TAR DNA binding protein 43 KD (TDP-43) is an essential gene that regulates gene transcription, mRNA splicing and stability. In amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two fatal neurodegenerative diseases, TDP-43 is fragmented, generating multiple fragments that include the C-terminal fragment of ∼25 KD. The role of these fragments in the pathogenesis of ALS and FTD is not clear. Here we investigated the aggregation propensity in various polypeptide regions of TDP-43 in mammalian cells and the effect of these fragments on cultured neurons. By expressing the full length and various TDP-43 fragments in motor neuron-derived NSC-34 cells and primary neurons, we found that both N- and C-terminal fragments of TDP-43 are prone to aggregate and the C-terminal end of RRM2 region is required, though not sufficient, for aggregation. The aggregation of the TDP-43 fragments can drive co-aggregation with the full-length TDP-43, consequently reducing the nuclear TDP-43. In addition, the TDP-43 fragments can impair neurite growth during neuronal differentiation. Importantly, overexpression of the full-length TDP-43 rescues the neurite growth phenotype whereas knockdown of the endogenous TDP-43 reproduces this phenotype. These results suggest that TDP-43 fragments, particularly the pathologically relevant C-terminal fragments, can impair neuronal differentiation by dominant-negatively interfering with the function of the full length TDP-43, thus playing a role in pathogenesis in ALS and FTD. TAR DNA binding protein-43 (TDP-43) has been identified as a major component of the ubiquitin-positive protein inclusions in neurons and glia in ALS, FTD as well as several other neurodegenerative disorders In both ALS and FTD, TDP-43 is fragmented, resulting in lower molecular weight C-terminal fragments of ∼35 to ∼25 KD To further understand the aggregation propensity of TDP-43 and the role of TDP-43 fragments in pathogenesis, we engineered a series of TDP-43 truncation fragments and expressed these fragments in cultured neurons. We found that both the C-terminal and the N-terminal fragments of TDP-43 can form aggregates as long as they contain the C-terminal end of the RRM2, which is necessary but by itself is insufficient for aggregation. The aggregation of the TDP-43 fragments can drive co-aggregate with the full-length TDP-43, thereby reducing nuclear TDP-43. Furthermore, the aggregation-prone fragments can impair neurite growth during neuronal differentiation. Importantly, this impairment can be rescued by an overexpression of full-length TDP-43 and can be reproduced by a knockdown of the endogenous TDP-43 expression. These results suggest a model where the C-terminal fragments of TDP-43, found in ALS and FTD patients, harm neurons by a dominant-negative mechanism.To understand the properties of fragmented TDP-43, we made a series of constructs that synthesize the N-terminal and C-terminal truncated TDP-43 fragments . We taggIn general, cells transfected with the C-terminal fragments 2, 3 and 4 had a high percentage of aggregate-containing cells than the cells transfected with the N-terminal fragments 6, 7 and 8 . In cellTo investigate the functional consequence of TDP-43 aggregation, we examined the cell death/viability. By several assays, including trypan blue staining, MTS assay, counting EGFP-fluorescent cells at 1, 2 and 3 days after transfection, TUNEL, activated caspase 3 staining and LDH release, we did not find a significant difference among the cells transfected with the constructs expressing EGFP and various TDP-43 fragments -expression construct that silences TDP-43 expression. This construct employed the CAG promoter to drive expression of a primary miRNA, which encode a red fluorescent protein (dsRed2) and a pre-miRNA targeting mouse TDP-43  [35]. ThTo confirm the neurite phenotype observed in NSC-34 cells, we transfected the constructs into the rat forebrain neural precursors and then differentiated them into neurons. The neurons transfected with full-length TDP-43-EGFP, the N-terminal fragments (construct 6-10) and the RRM2 fragment (construct 11) grew normal neurites that were not different from the cells transfected with EGFP alone . In contTo determine whether TDP-43 is required for neurite growth in primary neurons, we also conducted the knockdown experiments. Because the transfection efficiency was low, we first tested whether our two miRNAs (which were designed to silence mouse TDP-43) could knockdown rat TDP-43 by transfecting our constructs into rat-2 cells, which can be transfected with above 90% efficiency. Although both miR-TDP43a and miR-TDP43b matched rat TDP-43 mRNA sequence well, miR-TDP-43a did not knockdown rat TDP-43 (data not shown), probably due to the sequence differences in the regions flanking the miRNA target between the rat and mouse TDP-43 mRNA. miR-TDP43b knocked down rat TDP-43 efficiently . TherefoThe above data are consistent with the hypothesis that TDP-43 fragments that contain RRM2 are prone to aggregation and impairing neurite growth by dominant-negatively interfering with the normal TDP-43 function. The functional impairment could be mediated through the interaction between the TDP-43 fragments and the full-length TDP-43. To test this possibility, we cotransfected the EGFP-tagged TDP-43 fragments with a full-length TDP-43 that was tagged with a red fluorescent protein tdTomato (RFP). Unlike the EGFP-tagged TDP-43, this construct expressed the full-length fusion protein and unfused tdTomato was detected . When coTo determine whether the aggregation reduces the endogenous nuclear TDP-43, we transfected the cells with the EGFP-tagged fragments. To avoid the interference from the transfected TDP-43 fragments, we observed the endogenous TDP-43 using antibodies against the N- or C-terminal peptide. In cells transfected with wild type TDP-43, there was an increase in the nuclear TDP-43 staining compared with the untransfected cells , indicatTo rule out the possibility that the above observations were caused by the tags, we constructed untagged constructs and transfected them into NSC-34 cells. Like the tagged constructs, constructs 1 (the full-length TDP-43) and 11 did not form aggregates whereas the aggregation-prone constructs  formed aggregates . FurtherA well-known function of TDP-43 is modulation of mRNA splicing. A best characterized example is that it promotes exon 9 skipping during the CFTR mRNA splicing Our data show that both the C-terminal and the N-terminal fragments of TDP-43 can form aggregates in mammalian cells as long as they contain the C-terminal end of the RRM2 . The C-tConsistent with this model, in FTD and ALS patients, the C-terminal fragments are generated Our result that the RRM2 is necessary for TDP-43 aggregation agrees with the recent literature, where all fragments capable of forming aggregates in cells contain the RRM2 region The C-terminal half of TDP-43 is important for pathogenesis. In addition to the presence of the C-terminal fragments in the CNS tissues from the patients, most ALS-associated mutations are located in this region Our data indicate that the neurite growth can be an in vitro readout for the toxicity of TDP-43 fragments. Observations from other groups support the relevance and the reliability of this assay. In Drosophila, a loss of TDP-43 function leads to a decrease in dendritic branching during the development of larva nervous system While the data from our experiments and the literature are consistent with a dominant-negative effect of the C-terminal fragments in neurodegeneration, this does not preclude a role from other properties of TDP-43 in the pathogenesis. For example, numerous studies have shown that the full-length TDP-43 can drive aggregation and toxicity when TDP-43 is overexpressed in vitro and in vivo In summary, our results suggest a model where the C-terminal fragments cause neuronal toxicity by a dominant-negative mechanism. This model does not preclude a role of toxicity from the other properties of TDP-43 protein and is consistent with the data in the current literature. Many details in this model remain hypothetical and will require data from experiments to confirm. Nevertheless, this model may be a useful guide for designing future experiments.5′-AAGGTGTTTGTTGGACGTTGT-3′ for miR-TDP43a and 5′-ATGCTGAACCTAAGCATAA-3′ for miR-TDP43b. The sense and antisense strands of DNA encoding the miRNAs were synthesized, annealed and digested with KpnI and XhoI. They were then cloned into an intron in the 3′ untranslated region of the dsRed2 gene To construct the full-length and truncated TDP-43 tagged with EGFP, the open reading frame (ORF) of human TDP-43 was amplified by PCR using the forward primers with XhoI restriction site at the 5′ end and the reverse primers with BamHI restriction site at the 5′ end. The PCR product was digested with XhoI and BamHI and inserted into the pEGFP-N1 Vector (BD Biosciences Clontech) that was digested using the same restriction enzymes. To construct the full-length TDP-43 tagged with tdTomato (kindly provided by Dr. Roger Tsien 3 cells/cm2 in the medium consisting of 1∶1 DMEM and Ham's F12 (Gibco BRL) supplemented with 1% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mM glutamine. After 72 hrs, the cells were fixed using 4% paraformaldehyde and 0.1% glutaraldehyde in PBS.NSC-34 cells 6 cells per 100-mm dishes precoated with 15 µg/mL poly-L-ornithine (Sigma) and 1 µg/mL fibronectin , and treated daily with 10 ng/mL of basic fibroblast growth factor 2 (FGF2) (R&D Systems). At 5 d in vitro, cells were passaged and plated out at 0.8–1.0×106 cells per precoated plate and expanded as above for an additional 3–4 d . Cells were passaged again and plated out at 1.2–1.4×106 per precoated plate. The cells were then transfected with constructs using Lipofectamine 2000 (Invitrogen). Two days after transfection, FGF2 was removed to induce neuronal differentiation. After another 2 days, cells were washed once with PBS and fixed.For primary neuronal cultures, neural precursors were prepared from forebrain of embryonic day 14.5 (E14.5) SASCO SD rat embryos . Cells were plated out at 1.2–1.4×10HeLa cells (ATCC) were grown at 37°C in DMEM/10% FBS. The cells were then transfected with constructs using Lipofectamine 2000 (Invitrogen), cultured for 48 hours and then fixed.NSC34 cells were transfected with different constructs and 72 hours after transfection, cell viability/death assays were performed. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were performed using the In Situ Cell Death Detection kit, TMR red  according to the manufacturer's instructions. The 3--5-(3-caboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)-based cell proliferation assay (MTS assay) was carried out using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay , according to the manufacturer's instructions. Absorbance at 490 nm was measured in a microplate reader . After 72 hours transfection, the medium was collected for NSC34 cells and a LDH assay (Promega) was used to measure LDH levels as an indicator of toxicity. Cells were then fixed and immunofluorescent staining was done using an antibody against activated caspase-3  and an Alexa Fluor®568-conjugated goat anti-rabbit IgG .After fixation for 15 min at room temperature, cells were permeabilized by using 0.5% Triton in PBS for 5 min. Nuclei were stained with 4-, 6-diamidino-2-phenylindole (DAPI). To stain for TDP43, the fixed cells were incubated in the blocking buffer  for 15 min at room temperature and then incubated with a rabbit polyclonal antibody  at 1∶100 dilution or N- or C-terminal rabbit polyclonal antibody (Kind gifts from Dr. Virginia Lee) at 1∶1000 dilution in the blocking buffer at 4°C. After overnight incubation, cells were washed and incubated with Alexa Fluor®568-conjugated goat anti-rabbit IgG (invitrogen) at room temperature for 1 hour. To stain Tubulin, a mouse monoclonal anti-α-tubulin (1∶100) and Alexa Fluor®488-conjugated goat anti-mouse IgG (invitrogen) were used.The cells were visualized and photographed using a Nikon Eclipse Ti fluorescence microscope. For observation of nuclear aggregates, a series of optical sections were taken along the z-axis and processed by deconvolution  to ensure that the aggregates are within the bounds of the nucleus. For quantification of neurites, the cells with at least one neurite longer than 10 µm in NSC-34 cells or longer than 30 µm in primary neurons were counted as having neurites. Otherwise, the cells were counted as having no neurites. For illustration, some panels were adjusted for brightness and contrast as a whole.The endogenous nuclear and cytoplasmic TDP-43 was estimated by fluorescence density measurement. When the cells were transfected with the full-length or the C-terminal fragments, the antibody against the N-terminus of TDP-43 was used for staining. When the cells were transfected with the N-terminal fragments, the antibody against the C-terminus of TDP-43 was used. After staining the cells were visualized and photographed. For each cell, average density of TDP43 staining was measured in the nucleus and the cytoplasm by Nikon NIS-Elements software. The density ratio of the nucleus to the cytoplasm was calculated and then normalized to the average ratio of the EGFP control.Cells were collected by a hard plastic cell scraper from the plates and centrifugation at 400 g. The pellets were resuspended in RIPA buffer  plus protease inhibitors (Pierce). After clearing the lysate by centrifugation, protein concentration of cell lysates or tissue homogenates was measured using BCA assay . The samples were heated in Laemmli's buffer, and equal amounts of protein were loaded and resolved by SDS-PAGE. After transfer to nitrocellulose membranes, blots were blocked with 5% nonfat dry milk in PBST  for 1 h, and then incubated with primary antibodies overnight. After washing 3 times in PBST, the membrane was incubated with anti-rabbit IgG  or mouse IgG  conjugated to horseradish peroxidase for 1 hour. Membranes were washed three times and proteins were visualized after ECL (Pierce) treatment and detected using the LAS-3000 imaging system. The primary antibodies used were rabbit polyclonal anti-EGFP  and anti-TDP-43 ; and mouse monoclonal anti-α-tubulin  and anti-HA .5′-CAACTTCAAGCTCCTAAGCCACTGC-3′) and reverse Bra2 (5′-TAGGATCCGGTCACCAGGAAGTTGGTTAAATCA-3′) primers. PCR was performed at 95°C for 5 min, followed by 30 cycles of denaturing at 95°C for 30 s, annealing at 57°C for 30 s, extension at 72°C for 60 s. PCR products were visualized on agarose gels.The experiment was conducted based on a previously published method


In particular, genotoxic agents , PPARα agonists (bezafibrate and fenofibrate), and non-steroidal anti-inflammatory drugs  induced L1 promoter activity. To examine their effects on L1 retrotransposition, we developed a high-throughput real-time retrotransposition assay using a novel secreted Gaussia luciferase reporter cassette. Three compounds  produced a significant enhancement in L1 retrotransposition. This is the first study to report the effects of a wide variety of compounds on L1 transcription and retrotransposition. These results suggest that certain chemical- and drug-induced stresses might have the potential to cause genomic mutations by inducing L1 mobilization. Thus, the risk of induced L1 transcription and retrotransposition should be considered during drug safety evaluation and environmental risk assessments of chemicals.The Long interspersed element 1 (LINE1 or L1) retrotransposon constitutes 17% of the human genome. There are currently 80–100 human L1 elements that are thought to be active in any diploid human genome. These elements can mobilize into new locations of the genome, resulting in changes in genomic information. Active L1s are thus considered to be a type of endogenous mutagen, and L1 insertions can cause disease. Certain stresses, such as gamma radiation, oxidative stress, and treatment with some agents, can induce transcription and/or mobilization of retrotransposons. In this study, we used a reporter gene assay in HepG2 cells to screen compounds for the potential to enhance the transcription of human L1. We assessed 95 compounds including genotoxic agents, substances that induce cellular stress, and commercially available drugs. Treatment with 15 compounds increased the L1 promoter activity by >1.5-fold ( Long interspersed element 1 (LINE1 or L1) is a non-long terminal repeat retrotransposon. L1 constitutes 17% of the human genome 2O2L1 transcription and/or mobilization is upregulated by certain types of stress: for example, genotoxic stress, which is induced by benzo[a]pyrene, UV light Chemical- or drug-induced retrotransposition of L1 into a functional gene in germ cells or somatic cells might cause teratogenicity or disease, including cancer. However, it is still unclear what kinds of compounds affect L1 retrotransposition, and the risk of inducing retrotransposition by compounds has not been considered during drug or chemical safety assessment.In this study, we used a reporter gene assay in HepG2 cells to investigate compounds for their potential to enhance the transcription of human L1. We examined 95 compounds, each of which was categorized as a genotoxic agent, cellular stress agent, or drug. Of these compounds, 54 are now available commercially. Among the 95 compounds, 15 compounds  increased the L1 promoter activity. To examine their effect on L1 retrotransposition, we designed a retrotransposition assay using a novel secreted Gaussia luciferase reporter cassette. Importantly, we found that salicylamide not only increased L1 promoter activity but also slightly enhanced L1 retrotransposition in HeLa cells. Because this drug is widely used as an anti-pyretic analgesic, the data presented here suggest a new paradigm for drug assessment.5′-ATGCCTCGAGGGGGGAGGAGCCAAGATGGCCG-3′) that contains the XhoI restriction site and a reverse primer (5′-GCATAAGCTTCTTTGTGGTTTTATCTACTTTTGGTCTTTG-3′) that contains the HindIII restriction site. The PCR product was cloned into XhoI- and HindIII-digested linearized pGL4.11 (Promega). pCEP4/L1.3mneoI400/ColE1 pGL4.11-L1.3 5′ UTR contains the 5′ UTR of L1.3 upstream of a Firefly luciferase (FLuc) gene. The 5′ UTR was amplified by PCR using a forward primer  signal of the construct pORF5-Fcy::Fur (InvivoGen) was replaced with a thymidine kinase (TK) poly(A) signal. Next, an EcoICRI restriction site was introduced into the GLuc gene NcoI and NheI sites) of pORF5-Fcy::Fur, immediately downstream of the EF-1α/eIFg hybrid promoter. The chimeric mini-intron of the plasmid psiCHECK-2 (Promega) was isolated and cloned in the EcoICRI site of GLuc in the antisense orientation. The entire reporter cassette was then extracted with flanking PacI and AsiSI restriction enzyme sites, blunted, and cloned in a BstZ17I site in the 3′ UTR of the active L1RP contained within the vector 99-PUR 99-PUR-RPS-mGLucI is the JM111 mutant of 99-PUR-RPS-mGLucI. The JM111 mutant contains two missense mutations in L1RP ORF1 that abolish retrotransposition 99-PUR-JM111-pSV40-CLuc (New England BioLabs), which contains an SV40 promoter and Cypridina luciferase (CLuc) Ultimate ORF cDNA clones (Invitrogen) were V5-tagged on their N-termini by shuttling them from pENTR221 vector into pcDNA3.1/nV5-DEST using Gateway Technology (Invitrogen) as previously described l-glutamine, and 10% fetal bovine serum . Human adenocarcinoma HeLa cells (ATCC) and HEK293T cells (ATCC) were grown in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum . Cells were grown at 37°C in a humidified 5% CO2 atmosphere. Cells were grown in a T75 or T225 flask to ∼80% confluence and passaged using a 0.25% trypsin/0.02% EDTA solution (Sigma-Aldrich).Human hepatocellular carcinoma HepG2 cells (ATCC) were grown in minimum essential medium supplemented with 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 2 mM Most of the compounds were purchased from Sigma-Aldrich, Wako Pure Chemical Industries, Ltd., or EMD Millipore. All test compounds were solubilized in dimethyl sulfoxide (DMSO) and then stored at −20°C until use. They were diluted with DMSO to different concentrations and added to the culture medium at a 1/1000 (vol/vol) dilution just before use.4 cells/100 µl/well) or HeLa (0.5×104 cells/100 µl/well) cells were seeded on 96-well plates. After a 24-hour incubation, cells were cotransfected with 30 ng pGL4.11-L1.3 5′ UTR and 30 ng pGL4.74 with 0.18 µl FuGENE6 reagent (Roche) per well. At 24 hours post-transfection, cells were exposed to the various concentrations of test compounds. After 6 or 24 hours of treatment, FLuc and RLuc luminescence were measured by Multilabel Counter 1420 ARVO (PerkinElmer) using the Dual-Glo Luciferase Assay System (Promega).HepG2  control. If the addition of the compound reduced the RLuc activity to <50% of the DMSO control, the data at those concentrations were not used for analysis.-mGLucI or 99-PUR- JM111-mGLucI was cotransfected with 0.5 µg of empty vector (pcDNA3) or V5-tagged Ultimate ORF cDNA construct (four replicate wells each). At 24 hours post-transfection, 3 mls of media were replaced, and at subsequent time-points 50 µl of media was sampled from each well for time-course determination of luminescence. All readings for a single experiment were made at the same time using the BioLux Gaussia luciferase assay kit (New England BioLabs). Readings were adjusted for background luminescence of media alone.Initially, Gaussia luciferase L1 reporter constructs were tested for efficacy in HEK293T cells and 6-well plates. One µg of 99-PUR-RPS4 cells/100 µl/well) were seeded on a 96-well plate. After a 24-hour incubation, cells were cotransfected with 100 ng 99-PUR-RPS-mGLucI or 99-PUR-JM111-mGLucI and 2 ng pSV40-CLuc with 0.3 µl FuGENE6 reagent per well. At 24 hours post-transfection, the medium was replaced with fresh medium containing 1 µg/ml puromycin and various concentrations of test compounds. Untreated cells were included in each plate for normalization between plates and experiments. After a 2-day culture, cells were exposed to fresh medium containing only the test compounds. After an additional 3-day incubation, two 20-µl aliquots of the medium were sampled to measure GLuc and CLuc. Luminescence was measured with the Multilabel Counter 1420 ARVO using the BioLux Gaussia luciferase assay kit (New England BioLabs) and BioLux Cypridina luciferase assay kit (New England BioLabs).To screen chemical compounds for effects on retrotransposition, HeLa cells  control was calculated. After luminescence in the medium was measured for the L1 retrotransposition assay, cell viability was determined (see below). The concentration at which cell viability was >80% was used to evaluate L1 retrotransposition activity.mGLucI-tagged L1s, HeLa cells were harvested at four days post-transfection and their DNA were extracted with a DNeasy Tissue Kit (Qiagen). PCR was performed using primers that flank the intron: sc-F4 (CTTTCCGGGCATTGGCTTCC) and sc-R4 (CAAGCCCACCGAGAACAACG). Digesting the genomic DNA prior to PCR with restriction enzymes that cut within the intron (BanI or BspT107I) increased the amount of 149-base pair (bp) spliced product and reduced the amount of 282-bp unspliced product.To confirm removal of the mini-intron, and therefore genomic insertion of Immunofluorescence techniques have been described Cell viability was determined by measuring the amount of ATP present using the Cell Titer-Glo Luminescent Cell Viability Assay (Promega). Luminescence was measured with the Multilabel Counter 1420 ARVO. The percentage of luminescence in treated wells relative to that in control wells was used to determine cell viability. Cell viability was evaluated for four or six independent experiments.t-test between the control and treated wells. When two or more concentrations were used, Dunnett’s multiple comparison test was applied. Statistical analyses were performed with SAS 9.1.3 software (SAS Institute Inc.), with p-values <0.05 considered significant.Statistical analyses were performed with Student’s We wished to determine the effects of a wide selection of environmental, genotoxic, and therapeutic compounds, including many of common human exposure, on the activity of the LINE1 retrotransposon, an insertional mutagen linked to the etiology of human disease and possibly cancers. To begin, we cloned the 5′ UTR of L1.3, one of the most active human L1s p<0.05) after exposure for 6 hours and/or 24 hours (p<0.05) in L1 promoter activity listed in Using the system described above, we screened 95 compounds for effects on L1 promoter activity. Among these 95 compounds, 21 genotoxic agents with various mechanisms of action, such as DNA alkylation, DNA crosslinking, and DNA topoisomerase inhibition, were evaluated . Five ge24 hours . Among 9Benzo[a]pyrene, a DNA intercalator, increased the L1 promoter activity in a dose-dependent manner by >1.5-fold at 24 hours. This is consistent with a previous report, which showed that Benzo[a]pyrene activates mouse L1 transcription p<0.05) after exposure for 6 and/or 24 hours  stress . Four ce24 hours .Among the cellular stresses, oxidative stress is induced by diethyl malate, iodoacetamide, and phorone, all of which increased L1 promoter activity 1.2- to 2-fold. Cyclosporin A, which induced L1 promoter activity at 24 hours, inhibits mitochondrial membrane permeabilization p<0.05) after exposure for 6 and/or 24 hours (p<0.05) after exposure for 6 and/or 24 hours , isolated from the marine copepod RP in the opposite orientation. L1RP is one of the most active human L1s RP containing the GLuc retrotransposition cassette into an episomally replicating mammalian expression vector that expresses a puromycin resistance gene. The resultant plasmid was named 99-PUR-RPS-mGLucI.The secreted GLuc retrotransposition cassette consists of the GLuc gene interrupted by an antisense mini-intron, the EF-1α/eIF4g hybrid promoter (which is stronger than the CMV promoter), and the TK poly(A) signal . The GLu-mGLucI with empty vector or cDNA constructs whose protein products were previously determined to alter EGFP-reporter retrotransposition in HEK293T cell culture . Heterogeneous nuclear ribonucleoprotein K (hnRNPK) increased retrotransposition as previously reported All evidence indicates that Gaussia luciferase secreted into the media serves as an effective read-out of accumulated retrotransposition events. GLuc is expressed only when the intron is spliced from the chimeric L1-reporter RNA, the RNA is reverse transcribed, and its cDNA is inserted in the genome by retrotransposition. Testing initially for efficacy in 6-well plates, we cotransfected 99-PUR-RPS culture . At 24 hKroutter et al. noted that earliest L1 retrotransposition events are detected only at 32 hours post-transfection mGLucI  inhibits reverse transcription by becoming integrated into the elongating cDNA sequence. AZT inhibits L1 retrotransposition as measured with a neomycin cassette p<0.0001; p<0.0001; p<0.05; p<0.0001; We evaluated the effects of 26 compounds on L1 retrotransposition with the dual secreted luciferase assay. Twenty one of the test compounds were chosen from among the agents that enhanced L1 promoter activity in the L1 reporter gene assay. The remaining five compounds were randomly chosen from among those that did not enhance L1 promoter activity. Among the 21 compounds that elevated L1 promoter activity, etomoxir elevated L1 retrotransposition by 40.6% as compared with the control at 50 µM  significantly increased L1 promoter activity by >1.5-fold at 6 and/or 24 hours. We then assessed the effects of compounds on L1 retrotransposition. The results revealed that 3 compounds significantly increased L1 retrotransposition activity by 20–40.6% . The datin vitro, there is a possibility that the effects of these compounds on retrotransposition in vivo might be larger than those observed in vitro. Several factors suggest that this might be the case. First, benzo[a]pyrene, which is a DNA intercalator and an inducer of genotoxicity, induces both transcription Although our retrotransposition experiments were conducted in vitro versus those in vivo has to do with the L1. Here we used L1RP, which has very high retrotransposition activity RP, we may not have been able to detect enhanced retrotransposition activity, even if it was substantial. Thus, compounds that only slightly elevated L1 retrotransposition might induce retrotransposition more in vivo than would have been expected based on experiments in vitro. Moreover, it is estimated that 80 to 100 L1s remain potentially active for retrotransposition in the average diploid human genome A second factor that may lead to a difference in the effects seen in vivo and in vitro assays. In the case of the PPARα agonists, bezafibrate and fenofibrate enhanced L1 transcription and bezafibrate and WY-14643 slightly increased L1 retrotransposition. Long-term exposure of mice to WY-14643 decreases the DNA methylation in the liver of LINE1s, LINE2s, and intracisternal A particles (IAPs); the latter is an active retrotransposon in mice in vivo. A stable assay system that allows us to assess retrotransposition in vivo, including effects that result from epigenetics, will be useful to evaluate the effects of compounds on L1 retrotransposition.The third factor concerns the difference in conditions between neoRT reporter cassette, containing a neomycin phosphotransferase (neo) gene and an artificial intron placed between the promoter and neo coding sequence Cell culture assays for retrotransposition are the most important tools for studying LINE1 elements in humans and mice. These assays report the final step of retrotransposition, the number of cells in a total population of cells that receive an insertion event. The first assay to measure retrotransposition in cells was developed over 25 years ago neoRTmneoI) included neo interrupted by antisense γ-globin intron 2. Moran et al. inserted mneoI in opposite transcriptional orientation into the 3′ untranslated regions (UTRs) of L1.2 and LRE2 RPFreeman et al. redesigned mneoI assay requires 3 weeks for antibiotic selection. A blasticidin S-deaminase gene (bsr) reporter cassette cloned in L1RP and a vector that constitutively expresses puromycin cut antibiotic selection time in half Limitations exist for each of the L1 cell culture assays in widespread use. The None of the current assays permit easy study of retrotransposition kinetics and the performance of multiple assays in a single well. Different wells may be sampled sequentially to establish a time-course series mGLucI) containing a modified Gaussia luciferase gene. The JM111 mutant, which exhibits almost no retrotransposition activity because of the introduction of two missense mutations in ORF1 Cognizant of assay limitations, we designed a “fourth generation” cell culture retrotransposition assay that is easy to execute, sensitive, amenable to high-throughput analyses, and permissive for time-course sampling from a single transfected well. We constructed a novel retrotransposition cassette reporter . However, the risk of retrotransposition has not yet been considered as part of drug safety assessment, even though there are several reports that chemical-induced stress activates L1 transcription and retrotransposition and that the integration of retrotransposition into genes causes disease  and Renilla luciferase (RLuc) expression in the L1 reporter gene assay in HepG2 cells. Relative FLuc luminescence, relative RLuc luminescence, and relative normalized luciferase luminescence at 6 hours (A) and 24 hours (B) after treatment with vehicle control (DMSO) in the L1 reporter gene assay using HepG2 cells. FLuc and RLuc indicate L1 promoter activity and expression of the internal control, respectively. Data are the mean ± SD from four independent experiments. TK pro-R: pGL4.74; no pro-F: pGL4.11; L1 5′ UTR-F: pGL4.11-L1.3 5′ UTR.(TIF)Click here for additional data file.Figure S2FLuc and RLuc expression in L1 reporter gene assay in HeLa cells. Relative FLuc luminescence, relative RLuc luminescence, and relative normalized luciferase luminescence at 6 hours (A) and 24 hours (B) after treatment with the vehicle control (DMSO) in the L1 reporter gene assay using HeLa cells. FLuc and RLuc indicate L1 promoter activity and an internal control, respectively. Data are the mean ± SD from four independent experiments. TK pro-R: pGL4.74; no pro-F: pGL4.11; L1 5′ UTR-F: pGL4.11-L1.3 5′ UTR.(TIF)Click here for additional data file.Figure S3Data for the earliest sampling time-points of the time-course GLuc retrotransposition assay shown in . See legend for that figure.(TIF)Click here for additional data file.Figure S4GLuc and CLuc expression in the novel dual secreted luciferase L1 retrotransposition assay. GLuc and CLuc luciferase luminescence in the novel L1 retrotransposition assay 6 days post-transfection. Either 99-PUR-RPS-mGLucI or 99-PUR -JM111-mGLucI containing two missense mutations in L1RP ORF1 and pSV40-CLuc were co-transfected. GLuc can be expressed only when retrotransposition occurs. CLuc was used as an internal control. Data are the mean ± SD from six independent experiments. RLU: Relative Light Units.(TIF)Click here for additional data file.Dataset S1Results of L1 reporter gene assay in HepG2 cells. L1 promoter activity 6 and 24 hours after treatment was evaluated by luciferase reporter gene assay. L1 promoter activities are represented as a percentage relative to that of the vehicle control. Mean and SD from at least two independent experiments  are shown.(XLS)Click here for additional data file.Dataset S2Results of L1 reporter gene assay in HeLa cells. L1 promoter activity 6 and 24 hours after treatment was evaluated by the luciferase reporter gene assay. L1 promoter activities are represented as a percentage relative to that of the vehicle control. Mean and SD from two independent experiments  are shown.(XLS)Click here for additional data file.Dataset S3Results of L1 retrotransposition assay and cell viability. L1 retrotransposition activity and cell viability after treatment are represented as a percentage relative to that of the vehicle control. Mean and SD from four independent experiments are shown.(XLS)Click here for additional data file.


Graphene's unique optoelectronic properties are promising to realize photodetectors with ultrafast photoresponse over a wide spectral range from far-infrared to ultraviolet radiation. The underlying mechanism of the photoresponse has been a particular focus of recent work and was found to be either photoelectric or photo-thermoelectric in nature and enhanced by hot carrier effects. Graphene supported by a substrate was found to be dominated by the photo-thermoelectric effect, which is known to be an order of magnitude slower than the photoelectric effect. Here we demonstrate fully-suspended chemical vapor deposition grown graphene microribbon arrays that are dominated by the faster photoelectric effect. Substrate removal was found to enhance the photoresponse by four-fold compared to substrate-supported microribbons. Furthermore, we show that the light-current input/output curves give valuable information about the underlying photophysical process responsible for the generated photocurrent. These findings are promising towards wafer-scale fabrication of graphene photodetectors approaching THz cut-off frequencies. Graphene's high carrier mobility combined with its universal optical absorption from terahertz to the ultraviolet wavelength regime, as well as its large nonlinear optical coefficients, are promising towards novel applications in photodetection123478910111234512131415161718192021123915161718181920222324Practical applications require scalable approaches such as chemical vapor deposition (CVD) grown graphene photodetectors. The photophysics of CVD-grown graphene devices is however less understood, and it is a priori unclear to what extent inherent grain boundaries and other defects affect the mechanism of photocurrent generation. A recent time-resolved study of the photoresponse in CVD-grown graphene embedded in a coplanar stripeline circuit reveals that the time constant of the photo-thermoelectric effect is, at 130 ps, more than an order of magnitude slower than the photoelectric effect, at 4 ps12912151620Here we demonstrate fully-suspended CVD-grown graphene microribbon arrays that are dominated by the faster photoelectric effect. Removal of the substrate was found to enhance the photoresponse by four-fold compared to substrate-supported microribbons. We also show that the slope of the light-current input/output curves (L-I curves) gives valuable information about the underlying photophysical process responsible for time-integrated photocurrents. These findings are promising towards wafer-scale fabrication of graphene photodetectors approaching THz cut-off frequencies.2 and patterned using electron-beam lithography . The graphene quality was evaluated by Raman spectroscopy as well as optical transmission measurements and found to be monolayer graphene . Ultimately, IPC saturates at the highest pump power with values for suspended graphene being approximately 6-times smaller than those for supported graphene, as indicated by the arrow labeled “A” in PC ∝ (Popt)α. For supported graphene we studied five different devices and found an average value of α = 0.62 ± 0.05 at zero gate voltage. In stark contrast, the power exponent of the fully suspended device is with 0.9, near unity. The pronounced change in slope of the L-I input-output curve indicates that the underlying physical mechanism has changed.While demonstrating four-fold enhanced photocurrents in large-scale CVD-grown graphene photodetectors is of great technological importance, one would also like to understand the underlying photophysical mechanism to be able to optimize the detector device performance. The two main competing mechanisms for photocurrent generation in graphene are the photo-thermoelectric effect and the photoelectric effect. One way to tell these two effects apart is to characterize their time constant in optical pump-probe experiments which differ by at least an order of magnitudeTE) and the optical pump power Popt can be estimated and yields ITE ∝ (Popt)2/3, i.e. α = 0.66 (see 131415161718192021e underneath the laser excitation spot located in the center of the microribbon. These photoexcited carriers interact strongly with the surface polar phonons (SPP) of the substrate2223242533e in carrier temperatures. This ΔTe is the drive for the Seebeck effect and thus a strong photo-thermoelectric contribution  effect still partly contributing to the photocurrent in this device. In addition, Quantitatively, the photoelectric contribution depends linearly on photogenerated carrier density which is directly proportional to the rate of absorbed photons and thus the incident excitation power. Therefore, the photoelectric contribution Ie, driving the Seebeck effect before reaching contacts. It is thus evident that novel graphene photodetectors can be engineered in which either the photo-thermoelectric or the photoelectric effect dominates the photoresponse. Since the time constant of the photoelectric effect is, at 4 ps, over an order of magnitude faster than the time constant of the photo-thermoelectric effect, at 130 psIn contrast to our findings, a recent study of partially supported graphene shows that such a sample design yields a dominant photo-thermoelectric effectIn conclusion, we have demonstrated four-fold enhancement of the photoresponsitivity of fully suspended CVD-grown graphene photodetector devices over substrate-supported graphene. In addition, we have shown that the light-current input/output curves give valuable information on the underlying photophysical process responsible for the generated photocurrent. Finally, we have found that fully suspended CVD-grown graphene devices are dominated by the photoelectric effect, which is promising towards CVD-grown graphene photodetectors approaching THz cut-off frequencies.2 substrate confirms good quality monolayer graphene film . The devices were then wire bonded using 25 μm thick Au wires on a chip carrier for electrical measurements. For suspending graphene, we etched the 300 nm thick underlying layer of SiO2 using (6:1) buffered oxide etchant (BOE) followed by critical point drying (CPD) to avoid damage to the suspended graphene ribbon.Large area graphene was grown on high purity copper foil using a low pressure CVD furnace at 1000°C in a hydrogen, argon and methane environment. Raman spectrum of the graphene transferred onto 300 nm SiO−6 mbar. The photoresponse of the graphene ribbons was measured using a lock-in amplifier by positioning a 532 nm wavelength laser spot onto the ribbon. A spot of approximately 1 μm diameter (FWHM) was focused using a 50 × objective with working distance of 13 mm. The position of the laser spot was manipulated by using piezoelectric motor controlled scanning mirrors with nanometer precision in X and Y directions. The incident laser power, which was measured to be 20 mW at maximum, was controlled by using neutral density filters to sweep between 1 nW and 2.8 mW. The beam was modulated with a chopper wheel at 2 kHz. All current and voltage values were supplied by two Keithly 2400 source meters, while output was read from the lock-in amplifier. A moderate source-drain bias of 0.1 V was used for photocurrent measurements. Back-gate sweeps were performed with 1 mV with voltage ranges selected to center the Dirac point.All the measurements were performed at room temperature in a chamber with vacuum level of 10V.P., S.S. and E.H.Y. conceived and designed the experiments. V.P fabricated the suspended CVD-graphene microribbons. V.P. carried out the electro-optical experiments and analyzed the data. V.P. and A.C. carried out the Raman experiments. V.P., S.S. and E.H.Y. co-wrote the manuscript. E.H.Y. supervised the device fabrication and S.S. supervised the electro-optical measurements. All authors contributed to scientific discussions.SI


The mechanical behavior of graphene under various indentation depths, velocities, and temperatures is studied using molecular dynamics analysis. The results show that the load, elastic and plastic energies, and relaxation force increased with increasing indentation depth and velocity. Nanoindentation induced pile ups and corrugations of the graphene. Resistance to deformation decreased at higher temperature. Strong adhesion caused topological defects and vacancies during the unloading process. Graphene has received a lot of attention due to its good mechanical and electromagnetic properties -3, incluNovoselov and Geim  used graMany studies -25 have In this study, the effects of nanoindentation depth and velocity on the mechanical properties and contact behavior of graphene at various temperatures are investigated using molecular dynamics (MD) simulations. Adhesion, relaxation, defects, and deformation are discussed.Figure The Lennard-Jones potential function is employed to describe the interaction between the diamond tip and the graphene atoms. The Tersoff empirical potential energy function  is generFigure The tip then indented the graphene. The absorptive force gradually turns into a repulsive force. As the indentation depth increased, the stress wave spread out farther from the center, inducing ripples and corrugations. Figure The effect of indentation depth was examined. With all other conditions fixed, indentation depths of 0, 0.2, 0.4, and 0.6 nm were investigated. Figure 2) defects, which are composed of one octagonal ring and two pentagonal rings, during the adhesion pulling process. Double vacancies are referred to as 5-8-5 defects [2 defects [Figure  defects . Our sim defects , who inv defects .Figure Figure Indentation velocities of 25, 50, 75, and 100 m/s were tested by fixing the temperature at 300 K and the packing time at 15 ps. Figure Figure The indentation behavior of graphene was studied using molecular dynamics simulations. The following conclusions were obtained:1. The affected area, load, elastic and plastic energies, and relaxation force increased with increasing indentation depth.2. Nanoindentation-induced pile ups and corrugations of graphene were observed. Strong adhesion causes topological defects and vacancies.3. The average contact stiffnesses of the graphene at temperatures of 0, 200, 300, and 400 K are 58.7, 58.1, 49.48, and 36.6 N/m, respectively.4. At higher temperature, the kinetic energy among atoms increases, which weakens covalent bonds and thus decreases resistance to deformation. The load, elastic and plastic energies, and relaxation force decrease with increasing temperature.5. With a fast indentation, the graphene has insufficient time to respond, which leads to a high load, elastic and plastic energies, and relaxation force.The authors declare that they have no competing interests.The work presented here was carried out in collaboration between all authors. THF, THW and JCY defined the research theme. THF and JCY designed methods and analyzed the data, interpreted the results and wrote the paper. YJH co-worked on associated data collection, their interpretation, and presentation. All authors have contributed to, seen and approved the final manuscript.


Group I introns are intervening sequences that have invaded tRNA, rRNA and protein coding genes in bacteria and their phages. The ability of group I introns to self-splice from their host transcripts, by acting as ribozymes, potentially renders their insertion into genes phenotypically neutral. Some group I introns are mobile genetic elements due to encoded homing endonuclease genes that function in DNA-based mobility pathways to promote spread to intronless alleles. Group I introns have a limited distribution among bacteria and the current assumption is that they are benign selfish elements, although some introns and homing endonucleases are a source of genetic novelty as they have been co-opted by host genomes to provide regulatory functions. Questions regarding the origin and maintenance of group I introns among the bacteria and phages are also addressed. Tetrahymena thermophila   , and theas.edu/] -33. Whenas.edu/] . In caseas.edu/] ,36.Group I introns are removed from precursor RNA by an autocatalytic RNA splicing event that is mediated by the intron’s RNA tertiary structure. Base-pairing interactions between the 5′-end of the intron and flanking exon sequences define the location of the 5′ and 3′ splice sites. The Internal Guide Sequence (IGS), which is a short intronic sequence near the 5′-end that pairs with sequences of the upstream exon to form P1, determines the 5′ splice site Figure . The 3′ Azoarcus sp. BH72 pre-tRNAIle intron-exon complexes ,6710,67]Currently, there are six families of HEases, classified primarily on the basis of conserved amino acids that correspond to structural or active site residues; the LAGLIDADG, H-N-H, His-Cys box, GIY-YIG, PD-(D/E)xK, and EDxHD families -73. The trans-acting HEases have been described in T4 and related phages that can promote the homing of either group I introns lacking ORFs or group I introns that encode defunct (degenerated) HEases  to avoid loss. Alternatively, it is thought that group I introns can ‘escape’ to a new population of intronless alleles by transposition to new sites (ectopic integration) by reverse splicing. Reverse splicing is the reverse of the forward splicing reaction, and theoretically allows a group I intron RNA to insert into a RNA molecule with four to six complementary bases to the P1 stem of the intron RNA ,85. ThisAnother mechanism for ectopic integration or transposition relates to the relaxed specificity of many intron-encoded HEases. For instance, cleavage at a site similar to a HEase’s native target site may promote intron mobility, and it has been shown that the cleavage specificity of the I-TevI HEase can be influenced by oxidative stress . HoweverClostridium difficile as a feature of a two-component riboswitch that involves c-di-GMP as an allosteric activator [There are a few instances where group I introns or their components may have been domesticated by their host genomes, or by other types of mobile genetic elements. The bacterial DUF199/WhiA protein is a transcription factor and its N-terminal region contains the same protein fold as found in monomeric LAGLIDADG HEases encoded within group I introns ,90. Thisctivator . Here, ictivator . In esseC. difficile in the tcdA locus. The composite element, termed an IStron, is composed of a splicing-competent group I intron (IA2 subgroup) that has an insertion element  embedded within its 3′-end and encoding two transposases [A unique composite element has been described in some enterotoxin producing strains of sposases ,94. One sposases . TnpA casposases ,97. Thissposases . Althougsposases ,100.Bacillus species [recA and nrdE genes in various Bacillus species [nrdE genes [Within bacteria, group I introns are predominately inserted within structural RNA genes such as tRNA and rRNA genes ,101-107. species ,112, rec species ,113, anddE genes ,110. ThidE genes ,114-117.in vivo splicing rates, and that self-splicing RNA introns became extinct in the archaeal lineage [Interestingly, group I introns have so far not been discovered in archaeal genomes, although group I intron derived HEase sequences are sometimes associated with archaeal introns -122. TheBacillus cereus group suggested that some of the genomic recA, nrdE, nrdF introns are similar to phage introns, indicating that phage infection could serve as a vector system for the lateral movement of introns among different genomes [Tetrahymena into the E. coli 23S gene resulted in the reduction of the growth rate which was correlated with poor splicing of the Tetrahymena intron. Moreover, the intron RNA was shown to associate with the 50 S ribosomal subunit and possibly interfere with translation. Clearly, there are barriers to intron spread in bacteria [The persistence and spread of group I introns in prokaryotic genomes is dependent on a number of factors including (1) the phenotypic cost associated with the insertion of a group I intron, (2) the availability of intronless alleles for endonuclease-mediated homing, (3) the presence of efficient homology-based DSB repair systems, (4) the availability of DNA or RNA transfer mechanisms such as DNA uptake by natural transformation, conjugation and plasmid transfer, and phages. Interestingly, recent work on the  genomes . However genomes  showed tbacteria  that areet al. [sunY gene of the T4 phage the intron sequences flanking the HEase ORF (I-TevII) were similar to the exon junction sequences that comprise the I-TevII target sequence. Importantly, they were able to demonstrate that a synthetic construct that included the fused sequence composed of the up- and down-stream sequences that flank the I-TevII ORF was indeed cleaved by I-TevII. This result provided strong circumstantial evidence for the ‘endonuclease-gene invasion’ hypothesis whereby a free-standing HEase cut an intron sequence that fortuitously contained a similar HEase target site. During the recombination-based repair process, the endonuclease gene sequence was inserted into the cleaved intron sequence, thus generating a composite potentially mobile intron.One of the most intriguing questions about mobile group I introns concerns their evolutionary origin. The current consensus is that HEases and group I introns had distinct evolutionary origins, and that HEases have on multiple independent occasions invaded an endonuclease-free intron. The alternative scenario, that group I introns always possessed an endonuclease gene is problematic for a number of reasons, including the fact that many group I introns do not contain ORFs, and the notion that group I introns were direct descents of catalytic RNAs from the RNA world. Moreover, the finding that HEases can exist outside of the protective confines of introns, as so-called free-standing homing endonucleases, lent credibility to the hypothesis that these free-standing enzymes could be a potential source of the ‘invading’ endonuclease. Two mechanisms that would lead to the formation of such a composite mobile intron have been proposed. Loizos et al.  noted thRecent studies ,76 proviRegardless of the origin of mobile group I introns, one would assume that endonuclease invasion would have a deleterious effect on intron splicing. In this respect, it is interesting to note that many endonuclease ORFs are inserted in loops that presumably do not interfere with folding and splicing. It is also possible that the intron-encoded endonucleases and/or host factors were able to compensate by stabilizing the intron tertiary RNA structure or discouraging misfolding of the intron RNAs -132. ThiThis returns us to the enigma of why group I introns and their associated HEases have been successful in spreading among the organellar genomes of plants, protozoans, and fungi but have very limited representation among bacterial and phage genomes. Koonin  proposedThe mechanisms that promote and prevent group I introns from proliferating among bacterial genomes are poorly understood, as is the long-term impact of introns on organismal viability. When present, it is assumed that introns are phenotypically neutral, yet the co-opting of intron functions by a riboswitch or the domestication of intron-encoded homing endonuclease as a regulatory protein (WhiA) indicates that introns can be a source of genetic novelty. Future research efforts directed at understanding the effect of group I introns on host gene expression, mechanisms of mobility to ectopic sites and their spread among bacterial genomes and phages will lead to valuable insights regarding the dynamics and evolution of group I introns.bp: base pair; c-di-GMP: cyclic diguanylate; DSB: double-strand break; GTP: guanosine-5′-triphosphate; HEase: homing endonuclease; HEG: homing endonuclease gene; HUH: endonuclease motif; IGS: Internal Guide Sequence; IS: insertion element; ORF: open-reading frame; rRNA: ribosomal RNA tRNA, transfer RNA; SDSA: synthesis-dependent strand annealing.The authors declare that they have no competing interests.GH: conception and design, figure preparation, manuscript writing and final approval of manuscript. MH: conception and design, compilation of data for Figures


The results provide direct experimental evidence of ripple formation, sp2 to sp3 hybridized carbon transformation, electronic damage, Ar+ implantation, unusual defects and edge reconstructions in FLG, which depend on the irradiation energy. In addition, shadowing effects similar to those found in oblique-angle growth of thin films were seen. Reliable quantification of the transition from the sp2-bonding to sp3-hybridized state as a result of Ar+ ion irradiation is achieved from the deconvolution of the XPS C (1s) peak. Although the ion irradiation effect is demonstrated through the shape of the derivative of the Auger transition C KVV spectra, we show that the D parameter values obtained from these spectra which are normally used in the literature fail to account for the sp2 to sp3 hybridization transition. In contrast to what is known, it is revealed that using ion irradiation at large FLG sample tilt angles can lead to edge reconstructions. Furthermore, FLG irradiation by low energy of 0.25 keV can be a plausible way of peeling graphene layers without the need of Joule heating reported previously.Roughness and defects induced on few-layer graphene (FLG) irradiated by Ar Ion irradiation of materials subjected to different ion energies, ion doses and irradiation geometries such as angle of incidence of the beam, sample tilt angle and sample rotation has been widely studied ,2. In ad2/Si substrate exposed to 30 keV Ga+ ion irradiation pointed out the requirements for developing a more thorough understanding of graphene's ability to withstand prolonged ion irradiation. These include: (1) the absence of cascade collisions due to the 2-D graphene nature, (2) the possibility of C atoms not to be displaced from the lattice as the ion energy is completely transferred to the substrate underneath, (3) graphene open structure which facilitates the ion channeling, hence limiting the number of ion/target collisions and ejected carbon atoms, and (4) implanted ions which might cause a net positive charge to build up in graphene and electrostatically repel the subsequent incoming incident ions. In addition to the above issues, the importance of this study helps to understand the damage production mechanisms and types of defects created by the energetic ions in the sample for the efficient use of ion beams and optimization of the graphene cutting process [ex situ prepared graphene layers [sp2 bonding configurations and affect the graphene carrier mobility, this study shade light on defects associated with this process which are equally important. In this respect, applications and mechanisms based on defects in graphene such ferromagnetism [Experimentally, Lopez et al.  in their process . Further process , eliminae layers  and grapagnetism , creatioagnetism , porous agnetism , atmosphagnetism  and grap+ with energies from 0.25 to 5 keV at incidence angle of 45° and sample tilt angle of 66° to study the irradiation effects in FLG. In addition to defects found on flat irradiated areas, we show that FLG edge defects associated with sp2 to sp3 hybridized carbon transformation are most typical in the low energy range and irradiation geometry used. In addition, we show that the reliable quantification of the sp2/sp3 hybridized state ratio as a function of irradiation energy can be achieved from the deconvolution of the XPS C 1s envelope. This ratio, together with newly developed features in the Auger transition C KVV spectra and π − π* transition behavior as a function of irradiation energy provide insights on the FLG structural and electronic damage. Furthermore, we point out the possibility of a combined effect of ion implantation and ion reflection to be responsible for the low rate of sputtering of FLG.Here, we have employed low energy Ar+ ion irradiation for 30 min for energies ranging from 0.25 to 5 keV at incidence angle of 45° and sample tilt angle of 66° in the ultra high vacuum conditions.FLG samples were obtained by peeling layers from highly oriented pyrolytic graphite (HOPG ZYA) using adhesive tape. They were then fixed on steel substrates by double sided stick carbon tape. These substrates were then subjected to Ar−10 mbar. The chemical composition of the sample was extracted from the wide scan using CASA XPS software . Short scans were recorded at pass energy of 20 eV. In order to avoid charging effect during the scans, an electron gun flooding was used for charge compensation.XPS measurements were carried out using an Omicron Nanotechnology XPS system  using a monochromatic Al Kα radiation . The source voltage and emission current were 15 kV and 20 mA, respectively. The base pressure at which the scans were done was 10The nanoscale images presented were performed using Nanoscope V atomic force microscope (AFM) obtained in tapping mode using ultra high resolution cantilevers made of tungsten having radius of less than 1 nm and force constant of 46 N/m. During the imaging, both the scan rate and the imaging resolution were set at 0.5 Hz and 512 × 512 pixels, respectively.+ ion energies ranging from 0.25 to 5 keV. At first glance, roughness and well-known ripple structures of varying sizes and wavelengths [Ra) and RMS roughness (Rq) reveals that the roughness is not monotonically increasing as a function of irradiation energy; instead, a reduction in the roughness is found at 1.5 keV as indicated in Figure Figure elengths  are visif) with ripple wavelength (λ) of =24 nm. After irradiation at 0.25 keV, the PSD spectrum superimposes to that of the un-irradiated sample, indicating negligible damage of the FLG surface. Interestingly, a decrease in the graphene PSD intensity is observed at energies higher than 0.25 keV for the spatial frequencies between 60 and 120 μm−1 .The evolution of the large scale morphology was probed by two-dimensional power spectral density (PSD) analysis ,17. PSD λ = 72 nm that resulted from sputtering of both the first and the second graphene layers. It should be noted that the irradiation by 1.5 and 5 keV will only affect the first and the second FLG layers; hence, the assignment of ‘second layer’ and ‘first layer’ is used in Figure This suggests strong suppression, smoothing and lateral coarsening of ripple structures  at these energies. At low frequency regime, the spectra show a switching behavior where the PSD intensities are seen to be higher than that of un-irradiated and increase by a factor of 10 and 100 for the 1.5 and 5 keV irradiations, respectively. This increase is a sign of large amplitude ripple formation with long periodicity of K f−β where β and K are the spectral index and spectral strength, respectively [β1 = 6.0 and β2 = 6.5. Since β is related to α by the equation α = (β − d)/2 [d is 2, then α1 = (6–2)/2 = 2 and α2 = (6.5 − 2)/2 = 2.25. Here, α1 attributes to the roughness exponent associated to the first FLG layer and α2 could be assigned to roughness exponent of the second FLG layer.In order to further shed light on the morphology of the two layers after the 5-keV ion irradiation, the roughness exponent α has been determined from the 5-keV PSD spectrum in the high frequency region. It is evident that the spectrum shows bimodal intensity trend /2 , where ts), Ar (2p), and Ar (2s) core level excitations recorded from FLG samples after Ar+ ion irradiation at different energies. It is clear from the XPS spectra and from the inset of Figure + ions are implanted into the FLG and reached the saturation limit at energies ≥ 3 keV.Figure + irradiation might be the reason. In addition, the commonly observed π − π* transition in graphene at binding energy of 290 eV [sp2-bonding to sp3-hybridized state as a result of Ar+ ion irradiation.Also, variation in the effect of ion irradiation can be observed from the newly developed shoulder (shoulder labeled A) in the XPS spectra along with the increasing of the irradiation energy. The origin of shoulder A is unclear at the moment; however, complex defects previously theoretically predicted  to be asf 290 eV  seems to+ at 5 keV for 30 min) of FLG to the Ar+ ion irradiation stems from the combined effect of the Ar+ implantation as evident from Figure + ions build up, they electrostatically repel subsequent incoming incident ions, therefore reducing number of ions involved in collisions. This charge repulsion can be understood from the ion concentration saturation observed in the inset of Figure + ions can also take place and lead to the saturation observed. Auxiliary experiments at constant irradiation energy and at different tilt sample angles were carried out to confirm the effect of the reflection of the incoming ions on the resilience of FLG. Indeed, the results (to be reported elsewhere) obtained from the ion irradiation of FLG samples at constant energy of 1 keV, and different sample tilt angles (Φ) from 0° to 80° show that the amount of implanted Ar+ ions seems to decrease as the tilt angle is increased.It can be concluded that the resilience  XPS spectra of FLG samples for the un-irradiated and irradiated at energies 0.5 and 4 keV. The fitting of the spectra were done by Gaussian-Lorentzian functions with a Shirley background subtraction [sp2 and sp3-hybridized states, respectively [sp2 peak appears to be shifted to higher binding energies at high Ar+ ion irradiation energies.Figure traction . The fitectively . The sp2sp2/sp3 ratio along with the full width at half maximum (FWHM) of the sp2 and sp3 peaks as a function of irradiation energy is shown in Figure sp2 hybridization dominates  - a FLG characteristic of a two-dimensional sheet of sp2 bonded carbon atoms in a honeycomb lattice [sp3 in the un-irradiated sample is attributed either to the edge of individual graphene layers or to the intrinsic sp3 defects reported to exist in the FLG samples [sp2 to sp3 hybridization due to oxygen exposure of FLG [s peak in the XPS spectra.Quantification of the  lattice -27. Howe samples . The wele of FLG  is ruledsp2 to sp3 hybridization as seen in region I of Figure sp2 character due to the enhanced possibility of the damage of the edge of the graphene structure, consequently the formation of dangling bonds. An amorphous stable mixture containing equal sp2 and sp3 hybridizations  is observed (region II) when employing irradiation energy in the range from 1.5 to 4 keV. At high irradiation energies above 4 keV, the number of transformed sp2 bonds seems to be slightly less as depicted in region III of Figure sp2 contribution from this layer and the layers beneath up to approximately 2 nm of the expected XPS sampling depth in the geometry used. In the lateral direction and as seen from the AFM image at 5 keV in Figure sp2 character are expected to exist. The reduced number and the large size of these pits are reflected from the long periodicity of λ = 72 nm and two orders of magnitude increase in PSD intensity found in Figure On the other hand, the initial irradiation of the FLG causes fast transformation of D) between the most positive maximum and most negative minimum obtained from the first derivative Auger transition C KVV spectra recorded from FLG samples, as a function of Ar+ ion irradiation energy.To further account for the effect of the ion irradiation, we adapt the commonly used first derivative of C KVV spectra obtained from XPS data . The selD values are within the reported values of 21 to 23 eV for HOPG (multi-layer graphene) [sp2 to sp3 transition trend similar to that observed in Figure D parameter values obtained from these spectra fail to account for sp2/sp3 hybridization transition, and reliable quantification of sp2/sp3 ratio is only obtained from the deconvolution of the XPS C (1s) envelope as explained before and shown in Figure The raphene) , but notsp2 to sp3 transformation mechanism. For example, irradiation energy will cause lattice displacement in the FLG which affects the FLG layer cross-linking, therefore contributing in the formation of new sp3 bonds. In addition, bonds can be broken to form sp3 due to the collision impact. This is evident from the XPS reduction of π electron (π bond) intensity as the irradiation energy is increased. Furthermore, bending (to be discussed later) and breaking of the graphene planes are likely to contribute to the sp2 to sp3 transformation observed.From all the above findings, it is clear that more than one factor can contribute to the + ion irradiation on the FLG edges. In contrary to the damage found in the flat regions, the edges of FLG are found to be much rougher; with four distinct features as shown in Figure We turn our discussion to the effect of ArIn this case, the edges are seen to be lifted up and shadow the regions behind them; shadowing effects are seen from the cross section profile taken at a position shown by a solid line in Figure + ion irradiation of 1.5 keV. Few holes (enclosed by circles) can be clearly seen, suggesting the possibility of creating nanoporous graphene layers by ion irradiation for filtration applications. Increasing the ion irradiation to 4.5 keV, the peeling of graphene layers in the direction normal to the edge takes place which is associated with triangular-shaped FLG folding (enclosed by a circle) as shown in Figure During the ion irradiation, unusual defects and edge reconstructions were also observed; defects appear only after ion irradiation and were not found in the un-irradiated samples. Furthermore, these defects and edge reconstructions are similar to those reported by Zhang and Feng  during tsp2/sp3 ratio, sp2 and sp3 FWHM values and Auger transition C KVV spectra data analysis reveal that, using low energy of 0.25 keV Ar+ ion irradiation at large sample tilt angle at room temperature, is an alternative way of peeling graphene layers without the need of Joule heating.This type of folding has been observed on the surface of FLG resembling single wall nanotubes or double wall nanotubes due to the single- or double-graphene layer folding back to itself . The fol+ ion irradiation of FLG has been investigated in which ripple structure formation with varying amplitude and periodicity predominantly in the first two graphene layers was observed. In addition, sp2 to sp3 bonding transformation through the quantification of the deconvolution of the XPS C (1s) envelope along with electronic damage as deduced from the gradual disappearance of the π − π* transition in graphene, as a function of irradiation energy, was found. Although the shape of derivative of Auger transition C KVV spectra provided an indication of the ion irradiation effect, the D parameter values obtained from these spectra failed to account for sp2 to sp3 hybridization transition. Among others, energy-dependent edge wiggles, shadowing effects, defects (such as holes and FLG edge reconstructions) and edge folding which depend on the sputtering conditions were all found. The existence of these defects suggests the possibility of creating nanoporous FLG by ion irradiation and the development of high density nanobubble FLG surfaces for various applications. Furthermore, the combined effect of Ar+ implantation and sample tilt angle on FLG irradiation can explain the ion irradiation resilience of FLG. Although we looked specifically at Ar+ irradiation, our results can also provide insights into the response of FLG to irradiation by other noble gasses or other species. Despite the mixing of sp2 and sp3 is not desirable for the electronic properties of FLG, coming up with applications which depend on tuning sp2/sp3 ratio will be essential to fully appreciate the importance of the ion irradiation of FLG. In addition, future investigations at atomic scale for the edge reconstructions and other defects will be essential to support the results of this study.ArThe authors declare that they have no competing interests.SHA contributed in the overall project design and implementation. ME and MA contributed in the data analysis. AK contributed in the atomic force microscopy experiments. TH contributed in the few layer graphene sample preparation and sputtering. MTZM contributed in the X-ray photoemission spectroscopy experiment and manuscript preparation. MRA contributed in the manuscript preparation. All authors read and approved the final manuscript.SHA is an associate professor in Nanotechnology and Surface Science. ME is a professor in Theoretical Physics, doing first principle calculations. MA is an associate professor in Theoretical Physics and Computational Simulation Physics. AK is a masters degree student. TH is a Ph.D. student working on swift ions in materials. MTZM is a Ph.D. student working on nanotechnology. MRA is a professor in magnetism and head of the department.Effect of surface roughness on the nanobubble formation. Figure S1a,b show 2-D and 3-D AFM images of nanobubbles formed on the unsputtered FLG sample. The height and lateral size of the bubbles range from 2 to 5 nm and from 15 to 30 nm, respectively. It is also clear that the bubbles are well dispersed in a random fashion. However, agglomeration and an increase in their size and number density are prominent features of the bubbles grown on the irradiated FLG samples as seen from Figure S1 and , respectively.Click here for fileIrradiation features and roughness amplitudes after 3-keV ion irradiation. Figure S2. (a) 3-D image showing the effect of ion irradiation on the FLG edges after Ar+ ion irradiation of 3 keV. A, B and C denote the irradiated edges, cross sputtered areas at 53° from A direction and shadowing features, respectively. (b) PSD obtained from the image shown in (a) showing the amplitude of the surface's roughness of features A, B, and C as a function of the spatial frequency. FFT of the image shown in (a) revealing the presence of features A, B and C is shown in the inset of (b).Click here for file


Idiopathic pulmonary fibrosis (IPF) is a devastating interstitial pneumonia causing a loss of respiratory surface area due to a proliferative fibrotic response involving hyperplastic, hypertrophic, and metaplastic epithelium, cystic honeycomb change, septal expansion, and variable inflammation. Wnt (wingless) signaling glycoproteins are known to be involved in lung development and tissue repair, and are up-regulated in patients with IPF. Based on previous qRT-PCR data showing increased Wnt7B in lungs of IPF patients, a systematic, quantitative examination of its tissue site distribution was undertaken.Tissue samples from the Lung Tissue Research Consortium (LTRC) of 39 patients diagnosed with mild to severe IPF/usual interstitial pneumonia (UIP) and 19 normal patients were examined for the immunolocalization of Wnt7B.In normal lung, moderate Wnt7B reactivity was confined to airway epithelium, smooth muscle of airways and vasculature, and macrophages. IPF lung showed strong Wnt7B reactivity in fibroblastic foci, dysplastic airway and alveolar epithelium, and in highly discrete subepithelial, basement membrane-associated regions. All reactive sites were sized and counted relative to specific microscopic regions. Those in the subepithelial sites were found in significantly greater numbers and larger relative area compared with the others. No reactive sites were present in normal patient controls.The results demonstrate Wnt7B to be expressed at high concentrations in regions of active hyperplasia, metaplasia, and fibrotic change in IPF patients. In this context and its previously established biologic activities, Wnt7B would be expected to be of potential importance in the pathogenesis of IPF. Idiopathic pulmonary fibrosis/usual interstitial pneumonia (IPF/UIP) is a debilitating disease characterized by a loss of normal respiratory architecture and replacement with a heterogeneous population of myofibroblast-like cells and excess of fibrous connective tissue restricted to the lung ,2. IPF aThe signaling pathways involved are only partially understood. It is known that there is increased expression of TGF-β and α-smooth muscle actin (α-SMA) in progressive lesions of IPF reflecting the transition of fibroblasts to myofibroblasts with proliferation and collagen maintenance modulated by wingless (Wnt) glycoproteins -12. MousWnt glycoproteins have been found to be expressed at low levels in the normal lung and may be associated with epithelial turnover ,20. For Tissue blocks of formalin-fixed lung tissue samples were obtained from the Lung Tissue Research Consortium (LTRC). The samples had previously been placed into three groups see Table: group 1Sections were treated with citrate buffer for antigen retrieval and treated with a polyclonal goat anti-Wnt7B antibody  at a 1:100 dilution overnight at 4°C, followed by peroxidase-labeled secondary antibodies  and Nova Red . The Wnt7B antibody recognizes both precursor and mature forms of human origin. Control samples substituted normal goat serum for the primary antibody, or were pre-treated with 5% testicular hyaluronidase  for 30 minutes at 37°C to release Wnt7B bound to extracellular matrices , or trea2, 50–100 μm2, and >100 μm2; while their histologic sites were separated into one of three regional designations: 1) epithelium and underlying fibroblasts with or without extracellular matrix (EF), 2) subepithelial fibroblasts and ECM (SE) without epithelial staining (included most fibroblastic foci), and 3) interstitial fibroblasts and ECM (I) that were not directly subjacent to an epithelial surface. The number of sites corresponding to the respective categories was recorded. The total surface area, as defined by the external border of each tissue section, was determined using ImageJ , and the percent of the total area of each reactive site category relative to the total section surface area was calculated  for EF, SE, and I locations. Statistical significance was set at an alpha value of ≤ 0.05.Comparisons of the descriptive statistics of reactive sites were made using Analysis of Variance or Kruskal-Wallis non-parametric test for histologic locations in the same reactive site groups and within lung capacity category using SAS software  or Minitab . For example, for patients in FVC 50-80%, comparisons were made for reactive sites >100  had weak to moderate Wnt7B immunoreactivity in the cytoplasm of airway epithelium (ciliated cells) and smooth muscle of airways and arteries  and strong extracellular reactivity for Wnt7B, with most found adjacent to thickened airways , standard deviations, and minimum, median, and maximum sizes of reactive sites (in μm2) are presented in Tables Patients with FVC > 80% had few quantifiable Wnt7B reactive sites Figurea compareThe initiating cause(s) underlying the pathogenesis of idiopathic pulmonary fibrosis (IPF) is unknown. Much attention has focused on a failed repair of alveolar epithelium, and its impact on subsequent loss of pulmonary architecture. This failure manifests as the inability of epithelial surfaces to proliferate and differentiate effectively resulting in hyperplasia, metaplasia, and/or transdifferentiation into myofibroblast-like cells . Exactly2) that, like fibroblastic foci, had both cellular and extracellular matrix components. The loss of extracellular Wnt7B immunoreactivity after hyaluronidase digestion supports the matrix-associated localization, which is not surprising, as Reichsman, et al., [BMP-4 and Id2[The finding of strong Wnt7B immunoreactivity in the fibroblastic focus of UIP/IPF lungs would seem to fit readily with the above discussion on canonical Wnt signaling involving β-catenin. The demonstration of Wnt7B in dysplastic airway and alveolar epithelium and in myofibroblasts of the fibroblastic foci closely correlates with the site localization of active β-catenin in IPF lungs . However et al.,  have dem et al., . In the 4 and Id2. Further4 and Id2. It may 4 and Id2 where my4 and Id2. Coupled4 and Id2.2 (Figures The notion that Wnt7B may have a potentially contributive if not significant role in the development and/or progression of IPF draws on previous assumptions that the fibroblastic foci are central to the disease process and its prognosis ,40. The  Figures d2a3b-d. These observations draw attention to a specific Wnt signaling ligand, Wnt7B, which by virtue of its established roles in epithelial and mesenchymal proliferation and differentiation, procollagen production, and enhanced gene expression in IPF, would be expected to act as a significant contributor to the pathogenesis of IPF. This is supported not only by the strong expression of Wnt7B in fibroblastic foci but also by the numerous, small subepithelial sites that may represent early stages of developing fibroblastic foci as part of a larger, expanding network of fibrogenic tissue.There are no competing interests to report by any of the authors.TM – performed immunostaining, prepared photographic images, drafting and revision of manuscript; AH - performed immunostaining, KF - developed protocol staining and performed immunostaining; BG - performed immunostaining; JT – developed immunostainig protocol; DN - interpretation of data, drafting and revision of manuscript; SJ – analysis and interpretation of data, manuscript revision; MC - analysis and interpretation of data, statistical analysis; PS – conception and design of study, analysis and interpretation of data, preparation of photographic images, drafting of manuscript and revision; all authors read and approved of the final manuscript.


PD-L1, PD-L2 and other immune-related molecules regulate Th1 and Th2 immune responses. Tumor-expressed PD-L1, when bound to PD-1 or B7.1 on activated T cells, can mediate cancer immune evasion. While inhibiting PD-L1 receptor binding represents an attractive strategy to restore tumor-specific T-cell immunity, other immune-related factors and cell types within the tumor microenvironment affect anti-tumor T-cell responses. MPDL3280A, a human mAb containing an engineered Fc-domain designed to optimize efficacy and safety and to promote Th1-driven responses, is described here with PhI biomarker results. MPDL3280A was administered IV q3w in >300 pts with locally advanced or metastatic solid tumors. ORR was assessed by RECIST v1.1, including u/cCR and u/cPR. PD-L1 and CD8 were measured by IHC. The expression of ≈90 immune-related markers, including PD-L1 and PD-L2, were evaluated at baseline (BL) and on-treatment. BL tumor samples were available for 103 pts, and matched on-treatment samples were available for 26 pts. As of Feb 1, 2013, 140 pts enrolled prior to Aug 1, 2012 were evaluable for efficacy. Responses were observed in multiple tumor types including NSCLC (9/41), RCC (6/47), melanoma (11/38), CRC (1/4) and gastric cancer (1/1). An ORR of 21% (29/140) was observed in nonselected solid tumors with a duration of response of 1+ to 253+ days. Elevated pretreatment PD-L1 expression (IHC) was associated with response to MPDL3280A, and coordinated expression of PD-L1 and CD8+ T cells was seen. The ORR was 36% (13/36) for pts with PD-L1-pos tumors vs 13% (9/67) for pts with PD-L1-neg tumors. 81 pts had tumors evaluable for PD-L2. Median PD-L2 expression was ≈2x higher in PD-L1-pos tumors vs PD-L1-neg tumors. A T-cell gene signature  was associated with treatment response. On treatment, responding tumors had increased PD-L1 expression and a Th1-dominant immune infiltrate, evidence of adaptive PD-L1 upregulation. Nonresponders showed minimal tumor CD8+ T-cell infiltration and an absence of T-cell activation. Additionally, elevated expression of immune suppressors, including FOXP3 and RORC, correlated with lack of response. Both the presence of Th1-related CD8 biology at BL and adaptive tumor PD-L1 enhancement with MPDL3280A treatment correlated with tumor response, as did PD-L1 status. Updated data will be presented.


We conducted a metacommunity experiment to investigate the role of dispersal for bacterial community composition (BCC) and function of freshwater bacteria. Bacteria were dispersed from a common source pool into three different lake communities in their natural lake water. The experiment was conducted in dialysis bags to enable a decoupling between a change in the local environment and dispersal. BCC was determined by terminal restriction fragment length polymorphism (tRFLP) of the 16S rRNA gene. We show that the greatest changes in BCC occurred between 10% and 43% of dispersal of standing stock per day. Functioning, measured as growth rate, was also affected by dispersal in all three communities but the qualitative pattern differed between communities, sometimes showing a hump-shaped relationship to dispersal and sometimes decreasing with increasing dispersal. In all waters, functioning was related to BCC. Our results show that dispersal does affect BCC and functioning but that high dispersal rates are needed. Further, the effect of dispersal on BCC and function seem to depend on the quality of the habitat to which bacteria disperse into. Due to the concern of which consequences the loss of biodiversity will have on ecosystems, it is of considerable interest to evaluate the importance of biodiversity for ecosystem functions e.g. . The mecIn an experimental study, Venail et al. Bacterial communities can serve as practical models in ecology since experiments and field studies can relatively easy (in comparison to larger organisms) be designed over a range of relevant temporal and spatial scales Results obtained from studies of diversity-functioning relationships in bacterial communities have so far yielded disparate results. If different bacteria play different roles in ecosystems, for instance regarding their ability to degrade different organic molecules, changes in community composition (BCC) should lead to an altered functionality e.g. . On the In the present study we are conducting a laboratory experiment in which bacteria were dispersed into bacterial communities in dialysis bags kept in larger containers with natural water. Because the dialysis bags allow transportation of water and dissolved chemicals but not bacterial cells across the membrane, dispersal can be disconnected from a change in environmental conditions. The aim of this study was to evaluate 1) the magnitude of dispersal rates from a metacommunity that are needed to cause changes in local BCC, 2) whether dispersal from a metacommunity affects a functional trait, such as local bacterial growth rate, and 3) whether there is a relationship between BCC and the functional trait.−1, total organic carbon (TOC) = 5 mg L−1, Absorbance at 430 nm, a measure of humic matter content, (Abs430) = 0.073), Lake Fälaren is a humic lake , and Lake Funbosjön is an eutrophic lake . Lakes Fälaren and Funbosjön were sampled on July 27 2009, and Lake Ånnsjön July 25 2009. The experiment started on the 29 of July 2009 prior to which all water was filtered through a 1 µm glass fibre filter (Gelman A/E) to remove larger organisms than bacteria.We sampled water and the natural bacterial communities from three lakes with different characteristics to invoke large differences in environmental conditions between communities. No permits were required to sample these lakes. Lake Ånnsjön is an oligotrophic clear-water lake  = 15 µg LFrom each lake we filled three 10 L buckets (replicates) with filtered water, i.e. in total there were nine buckets. In each bucket we placed five 10 mL dialysis bags  containing the natural bacterial community from that lake . In addition we also added one dialysis bag with equal parts from the three different lakes (Mix) that served as a treatment with no continuous dispersal but where dispersal limitations between communities was eliminated. Thus, for the experiment we had 9*6 = 54 dialysis bags. In addition we also had one negative control dialysis bag of autoclaved MQ water in each bucket. The water in the buckets was mixed by vigorous air bubbling. The dialysis bags were pre-wetted in MQ water for one day before the initiation of the experiment.Three times a day we dispersed bacteria into the communities in the dialysis bags by pipetting. At each dispersal event a metacommunity was first constructed from a mixture of equal volumes of the three original lake bacterial communities. That is, the three natural lake communities were kept in separate 1 L glass bottles under the same conditions as the experiment was run (see below). At dispersal, equal volumes were taken out from the three lake communities and mixed into a metacommunity which we used for dispersion of bacteria into the dialysis bags. See 3, where X is volume transferred in and out of a dialysis bag per dispersal event. Thus, for the 1.7 mL and 3.3 mL treatments the approximate dispersal rates were 43% and 70% per day, respectively. The 0 dispersal and the “Mix” treatment received no dispersal during the course of the experiment, but were opened and closed to mimic the handling of the dispersal treatments.Empirical estimates about how high dispersal rates are needed to affect bacterial communities in lakes are scarce, but there are indications that high dispersal rates are required In nature, lakes exist with water retention times of one day (i.e. 100% replacement per day), and in Sweden the great majority of lakes has a water retention time of less than 100 days The experiment was run for 5 days at 15°C in darkness. At the end of the experiment samples were taken from each dialysis bags for analyses of bacterial community structure, growth rate and abundance. Samples for community composition and abundance were also collected from the three start communities and the three communities making up the metacommunity from which dispersal occurred, at the end of the experiment.2HPO4 (1g L−1). The bag was put in a beaker with deionized water and we recorded conductivity in the beaker for up to 4 hours. The conductivity in this beaker was compared with the conductivity in a beaker where the same amount of salt had been added directly into the same amount of deionised water.Dialysis bags prevent movement of bacteria out of the bag but allow the local environmental conditions in the bags to be close to the conditions in the buckets, independent of dispersal. Compared to previous experiments on microbial communities e.g.  we were HaeIII and HinfI.Bacterial community composition (BCC) was determined by terminal restriction fragment length polymorphism (tRFLP) of the 16S rRNA gene as described by Lindström et al. Peaks (tRFs) in lengths greater than 447 bp were eliminated from the analysis. Peaks with a relative area less than 0.5% were also eliminated from the analysis as these are difficult to separate from noise. The average relative peak area of restriction digest duplicates was used for further statistical analysis. In five samples, among which 4 were experimental negative controls (i.e. autoclaved MQ water), the amount of DNA extracted was too low to enable tRFLP analysis.1 mL samples for bacterial abundance were collected and preserved with 4%  formaldehyde. Bacterial abundance was determined by flow cytometry (Partec) after staining with Syto13 at a final concentration of 1.25 µM Bacterial growth rate was determined using the leucine incorporation method Bray-Curtis (relative peak areas) as well as Sørensen (presence-absence of peaks >0.5%) dissimilarities 2,45 = 15, P<0.001, r2 = 0.33; Sørensen: F2,45 = 26, P<0.001, r2 = 0.46) and dispersal . The importance of lake identity for BCC among communities receiving the same dispersal treatment declined with increasing dispersal rate  showed greater similarity to each other . For botsal rate . Lake idsal rate . The expsal rate . The impsal rate . When th−1) differed depending on dispersal treatments , lakes  as well as on the interactions between them . In the oligotrophic lake (Ånnsjön) and humic lake (Fälaren) the bacterial growth rate peaked at intermediate dispersal, i.e. at 43% per day  and growth rate per cell  in the negative controls were lower than in all other treatments in the oligotrophic and humic lakes, while in the eutrophic lake two of the negative controls had higher growth rate than the two highest dispersal treatments. In the five (of the nine) negative controls where DNA extraction was possible, >84% of the tRFLP peak area corresponded to peaks also found in the start communities or source communities of the respective lakes. Thus, there appears to have been, in some cases, inflow of viable bacteria from the buckets, either over the dialysis membranes or during handling. This may in some cases have had a marginal impact on the growth rate, and may have affected BCC. Thus, this inflow can have exaggerated the role of the local environment for BCC slightly, especially in low dispersal treatments. An important point is though that there was no abundant “alien” bacterium growing within the bags confounding the results.By the end of the experiment water colour at 430 nm was similar in the buckets and the dialysis bags receiving the highest dispersal in the humic  and in the eutrophic  lake. However, in the lake with the clearest water, colour had increased in the highest dispersal treatment by almost 70%, since absorbance was 0.073 in the bucket and 0.124 in the dialysis bag. At the end of the experiment, salt was added in a dialysis bag from the most eutrophic lake (Funbosjön). After 4 h dialysis the salt concentration in the surrounding beaker was 83% of the control beaker. Thus, the diffusion of inorganic ions through the membrane was still efficient at the end of the experiment.Our results show that both the local environment (i.e. species sorting) and dispersal (i.e. mass effects) affected BCC, which in turn altered bacterial functioning. Low dispersal communities (i.e. 0–10% of standing stock per day) were similar to the start communities of the respective lakes, while the high dispersal communities (i.e. 43 and 70% per day) became more similar to the same treatments from other lakes. The Mixed communities, i.e. identical start communities, kept in the different lake environments but receiving no further dispersal, showed variable results. Thus, high and continuous dispersal rates were necessary to consistently change community composition.Clearly, the relative importance of species sorting decreased with increasing continuous dispersal rates. However, even at the highest dispersal treatments the local environment (i.e. the lake water) explained a great share of the variation in community composition, and, consequently, the high dispersal communities in the three environments were not identical. Hence, at least parts of the communities were affected by species sorting processes rather than mass effect also at very high dispersal. This conclusion is not likely to change regarding that organic carbon increased with dispersal in one lake. At the opposite, differences in BCC could have been even larger if the environment would have been kept even more similar to that of the bucket.Results from field studies have shown that not only the immigration rate determines if a community is shaped by mass effect or species sorting. For instance, the position of the habitats of the sink and source communities in the landscape may be of importance In the oligotrophic and the humic lakes the bacterial growth rates peaked at intermediate dispersal levels (43%), i.e. suggesting that also in these lakes variation in BCC imposed by dispersal affected functioning. It should be noted that dispersal increased concentration of organic compounds in the communities from the oligotrophic Lake Ånnsjön, which could have influenced bacterial growth rates. However, since the relationship between dispersal and growth rate was not linear, but rather hump-shaped, this effect did not seem to be a consequence of increased nutrient levels. The close to identical results obtained when using dpm directly or dpm per cell as a measure of growth rate also show that the results were not dependent on changes in cell abundances caused by the dispersal. Thus, we conclude that dispersal appear to have changed growth rate via a change in BCC.Previous studies investigating the role of microbial dispersal for ecosystem function  also found highest biomass Therefore, our results show that dispersal can change community function via a change in community composition but that the exact outcome may differ depending on the characteristics of the habitats studied. Future studies therefore need to address how different environments affect the interplay between dispersal, BCC, and functioning. It should also be fruitful to evaluate the possibility that dispersal and establishment may vary among taxa, which would lead to that different populations are affected by different forces. With the future development of techniques for the study of microbial diversity, i.e. sequencing technologies such as 454-sequencing e.g. , OTU defFuture experimenters should also seek for means to investigate more long-term effects of dispersal, which our design did not allow for technical reasons. The start and end communities of Lake Ånnsjön and Lake Fälaren inocula were similar suggesting that community compositions were close to equilibria. In contrast, the start and end communities of the Lake Funbosjön inocula had shifted over the study period . How thiOur results together with the observations made by Venail et al. Figure S1There were three buckets (replicates) of lake water from each of the three lakes. The source community (metacommunity) was constructed by equal volumes of water from the three lakes. This source community was then dispersed into the six dialysis bags in each bucket, with different rates, three times per day. The dispersal treatments were replacement of 0, 1, 10, 43 and 70% of the dialysis bags volumes per day. The ”Mix” treatment was a mixture of equal volumes of the start communities from each lake, which thereafter received no further dispersal. All dispersal treatments were made in all buckets.(TIFF)Click here for additional data file.