80 °C freezer condition for up to 3 years  have been found to be beneficial for neurodevelopment and vision in infants and young children in some studies, but the evidence is not conclusive , 2, 18. A limitation of this validation study was the use of DMS samples that had been frozen as liquid milk, thawed then spotted onto the filter paper rather than spotted fresh. Due to the timing of the initiation of our collaboration with the research team, it was not possible to do the analyses on fresh samples. However, FA in liquid milk samples stored at -80C are stable and simulating the DMS using thawed milk and storing for 1 week at room temperature still demonstrates the value of DMS compared to traditional collection and storage methods.Difficulties in collecting, storing and transporting milk samples, particularly in field research, have limited advancements in this area. The method described here had the potential to overcome some of these challenges. One limitation of the DMS method is that (at present), the concentration of FA in milk  cannot be measured, only the percent composition. However, few researchers in this field would consider this a limitation since the vast majority of milk FA researchers have used the percent composition approach. Its appeal lies in the robustness of the analytical method, the long tradition of use, and the ability to compare results between studies. In addition, since milk percent FA composition is relatively stable during a feed and throughout the day , 22, samblood spot samples have been used for the determination of blood FA composition for many years [Logistic challenges in collection, storage and transport of samples have impeded studies where simply measuring milk FA composition is the goal. The ability to collect samples as DMS instead of liquid milk and to store and ship at ambient temperature provides a cost-effective, practical alternative without compromising the quality of the FA data. In a parallel setting, dried ny years , 10 and ny years , particuny years , 27 and ny years . The abiny years . The abiThe datasets supporting the conclusions of this article are included in the Additional file


The graphene films with low sheet resistance were grown by chemical vapor deposition. The BMN thin films were fabricated on graphene by using laser molecular beam epitaxy technology. Compared to BMN films grown on Au, the samples on graphene substrates show better quality in terms of crystallinity, surface morphology, leakage current, and loss tangent. By transferring another graphene layer, we fabricated flexible and transparent capacitors with the structure of graphene-BMN-graphene. The capacitors show a large dielectric constant of 113 with high dielectric tunability of ~40.7 % at a bias field of 1.0 MV/cm. Also, the capacitor can work stably in the high bending condition with curvature radii as low as 10 mm. This flexible film capacitor has a high optical transparency of ~90 % in the visible light region, demonstrating their potential application for a wide range of flexible electronic devices.We report a kind of electric field tunable transparent and flexible capacitor with the structure of graphene-BiThe online version of this article (doi:10.1186/s11671-015-0974-4) contains supplementary material, which is available to authorized users. Th. Th17]. graphene , 21.Fig −1, respectively [2g mode, and the 2D band is the result of a second-order process involving two phonons with opposite momentum [I2D/IG is larger than 2.0 and the full width at half maximum of 2D peak is ~30 cm−1, which can be interpreted as the structure of monolayer graphene [−1 corresponds to the breathing mode of sp2 hexagonal carbon but only gets activated by a defect with the help of a double resonance in the inter-valley scattering [IG/ID in an area of 50 × 50 μm2  of graphene on BMN film  decreases and the secondary phase MgNb2O6 (JCPDS card number 33–0857) is detected [2O6 can be ascribed to the volatilization of Bi element at high temperature. The lattice constant of the BMN films calculated from the XRD patterns is about 10.529 Å, which is similar to the value of BMN thin films on Pt-coated sapphire substrates [Figure detected . The emebstrates . Figure bstrates .Fig 4ComI{1100}/I{2110} > 1.Figure Surface roughness of the BMN films was evaluated by AFM. Figure As a kind of electrode materials, the electrical properties of graphene are an important parameter in device fabrication. The spatial distributions of sheet resistances tested on a 30 × 30 mm graphene on top and bottom of the BMN film are shown in Fig. ε is calculated from the capacitance C:Figure d is the BMN film thickness, ε0 is the dielectric constant in vacuum, and A is the capacitor area. The maximum dielectric constant reaches about 113 for the sample grown at 750 °C. The tunability of the thin films is defined as [where fined as –36:2\docε(0) and ε(v) are the dielectric constant at zero and a certain bias field, respectively. For the films grown at 550 °C, the tunability of the dielectric constant is very low of ~13.5 % at a bias field of 1.0 MV/cm, which is consistent with their relative poor crystallized phase. The tunability of the film increases with growing temperature from 550 to 750 °C, possibly due to the enhancing crystallization of the films. For the films grown at 750 °C, the maximum tenability of ~40.7 % is achieved at a bias field of 1.0 MV/cm. Compared to the BMN thin film grown at 750 °C, the sample grown at 800 °C shows a decrease of tunability, which can be ascribed to the appearance of additional phase of MgNb2O6 as illustrated in XRD patterns in Fig. 2O6 phase has a lower ε of ~19.9 and may also disorders the cubic pyrochlore structure of BMN, reducing the polarization of BMN [2O6 as shown in Fig. −5 A/cm2 at a bias voltage of 7.0 V. In contrast, the sample grown on graphene shows a low leakage current density of ~4.0 × 10−6 A/cm2 at a bias voltage of 7.0 V. The difference of the leakage current is thought to be associated with both surface roughness and crystallinity of the BMN films. For the film grown at Au, its surface has a relatively high roughness of 6.38 nm. The local electric field will vary from place to place due to the fluctuation of the film surface. At the valley, the electric field is larger than that at the peak, which increases the leakage current density of film [where n of BMN . The los of film . Moreove of film , 44. TheWe fabricated a kind of electric field tunable transparent and flexible capacitor by using graphene as electrodes. The graphene film was grown by CVD method, and the BMN thin films were fabricated on graphene by using LMBE technology. The comparison of BMN films grown on graphene with Au substrates shows that the graphene can obviously improve BMN film quality in terms of crystallinity, surface morphology, leakage current, and loss tangent. By using transfer method, we fabricated flexible and transparent capacitors with the structure of graphene-BMN-graphene. The capacitors show the largest dielectric constant of 113 and the largest dielectric tunability of ~40.7 % at 1.0 MV/cm and can work stably in the high bending condition with curvature radii as low as 10 mm. This film capacitor has a high optical transparency of ~90 % in the visible light region, making tunable capacitor highly promising for integration into flexible optoelectronic devices.


Purpose. To determine the feasibility of using 25-gauge microincision vitrectomy surgery (25GMIVS) to treat vitreoretinal disease in glaucomatous eyes which have previously undergone trabeculectomy (TLE). Methods. A consecutive, interventional case series. We performed 25GMIVS in 15 glaucomatous eyes that had undergone TLE. Follow-up period was 11.5 months. Results. 25GMIVS was successfully used and led to improvement in visual acuity (P < 0.01). We performed 25GMIVS for proliferative diabetic retinopathy with neovascular glaucoma in 53% of eyes (8 of 15). Although 3 eyes needed further TLE following 25GMIVS, final IOP was below 21 mmHg in all eyes except one eye (93%) and was comparable to pre-25GMIVS IOP (P = 0.20) without an increase in the number of glaucoma medications (P = 0.14). Conclusions. 25GMIVS is a feasible treatment for vitreoretinal disease in eyes with preexisting TLE, effective in both significantly improving BCVA and preserving the filtering bleb, while not excluding further glaucoma surgery. Trabeculectomy (TLE) is a procedure most often performed when drug-based therapies for glaucoma have been ineffective. It can effectively reduce intraocular pressure (IOP) over the long term –3, but cTwenty-five-gauge microincision vitrectomy surgery 25GMIVS) was first reported in 2002, and this procedure is now commonly used worldwide GMIVS was. One of Thus, the purpose of this study was to determine the feasibility of using 25GMIVS to treat vitreoretinal disease in glaucomatous eyes that have undergone TLE.This was a retrospective, consecutive, interventional case series performed at a single center. Fifteen consecutive post-TLE eyes of 15 patients with retinal diseases who underwent 25GMIVS were studied. The inclusion criterion was any retinal vitreous disease causing visual dysfunction in eyes that had previously undergone TLE, . The exclusion criteria were prior scleral buckling, prior trauma, and a follow-up period of less than 3 months. The preoperative demographics and postoperative courses of the patients are shown in All surgeries were performed under retrobulbar anesthesia using the oblique sclerotomy technique and were performed using the Accurus Vitrectomy System . First, an infusion cannula was inserted through the inferotemporal sclera followed by the insertion of two cannulas through superotemporal and superonasal sites. The insertion point of the cannulas was shifted as necessary to avoid disturbing the conjunctiva adjacent to the filtering bleb. Next, a 2.4 mm superotemporal corneal incision was made, followed by phacoemulsification, aspiration (PEA), and intraocular lens (IOL) implantation before the vitrectomy, if the eye had a cataract. After resecting the vitreal core, 4 mg of triamcinolone acetonide  was injected into the vitreous cavity to determine if a posterior vitreous detachment (PVD) was present. If a PVD was not present, we created one with a 25-gauge cutter. After shaving the peripheral gel, the proliferative membrane was removed, and fluid air exchange and endophotocoagulation were performed if needed. The exact surgical procedures varied according to the type of vitreoretinal disease. Additional TLE was also performed under retrobulbar anesthesia, with a fornix-based conjunctival flap. A half-thickness 4.0 by 4.0 mm rectangular scleral flap was made in the superior area. Mitomycin C (MMC) was used with a concentration of 0.04% and an exposure time of 5 minutes. The area was irrigated thoroughly with 200 mL of balanced salt solution. Trabeculectomy was then performed, followed by peripheral iridectomy. The scleral flap and conjunctiva were closed with a 10-0 nylon suture. Postoperatively, antibiotics and corticosteroids were injected subconjunctivally.We evaluated best-corrected visual acuity (BCVA), IOP, number of glaucoma medications, intraoperative subconjunctival hemorrhage, intraoperative suturing at the sclerotomy site, and additional TLE (after 25GMIVS). BCVA was measured using the Landolt C visual acuity chart, and the decimal BCVA was converted to logarithm of the minimal angle of resolution (LogMAR) units for statistical analysis. Success of the 25GMIVS procedure was defined as improvement or maintenance of BCVA and maintenance of IOP ≤21 mmHg with the use of topical glaucoma medication, with no need for additional TLE. The procedure was recorded as a failure if there was a decrease in BCVA, additional TLE was required, or IOP could not be maintained ≤21 mmHg with the use of topical glaucoma medication. t-test. For the statistical analysis, “count fingers” visual acuity was set as 2.0 logMAR units, and “hand motion” acuity was set as 3.0 logMAR units. The significance of the difference in IOP before TLE, after TLE, and after 25GMIVS was determined by the Friedman test, and the significance of the difference between the IOP after TLE and after 25GMIVS was determined by the Scheffe's test. The significance of the difference in the pre-25GMIVS number of glaucoma medications and the final number was also determined by the two tailed paired t-test. A P value of less than 0.05 was considered to be statistically significant.The data are presented as the mean ± standard deviation. The significance of the difference between the pre-25GMIVS BCVA and final BCVA in logMAR units was determined by the single tailed pairedA summary of the patients' characteristics and pre-25GMIVS course is shown in P = 0.01). Pre-TLE IOP, pre-25GMIVS IOP, and final IOP were 36.0, 11.9, and 15.7 mmHg, respectively. There were significant IOP differences pre-TLE, pre-25GMIVS, and post-25GMIVS (P < 0.001), but no significant difference between the pre-25GMIVS IOP and final IOP (P = 0.20). There was no difference in the pre-25GMIVS and final number of glaucoma medications (P = 0.14). Subconjunctival hemorrhage occurred in 5 eyes (33%); however, in 3 of these eyes, there was no hemorrhage invasion into the filtering bleb. Four eyes (27%) had intraocular pressure >20.0 mmHg after 25GMIVS, and 3 of these eyes needed additional TLE. It should be mentioned that this additional TLE was a technically simple procedure, because 25GMIVS had been performed without any sutures. Vitreoretinal diseases in all 15 eyes with preexisting TLE were successfully treated with 25GMIVS. We achieved success with 25GMIVS in 10 cases (67%) and did not observe surgical complications, such as bacterial endophthalmitis, associated with either TLE or 25GMIVS in any of the cases. The mean follow-up period was 11.5 ± 7.7 months with a range of 6 to 34 months.A summary of the patients' characteristics and post-25GMIVS course is shown in We set out to evaluate the feasibility of using 25GMIVS to treat vitreoretinal disease in glaucomatous eyes that had undergone TLE. The most common vitreoretinal disease in eyes with preexisting TLE we treated with this technique was PDR complicated by NVG . In spite of the relatively high incidence of such a severe disease, mean BCVA improved significantly after 25GMIVS. Additionally, although 3 eyes needed further TLE following our 25GMIVS procedure, the final measurement of IOP was statistically comparable to IOP before 25GMIVS, without an increase in the number of glaucoma medications. Furthermore, because of our use of sutureless 25GMIVS, the additional TLE procedure itself, following 25GMIVS, was not more difficult than a standard TLE procedure.Our results confirm existing data that, in about one-third of cases, eyes will develop elevated pressure if they undergo vitrectomy after TLE . About 3We speculate that subconjunctival hemorrhage during 25GMIVS affects the preexisting filtering bleb. Many earlier reports have demonstrated the advantages and disadvantages of using autologous blood injection to treat overfiltering or leaking blebs after glaucoma surgery –33. ThusThere were limitations to our study, including the retrospective nature of the analysis, a short follow-up period, and a small number of patients. We did not discuss filtering bleb function in detail because our study included eyes with blebs whose function before 25GMIVS was doubtful (these eyes continued to need glaucoma medication after TLE). Furthermore, we did not compare 25GMIVS and 20GPPV. A comparative, prospective study in post-TLE eyes would provide valuable insights into the relative value of these techniques but is impossible due to ethical considerations, making an experimental analysis using an animal model perhaps the most useful approach for such a future study. Nevertheless, as post-TLE eyes that require glaucoma medication for IOP control before vitrectomy are commonly observed clinically and indeed comprised about 50% of the cases in our study, we believe that this is a valuable study of a useful treatment for post-TLE eyes with various retinal conditions. Further investigation is needed to evaluate postoperative visual quality and complications in the late postoperative period before a final determination can be made of the efficacy of this procedure.In conclusion, 25GMIVS is technique that can feasibly be used to treat vitreoretinal disease in glaucomatous eyes that have undergone TLE. Regardless of a high incidence of PDR with NVG in our case series, we were able to achieve good final results for BCVA and IOP with 25GMIVS, without increasing the number of glaucoma medications. Though there were a few cases that needed additional TLE following 25GMIVS, the additional TLE procedure was not more technically difficult than usual, because of the good condition of the conjunctiva after sutureless 25GMIVS. Our results showed that 25GMIVS was effective in preserving the filtering bleb and the other quadrant conjunctiva in eyes with glaucoma and did not exclude further surgical intervention for this disease.


Graphene photo-detectors functionalized by colloidal quantum dots (cQDs) have been demonstrated to show effective photo-detection. Although the transfer of charge carriers or energy from the cQDs to graphene is not sufficiently understood, it is clear that the mechanism and efficiency of the transfer depends on the morphology of the interface between cQDs and graphene, which is determined by the shell of the cQDs in combination with its ligands. Here, we present a study of a graphene field-effect transistor (FET), which is functionalized by long-ligand CdSe/ZnS core/shell cQDs. Time-resolved photo-luminescence from the cQDs as a function of the applied gate voltage has been investigated in order to probe transfer dynamics in this system. Thereby, a clear modification of the photo-luminescence lifetime has been observed, indicating a change of the decay channels. Furthermore, we provide responsivities under a Förster-like energy transfer model as a function of the gate voltage in support of our findings. The model shows that by applying a back-gate voltage to the photo-detector, the absorption can be tuned with respect to the photo-luminescence of the cQDs. This leads to a tunable energy transfer rate across the interface of the photo-detector, which offers an opportunity to optimize the photo-detection. In recent years, graphene has been widely used for the development of new electro-optical devices such as transparent electrodes45789121314151618et al.In general, the transfer consists of two parts: the charge transfer/Dexter transfer21222425Herein, we first built up a graphene field-effect transistor (FET) via a simple laser ablation method, which avoids a chemical photo-lithography process. We measured the time-resolved photo-luminescence (TRPL) on a graphene FET functionalized with CdSe/ZnS QDs at different back-gate voltages. A Förster transfer model is elucidated from the back-gate dependence of the PL lifetime. The optimization of the back-gate voltage for an efficient energy transfer is discussed together with a consideration of the device’s responsivity.τ1 = 30 ± 1 ns and a slow decay with τ2 = 7.0 ± 0.5 μs. A graphene FET was fabricated using laser ablation and electrical contacts (Ti/Au:20 nm/200 nm) were deposited using metal evaporation in combination with a laser-cut shadow mask. The cQDs employed exhibit a PL peak at 580 nm and the first exciton absorbance peak at 560 nm. The characterization of absorbance as well as the photo-luminescence of these cQDs in solution are provided in the To obtain insight into the transfer dynamics, three independent measurements with different but similar transistors were performed for the statistics, which demonstrate qualitative accordance. Indeed, those devices, which are based on the same design and are the result of the same fabrication technique, exhibited a similar structure as well as cQD coverage within fabrication tolerances cf. . The recP. We can observe a shift of the response curve at functionalization as well as at different light intensities, as shown in et al.Iph − Idark)/(P), with Iph representing the current at given illumination (P), and Idark the FET current in the dark, respectively. Such responsivity curve is plotted in et al.Furthermore, the shift of the Fermi level depending on the optical power was investigated for one of the transistors representatively. The resistance-response curve was therefore measured at different optical powers ωFRET is therefore proportional to the integral of the photo-luminescence of the quantum dots PLQD (a measure of the number of excited cQDs) and the absorbance of graphene αGr (see Since we have been investigating a system with long ligands (oleic acid), we expect the transfer to be dominated by a Förster-like energy transfer αGr see :VBG is changed, the Fermi level EF in the graphene is being tuned and the absorbance changes accordingly. In other words, by changing the back-gate voltage, one shifts the absorbance of graphene with respect to the fixed radiative transition energy, i.e. PL line, of the cQDs. The absorbance of graphene α(ω) consists of two parts, as ω)-dependent intraband absorbance αintra(ω) and the interband absorbance αinter(ω).This means that the transfer rate peaks, when the high absorbance of graphene overlaps with the PL energy of cQDs, and decreases, when the absorbance of graphene mismatches the PL energy of QDs at a certain energy detuning. If the back-gate voltage q = −1, Eres = 5.02eV. The interband absorbance can be modelled by equation (3)EF relative to the Dirac point in the frequency-dependent optical sheet conductivity σ(ω), with (π2e2)/(ch) comprising of natural constants  and kBT the thermal energy at room temperature:To include the effect of the exciton resonance, the interband absorbance has been multiplied by the fanofunction according to ref. τs = 50 fs representing the momentum relaxation time at charge neutralityThe intraband part can be modelled by a Drude modelσExp0, –conductivity at the Dirac point, represented by 1/RExp(0V) –has been normalized to the theoretical value RExp(VBG) − RContact), which takes into account the estimated contact resistance of each device, is particularly used to estimate the uncertainty of the model values extracted for the absorbance.The measured conductance α(ω) given by λ = 2πc/ω in the range of 530 nm to 640 nm (corresponding to the cQD emission line) and is plotted in RSD paired with Fermi energies. In addition, the displayed chart in RExp(VBG) are shown in −log(I/I0) in units of πα = 0.023. Here, the relative absorbance only considering interband transisions amounts to 1, while α of The absorbance τeff consisting of two parts similar to Niebling et al.τQD,pure of the cQDs  on silicon, similar to Kim The time-resolved photoluminescence (PL) measurements were performed using a frequency-doubled Nd:YAG laser (Quantel Brilliant) at 532 nm and 10 Hz repetition rate with 1.5 μJ per Pulse. The optical signal was collected and focused onto a spectrometer (Oriel Instruments MS-257) with a blaze grating of 300/500. Using a gated intensified charge coupled device (ICCD), transients of PL were measured. For current and bias voltage control, respectively, two current meters (Keithley 617) were employed. One of them was used to apply a 50 mV source-drain bias voltage to monitor the resistance of the graphene channel, while the other device was used to set the back-gate voltage. The gold contact pads on the sample were electrically contacted by tungsten needles cf. . For theHow to cite this article: Li, R. et al. Gate Tuning of Förster Resonance Energy Transfer in a Graphene - Quantum Dot FET Photo-Detector. Sci. Rep.6, 28224; doi: 10.1038/srep28224 (2016).


A rising need for workers in science, technology, engineering and mathematics (STEM) fields has fueled interest in improving teaching within STEM disciplines. Numerous studies have demonstrated the benefits of active learning approaches on student learning outcomes. However, many of these studies have been conducted in experimental, rather than real-life class, settings. In addition, most of these studies have focused on in-class active learning exercises. This study tested the effects of answering questions outside of class on exam performance for General Biology students at the University of Minnesota. An online database of 1,020 multiple-choice questions covering material from the first half of the course was generated. Students in seven course sections (with an average of ∼265 students per section) were given unlimited access to the online study questions. These students made extensive use of the online questions, with students answering an average of 1,323 questions covering material from the half of the semester for which the questions were available. After students answered a set of questions, they were shown the correct answers for those questions. More specific feedback describing how to arrive at the correct answer was provided for the 73% of the questions for which the correct answers were not deemed to be self-explanatory. The extent to which access to the online study questions improved student learning outcomes was assessed by comparing the performance on exam questions of students in the seven course sections with access to the online study questions with the performance of students in course sections without access to the online study questions. Student performance was analyzed for a total of 89 different exams questions that were not included in the study questions, but that covered the same material covered by the study questions. Each of these 89 questions was used on one to five exams given to students in course sections that had access to the online study questions and on three to 77 exams given to students in sections that lacked such access. Data from over 1,800 students in sections with access to the online study questions show that those students scored a statistically significant average of 6.6% points higher on the exam questions analyzed than students in sections without access to the study questions. This difference was greater than the average amount necessary to raise students’ exam grades by one grade . In addition, there was a higher correlation between number of questions answered and success on exam questions on material related to the study questions than between number of questions answered and success on exam questions on material unrelated to the study questions. The online study question system required substantial effort to set up, but required minimal effort to maintain and was effective in significantly raising average exam scores for even very large course sections. Due to a rising need for highly trained workers in science, technology, engineering and mathematics (STEM) fields , and forAlthough intriguing, these studies leave open the question of the extent to which providing students with access to study questions can improve overall student performance in a real classroom. To address this question, students in seven sections of BIOL 1009 General Biology at the University of Minnesota Twin Cities were provided with unlimited access to an online study question database. This online database contains 1,020 multiple-choice questions on material taught during the first half (14 lectures) of the course. After students answered a set of questions, they were shown the correct answers for those questions. More specific feedback describing how to arrive at the correct answers was provided for the 73% of the questions for which the correct answers were not deemed to be self-explanatory. The effects of access to the online study questions were assessed by comparing the performance of students with access to the online study questions to the performance of students without access to the online study questions on in-class exam questions. Use of the online study questions was found to significantly improve student performance on exams.BIOL 1009 General Biology is a one-semester (14–15 week) course. It is taught in multiple sections each term on the University of Minnesota Twin Cities campus. This course is intended for students who are not within the College of Biological Sciences, but who do have a specific need to learn biology . Course sections meet twice per week for 75 min each time. Course sections meet an additional time each week for a 2-h laboratory session. The course as a whole (lecture plus lab) is a four-credit course. Most course instructors, including the author, teach the course using primarily a traditional lecture format, but include some in-class active learning exercises as well. These in-class active learning exercises may include answering a few questions, think-pair-share or similar activities. All instructors for BIOL 1009 use the same textbook (Campbell Biology). The author taught the first half (lectures one through 14) of BIOL 1009 section 30 in 2012 and sections 1 and 30 in 2013–2015. The sections taught by the author had approximately 220 to 320 students per section.https://moodle.org/). The question bank contains a total of 1,020 questions covering material taught by the author during the first 14 lectures  of BIOL 1009 General Biology. There are an average of 73 questions per lecture, with a range of 47–98. All of the questions in the question bank are multiple-choice questions. Each question has a single “best” answer for which full credit is assigned and three distractors for which no credit is assigned. Most of the questions fit into one of the three lower levels  of a revised version (http://www.pearsonmylabandmastering.com/northamerica/masteringbiology/) that cover material from Campbell Biology Ninth Edition  in answering quiz questions. Students were allowed to attempt each quiz a maximum of three times, with only their highest score for each quiz being counted. In addition, the lowest two quiz scores were dropped for each student. These quizzes, as a whole, accounted for 10% of students’ course grades for the first half of the semester, or 5% for the course as a whole.The Biology Program at the University of Minnesota Twin Cities established a database of exam questions for BIOL 1009 General Biology in fall 1978. Each term new questions are added to this database by the different faculty members teaching the course. This database currently contains 2,284 active questions covering material from the entire course. Records are maintained for each question. These records indicate each course section for which that question was used on an exam and the percentage of students that answered the question correctly each time it was used on an exam. Using this information, an “expected” exam score can be calculated for each group of questions chosen from the exam question database. These “expected” exam scores are calculated by averaging the average facility indexes for the chosen questions. For this work, “expected” scores were calculated using only results from questions that had been used at least three times in exams for course sections for which online study questions were not available, and that were not honors sections. There were a total of 89 such questions. On average, the questions from the exam question database analyzed as part of this work were used in 24.8 exams for course sections for which online study questions were not available and that were not honors sections . In totaThere were three course exams. All exams took place in class and lasted for a maximum of 75 min (exams 1 and 2) or 120 min . Students were not allowed to use any resources  during exams. All exams consisted of multiple-choice questions with one “best” answer and three distractors. There were 40 questions each for exams 1 and 2 and 50 questions for the final exam. Exams 1 and 2 each accounted for 16.67% of students’ final course grades and the final exam accounted for 20.83% of final course grades. Exam 1 covered material from lectures one through eight. Questions for exam 1 were drawn from two sources. For each exam 1, 20–21 questions were chosen by the author from the online study questions. The remaining 19–20 questions  were choThe University of Minnesota Twin Cities IRB: Human Subjects Committee determined that the referenced study (study number 1508E77489) is exempt from review under federal guidelines 45 CFR Part 46.101(b) category #4 EXISTING DATA; RECORDS REVIEW; PATHOLOGICAL SPECIMENS. The author received this exempt study notification from the IRB via email.t-test. Correlations between numbers of questions answered versus performance on different groups of exam 2 questions were calculated as the Pearson product moment correlation coefficient, r. Calculations of the p-values for different r values were performed using an online calculator .“Actual” versus “expected” scores for questions chosen from the Biology Program exam question database were analyzed using a two-tailed, paired Student’s A database of 1,020 questions covering material from the first half (first 14 lectures) of BIOL 1009 General Biology was developed in 2012. These questions were made available online to students in seven course sections from 2012 to 2015. Students answered questions from this database both as sets of optional online “study questions” and as mandatory online quizzes. Immediately after answering a set of study questions or a quiz, students were shown the correct answers for each question. Questions they had answered incorrectly were also indicated. More detailed feedback explaining how to arrive at the correct answer or why a particular distractor is incorrect was provided for 73% of the questions. The answers for the remaining 27% of the questions were self-explanatory. This form of delayed feedback  has been shown to be particularly effective in promoting learning . A conceFrom 2012 to 2015, students answered an average of approximately 1,323 questions (an average of 94.5 questions per lecture) for the 14 lectures for which online questions were available . As a reThe majority of the questions answered were optional study questions for which no credit was available, rather than quiz questions for which credit towards final course grades was available. In 2012, for example, 76% of the questions answered by students were study questions . The aveThe Biology Program at the University of Minnesota maintains a database of questions that have been used on BIOL 1009 General Biology exams. Records are kept for each question in the exam question database. These records indicate the exams on which the question was used and the fraction of students who answered the question correctly (the facility index) on each exam. Using this information, it is possible to calculate an expected exam score for questions chosen from this exam question database. For the seven sections of BIOL 1009 that are the focus of this study, 19–20 questions from the Biology Program database were chosen for exam 1 each year and 10–13 questions were chosen for exam 2 each year. The author used the same exams for both sections of the course taught in the same year. In addition, some questions were used on exams in more than one year. To help ensure the accuracy of the results, only questions that had been used by a minimum of three course sections for which online study questions were not available (and that were not honors sections of the course) were analyzed. Consequently, a total of 89 questions (50 exam 1 and 39 exam 2 questions) chosen from the Biology Program exam question database were analyzed. These questions were used on an average of 24.8 exams, with a range of 3–77 exams, for courses for which study questions were not available .The actual scores achieved by students in the seven course sections for which online study questions were available were consistently higher than expected . The comr) of 0.36 . This study therefore indicates the desirability of providing students with opportunities for more active forms of learning outside of class. The online study question system analyzed in this work provides an effective means of achieving this goal. It requires substantial effort to set up in the first year, but requires minimal effort to maintain in subsequent years. In addition, it is effective in significantly raising average exam scores for even very large course sections.10.7717/peerj.1322/supp-1Table S1This table summarizes information on the exam questions from the University of Minnesota Biology Program exam question database for BIOL 1009 that were analyzed as part of this work. “Mean actual score” refers to the percentage of students who correctly answered the indicated question on the indicated exam for the indicated year and course section. “Mean expected score” refers to the percentage of students who correctly answered the indicated question on all exams in which the question was used, excluding exams from one of the seven course sections being analyzed as part of this work  and from honor’s sections. ”# of other exams on which question was used” refers to the number of times the indicated question has been used on a BIOL 1009 exam at the University of Minnesota, excluding exams from one of the seven course sections being analyzed as part of this work and exams from honor’s sections of BIOL 1009.Click here for additional data file.10.7717/peerj.1322/supp-2Table S2The numbers of study questions, quiz questions and total questions (study plus quiz questions) answered by each student in preparation for exams 1 and 2 during 2012 are shown. The overall exam 1 and 2 scores for each student are also indicated. In addition, the scores on different groups of exam 2 questions are shown. “Exam 2 study questions” refers to questions on exam 2 that were taken directly from the online study questions. “Questions on same material” refers to questions covering the same material covered by the online study questions; these questions were chosen by the author from the exam question database maintained by the Biology Program at the University of Minnesota (thus these questions were not written by the author). “Questions on other material” refer to questions on material not covered by the study questions; these questions were chosen by a different faculty member.Click here for additional data file.10.7717/peerj.1322/supp-3Table S3The total numbers of questions (study questions plus quiz questions) answered by each student in preparation for exams 1 and 2 in 2012 and 2013 are indicated. The average number of questions answered per lecture for each student in preparation for exams 1 and 2 are also shown. The questions in preparation for exam 1 covered material from eight lectures and the questions in preparation for exam 2 covered material from six lectures.Click here for additional data file.10.7717/peerj.1322/supp-4Table S4In 2012 through 2014, questions for exam 2 were drawn from three sources. 13–15 questions were chosen by the author from the online study questions, 11–13 questions that covered the same course material covered by the online study questions were chosen by the author from questions in the exam question database maintained by the Biology Program at the University of Minnesota  and 13–16 questions were chosen by another faculty member and covered material taught by that faculty member that was not covered by the online study questions . The number of questions answered by each student in preparation for exam 2 and the scores obtained by that student on the three groups of exam 2 questions and exam 2 overall are indicated.Click here for additional data file.


Interestingly, RCMGO-24 demonstrated a higher specific capacitance and excellent cycle stability due to its residual oxygen functional groups that accelerate the faradaic reactions and aid in faster wetting. This non-annealed strategy offers the potential for simple and cost-effective preparation of an active material for a supercapacitor electrode.An efficient active material for supercapacitor electrodes is prepared by reacting potassium hydroxide (KOH) with graphene oxide followed by chemical reduction with hydrazine. The electrochemical performance of KOH treated graphene oxide reduced for 24 h  exhibits a specific capacitance of 253 F g Supercapacitors or electrochemical capacitors, powerful energy storage systems used for a wide range of energy capture and storage applications, provide clean energy with almost zero waste emission -3. HowevGraphene can be prepared by the chemical reduction of graphene oxide (GO) -18, epitHerein we report on the preparation, through simple reactions of KOH with graphene oxide followed by hydrazine reduction, of an active material of graphene with adequate dispersibility and high storage capacity. The reactions of KOH induce the formation of oxygen functional groups and defects on graphene sheets. The reduced KOH-treated graphene oxide was analyzed with thermal gravimetric analysis (TGA), Raman, Fourier transform infrared spectroscopy (FT-IR), and electrochemical charge/discharge cycles to elucidate the physical and electrochemical performances. KOH-treated reduced graphene oxide exhibits adequate dispersibility in an aqueous medium; consequently, it induces good wettability in an aqueous electrolyte solution. Due to the presence of residual oxygen functional groups for pseudocapacitance in this KOH-treated graphene oxide, higher storage capacitance and good electrochemical stability is achieved.2SO4), potassium permanganate (KMnO4), hydrochloric acid (HCl), and hydrogen peroxide (H2O2) were purchased from Samchun Chemical . Hydrazine monohydrate (N2H4∙H2O) and KOH was purchased from Sigma-Aldrich . All chemicals were used as received without further purification.Expandable graphite  was kindly provided by Asbury Carbon. Concentrated sulfuric acid  was added and stirred for each reaction time. The reduced GO was filtered and washed with water, then dried in an oven at 80°C for 24 h. The reduced CMGO (RCMGO) sample was also prepared by applying same reaction conditions of RGO to CMGO. The sample of GO reduced for 6 h, 24 h without KOH treatment, and CMGO reduced for 24 h were named RGO-6, RGO-24, and RCMGO-24, respectively.-1. Cyclic voltammetry (CV) was conducted at 10 mV s-1 and electrochemical impedance spectra (EIS) was measured at frequencies from 100 kHz to 0.01 Hz.All electrochemical measurements were conducted in a three-electrode cell assembly equipped with a reference electrode (Ag/AgCl), platinum wire as a counter electrode, and a glassy carbon working electrode. To fabricate the electrode, 85% active material, 10% super P, and 5% polytetrafluoroethylene were mixed with ethanol and then dried to form a paste. Electrochemical performance was measured in a 2 M aqueous sulfuric acid solution as an electrolyte. The specific capacitances of RGO-6, RGO-24, and RCMGO-24 were measured at 0.2, 0.5, and 1 A gθ range from 2° to 40°. X-ray photoelectron spectroscopy (XPS) was taken on a Thermo Fisher instrument using Al Kα radiation (energy range 200 to 3 keV). Galvanostatic charge/discharge and CV were characterized using a Won A Tech (WBCS 3000) battery tester. EIS was measured using BioLogic Science Instruments .Morphologies of the RGO-24 and RCMGO-24 were characterized via field emission scanning electron microscopy . Thermal properties were characterized using thermal gravimetric analysis . The electrical conductivity of the graphene pellet was determined by the four-point probe method . The functional groups of the RGO were determined by FT-IR on a Nicolet IR 200 FT-IR spectrometer  in transmission mode, and Raman  with 633-nm wavelength incident laser light was used to characterize the degree of reduction on the graphene sheets. X-ray diffraction (XRD) analyses were performed on a high-power X-ray diffractometer  at 2As shown in Figure -1. The electrical conductivity of RGO-24 was measured to be as high as 1,980 S m-1. On the other hand, the residual functional groups obtained from KOH reaction diminish electron transport of RCMGO-24 and thus the electrical conductivity decreases to 431 S m-1. Nevertheless, there is enough electrical conductivity for charge transport during charge/discharge cycles.TGA was carried out to observe the presence of functional groups on the graphene sheets. As shown in Figure -1 originates from the stretching vibration of OH groups, and a peak at 1,727 cm-1 is due to the C = O stretching vibrations of carbonyl and carboxyl groups. The C = C of an un-oxidized graphitic domain gives a skeletal vibration at 1,590 cm-1. The peaks at 1,230 and 1,050 cm-1 are characteristics of carboxyl C-O deformation and alkoxy C-O stretching vibrations, respectively. As seen in the figure, the peak intensities of RGO-24 and RCMGO-24 were lower than those of GO, suggesting effective removal of functional groups by hydrazine. However, these graphene samples still have some surface functional groups over the basal and edge planes of the graphene sheets, which were observed at 3,400, 1,560, and 1,205 cm-1 for residual -OH, C = C, and C-O groups, respectively. The broad peak observed at 1,205 cm-1 for RCMGO-24 denotes the greater presence of residual oxygen functional groups.As shown in FT-IR spectra Figure b the bro3/2 and K2p1/2 peaks at 292.9 and 295.5 eV, respectively  yielded a value very near to that of GO. Interestingly, the reactions of CMGO at higher temperature accelerated the formation of new oxygen functional groups, so that the D band of RCMGO-24 was observed at a similar level to that of GO. Moreover, the degree of reduction and defects were also determined by calculating the ID/IG ratio of GO, RCMGO-24, and RGO-24. The ID/IG ratio of GO was calculated to be 0.97. After the reduction, the D band of GO increased to more than that of the G band (ID/IG = 1.4), suggesting that the reaction of hydrazine formed a higher reduction and larger number of defects over the graphene sheets [ID/IG ratio of RCMGO-24 is much smaller than that of RGO-24. The ID/IG ratio of RCMGO-24 was calculated to be 1.15; this small number results from the formation of a large number of oxygen functional groups on the defected areas. From XRD analysis  restored its graphitic structure, and then formed a stacked, multilayer graphene.Raman spectroscopy was used to characterize the structures of graphene by comparing G and D bands. The G band corresponds to the Er number . For RGOe sheets ,29. It is Figure , compare-1 under a potential window of 0 to 1 V. The slope of a full discharge curve  drop) was chosen to measure the specific capacitance of RGO-24 and RCMGO-24. The specific capacitance of graphene was determined by using the following equation:Next, we performed the analysis in a supercapacitor electrode to evaluate the storage capacitance and cyclic stability of RGO-24 and RCMGO-24. We believe that the crumpled structure and residual oxygen functional groups obtained from KOH treatment can effectively increase the specific capacitance of graphene sheets. Galvanostatic charge/discharge was conducted at current densities of 0.2, 0.5, and 1 A gCspec is the specific capacitance of an electrode, I is the current density (A), m is the mass of the active material (g), and dv/dt is the slope of the discharge curve. Figure -1 measured after the 10 CV (10 mV s-1) cycles. The discharge curves are close to linear over the whole potential range, implying perfect capacitance behavior [-1. The maximum specific capacitances of RGO-24 and RCMGO-24 were observed at 141 and 253 F g-1at 0.2 A g-1, respectively. The specific capacitance of RCMGO-24 is comparable to the value reported for reduced graphene supercapacitors [where behavior . Secondlpacitors ,24. The -1  represents ESR and it corresponds to the internal charge, charge/discharge rate, and wettability of the electrode, [y-axis in comparison to RGO-24, thereby confirming the contribution of higher EDLC characteristics. These interesting characteristics of RCMGO-24 indicate that the KOH treatment decreased electrical conductivity, but it significantly increased the electrode wetting, electrolyte diffusion, EDLC, and more pseudocapacitance in comparison to RGO-24.Figure ectrode, ,32,33. TA non-annealing strategy of KOH-treated GO was developed for preparing RCMGO-24 containing residual functional groups. The reactions of KOH efficiently created the oxygen functional groups and were involved in the pseudocapacitance effect; the same characteristic features were observed using a solvothermal method at elevated temperature (150°C) . The cruWe have investigated the effect of KOH treatment of GO on supercapacitor performance at various current densities. The Raman, TGA, FT-IR, and XPS spectra of RCMGO-24 confirmed that KOH functionalization efficiently prevents complete reduction and supplies residual functional groups on graphene sheets. The excellent performance of supercapacitor electrodes obtained with RCMGO-24 illustrated the key role of the KOH treatment. It promoted faster wetting of the electrode, more pseudocapacitance, and the permeation of an electrolyte solution. The electrochemical stability of RCMGO-24 was sufficient and maintained capacitance retention for up to 3,000 cycles, demonstrating that it is an excellent active material for supercapacitor electrodes.CV: cyclic voltammetry; EIS: electrochemical impedance spectra; ESR: equivalent series resistance; EDLC: electrical double-layer capacitor; FT-IR: Fourier transform infrared spectroscopy; GO: graphene oxide; IR: internal resistance; RGO: reduced graphene oxide; RCMGO: reduced chemically modified graphene oxide; FE-SEM: field emission scanning electron microscopy; TGA: thermal gravimetric analysis; XRD: X-ray diffraction; XPS: X-ray photoelectron spectroscopy.The authors declare that they have no competing interests.BR designed the work, carried out the experiment, and finalized the manuscript. JSC supervised the work and revised the manuscript. All authors read and approved the final manuscript.BR is a PhD student at the University of Ulsan (UOU). JSC has a PhD degree and is a professor at UOU.


Genetic and epigenetic abnormalities have been reported by Wissing and colleagues to occur during hiPSC derivation, including mobilization of engineered LINE-1 (L1) retrotransposons. However, incidence and functional impact of endogenous retrotransposition in hiPSCs are yet to be established. Here we apply retrotransposon capture sequencing to eight hiPSC lines and three human embryonic stem cell (hESC) lines, revealing endogenous L1, Alu and SINE-VNTR-Alu (SVA) mobilization during reprogramming and pluripotent stem cell cultivation. Surprisingly, 4/7 de novo L1 insertions are full length and 6/11 retrotransposition events occurred in protein-coding genes expressed in pluripotent stem cells. We further demonstrate that an intronic L1 insertion in the CADPS2 gene is acquired during hiPSC cultivation and disrupts CADPS2 expression. These experiments elucidate endogenous retrotransposition, and its potential consequences, in hiPSCs and hESCs.Human induced pluripotent stem cells (hiPSCs) are capable of unlimited proliferation and can differentiate  Genetic and epigenetic abnormalities have been found to result from reprogramming of differentiated cells into human induced pluripotent stem cells (hiPSCs). Here, Klawitter et al. identify endogenous L1, Alu and SVA mobilization during reprogramming, highlighting the risk of insertional mutagens in hiPSCs. Unlike human embryonic stem cells (hESCs), hiPSCs are a potential source of autologous cells compatible with the immune system of transplant recipientsin vitro2345Human induced pluripotent stem cells (hiPSCs) hold substantial promise for biomedical applications and as Alu and SVA retrotransposition, has caused widespread genome structural variation in human populations101213De novo retrotransposition events can profoundly alter gene structure, expression and function, and drive pathogenesis15168LINE-1 (L1) retrotransposons  are mobi910Alu and SVA mobilization in hiPSCs, and use an exemplar de novo L1 insertion in CADPS2 to demonstrate the potential impact of endogenous retrotransposition in pluripotent stem cells.Nonetheless, epigenome-wide remodelling20132023de novo in descendant hiPSCs. As additional controls, we used three hESC lines as benchmarks of L1 expression and pluripotency  using several combinations of reprogramming factors, as well as integrating and non-integrating delivery systems . Extensi2526ipotency .et al.P<0.05–P<0.0001, analysis of variance (ANOVA)), that peaked in earlier passages of cell lines hiPS-SB4 and hiPS-SB5  and resembled levels observed in hESCs . Consistently, L1 ORF1p was abundant in hiPSCs, based on immunoblot  to map the genomic integration sites of Alu, 53 SVA and 14 LTR non-reference genome insertions per hiPSC and hESC sample (de novo in pluripotent cells if they were not (i) reported previously in non-reference retrotransposon insertion databases9123334353637Alu and two SVA putative de novo insertions  known to be active in humans contributed putative de novo insertions1011Alu and one SVA insertion confirmed as de novo in hiPSCs and a single Alu insertion (Alu-2) in hESCs  were detected by PCR in parental cells or an earlier hESC passage, suggesting that these variants were present but were not de novo. Next, we determined the entire nucleotide sequence of 10/11 confirmed de novo retrotransposon insertions . Thus, we concluded that although RC-seq reliably detected high variant allele fraction retrotransposon insertions, a large pool of low variant allele fraction events may have been overlooked at the RC-seq thresholds used here. This would be particularly acute in the chosen hESC lines where, unlike iPSCs, cells had not undergone a recent population bottleneck in vitro. Using these parameters and the observed de novo L1 insertion counts, we estimated that hiPSC lines carried 3.7 de novo L1 insertions with allele frequencies ≥5%, on average, extrapolating to ∼1 de novo L1 insertion per cell (see Methods). However, the low number of insertions identified precluded similar estimates for hESC lines.The rate of L1-mediated retrotransposition occurring in pluripotent stem cells was difficult to accurately assess given the unknown genomic heterogeneity of each population. However, by estimating the sensitivity of RC-seq, we were able to determine the approximate L1 mobilization rate in hiPSCs. First, we identified that the overall RC-seq false positive rate was 1.5%, based on our recent PCR validation rate of 98.5% for insertions found by RC-seq in a cohort of hepatocellular carcinoma patientsng depth . At 50% de novo L1 insertions detected by RC-seq were absent from the earlier hiPSC passage surveyed and therefore may have arisen after reprogramming. To better establish the temporal dynamics of L1 retrotransposition in hiPSCs, we performed multiplex TaqMan qPCR incorporating a 5′ junction-spanning probe  of hiPS-SB4 isolated well before the later passages analysed by RC-seq (p43 and p53)  . L1-dn13and p53) . Hence, nsertion , can leade novo L1 insertions were full length, a surprising result given that most preexisting genomic L1 retrotransposition events are 5′ truncated3350de novo L1s  revealed no deleterious nonsense mutations in their ORFs  and nephron (SLC12A1) biology, as well as genes with established and predicted roles in cell cycle regulation and oncogenesis . Insertions showed a marked bias for the 5′ end of genes, with insertions falling on average in the 20th percentile of gene length measured from the annotated RefSeq transcription start site (TSS), a significant deviation from random expectation . Albeit based on a small sample of insertions, this outcome could be explained by L1 endonuclease preference for open chromatinSix CADPS2 and, as noted above, exhibited copy-number variation during hiPSC cultivation , (ii) 423 bp spanning the same region but in this case containing the 389 bp L1-dn13 insertion and its TSDs to produce a 825 bp sequence (pSHM06_02) and (iii) the 423 bp sequence on its own (pSHM06_03). We cloned each of these fragments into the triose phosphate isomerase/Renilla reporter cassette and quantified their effect on luciferase activity (CADPS2 intron sequence harbouring L1-dn13 (pSHM06_03) had the strongest inhibitory effect and reduced luciferase activity by 62%, a significant decrease beyond the constructs lacking L1-dn13 (P=0.022).To further test this possibility, we employed a human triose phosphate isomerase/Renilla luciferase reporter assay developed to monitor the effects of different introns on mammalian gene expressionactivity . InteresCADPS2 expression was ∼95% lower in hiPS-SB4_D than in hiPS-SB4_B  where L1-dn13 was identified by RC-seq, one carrying L1-dn13 (hiPS-SB4_D) and the other not carrying L1-dn13 (hiPS-SB4_B) . The idePS-SB4_B . ConsistPS-SB4_D , as distHere we have demonstrated that endogenous L1-mediated retrotransposition can occur in hiPSCs and hESCs, building upon earlier reports of engineered L1 retrotransposition in stem cells1320222333de novo L1 insertion, with the notable caveat that this calculation was based on a small number of observed events. Nonetheless, this is a much lower rate than recently found for human hippocampal neurons and glia de novo insertions with a variant allele fraction of <5% in hiPSC and hESC populations were overlooked by RC-seq at the detection thresholds used here, and these were not included in the above rate estimate. This is a major consideration in concluding whether parental cell type or choice of reprogramming vector affects endogenous retrotransposition activity in hiPSCs. Low frequency or subclonal retrotransposition may indeed occur in our hCBEC-derived hiPSC lines, that were reprogrammed via lentiviral systems, and escaped detection by RC-seq here. Therefore, we would propose that additional experiments are required to better define how these and other considerations  affect L1 activity. Indeed, one explanation for the low number of insertions characterized in hESCs is that these cell populations were not clonally derived and were therefore likely to present more extensive genomic heterogeneity than hiPSCs. The lone Alu insertion found here in H9 cells is nonetheless the first endogenous retrotransposition event reported in hESCs, reinforcing evidence that L1-mediated mobilization can occur in early human development13We estimated that hiPSCs each carried ∼1 per se may activate the expression of cellular L1 restriction factors such as APOBEC proteins22L1 activity was highly dynamic during reprogramming and hiPSC cultivation. Parental cells, early hiPSC passages, later hiPSC passages and re-differentiated cells presented grossly different levels of L1 expression. As corroborated by RC-seq, genotyping PCR and qPCR, the majority of retrotransposition in hiPSCs likely took place during or immediately after reprogramming, where we observed a peak in expression of the L1 mobilization machinery. As a result, each detected variant could affect substantial hiPSC subpopulations. Interestingly, major induction of L1 mRNA and protein expression, far in excess of that seen in hESCs and neural stem cells13de novo L1 insertions reported here were full-length was consistent with 2/3 of the engineered L1 de novo insertions characterized by Wissing et al. also being full-lengthde novo insertions in hiPSCs is unexpected as only ∼15% of L1 copies in the human reference genome and <1% of somatic L1 insertions identified in tumours are full length333650de novo L1 insertions found in hiPSCs. The mechanism of L1 5′ truncation is not fully understood. On one hand, the preponderance of 5′ truncated L1 copies in the genome has long been explained by an inability of the L1 reverse transcriptase encoded by L1 ORF2p to copy the entire template L1 RNA, either due to premature dissociation of the L1 reverse transcriptase from its RNA or competition from an unknown cellular RNase that digests the L1 RNA before completion of reverse transcriptionde novo L1 insertions in human neural stem cellsThat 4/7 of the de novo L1 insertion reported here integrated in a protein-coding gene expressed in pluripotent cells. In one case, we identified an L1 insertion (L1-dn13) that arose during hiPSC cultivation and integrated into an intron of the gene CADPS2. It remains to be determined whether acquisition of L1-dn13, and a concurrent reduction in CADPS2 expression, imbued carrier hiPSCs with a selective advantage in vitro. Furthermore, it remains unclear why transcription of the CADPS2 allele lacking L1-dn13 was reduced by >90%. To speculate, it is possible that CADPS2 expression involves a direct or indirect positive feedback loop where, for example, transcription from CADPS2 reinforces open chromatinCADPS2 expression caused by L1-dn13 could hence have a strongly negative effect on transcription from the wild-type CADPS2 allele.Each Alu and SVA insertions can alter cellular phenotype and are associated with numerous instances of human disease2345In closing, it is notable that intronic L1, OCT4, SOX2, KLF4 and c-myc) or OSKML (OSKM+LIN28) expression cassettes5 cells per well) were transfected by nucleofection (Lonza) according to the manufacturer’s instructions. In each transfection, 2 μg of transposon plasmid (pT2-OSKM or pT2-OSKML) and 0.2 μg of CMV- SB100X vector , 20% Knockout Serum Replacement (Gibco), 1 mM L-glutamine (Biochrom AG), 50 μM β-mercaptoethanol and 0.1 mM nonessential amino acids. The medium was replaced every day. Newly formed hiPSC colonies were picked, transferred to Matrigel-coated 24-well plates, and expanded for 4–6 days in MEF-conditioned ESC medium. Subsequently, cells were trypsin dissociated, plated onto feeder cells and cultivated in ESC medium.hiPSC lines hiPS-SB4 (hFF-T2-OSKM) and hiPS-SB5 (hFF-T2-OSKML)/hiPS-SB5.1 (hiPS-OSKML#6) were generated by reprogramming HFF-1 cells (ATCC-Number: SCRC-1041) using Sleeping Beauty (SB) transposon-based plasmids pT2-OSKM or pT2-OSKML which contain polycistronic OSKM  based on their positivity for SSEA4. Six SB-OSKML hiPSC clones and one SB-OSKM hiPSC clone were then characterized for pluripotency and differentiation potential as describedThe lines hiPS-CRL1502 ref. , hiPS-CRhttp://www.geneastemcells.com.au). It formed well-defined colonies with compact cells displaying a high nuclear to cytoplasmic ratio and prominent nucleoli. Karyotype analysis  did not uncover any abnormalities at passage 42. HESG cells express pluripotency markers Nanog, Oct-4, Tra1-60 and SSEA4, stain positive for alkaline phosphatase and form teratomas. As for hiPSCs, hESCs were grown on gelatin-coated six-well plates (Greiner) on inactivated mouse embryonic fibroblasts . MEFs were expanded and mitotically inactivated by γ-irradiation with a Cesium source with 30 Gy after 3–7 passages, and stored in liquid nitrogen until further use. After thawing, MEFs were seeded at a density of 6 × 105 cells per well of a six-well plate. hESC medium was replaced daily and cells were passaged at a 1:2 dilution every 5 days using splitting medium  in KO-DMEM).hESC lines H1, H9 and HES-3 were purchased from the WiCell Research Institute  and Cythera Inc. . The H1 line was used exclusively for the isolation of cell lysate that was loaded as positive control of the immunoblot analysis in hiPSCs were washed once in PBS containing 0.5% bovine serum albumin, and incubated for 30 min with allophycocyanin-conjugated anti-human SSEA4 antibody (R&D Systems). In all samples an anti-mouse Sca-1 (Ly-6A/E)  antibody was employed, for gating out the positively labelled mouse feeder cells. Samples were analysed and sorted using an Aria High Speed Cell Sorter (Becton-Dickinson).−1; Gibco) per well of a six-well tissue culture plate. Next, cells were resuspended in 750 μl of ESC medium, transferred to a 15 ml conical tube and centrifuged at 800 r.p.m. in a Heraeus Multifuge 4KR for 3 min at room temperature. Subsequently, medium was removed, cells were resuspended in 3 ml of ESC medium without FGF2 and cultured for 1–16 days in T25 flasks (Greiner) containing 10 ml of ESC medium without FGF2. At the indicated time, embryoid bodies were harvested and cytoplasmic RNA was isolated as described below. Passage 10 of the hiPSC line hiPS-SB5.1 was cultured in one well of a GeltrexTM-coated six-well culture dish, and treated with collagenase IV (1 mg ml−1) for 5 min. Cells were washed with warm PBS twice, and fed with 1 ml embryoid body formation medium ) and split into small cell clumps. hiPSC colonies were then dissociated with collagenase IV (1 mg ml−1) for 5 min, and split into small cell clumps. Cell clumps were transferred into three 10-cm low-attachment dishes and fed with embryoid body medium. The medium was changed every 2 days. Embryoid bodies were cultured for 8 days in total. Embryoid bodies were collected by sedimentation under gravity from three dishes on day 0 (undifferentiated hiPSCs), 2, 4, 6 and 8, respectively . For each time point and transcript to be quantified, qRT–PCR analyses were done in triplicate. qRT–PCR for pluripotency/differentiation markers was carried out using Power SYBR Green PCR Master Mix (Applied Biosystems) on the ABI7900HT sequence detector (Applied Biosystems), and data was normalized to GAPDH expression. qRT–PCR for L1 was performed with ABsolute QPCR Mix (ABgene), and data was normalized to 18S rRNA expression.6 to 3 × 107 somatic cells, hiPSCs or embryoid body cells using the RNeasy Midi Kit  according to the manufacturer’s instructions. Cytoplasmic RNA (0.5–1 μg) was incubated with 2 U of RNAse-free DNaseI  for 30 min at room temperature. DNAseI digestion was stopped by adding 2 μl of 25 mM EDTA and incubation for 10 min at 65 °C. DNAseI-digested cytoplasmic RNA (0.1–0.5 μg) was used for cDNA synthesis using the SuperScript III First-Strand Synthesis Kit (Invitrogen) in combination with a Random Hexamer Primer  according to the manufacturer’s instructions. Quantitative real-time PCR was carried out in ABgene plates using an Applied Biosystems 7900HT Fast Real-Time PCR System. The primer and probe combination L1 5′UTR#2  spanning exon sequences (Cytoplasmic RNA was extracted from 5 × 10R#2 ref.  was usedequences . Cycling+-Nylon membrane (Amersham) by overnight capillary transfer using 10 × SSC as transfer buffer. A total of 4 μl RiboRuler High Range RNA ladder  were loaded as size marker. After crosslinking the RNA onto the membrane by ‘baking’ at 80 °C for 2 h, the membrane was prehybridized overnight in 50% Formamide/4xSSC/1%SDS/2 × Denhardt’s at 42 °C. The full-length L1 mRNA-specific probe was generated by PCR amplification of a 1299-bp L1 fragment ranging from position numbers (pos.) 58–1356 of a full-length L1 element by using primers L1_FW1 and L1_RV1 (RPΔCMV32P]dCTP by applying the Nick Translation System (Invitrogen) according to the manufacturer’s instructions.Total RNA was isolated from the cell lines HFF-1, 2102Ep ref. , HES-3 ad L1_RV1  and pJM1V1 RPΔCMV and plasV1 RPΔCMV as templAfter denaturing the probe for 10 min in boiling water and subsequent incubation for 10 min in ice water, the probe was added to the hybridization buffer (50% Formamide/4 × SSC/1% SDS/1 × Denhardt’s) and the membrane was incubated in the probe-containing hybridization buffer overnight at 42 °C. Subsequently, the membrane was subjected to two 5 min low-stringency washes (2 × SSC) at room temperature and one 30 min high-stringency wash (2 × SSC/0.5%SDS) at 65 °C. The membrane was stripped by being boiled for 30 min in a solution of 10 mM tris-HCl (pH 7.5)/1 mM EDTA/1 mM SDS. The hybridized membrane was exposed to X-ray films for 5–10 days with intensifying screens.http://www.repeatmasker.org/cgi-bin/WEBRepeatMasker. Next, the fraction of unmethylated CpG sites was calculated by comparison to a consensus L1-Ta sequence. In addition, each individual sequence was compared to L1.3 and only the sequences with the highest homology to this sequence were used to plot methylation data in single clones .Bisulfite DNA sequencing analyses were performed as previously described20e clones . The pro−1 collagenase type IV/DMEM and washed subsequently in 1 × PBS. Cells were spun down, resuspended in lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 10% Glycerin, 1% Triton X-100; 2 mM EDTA, 2 mM EGTA, 40 mM β-Glycerolphosphate disodium salt hydrate, 50 mM NaF, 10 mM Na4P2O7, 200 μM Na3VO4, 2 mM DTT; 1 × complete protease inhibitor cocktail (Roche Applied Science)), homogenized by passing the lysate ten times through a 26 G needle, and lysates were cleared by centrifugation. A total of 50 μg of each protein lysate were boiled in 3 × SDS sample buffer (NEB), loaded on 4–12% Bis/Tris gels (Invitrogen), subjected to SDS–polyacrylamide gel electrophoresis, and electroblotted onto nitrocellulose membranes. After protein transfer, membranes were blocked for 2 h at room temperature in a 10% solution of non-fat milk powder in 1 × PBS-T ), washed in 1 × PBS-T, and incubated overnight with the respective primary antibody at 4 °C.hiPSC colonies were detached from their tissue culture dish by incubation with 250 μl of a 1 mg mlL1 ORF1p and Oct-4 proteins were detected using the polyclonal rabbit-anti-L1 ORF1p antibody #984 ref.  at a 1:2hiPSCs as well as their parental HFF-1 or hCBEC cells were grown on glass cover slips in 12-well plates. Cells were washed with 1 × PBS, fixed with 4% paraformaldehyde in 1 × PBS (pH 7.4) for 15 min at room temperature and permeabilized with 1% Triton X-100 (Sigma) in 1 × PBS for 10 min at room temperature. Subsequently, cells were washed thrice for 2 min in 1 × PBS. Cells were blocked by incubation with 5% (w/v) BSA/0,1% Triton X-100/1 × PBS (pH 7.4) for 30 min at room temperature followed by incubation with the respective primary antibodies, which are listed in 6 cells from each hESC and hiPSC line and their respective parental cells using DNAzol Genomic DNA Isolation Reagent  according to the manufacturer’s instructions. RC-seq and subsequent computational analyses were performed as described using the hg19 reference genome sequencede novo insertions supported by at least two unique amplicons separated by ≥5 nt (the minimum threshold for reporting) is provided in de novo L1 insertion with ≥2 unique reads at a given sampling depth, normalized to the corresponding false negative rate identified above and then determined the cumulative sum of this distribution for frequencies of 5–100%, leading to an estimate of ∼1 de novo L1 insertion per hiPSC. We did not consider de novo L1 insertions carried by fewer than 5% of hiPSCs in this estimate as none of the validated examples were routinely identified at that sampling depth. We also did not analyse the L1 mobilization rate in hESCs or the Alu or SVA rate in hiPSCs or hESCs due to the small number of confirmed true positive examples.Genomic DNA was isolated from 1 × 10de novo L1 insertions at the 5′ end of genes was performed by random sampling of genomic coordinates, with respect to RefSeq annotations. 1 × 106 permutations were performed and in 6,000 instances the average position was less than the 20th percentile of gene length, indicating P<0.006.A permutation test showing enrichment for validated de novo insertions  detected by RC-seq were first assayed with PCR using a standard empty site/filled site genotyping assay. Primers were positioned on either side of the insertion site so that the predicted PCR product of the empty site covered <300 bp. Additional retrotransposon specific primers were designed and paired with the existing insertion site-specific primers if required. In cases where an insertion was detected by RC-seq at one terminus only, PCR and capillary sequencing were applied to the remaining end to resolve integration site structure. PCR reactions contained 0.125 μl Crimson Taq (New England Biolabs), 5 × PCR-buffer, 10pMol of each Primer, 10 mM dNTPs and 10–20 ng genomic template DNA in a total volume of 25 μl. The following cycling conditions were used: 95 °C for 2 min, then 35 cycles of 95 °C for 30 s, 58 °C for 30 s, 68 °C for 40 s, followed by a single extension step at 68 °C for 5 min. Optimization in some cases required adjusted annealing temperatures and cycle number. PCR products of the correct size n repeat and Alu-like region, respectively. Combinatorial use of these genome-SVA primer pairs did either not result in a PCR product or generated non-specific products. For a complete list of used primers see de novo insertions  were confirmed by PCR as de novo. Six additional insertions were determined as germline insertions, already present in the parental cell line or an early hESC passage. Control genotyping PCR of the single-copy gene GAPDH in genomic DNA preparations of parental and hiPSC lines used for RC-seq and PCR validations of de novo insertions is presented in Seventeen ect size  that wer2 3′ end , at the de novo insertions L1-dn13 and L1-dn14 within the hiPS-SB4 culture, we applied real-time qPCR using TaqMan fluorogenic probes. To this end, genomic DNA was isolated using 1 ml DNAzol Genomic DNA Isolation Reagent  from 1 × 106 cells, according to the manufacturer’s instructions. A total of 100 ng of genomic DNA was used for quantitative real-time PCR (qPCR). Primer and probe combinations specific to the genomic 5′ junctions of the de novo insertions L1-dn13 and L1-dn14  was used. Cycling conditions were: 95 °C for 15 min (one cycle), 95 °C for 15 s and 60 °C for 1 min (40 cycles). For analysis of real-time and end point fluorescence, the software SDS version 2.3 as well as RQ manager 1.2 (Applied Biosystems) were used.To determine relative copy numbers of  L1-dn14  were usede novo retrotransposition event, were magnetically separated from feeder cells by applying a Feeder Removal Kit  according to the manufacturer’s instructions. hiPSCs were counted and seeded on feeder-coated 96-well plates  at a cell density of one cell per well or 0.3 cells per well. hiPSCs were grown for 24 h in the presence of 10 μM ROCK inhibitor . Subsequently, cells were cultivated until they formed a single colony per well. Single colonies were transferred to feeder-coated 12-well plates (Thermo Fisher/Nunc) and further expanded. To isolate genomic DNA from each clone, cells were harvested after collagenase IV treatment, centrifuged, washed and pelleted again. Genomic DNA was isolated as described in the previous paragraph. Genotyping PCR conditions applied to screen for the presence of the L1-dn13 insertion and to demonstrate its presence/absence  and primers located 50 bp upstream/downstream the insertion site (available upon request). For each PCR we used: 0.3 μl Expand Long Template Taq (Roche), 1 × buffer#1, 400 μM dNTPs, 1 μM each Primer and 300 ng genomic DNA in 50 μl per tube. Cycling conditions were: 95 °C for 5 min, then 30 cycles of 95 °C for 1 min, 56 °C for 30 s, 68 °C for 6 min, followed by a single extension step at 68 °C for 10 min. To avoid the generation of mutations that may lead to retrotransposition defective elements, we conducted at least four independent PCRs per L1. PCR products were resolved on 0.9% agarose gels, and fragments of the expected length of ∼6 kb representing potential full-length L1 elements were excised and purified using a Qiaquick kit (Qiagen) and cloned in the Topo-XL plasmid (Invitrogen). Each of the cloned PCR products carrying full-length L1 elements L1-dn4, L1-dn6 and L1-dn14 were sequenced  supplemented with L-glutamine, Penicillin/Streptomycin, and 10% fetal bovine serum  and passaged using Trypsin 0.05% (GIBCO-Invitrogen). 104 HeLa cells per well were plated in triplicate using six-well tissue culture plates. After 18 h, cells were transfected with 1 μg per well of plasmid using 3 μl of Fugene6 (Promega) following manufacturer’s instructions. Next day, medium was replaced and cells cultured for five additional days. Six days after transfection, Blasticidin-S (Invitrogen) was added to a final concentration of 10 μg ml−1 and cells were cultured for seven days in the presence of the antibiotic. Next, plates were fixed and stained with crystal violet, and foci counted manually.Retrotransposition assays in HeLa cells were conducted as described previously94652R2=0.79). Analyses were performed with SAS/STAT software, version 9.2 SAS system for Windows.The statistical evaluation of relative L1 mRNA levels determined by qRT–PCR was performed by ANOVA, using Bonferroni correction for multiple comparisons with the same control group. Reduction in full-length transcript levels in the embryoid body time kinetics experiment was evaluated by means of Linear Regression for data from day 0 to day 8 (Accession codes: The RC-seq FASTQ data have been deposited in the Sequence Read Archive (SRA) under accession code PRJEB3191.How to cite this article: Klawitter, S. et al. Reprogramming triggers endogenous L1 and Alu retrotransposition in human induced pluripotent stem cells. Nat. Commun. 7:10286 doi: 10.1038/ncomms10286 (2016).Supplementary Figures 1-15, Supplementary Tables 1-3, Supplementary Methods and Supplementary ReferencesEndogenous retrotransposon insertions in analyzed hiPSC and hESC lines identified by RC-seq


This study, and in particular the suggestion of a mechanism for epitaxial growth of oxides on graphene, offers new directions to exploit the development of oxide/graphene multilayer structures and devices.The transfer process of graphene onto the surface of oxide substrates is well known. However, for many devices, we require high quality oxide thin films on the surface of graphene. This step is not understood. It is not clear why the oxide should adopt the epitaxy of the underlying oxide layer when it is deposited on graphene where there is no lattice match. To date there has been no explanation or suggestion of mechanisms which clarify this step. Here we show a mechanism, supported by first principles simulation and structural characterisation results, for the growth of oxide thin films on graphene. We describe the growth of epitaxial SrTiO Therefore, it is necessary to develop a method for deposition of atomically uniform oxide layers without damaging the underlying graphene and without creating high interface defect density between the graphene and oxide materials3 (STO) thin films on graphene, which were prepared by chemical vapour deposition (CVD) and subsequently transferred onto STO and MgO substrates. The suggested mechanism is supported by density functional theory (DFT) modelling and verified by structural and electrical characterisations.Herein, we propose a growth mechanism that explains the formation of epitaxial SrTiO3 (Gr/STO) interface was modelled in a 2D-periodic square cell of side 8.85 Å containing 10 STO formula units representing four atomic layers of the (100) surface in a 2 terminations in a 2 surface layer. The average binding energy of the graphene to the oxide surfaces is 56 meV per C-atom. Calculation of the electrostatic field above the sheet establishes that the corrugation in the electrostatic potential is negligible suggesting that a complete graphene sheet will screen oxide overlayers from the direct influence of the substrate. This is confirmed by an explicit calculation of the corrugation potential computed by adding a periodic SrO sheet above the graphene layer and computing its energy on a 10 × 10 grid of points as it slides across the surface (perpendicular atomic coordinates optimised for each configuration). The resultant energy surface has a corrugation of less than 1 meV per carbon atom. Undisturbed through-graphene layer epitaxial growth mediated by wrinkling of the graphene sheet or electrostatic interactions is therefore highly unlikely.The graphene/SrTiOnterface . In the The ease with which the sheet slides across the oxide surface suggests an alternative mechanism for epitaxial growth. Defects in the sheet introduced during the graphene growth or transfer provide reaction centres for the adsorbing oxide and may allow bridge structures to form between the substrate and the oxide overlayer. In this scenario, an oriented single crystal STO film on a graphene layer might be supported by epitaxial pillars grown through the defects in the graphene layer (e.g. grain boundaries) . Within −8 Torr). The thin film growth was monitored using reflection high energy electron diffraction (RHEED). The specular RHEED intensity shows a sharp decrease and a recovery during deposition of the first unit cell, illustrated in In this study, 100 nm STO thin films were grown on Gr/MgO and Gr/STO by PLD at 850 °C in ultra-high vacuum (~10l) peaks from 2θ of 20° to 120° (not fully shown here). This indicates that STO was epitaxially grown on Gr/STO. To further prove the growth of STO on graphene, STO films were deposited on Gr/MgO. The (002) STO peak clearly appeared on the XRD pattern of STO/Gr/MgO, shown in D, G and 2D) as shown in D peak intensity (ID) at ~1350 cm−1 is related to the amount of disorder in graphene layers22−1, denoted by G, is caused by the doubly degenerate zone centre photon E2g mode242D peak located at around 2690 cm−1, is induced by a double resonance electron-phonon scattering processG peak and 2D Peak) on the Raman spectrum of Gr/MgO. The absence of D peak reveals a higher crystalline quality of the graphene layer2D peak intensities, I2D/IG, on Gr/STO and Gr/MgO are approximately 2, which are characteristic for monolayer graphene.Raman spectroscopy of random regions on the samples was undertaken to confirm the presence of graphene after STO growth. The Raman spectrum of Gr/STO has three prominent peaks  or even to less than 1 (STO/G/STO sample), see more details in 2 molecules27282931G and 2D peaks along with decrease of the full width at half maximum (FWHM) value of G peak27I2D/IG ratio is attributed to the compressive strain in the graphene and formation of multilayer graphene caused by the construction of STO pillar structures on the defects.Raman spectra of STO/Gr/STO and STO/Gr/MgO samples are not very different from those of samples without STO thin films. No appreciable peak position shift is detected. But the full width at half maximum (FWHM) values of film/Gr/STOsubstrate structure. The calculated geometry suggests that a distance about 0.3 nm is optimal. Also, there are well detected areas  thin films on graphene transferred onto STO and MgO substrates was revealed. The initial local defects in the graphene layer (e.g. grain boundaries) act like bridge-pillar spots that enable the epitaxial growth of STO thin film over the graphene layer. The growth of epitaxial STO pillar structures increases the interfacial sheer stress, which results in partial folding of the graphene layer. The results were confirmed by DFT modelling, HRTEM studies, XRD and Raman spectroscopy. The I-V measurement of the graphene after the STO film growth explicitly confirmed the conservation and the existence of the graphene layer after the deposition process. The evidence provided suggests that it is indeed possible to grow epitaxial oxide films on graphene.2 gas for 10–60 minutes. The growth was conducted in a high temperature tube furnace by introducing 40 sccm of CH4 gas at a total pressure of ~8 mbar for 10–30 minutes. Samples were unloaded from the quart tube after cooled to RT. Subsequently, a Poly(methyl methacrylate) (PMMA) layer was spin-coated on graphene to facilitate the transfer of graphene onto STO and MgO substrates. Then, the copper foils were etched. Finally, the PMMA/graphene layers were transferred onto STO and MgO substrates.Single layer of graphene was grown on copper foils at temperatures up to 1000 °C by CVD. Before growth, the copper foils (25 μm thickness) were pre-treated with acetic acid solution (50%) for 20 minutes and annealed at 1000 °C under 80 sccm H2 termination.However, before transferring graphene onto STO substrate, the surface of STO substrate was thoroughly cleaned in ultrasonic bath using acetone, isopropanol, and deionized water, then dipped in buffered HF etchant. The etched STO substrates were annealed at 1000 °C for 1 hour before transfer. The treated surface of the STO substrate is believed to be atomically flat and dominated by TiOAfter the graphene transfer, it is vital to eliminate the deleterious effects of the additional PMMA layer on top of the graphene layer. Therefore, as transferred samples were heated to 400 °C for 1 hour in ultrahigh vacuum (UHV) chamber to remove undesired PMMA and chemisorbed impurities, such as oxygen, carbon dioxide, carbon monoxide and water.−8 Torr) using a KrF excimer laser (λ = 248 nm). A stoichiometric single phase STO target was ablated with a laser fluency of 2 J/cm2 at 2 Hz. After deposition, as grown film was slowly cooled down to 600 °C at a rate of 10 °C/min in UHV and was subsequently in-situ post-annealed at 300 Torr O2 pressure for 1 hour to compensate the potential oxygen loss during the growth.Gr/STO and Gr/MgO samples were annealed at 850 °C in vacuum before deposition. To prevent destruction of the graphene layer via thermal oxidation, STO thin films (100 nm) were grown on Gr/STO and Gr/MgO at 850 °C by PLD in UHV .The surface of graphene was investigated with the aid of a high resolution field emission scanning electron microscope (FEG-SEM) LEO GEMINI 1525 FEG-SEM with an accelerating voltage of 5 kV and working distance of ~6.5 mm. Cross-sectional samples were prepared using an FEI Helios dual-beam and final thinning was done at 500 eV with an Ar ion beam on a Fischione Nanomill. Transmission electron micrographs were taken using an aberration-corrected (at the image plane) FEI Titan 80–300 scanning/transmission electron microscope (S/TEM). All analysis was done with electron energy of 80 keV.How to cite this article: Zou, B. et al. Growth of Epitaxial Oxide Thin Films on Graphene. Sci. Rep.6, 31511; doi: 10.1038/srep31511 (2016).