Surprisingly, the observed K+/Cl− selectivity persists in pores even as large as about 20 nm in diameter, suggesting that high throughput, highly selective graphene electrodialysis membranes can be fabricated without the need for subnanometer control over pore size.As population growth continues to outpace development of water infrastructure in many countries, desalination  at high energy efficiency will likely become a vital source of fresh water. Due to its atomic thinness combined with its mechanical strength, porous graphene may be particularly well-suited for electrodialysis desalination, in which ions are removed under an electric field via ion-selective pores. Here, we show that single graphene nanopores preferentially permit the passage of K Sub-nanometer graphene nanopores are usually required to create graphene-based reverse osmosis membranes. Here, Rollings et al. show that membranes with larger pores are highly ion selective and a hundred times more permeable to potassium ions than to chloride ions, making them useful for electrodialysis. Atomically thin graphene membranes have generated considerable interest for use as filtration membranes because their atomic thickness presents minimal resistance to fluid or ion flow while retaining high structural integrity. Recent investigations have suggested that porous graphene membranes can attain orders of magnitude higher flow rates than commercial reverse osmosis (RO) membranes, while still providing excellent salt rejection123456681011713Here, we examine ion selectivity  of single graphene nanopores with an emphasis on the relationship between ion selectivity and pore size. These experiments not only allow us to evaluate porous graphene membranes as electrodialysis membranes, but also shed light on the chemical structure of graphene pore edges and ion-specific interactions with graphene membranes.in situ enlargement of the nanopore16171819151621Graphene nanopores were fabricated using a recently reported electrical pulse method that enables rapid fabrication of very small single nanopores, as well as controllable, G is the pore conductance, σ is the solution conductivity (105 mS cm−1), t is the effective thickness of the graphene membrane  curves were performed with a variety of KCl concentration gradients across the pore (+ and Cl− ions were selected as a representative cation/anion pair because they have very similar bulk mobilities (see I-V curves for a 3 nm pore is shown in V=0) both K+ and Cl− ions diffuse from high to low concentration, and a net current  is produced only if one ion diffuses at a higher rate than the other through the pore . The lower horizontal axis is the measured conductance in 1 M KCl, while the upper axis shows estimated pore diameter based on +/Cl− selectivity ratios at pH 8 were generally above 100, values comparable to the biological ion channels and polymer membranes many microns thicket al.SGHK=1.3. It is possible that the high K+/Cl− selectivities measured here are unique to electrically pulsed pores, or that a small number of large pores or tears in the centimeter-scale membranes of O'Hern et al.SGHK<5, SGHK≅2 , where the edge charge is screened out by mobile counterions251621+/Cl− selectivity measurements.While the presence of negatively charged groups on the pore edge can explain strong K− anions at pH 8 is negligible for small pores (as shown in the previous section). It is important to note, however, that different cations have significantly different electrophoretic mobilities, which result in differences in conductivity in bulk solution  asTo examine how inter-cation selectivity depends on pore size, we measured normalized cation conductances for a pore during the sequential stages of electrical pulse enlargement  carries a pH-dependent surface charge due to deprotonatable oxygen-containing chemical groups on the graphene surface (oxidized graphene), or attached to the hydrocarbon contaminants on the graphene surface34The persistence of strong KTo test the plausibility of this hypothesis, we measured pore conductance with various KCl concentrations at pH 2 and 8 . These mσ=−0.6 C m−2 . As a result, the K+ current density is elevated and the Cl− current density reduced over the surface path  equations for a 2D axisymmetric pore geometry with a variable surface charge on the suspended graphene surface  as a function of salt concentration (chigh) and compared the results with predictions from the PNP model  is still needed to complete our understanding of the mechanism responsible for ion selectivity. Modifying the surface or using different thin materials may also allow nanopores to select for anions instead of cations. Although we have limited this study to single pores, we expect that large-area porous membranes containing many nanopores, 20 nm or smaller, will retain high selectivity while supporting orders of magnitude larger ionic currents. Previous investigations of the desalination potential of graphene have focused on subnanometer diameter pores for RO, but the results shown here suggest that such strict fabrication limitations are not necessary. The loosening of the pore size upper boundary from around 1 to 20 nm means that existing techniques for creating porous graphene membranes can likely be used to create highly effective cation exchange membranes for electrodialysis. Furthermore, these surprising observations indicate that atomically thin nanopores can behave quite differently than their thicker counterparts, and should continue to be a rich platform for studying nanoscale mechanisms of ion transport.In summary, we have shown that graphene nanopores up to about 20 nm in diameter show K2 and 4 sccm CH4 for 40 min. Graphene was transferred to an approximately 150-nm diameter aperture in a 300-nm thick low-stress LPCVD silicon nitride (SiNx) membrane using established wet transfer techniquesx membrane was prepared using standard techniques, including photolithography and anisotropic KOH etching of the silicon substrate. The 150-nm diameter apertures were milled using an FIB . Once transferred, samples were annealed at 250 °C for at least 2 h under 200 sccm H2 and 400 sccm Ar flow to remove surface contamination and were stored in a nitrogen-flushed drybox. Before wetting, the graphene membrane samples were annealed at 700 °C for 2 h under 200 sccm H2 and 400 sccm Ar to further remove surface contamination.Single-layer graphene was grown on Cu foil (Alpha Aesar) at 1,000 °C with a flow of 10 sccm H2 was flowed through the flow-cell for 3 min to purge out any air. The fluidic cell was then flushed on both sides of the membrane with 1 M KCl, pH 10, which reacts with CO2 gas to form soluble carbonate anions, removing all gas in the fluidic cell and fully wetting the graphene membrane. The fluid reservoirs on each side of the fluidic cell were contacted via Ag/AgCl electrodes in 1 M KCl via agarose salt bridges to eliminate the potential generated from redox reactions on electrodes in asymmetric salt conditions. An Axopatch 200B patch clamp amplifier was used to apply d.c. voltage biases and measure ionic current. Pores were nucleated and enlarged using a pulse generator (HP8110A) as previously describedSamples were loaded into an airtight PEEK fluidic cell with PDMS gaskets, and 99.999% pure COI-V measurements. Selectivity SGHK was estimated by nonlinear least squares regression to the measured reversal potential using equation SGHK depends exponentially on reversal potential and for the extremely high selectivities measured here (SGHK>100), small deviations in reversal potential produce large, nonlinear changes in SGHK. Error estimates in SGHK. Reported error bars in selectivity ratio Si in Reversal potentials were determined from interpolated After electrical pulse fabrication and solution-based experiments, samples were removed from the fluidic cell and stored in deionized water. To avoid membrane damage due to surface tension, samples were critical-point dried before imaging. The nanopores were imaged in a JEOL 2010 F TEM operating at 200 kV. Efforts were made to minimize the beam exposure during imaging, because 200 kV electrons at high doses are capable of creating defects in graphene membranes. The yield for TEM imaging of electrical pulse-fabricated nanopores was very low (about 1 in 10) due to contamination of the membrane during drying and imaging. Contaminants from the solutions and from the air can easily cover the nanopores, especially under the electron beam, which can cause further deposition of contaminants. It seems that membranes that have been exposed to solution are more contamination-prone than membranes that have never been wet.+ and Cl− ions and the electric field using the steady-state Poisson equation and the Nernst–Planck equation for each ion individually . The model solves for concentrations of Kally see . The negHow to cite this article: Rollings, R. C. et al. Ion selectivity of graphene nanopores. Nat. Commun. 7:11408 doi: 10.1038/ncomms11408 (2016).Supplementary Figures 1-3, Supplementary Table 1, Supplementary Note 1 and Supplementary References


Present in the genomes of bacteria and eukaryotic organelles, group II introns are an ancient class of ribozymes and retroelements that are believed to have been the ancestors of nuclear pre-mRNA introns. Despite long-standing speculation, there is limited understanding about the actual pathway by which group II introns evolved into eukaryotic introns. In this review, we focus on the evolution of group II introns themselves. We describe the different forms of group II introns known to exist in nature and then address how these forms may have evolved to give rise to spliceosomal introns and other genetic elements. Finally, we summarize the structural and biochemical parallels between group II introns and the spliceosome, including recent data that strongly support their hypothesized evolutionary relationship. Investigating the evolution of mobile DNAs involves unique challenges compared to other evolutionary studies. The sequences of mobile DNAs are usually short and evolve rapidly, resulting in limited phylogenetic signals. The elements often transfer horizontally, which prevents the linkage of their evolution to that of their host organisms or other genes in the organism. Finally, many mobile elements themselves consist of multiple components that may have different evolutionary histories. All of these complicating factors apply to group II introns and must be considered when trying to understand their evolutionary history.in vitro, while the intron-encoded protein (IEP)’s open reading frame (ORF) sequence is contained internally within the RNA sequence and encodes a reverse transcriptase (RT) protein [Group II intron retroelements consist of an RNA and a protein component. The RNA is a ribozyme  that is capable of self-splicing  protein -6. The t protein ,8.Group II introns have attracted considerable attention, in part due to their hypothesized relationship to eukaryotic pre-mRNA introns. The purpose of this review is to carefully consider the evidence available regarding the evolutionary history of group II introns. We present a summary of the multiple types of group II introns known to exist in nature and discuss a model for how the variant forms arose and subsequently evolved into spliceosomal introns and other elements.in vitro under appropriate reaction conditions, typically with elevated concentrations of magnesium and/or salt.The biochemical and genetic properties of group II introns have been described in depth elsewhere ,5,6,9-14The IEP is encoded within the loop of the RNA domain IV Figure  and is tin vivo. The translated IEP binds to the unspliced intron structure via the RT and X domains, which results in RNA conformational adjustments leading to splicing  ,31,40-44Relevant to this review is a key distinction in the character of group II introns in bacteria compared to introns in mitochondria and chloroplasts. In bacteria, the introns behave mainly as mobile DNAs that survive by constant movement to new genomic sites, whereas in organelles, they are less mobile ,49,50. TThe varieties of group II introns can be classified either according to their RNA or IEP components. Group II introns were initially classified as IIA or IIB based on the RNA sequence and secondary structure characteristics of introns in mitochondrial and chloroplast genomes . A thirdAlternatively, group II introns can be classified according to phylogenetic analysis of their IEP amino acid sequences. Eight IEP classes have been defined: mitochondrial-like (ML), chloroplast-like (CL), A, B, C, D, E, and F ,50,56. TReconstructing the evolution of group II introns requires an accounting of all known intron forms and their distribution. Here, we describe the range of variants that differ from the ‘standard’ retroelement form diagrammed in Figure Approximately a quarter of group II intron IEPs in organelles and over half in bacteria lack an En domain ,50,51, iattC motifs of integrons (imperfect inverted repeat sequences that are recognized by the integron’s integrase) [With regard to mobility mechanisms, En-minus introns are unable to form the bottom strand primer and require an alternative pathway. It has been shown for these introns that the primer is provided by the leading or lagging strand of the replication fork during DNA replication ,59-62. Stegrase) ,52,63-69trnKI1 in plant chloroplasts, nad1I4 in plant mitochondria, and psbCI4 in Euglena chloroplasts)  or lost alignability altogether to some of the conserved RT motifs  Figure C [27,28,trnKI1, which is located in an essential tRNALys gene. The IEP encoded by this intron, MatK, aligns with other RTs only across motifs 5 to 7, with the upstream sequence being unalignable with motifs 0 to 4; however, domain X sequence is clearly conserved, suggesting the maintenance of the maturase function [A well-studied example is the chloroplast IIA intron function ,44. MatKfunction ,72.Oceanobacillus iheyensis, which encodes an IEP of the ML class that lacks the YADD and other motifs. The fact that the ORF has not accumulated stop codons suggests that it retains maturase activity, particularly because its exons encode the DNA repair protein RadC [In bacteria, degenerations of the IEP sequences are rare because the great majority of non-truncated intron copies are active retroelement forms. The only known example is O.i.I2 of ein RadC .Thiomargarita namibiensis [Ustilago and Leptographium were shown biochemically to cleave intronless target sequences [Leptographium did not promote splicing of the host intron, as sometimes occurs for some group I intron-encoded LHEs [A small set of group II introns do not encode RT ORFs but instead encode proteins of the family of LAGLIDADG homing endonucleases (LHEs) and are presumably mobile through a distinct pathway that relies on the LHE Figure D. LHEs iibiensis -76. LHEsibiensis . Consistequences ,78. Howeded LHEs ,79. To dded LHEs ,80.Group II introns without IEPs have lost retromobility properties and exist as splicing-only elements Figure E. They atrans on the ORF-less introns [trans has in fact been demonstrated experimentally for an IEP in a cyanobacterium [Clostridium tetani, for which no IEP-related gene is present in its sequenced genome. C.te.I1 self-splices robustly in vitro, and it was speculated that the intron might not require splicing factors in vivo [In bacteria, ORF-less group II introns are rare. Among the known examples, the ORF-less introns nearly always reside in genomes containing related introns whose IEPs may act in  introns . Splicinacterium . The sol in vivo ,89. This in vivo .in vitro.Many group II introns in mitochondria and chloroplasts have defects in conserved ribozyme motifs, such as mispaired DV or DVI helices or large insertions or deletions in catalytically important regions Figure F 15,44,,91,92. FatpFI1), whereas others  aid in splicing multiple introns, which are usually structurally related [For introns with compromised ribozyme structures, splicing relies heavily on host-encoded splicing factors ,93,94. T related -100. The related ,101. Add related ,102-105.These examples illustrate that group II introns have repeatedly lost their splicing capability in organelles. To compensate, cellular splicing factors have evolved independently in different organisms to enable efficient splicing of the introns that lie in housekeeping genes. Similar to the case of ORF-less group II introns, there has been a conversion from retromobility to splicing-only function, and splicing is under the control of the host nuclear genome.Euglena gracilis chloroplasts , a number of group II introns have attained plasticity that allows them to splice at other points , which result in two protein products, one consisting of the upstream exon ORF alone and the other a fusion of upstream and downstream ORFs [C. tetani intron C.te.I1 utilizes four 3′ splice sites, each specified by a different DV/VI repeat. Each resulting spliced product is a distinct fusion protein between the 5′ exon-encoded ORF and one of four downstream exon-encoded ORFs [In bacteria, alternative splicing at the 3′ splice site was found for B.a.I2 of eam ORFs . In a moded ORFs . The latEuglena chloroplasts where they were first described, and where approximately 30 of its 160 introns are in twintron arrangements [Methanosarcina acetivorans have a particularly complex arrangement [A twintron is an intron arrangement in which one group II intron is nested inside another intron as a consequence of an intron insertion event Figure L. For a ngements . Severalngements ,122. Twiangement . There aEuglena chloroplasts [Rhodomonas salina) [Chlamydomonas) [Chattonella [Kluyveromyces lactis [Wolbacchia endosymbionts, because the resident introns are of different classes [While there has been much speculation about intron evolution, it remains difficult to obtain direct evidence for specific models. For group II introns, clear phylogenetic conclusions can only be drawn when analyzing closely related introns. This is because only closely related sequences allow the extensive alignments needed for robust phylogenetic signals. Such analyses have indicated multiple cases of horizontal transfers among organisms. Some of the inferred examples are as follows: from an unknown cyanobacterial source to roplasts ; from un salina)  or a gredomonas) ; betweenttonella ; and fros lactis ,128. In  classes . More br classes ,130,131.Beyond identification of horizontal transfers, unfortunately, global phylogenetic analyses result in poor phylogenetic signals because the number of characters available  decrease to at most approximately 230 aa for the ORF and approximately 140 nt for the RNA . With suThe limited phylogenetic resolution for group II introns was attributed to several potential factors . First, Over a decade ago, it was noticed that there is a general pattern of coevolution among group II intron IEPs and their RNA structures ,133. SpeThe principle of coevolution is a central principle to deciphering the history of group II introns. Importantly, it simplifies the reconstruction from two independent histories to a single history. Based on the pattern of coevolution, a model was set forth to explain the history of group II introns, which was called the retroelement ancestor hypothesis ,133. ThePhylogenetic analyses have in general supported the initial observation of coevolution, because both RNA and IEP trees define the same clades of introns, thereby excluding extensive exchanges between ribozymes and the different classes of IEPs . HoweverThe retroelement ancestor model continues to be consistent with available data and is elaborated here to show how it can explain the emergence of the known forms and distribution of group II introns Figure . The ancattC sites) [Of the three target specificities known for bacterial introns  ,65, any C sites) ,138,139.C sites) ,119,123.Introns belonging to the lineages CL, D, and E migrated from Eubacteria to archaebacteria ,123. TheWithin mitochondria and chloroplasts, the character of group II introns changed to become more genomically stable and less selfish. The introns took up residence in housekeeping genes, which necessitated efficient splicing, and which was enabled by host-encoded splicing factors ,93-96. Wtrans-splicing due to genomic rearrangements that split intron sequences [Euglena chloroplasts, the introns degenerated on a spectacular scale to become group III introns. The earliest euglenoids are inferred to be intron-poor while the later branching euglenoids harbor more introns, pointing to a process of intron proliferation within Euglena chloroplasts [In fungi, a small fraction of ORF-less introns acquired an IEP of the LAGLIDADG family, which permitted mobility through the homing endonuclease mechanism. In mitochondria and chloroplasts, introns sporadically became equences ,112,113.roplasts ,141.trans on the ORF-less intron. A single independent, ORF-less group II intron was found out of 225 genomes surveyed. Hence, it seems safe to predict that relatively few ORF-less introns have been overlooked in bacteria, unless they have domain V structures unlike those of known group II introns.It should be kept in mind that this model is contingent upon the available sequence data. One cautionary note is that our picture of group II introns in bacteria may be skewed, because for the data available the introns were identified bioinformatically in genomes based on the RT ORF. This may result in some oversight of ORF-less group II introns; however, the numbers of those introns do not appear to be large. In a systematic search of bacterial genomes for domain V motifs, nearly all introns identified were retroelement forms . There wIf the ancestor of extant group II introns was a retroelement, where did that retroelement come from? The simplest scenario is that pre-existing ribozyme and RT components combined into a single element, creating a new mobile DNA. An interesting alternative possibility is that a self-splicing RNA might have arisen at the boundaries of a retroelement to prevent host damage by the mobile DNA .There are many potential sources for the ancestral RT component, because a myriad of uncharacterized RTs exist in bacterial genomes, most of which could potentially correspond to forms that were co-opted by the primordial group II intron . BecauseSimilarly, there are many structured RNAs in bacteria that could have given rise to the ancestral group II ribozyme, including noncoding RNAs, riboswitches, or even a fragment of the ribosome -146. TheWhich class of modern group II introns best represents the ancestral group II intron retroelement? It is often claimed in the literature that IIC introns are the most primitive form of group II introns ,18,147. The concept that group II introns were the ancestors of spliceosomal introns emerged shortly after the discovery of multiple intron types  -151. SinThe major similarities and parallels for the two intron types are summarized here. In terms of splicing mechanisms, the overall pathways for group II and spliceosomal introns are identical, with two transesterifications and a lariat intermediate Figure A. The chStructurally, there are many parallels between group II intron RNAs and spliceosomal snRNAs, which run the gamut from being clearly analogous to being speculative. The most obvious parallel is the branch site motif that presents the 2′OH of a bulged A to the 5′ splice site for the first step of splicing. For group II introns, the bulged A is contained within a helix of domain VI; in the spliceosome the same bulged structure is formed by the pairing of the U2 snRNA to the intron’s branch point sequence  located at the heart of the spliceosome. A region of Prp8 cross-links to the 5′ and 3′ exons and also to the intron’s branch site, indicating its proximity to the spliceosome’s active site. Surprisingly, the crystal structure of a major portion of yeast Prp8 revealed that the cross-linking portion is composed of a reverse transcriptase domain fold . In facttrans that had either the native bI1 stem-loop sequence or the loop 1 sequence of the U5 snRNA [Additional parallels between group II intron and spliceosomal intron RNAs are credible but less clear. The loop 1 structure of U5 snRNA is predicted to be analogous the EBS1 loop of group II introns, a substructure that forms base pairs with the 5′ exon of group II introns, thereby delivering the 5′ exon to the active site  to the distal stem of domain V Figures  and 5. TGAGA box and the last nucleotide (G) of the intron.It is important not to ignore features that are not shared between group II and spliceosomal introns, in the rush to pronounce the two types of introns equivalent. Each type of intron has features not found or reported in the other. For example, the γ-γ′ interaction of group II introns is a Watson-Crick base pair between a J2/3 nucleotide and the last position of the intron, but it has not been reported for spliceosomal introns Figures  and 5. TTwo critical pairings that occur in the spliceosome but not in group II introns are temporal pairings formed during spliceosome assembly but not catalysis . The U1 On the other hand, Helices Ia and III of the U2-U6 structure Figure  occur duA modest discrepancy involves the secondary structure of the ISL of U6 and the DV structure of group II introns. The secondary structure of the ISL is usually drawn with an AU bulge opposite an unpaired C  domain, and possibly other domains that form portions of the modern 280-kDa Prp8 protein ,192. AddOne intriguing model for the emergence of the spliceosome predicts that proliferation of mobile group II introns was the driving force for invention of the nuclear membrane ,193. Thetrans-splicing intron-in-pieces. It was shown that a retromobile IIA intron could be split into multiple functional trans-splicing RNA transcripts, with the break points distributed throughout the sequence and not only in domain IV as occurs for nearly all natural trans-splicing introns [Several studies have experimentally addressed evolutionary issues of group II introns. One series of studies sought to reproduce the fragmentation of a group II ribozyme into a  introns ,194,195. introns ,196,197.In addition to spliceosomal introns, group II introns are believed to be the ancestors of non-LTR retroelements, a major class of mobile DNAs in eukaryotes . The RTscas1 gene (denoted G2L1 and G2L2 (group II-like 1 and 2) [cas1 gene encodes a nuclease that helps integrate short sequences of phage or plasmid into CRISPR arrays, lending cellular immunity to DNAs containing those sequences [Moreover, it is clear that group II introns spawned other RT-containing units. A subset of CRISPR/Cas elements contain an RT gene, either as a free-standing ORF or fused to a 1 and 2) ,201). By1 and 2) . The casequences . The RT Three additional types of group II-related RTs exist in bacteria, denoted G2L3, G2L4, and G2L5 . These aGroup II introns are compact and versatile retroelements that have successfully colonized genomes across all domains of life and have given rise to many variant forms. Current data are consistent with the model that the retroelement form (that is, the form diagrammed in Figure With regard to the evolutionary pathway of group II introns into spliceosomal introns, important insights over the past 2 years have largely erased doubts about the long-standing hypothesis that the spliceosome descended from group II introns. Indeed, there are no credible competing hypotheses for the origin of the spliceosome. Still, the specifics of the pathway and the full scope of mechanistic parallels remain to be resolved. Additional insight may be forthcoming from structural elucidations of the spliceosome and comparisons to group II intron structures, as well as genomic comparisons of early branching eukaryotes, which may give information about introns in the LECA and potentially suggest evolutionary intermediates or pathways. Overall, the elucidation of group II intron biology, structure, and evolution remains an important facet in understanding the evolution and dynamics of eukaryotic genomes.


Continuous-flow culture models are one tool for studying complex interactions between members of human fecal microbiotas because they allow studies to be completed during an extended period of time under conditions where pH, nutrient availability, and washout of waste products and dead cells can be controlled. Because many of the existing well-validated continuous-flow models are large and complex, we were interested in developing a simpler continuous-flow system that would allow microbial community dynamics to be examined in higher throughput while still maintaining complex microbial communities. To this end, we developed minibioreactor arrays (MBRAs), small volume bioreactors (15 ml) that allow simultaneous cultivation of up to 48 microbial communities in a single anaerobic chamber.We used MBRA to characterize the microbial community dynamics of replicate reactors inoculated from three different human fecal donors and reactors seeded with feces pooled from these three donors. We found that MBRA could be used to efficiently cultivate complex microbial communities that were a subset of the initial fecal inoculum . After an initial acclimation period of approximately 1 week, communities in each reactor stabilized and exhibited day-to-day variation similar to that observed in stable mouse fecal communities. Replicate reactors were predominately populated by shared core microbial communities; variation between replicate reactors was primarily driven by shifts in abundance of shared operational taxonomic units (OTUs). Consistent with differences between fecal donors, MBRA communities present in reactors seeded with different fecal samples had distinct composition and structure.From these analyses, we conclude that MBRAs can be used to cultivate communities that recapitulate key features of human fecal communities and are a useful tool to facilitate higher-throughput studies of the dynamics of these communities.The online version of this article (doi:10.1186/s40168-015-0106-5) contains supplementary material, which is available to authorized users. The gastrointestinal microbiome plays an important role in health and disease (reviewed in –8). AlthContinuous-flow culture models are beneficial for studying the complex interactions between members of the host microbiota in vitro because they allow for studies to be completed during an extended period of time under conditions where pH, nutrient availability, and washout of waste products and dead cells can be better controlled (reviewed in –25). AltTo this end, we developed minibioreactor arrays . MBRAs Clostridium difficile unless perturbed by antibiotics ).Having identified the window in which communities stabilize within each reactor days 8–21), we next wanted to examine how these communities varied across replicates from the same fecal donor. We determined the mean similarities between replicate reactors inoculated from the same donor on days 8–21 and compared this to the mean similarities observed within each reactor from day-to-day on days 8–21  and used data generated by pyrosequencing the V3–V5 region of the 16S rRNA gene, we reanalyzed a subset of their data, which was generated by Illumina sequencing of the V4 region and used to cross-validate a new dual-indexing sequencing approach for community analyses [Because their original work analyzed stability with a different dissimilarity measure . Flores et al.  examinedAnother possible contributing factor to the lower variation observed in mouse data is that communities colonizing these mice are highly adapted to co-existence in the murine GI tract, where selection can be imposed by interactions with host cells, other members of the microbiota, and nutrients from the host diet. In contrast, cultivation in MBRAs could allow organisms with functional redundancy under MBRA cultivation conditions to fluctuate stochastically during stabilization until stable communities are reached.To gain further insights into the composition and structure of MBRA communities, we identified those OTUs that were conserved across samples . We identified core communities on three different levels: OTUs found conserved from day-to-day over days 8–21 within each single reactor , OTUs shared from day-to-day over days 8–21 between replicate reactors of the same fecal donor , and OTUs common to all reactor communities over time . We also compared the composition of these core communities to their starting fecal inocula.OTUs were designated as members of a core community if they were present in at least 90 % of daily samples collected from a single reactor between days 8–21. Using these criteria, we found that the core communities maintained from day-to-day within each reactor varied from 55 to 72 OTUs  and were not present in any of the individual core communities  or donor B . Growth under MBRA cultivation conditions led to a decrease in the ratio of Firmicutes/Bacteroides in reactor communities seeded from donor C (mean ratio in MBRA communities from donor C: 56 % Firmicutes/32 % Bacteroidetes), which was more similar to that observed for MBRA communities seeded with other fecal samples. The phylogenetic distribution of the pooled fecal inoculum was unexpected, with only 2 % of sequences from Bacteroidetes, 81 % of sequences from Firmicutes, 3 % of sequences from Actinobacteria and 4 % of sequences from unclassified bacteria. The levels of Bacteroidetes were 5–14-fold lower than observed in any of the individual fecal samples used to generate the pool; whereas the levels of Actinobacteria and unclassified bacteria were 15–37-fold and 3–15-fold higher, respectively, than any of the individual fecal samples used to generate the pool. (Levels of Firmicutes (81 % of sequences), Proteobacteria (2 % of sequences), and Verrucomicrobia (4 % of sequences) were less than twofold different from at least one of the individual inocula.) This distribution could have been caused by heterogeneity in the individual fecal samples used for preparation of the pooled fecal sample and was not reflected on pooled MBRA communities, which looked more similar to other MBRA communities.At the phylum level, MBRA communities exhibited similar trends across replicate reactors and community types Fig. . SequencAkkermansia OTU (classified using the Greengenes reference taxonomy as Akkermansia muciniphila). From these data, we conclude that stable MBRA communities represent a subset of their fecal inoculum. The percent of the initial fecal inoculum present in core communities vary (from as little as 25 % of sequences (15 % of OTUs) in donor B communities to as much as 49 % of sequences (22 % of OTUs) in donor A communities). The amount of overlap observed was dependent upon the composition of the community present in the initial inoculum and could reflect both differences in abundance of obligate anaerobes that were lost prior to cultivation as well as differences in ability to grow under our cultivation conditions.The variation between reactor communities and their fecal inocula was more apparent when OTU distributions within specific phyla were examined Fig. ; these dFigure Faecalibacterium, Roseburia, some Bacteroides, and unclassified Lachnospiraceae; indicated OTUs are identified by red text in Fig. Flavonifracter, Akkermansia, Bacteroides #4, Ruminococcaceae #5; indicated OTUs are identified by blue text in Fig. Roseburia OTUs were lost from donor A and B communities upon cultivation but persisted in communities inoculated with samples from donor C  Of OTUs present in the fecal sample, ~20 % were lost or decreased >5-fold during the first several days in culture . Although many Enterobacteriaceae are facultative organisms, these organisms are unlikely to be respiring aerobically because oxygen levels in the anaerobic chamber are kept below 20 ppm. Organisms from the two Enterobacteriaceae OTUs could be respiring anaerobically, using alternative electron acceptors such as TMAO  or fumarate (likely metabolic byproduct in reactor), or could be fermenting available carbohydrates.In this paper, we demonstrated that MBRAs can be used to efficiently cultivate distinct communities from multiple fecal donors. Within the first week of cultivation, distinct microbial communities capable of metabolizing the available nutrients developed from the different starting inocula. Adaptation to growth in culture shifted the community structure. Although some community members persisted at similar abundances to the fecal inocula, other rare members of the inoculum increased in abundance and a subset of the initial inoculum was lost. This adaptation followed similar trends across replicates, with stable communities obtained by day 8 in culture .C. difficile in the absence of perturbation .Day-to-day variation within MBRA communities from single reactors were similar to those observed in stable mouse fecal communities. In contrast, replicate reactors from the same fecal donor exhibited slightly higher variation than was observed between replicate mice. This variation appeared to partly be driven by differing abundances of shared OTUs between replicates and could be indicative of functional redundancy of organisms under the MBRA cultivation conditions. This functional redundancy could allow abundances of OTUs to fluctuate stochastically until stable communities are reached. Further work will be needed to investigate functions of these different communities, although we know that communities formed from all three fecal donors as well as the pooled fecal sample are capable of resisting colonization by The composition and structure of MBRA communities are similar to those reported in other in vitro models of human fecal communities , 28, 39,C. difficile . Althoue . We alse . We . We C. de growth  and prote growth .Akkermansia muciniphila [Akkermansia in the absence of mucin.Although our model does not include a surrogate for mucosal surface as in M-SHIME, our phylogenetic distributions were more similar to the luminal contents of the M-SHIME model than to the original L-SHIME model , 39, witiniphila  were preFaecalibacterium prausnitzii  as well as several other less abundant Clostridiales species. Work from the Flint laboratory has demonstrated that both F. prausnitzii as well as certain members of Clostridium cluster XIVa require acetic acid as a cofactor for metabolism , which A second limitation of our MBRA model is the inability to study interactions with host cells, both to assess how the microbiome impacts the host and to determine feedback of the host upon the microbiome. Although this limitation can be overcome by coupling MBRA studies with follow-up studies in humanized microbiota mice , it woulw/v in anaerobic phosphate buffered saline in the anaerobic chamber, vortexed for 5 min, and centrifuged at 201×g. For the pooled sample, equal amounts of each fecal sample (by mass) were combined prior to vortexing.Fecal samples from three healthy individuals were collected into sterile containers, sealed, and transferred to an anaerobic chamber within 1 h of defecation. Samples were manually homogenized and subdivided into sterile vials, which were stored at −80 °C until use. Prior to MBRA inoculation, fecal samples were resuspended at 25 % In order to analyze the impact of freezing upon MBRA cultivation, one fecal donor (donor A) provided a second sample ~3 months post initial donation (donor A2). This sample was collected and transferred to the anaerobic chamber within 1 h of defecation. Following manual homogenization and subdivision into sterile vials, a portion of the sample was flash frozen in liquid nitrogen for 45 min (frozen), whereas the other sample was maintained in the anaerobic chamber until inoculation. Both fresh and frozen samples were then inoculated into triplicate reactors and analyzed as described below. Analysis of this data revealed that there was little impact on communities cultivated from frozen samples compared to freshly voided samples . (3) Bovine bile is significantly less expensive than taurocholate (>10-fold lower cost).MBRA were prepared for use as previously described  and inocg. Supernatants were discarded, and pellets were stored at −80 °C until further processed.After inoculation, fecal bacteria were allowed to equilibrate for 16–18 h prior to the initiation of flow. After equilibration, a 1-ml sample was removed (day 1 sample) and flow commenced at 1.875 ml/h (8-h retention time). Reactors were then sampled daily for 20 additional days (days 2–21). Cells were pelleted from samples by centrifugation at 21,000×Fecal sample collection was reviewed and approved by the Institutional Review Board from Michigan State University. All individuals donating samples provided informed consent prior to donation.C. difficile genes from samples that had been disrupted by bead beating without further purification [Previously, we had success amplifying fication . Furtherfication . We wereWe resuspended our samples in a 0.5-ml sterile water and transferred them to bead beating tubes. (Our bead beating tubes were prepared by transferring ~200 μl 0.1 mm silica beads (Biospec Products) and 100 μl sterile water into 2-ml screw cap tubes and autoclaving these tubes for at least 20 min prior to use.) Samples were homogenized in a mini-beadbeater-96 (Biospec Products) for 2 min, centrifuged at 8000×g for 1 min, then supernatants were transferred to new tubes, which were stored at -20 °C prior to amplification.The V4 region of the 16S rRNA gene was amplified with primers F515/R806, using a dual-indexing approach . The 96-indexed R806 primers used were previously described . Forward and reverse reads were paired, quality-trimmed, aligned to a Silva 16S rRNA gene reference database, trimmed to ensure overlap to the same region of the 16S rRNA gene (position 534-786 of Escherichia coli 16S rRNA gene), and pre-clustered to clusters with ≥99 % identity as described. Potential chimeric sequences were identified with the mothur-implementation of uchime and removed. Sequences were then classified with the Bayesian classifier in mothur, using the mothur-formatted ribosomal database project version 9 database from August 2013. Sequences were clustered from a distance matrix using the average-neighbor algorithm in mothur. Taxonomic assignments for each OTU were determined in mothur and are the majority consensus taxonomic assignments for each sequence within the OTU.Sequences were analyzed in Mothur versions 1.31, 1.33, and 1.34 essentially as described . MiSEQ SThe mean and SD of inverse Simpson diversity, Simpson evenness, Shannon diversity, and number of observed species were calculated in mothur from data randomly subsampled to 10,000 sequences over 100 iterations. A single iteration of subsampling to 10,000 sequences was used for determination of Bray-Curtis and Sorenson dissimilarity measures. Bray-Curtis and Sorenson dissimilarity values were calculated on untransformed data, both in mothur and with vegan package of R . Sequences were processed through mothur as described above, then subsampled to 3500 sequences prior to calculation of beta-diversity measures. Data were from three female  and three male  mice on days 1–9 (unstable communities) and days 141–150 (stable communities).For the mouse stability studies, paired Illumina reads were downloaded from the Schloss lab website (The sequence data described in this manuscript is can be accessed from the short read archive at NCBI (SRP059604).


L2 and L1 regularization, which can avoid overfitting and eliminate the influence of noise, (iii) recursive least squares, which can eliminate matrix-inversion operations and reduce computational complexity, (iv) a sliding-window approach, which can avoid caching all history samples and reduce the computational cost, and (v) the fixed-point subiteration and online pruning, which can make L1 regularization easy to implement. Finally, simulation results on two 50-state chain problems demonstrate the effectiveness of our algorithms. By combining with sparse kernel methods, least-squares temporal difference (LSTD) algorithms can construct the feature dictionary automatically and obtain a better generalization ability. However, the previous kernel-based LSTD algorithms do not consider regularization and their sparsification processes are batch or offline, which hinder their widespread applications in online learning problems. In this paper, we combine the following five techniques and propose two novel kernel recursive LSTD algorithms: (i) online sparsification, which can cope with unknown state regions and be used for online learning, (ii)  For simplicity of presentation, we assume that 𝒮 and 𝒜 are finite. Given an MDP ℳ and a policy π : 𝒮 → 𝒜, the sequence s1, r1, s2, r2,… is a Markov reward process ℛ = 〈𝒮, Pπ, Rπ, γ, d〉, where Pπ = ∑a∈𝒜π(a∣s)P and Rπ(s) = ∑a∈𝒜π(a∣s)∑s′∈𝒮Pr.In RL and dynamic programming (DP), an underlying sequential decision-making problem is often modeled as a Markov decision process (MDP). An MDP can be defined as a tuple r, γ, d〉 , where 𝒮Vπ(s) to evaluate how good the policy π is for the agent to be in state s. For an MDP, Vπ(s) can be defined as Vπ(s) = Eπ[∑t=0∞γtrt∣s0 = s], which must obey the Bellman equation T is a |𝒮 | × m feature matrix. Unfortunately, when approximating Vπ in this manner, there is usually no way to satisfy the Bellman equation exactly, because Rπ + γPπΦw may lie outside the span of Φ T denote the corresponding feature matrix. By the Representer Theorem T and β = T are the coefficient vector of wt and u, respectively. Then, from  = ϕ(si)Tϕ(sj). Thus, we can define Kt = ΦtΦtT, First, we use the kernel trick to kernelize . Suppose Theorem , wt anden, from , we have Theorem , the innn, from  is a regularization parameter. Let wt = u∗, we easily have αt = β∗ from  = T and Δkt = kt(si) − γ kt(si′).Second, we try to derive the ution of . Add an lty into ; that isβ∗ from . Then, tAt−1 and αt. Under online sparsification, there are two cases: (1) 𝒟t = 𝒟t−1, nt = nt−1, kt(·) = kt−1(·), Δkt = Δkt−1, and It = It−1; (2) 𝒟t = 𝒟t−1 ∪ {st}, nt = nt−1 + 1, kt(·) = T, Δkt = T, where Δkt = k − γk, and It is expanded ast−1 is the nt−1 dimensional zero vector.Third, we derive the recursive formulas of At−1, we getFor the first case,  can be ron lemma  for AtTst,st′,bt=bt−1+k1, we getAt−1=At−1plugging , we obtaAt and bt when the feature dictionary keeps unchanged, ht = ∑i=1tΔktkt−1(si), gt = ∑i=1tkΔkt−1, pt = ∑i=1tkΔkt + η, and qt = ∑i=1tkri. However, computing ht, gt, pt, and qt requires caching all history samples, and the computational cost will become more and more expensive as t increases. Inspired by the work of Van Vaerenbergh et al. . In addition, the variance matrix C0 of RLSTD is initialized to 0.4I, where I is the 20 × 20 identity matrix. (iv) In the simulations, each algorithm performs 50 runs, each run includes 100 episodes, and each episode is truncated after 100 time steps. In particular, the SKRLSTD algorithm requires an extra run for offline sparsification before each regular run.In the implementations of all tested algorithms for both chain problems, the settings are summarized as follows: (i) For all OSKRLSTD algorithms, the Mercer kernel is defined as Vπ(s) is solved by (jth run. From L2 and OSKRLSTD-L1 can obtain the similar performance as RLSTD and converge much faster than SKRLSTD. (ii) Without regularization, the performance of OSKRLSTD-0 becomes very poor, especially in the noise chain. In contrast, OSKRLSTD-L2 and OSKRLSTD-L1 still perform well. (iii) The performance of OSKRLSTD-Lu1 is only slightly better than that of OSKRLSTD-L1, which indicates that online pruning has little effect on the performance. Vπ(s) of the noise chain. L1 yields a much sparser dictionary. L1 and OSKRLSTD-Lu1. As episodes increase, the subiterations decline gradually. In addition, online pruning can reduce the subiterations significantly. Even in the noise chain, the subiterations are small. Finally, the main simulation results of all tested algorithms at the final episode are summarized in We first report the comparison results of all tested algorithms with the simulation settings described in RMSE=/50∑j=150Lu1 with M = 1,5, 10,…, 45,50. The logarithmic RMSEs of each algorithm at the final episode are illustrated in M. From L1 and OSKRLSTD-Lu1 obviously become worse rather than better as the window size increases, and OSKRLSTD-L2 has a strong adaptability to different window sizes. The reason for this result is analyzed as follows: From the derivation of our algorithms, the influence of the window size is mainly manifest in At−1. Since here At−1 is calculated by recursive update instead of matrix inversion and samples are used one by one, using too many history samples together may increase the calculation error. In OSKRLSTD-L2, a moderate regularization parameter η can relieve the influence of this error. In contrast, in OSKRLSTD-L1 and OSKRLSTD-Lu1, the subiteration may expand the influence. Especially for OSKRLSTD-L1, online pruning can introduce the new error, which further worsens the convergence performance. To verify the above analysis, we reset η = 0.6, ξ = 0.3, and N = 1 for OSKRLSTD-L1 and OSKRLSTD-Lu1 and reevaluate the effect of the window size. The new results are illustrated in L1 and OSKRLSTD-Lu1 can also adapt to M. Nevertheless, there is still no proof that a big window size can help improve the convergence performance of OSKRLSTD-L2 and OSKRLSTD-L1. Thus, as stated in M is suggested to be set to a small integer in practice.Next, we evaluate the effect of the sliding-window size on our proposed algorithms and OSKRLSTD-L2 and L1 regularization, that is, OSKRLSTD-L2 and OSKRLSTD-L1. By using Bellman operator along with projection operator, our derivation is more simple. By combining online sparsification, L2 and L1 regularization, recursive least squares, a sliding window, and the fixed-point subiteration, our algorithms not only can construct the feature dictionary online but also can avoid overfitting and eliminate the influence of noise. These advantages make them more suitable for online RL problems with a large or continuous state space. In particular, compared with the OSKRLSTD-L2 algorithm, the OSKRLSTD-L1 algorithm can yield a much sparser dictionary. Finally, we illustrate the performance of our algorithms and compare them with RLSTD and SKRLSTD algorithms by several simulations.As an important approach for policy evaluation, LSTD algorithms can use samples more efficiently and eliminate all step-size parameters. But they require users to design the feature vector manually and often require many features to approximate state-value functions. Recently, there are some works on these issues by combining with sparse kernel methods. However, these works do not consider regularization and their sparsification processes are batch or offline. In this paper, we propose two online sparse kernel recursive least-squares TD algorithms with There are also some interesting topics to be studied in future work: (i) How to select proper regularization parameter should be investigated. (ii) A more thorough simulation analysis is needed, including an extension of our algorithms to learning control problems. (iii) Eligibility traces would be combined for further improving the performance of our algorithms. (iv) The convergence and prediction error bounds of our algorithms will be analyzed theoretically.


Supplementation of human blood with exogenous melatonin or melatonin receptor antagonist during the in situ perfusion establishes a receptor-mediated action of melatonin on L1 expression. Combined tissue culture and in vivo data support that environmental light exposure of the host regulates expression of L1 elements in tumors. Our data imply that light-induced suppression of melatonin production in shift workers may increase L1-induced genomic instability in their genomes and suggest a possible connection between L1 activity and increased incidence of cancer associated with circadian disruption.Expression of long interspersed element-1 (L1) is upregulated in many human malignancies. L1 can introduce genomic instability via insertional mutagenesis and DNA double-strand breaks, both of which may promote cancer. Light exposure at night, a recently recognized carcinogen, is associated with an increased risk of cancer in shift workers. We report that melatonin receptor 1 inhibits mobilization of L1 in cultured cells through downregulation of L1 mRNA and ORF1 protein. The addition of melatonin receptor antagonists abolishes the MT1 effect on retrotransposition in a dose-dependent manner. Furthermore, melatonin-rich, but not melatonin-poor, human blood collected at different times during the circadian cycle suppresses endogenous L1 mRNA during  Both thposition ,5. L1 moposition  followedposition . As a reL1 ORF1p forms trimers that directly bind to L1 mRNA  and has de novo insertions, DSBs and non-allelic recombination, Figure Because L1 also provides the retrotransposition machinery for the parasitic Alu and SVA elements, it is the source of the entire mutagenic burden on the human genome imposed by active retrotransposons ,19,20. Ain vivo because most L1 biology is studied in cultured cells lacking multiple regulatory mechanisms that are unique to living organisms. One such regulator is the circadian system that synchronizes the timing of biological processes ranging from metabolism to DNA damage response  and NIH 3T3 cells were cultured in modified Eagle's Medium (MEM) with 10% fetal bovine serum (FBS), 100-mM sodium pyruvate, 1X non-essential amino acids and 200-mM Plasmids CMV5′UTR L1Neo, ΔCMVL1Neo, and untagged CMV5′UTR L1 are from , untaggeNetPhos 2 and CKSAAP prediction programs were used to identify putative phosphorylation and ubiquitination sites in the ORF1p sequence ,55.HeLa cells (500 000) were transfected 18–24 h after seeding by lipofectamine/Plus reagent (Invitrogen) with 0.4 µg of Neo-tagged L1 expression vector  and 0.1 6) were transfected by lipofectamine/Plus reagent with 0.3 µg of Neo-tagged L1 expression vector and 0.06 µg of pCDNA or MT1 expression vectors 18–24 h after plating. For toxicity of L1 activity, melatonin receptor antagonist, melatonin receptor or their combination, L1 notag, or MT1 expression plasmids were used with 0.8 µg of pIRES (has Neo-resistance). Selection with 0.4 (HeLa) or 0.22 (PC3) mg/ml G418 was initiated 24 h after transfection and maintained for 2–3 weeks. Cells were treated with 10−7, 10−6, 10−5 M of melatonin receptor antagonist S20928  at the time of transfection cocktail removal, at the time of G418 addition 48 h after transfection and the following day.PC3 cells , NIH 3T3 and immortalized human fibroblasts were seeded and transfected with L1 and MT1 expression plasmids as described for RNA analysis below. Total protein lysates were harvested 24 h after transfection. For analysis of MT1 effect on wild-type and mutant ORF1p, cells were transfected with 3 and 0.5 μg of ORF1 and MT1 expression plasmids, respectively. Western blot analysis was preformed as previously described . BrieflyrnuFoxn1) were implanted with PC3 human prostate cancer xenografts in a tissue-isolated manner as previously described  were perfused with human blood as previously described  and polyA selected as previously described . RNA was6 per T75 flask, were cotransfected 18–20 h after plating with 6 µg of the L1 expression plasmid and 0.5 µg of empty or MT1 expression plasmid  per well (six-well plate) were transfected 18–24 h after plating with 0.2 µg of luciferase expression plasmids [luciferase expression is driven by either CMV  or L1 5′UTR promoters ], 0.02 µde novo L1 retrotransposition events in HeLa . Treatment with increasing doses of MT1 antagonists was performed once a day for the first 3 days of the retrotransposition assay starting at the time of transfection cocktail removal Figure . This exThe same approach was used to test the effect of melatonin receptor antagonist treatment on L1-driven Alu retrotransposition . Similar to L1, treatments with luzindole increased L1-driven Alu retrotransposition in a dose-dependent manner  without any adverse effect on cell viability and colony formation .To understand the mechanism of MT1 downregulation of L1 retrotransposition, we tested the effect of MT1 overexpression on the steady-state levels of L1 mRNA and ORF1p. L1 mRNA was measured in HeLa, PC3 and NIH 3T3 cells cotransfected with the untagged human L1 expression plasmid and either empty or MT1 expression plasmids Figure . NortherFluc activity driven by either promoter  driven by the L1  or CMV  (in situ as described (To test circadian regulation of L1 12L:12D)  and perfescribed  with humescribed   blood samples, melatonin receptor antagonist S20928 was added during In 2007, the World Health Organization recognized light exposure at night as a probable carcinogen . This rede novo L1 retrotransposition in several human cancers demonstrated that some of these inserts occurred early in cancer development , which is genomic instability. Genomic instability in the form of point mutations, insertions and deletions, as well as large chromosomal rearrangements is responsible for cancer initiation, progression and heterogeneity [reviewed in ]. Genomielopment ,22,24,76g tumors ,22 of thg tumors ,78 or geg tumors ,79. The in situ tumor perfusion (Figure in situ perfusion restores suppression of endogenous L1 mRNA, suggesting that the effect of light exposure at night on L1 mRNA in tumors can be mitigated by melatonin supplementation.We demonstrate that expression of endogenous L1 elements in a tissue-isolated model of prostate cancer is suppressed by melatonin circulating in human blood Figure . Indeed,n Figure . Consistin vivo observation, overexpression of MT1 receptor decreases steady-state levels of L1 mRNA and L1 ORF1p in cultured cells (Figure Consistent with our s Figure . Adminiss Figure  as Alu rs Figure . Furthers Figure . In contin vivo will be needed to determine whether the genomes of shift workers or other individuals who have experienced extensive LEN accumulate increased amounts of L1-induced damage.Many signaling pathways, including melatonin signaling, activate a cascade of protein kinases that decrease amounts of cellular proteins by targeting them for degradation or change their ability to associate with their interacting partners ,80. Indein vivo data and tissue culture-based results support the following model of light-dependent regulation of L1 in cancer. Under normal light exposure, nocturnal melatonin production activates melatonin receptors, leading to suppression of L1 mRNA and ORF1p (Figure in vivo, leading to accumulation of de novo integration events in the tumors of shift workers or individuals with disrupted melatonin production. While specific molecular events controlling regulation of L1 by melatonin have yet to be identified, this finding provides a tantalizing first glimpse into the complex regulation of L1 activity in vivo. The connection between the circadian system and L1 suggests that L1-induced damage is likely increased in tumors originating in shift workers and the elderly. Both subpopulations have reduced nocturnal melatonin levels and increased incidence of cancer (Combined, our p Figure . Nighttip Figure . This cof cancer ,44,86,87f cancer ,23.Our findings also raise important questions about the control of L1 expression in normal tissues. We have previously detected various amounts of endogenous L1 mRNA in a broad spectrum of normal human tissues . This vaThe implications of circadian regulation of L1 expression and activity expand beyond its impact on the genomes of shift workers. Our findings suggest a possibility of an increase in L1-induced genomic instability with age. An age-dependent decline in melatonin production and melatonin receptor expression may result in upregulation of L1 expression, leading to an increase in L1 activity and the mutagenic burden associated with its expression. Consistent with this idea is a recent study on somatic L1 retrotransposition in colorectal cancers, which found a strong correlation between L1 retrotransposition and age . FurtherOverall, our finding of receptor-mediated melatonin-induced suppression of L1 expression and activity adds an important component to the numerous existing cellular pathways known to downregulate L1 expression and activity. It establishes a link among the host circadian system, environmental light exposure and genomic instability. Furthermore, this discovery has important evolutionary implications. Melatonin is one of the most evolutionarily conserved molecular entities, estimated to originate some 2.5 billion years ago . Transpoin vivo should be considered as a dynamic, probably age-dependent, entity with effects consequential to the whole human body, but likely unique to individual tissues, genetic backgrounds and environmental light exposures.In conclusion, L1 elements and their damage Supplementary Data are available at NAR Online.


Graphene is the most recent superior material which could potentially initialize another new material Age. However, while being exploited to its full extent, conventional processing methods fail to provide a link to today’s personalization tide. New technology should be ushered in. Three-dimensional (3D) printing fills the missing linkage between graphene materials and the digital mainstream. Their alliance could generate additional stream to push the graphene revolution into a new phase. Here we demonstrate for the first time, a graphene composite, with a graphene loading up to 5.6 wt%, can be 3D printable into computer-designed models. The composite’s linear thermal coefficient is below 75 ppm·°C Tg) and extruded through the 3D printer’s nozzle, whose diameter defines the printing resolution. Computer-designed 3D objects are constructed through successively stacking thermoplastic layers. The printing process not only is easy and rapid, but also can realize exotic topological structures which challenges the traditional casting and welding techniques.Although 3D printing technology has been around since the 1980 s, it hasn’t gained enough momentum to be commercialized until recently12 carbon crystal, has never been used as a 3D printing feedstock. Its two dimensional honeycomb structure endows it with many unique properties9102/g)2 S/m for reduced graphene oxide monolayers and 2 to 3 orders of magnitude higher for pristine graphene)Driven by new applications, the printable category keeps expanding into many frontier scientific and engineering fields345673 C-C bonds242 bonds in graphitic domains. Chemical reduction of GO is a cost-effective strategy to prepare graphene sheets by utilization of reductant. Hydrazine has been commonly used to high yield production of reduced GO (rGO) owing to its ability to in situ reduce oxygenated functional groups. It was reported that rGO restores 80% sp2 in its structure, with the rest of sp3 bonds derived from residual oxygen (C:O atom ratio 12.5:1) after the reduction with hydrazineStarting with the graphene composites, graphene sheets have been successfully added into polymers to explore novel applications in mechanical reinforcement, flame retardant or electrical conductive composites16189202122in-situ formed without obvious phase separation. To fractionally precipitate G-ABS composites from NMP, the dispersion was added into water. The precipitated G-ABS composite is shown in Tg in the heated build chamber (HBC). Subsequently, based on layer-by-layer manufacturing technique1These graphene-polymer composites were further extruded into 1.75 mm diameter filaments to fit the commercialized 3D printer. As shown in σc) was investigated on hot pressed rectangular composite models using a four-probe method . Φc in previous graphene-based polymer composite was reported to be around 0.1 vol%303229The graphene dispersion’s homogeneity directly affects the composite’s printing ability and other physical properties. With scanning electron microscopy (SEM), graphene individuals can be captured from the polymer matrix with different contrast and detailed morphologies. SEM images were taken from both the surface and the cross section of the composite to give a spatial perspective of the graphene dispersion inside composites . Graphene method  and plote method  At 0.4 war model , its meailaments  may acco−1 and a G peak at ~1600 cm−1, related to the defects and sp2 structures in graphene~2240 cm−1 can be ascribed to the C≡N stretch in ABSRaman and ultraviolet-visible (UV-vis) analysis were used to further verify the presence of graphene in composites. A laser excitation with a wavelength of 785 nm was used to record Raman spectra from the samples . The repTg and then cool down from it to room temperature (RT). Therefore, before any 3D printing, Tg is the first and most vital parameter to be determinedTg values can be calculated and listed in Table S3Tg value close to ~105.8 °C. This value slightly shifted to ~110 °C with incremental graphene loadings , dynamic mechanical analysis (DMA), thermogravimetric analysis (TGA) and thermomechanical analysis (TMA). In FDM printing, printing materials need to be heated above Tg and thermal stability of the composites as compared to the pure ABS polymersIt was expected that polymer’s viscoelasticity would change after the incorporation of graphene sheets. The loss factor (tan δ), ratio of loss and storage modulus, is usually a characteristic indicator of how much energy dissipates in the polymer matrix322 atmosphere, the degradation of the samples occurred with a sharp weight loss around 400 to 480 °C (onset) is defined as the temperature when testing sample loses 5% of its total weightonset, the composite materials simply melt into viscous fluids rather than decomposing. The set temperature was also sufficiently higher than the G-ABS’s Tg, ensuring the filaments smoothly printed through the 3D printer.The HBC needs to get hot enough to soften the composites22o 480 °C , accompaTg. CTE values were extracted in the same temperature range, according to CTE = (dL/L0)·(1/dT), where dL/L0is the dimensional deformation along the vertical axis and dT is the temperature change. All G-ABS samples’ CTE values were below 75 ppm·°C−1, which exhibited no significant variation from pure ABS’s 78.25 ppm·°C−1, suggesting suitable for the 3D printing. With Tg values ranging from 104 to 110 °C, by calibrating the HBP temperature at ~80 °C, 20 to 30 °C below Tg and maintaining the nozzle and HBC temperature at 130 °C and 230 °C, separately, we can efficiently avoid thermal deformation during the printing process  with a printing area of 140 mm x 180 mm x 135 mm was used to print our graphene related materials. The nozzle can move back and forth at a rate of ∼20 mm/s with a horizontal resolution of 200 μm or 400 μm (depending on the inner diameter of the nozzles) and vertical resolution of 200–400 μm to print out 3D models. The temperatures of HBC, HBP and nozzle were set at 230, 80 and 130 °C, respectively, for G-ABS 3D printing. They were changed to 190, 60 and 130 °C, respectively, for G-PLA 3D printing.Full Methods and details of characterization for G-ABS and G-PLA composites are given in the How to cite this article: Wei, X. et al. 3D Printable Graphene Composite. Sci. Rep.5, 11181; doi: 10.1038/srep11181 (2015).


It requires the ability to process on-going turns at talk while planning the next, and to launch this next turn without considerable overlap or delay. Recent research has investigated the eye movements of observers of dialogs to gain insight into how we process turns at talk. More specifically, this research has focused on the extent to which we are able to anticipate the end of current and the beginning of next turns. At the same time, there has been a call for shifting experimental paradigms exploring social-cognitive processes away from passive observation toward on-line processing. Here, we present research that responds to this call by situating state-of-the-art technology for tracking interlocutors’ eye movements within spontaneous, face-to-face conversation. Each conversation involved three native speakers of English. The analysis focused on question–response sequences involving just two of those participants, thus rendering the third momentarily unaddressed. Temporal analyses of the unaddressed participants’ gaze shifts from current to next speaker revealed that unaddressed participants are able to anticipate next turns, and moreover, that they often shift their gaze toward the next speaker before the current turn ends. However, an analysis of the complex structure of turns at talk revealed that the planning of these gaze shifts virtually coincides with the points at which the turns first become recognizable as possibly complete. We argue that the timing of these eye movements is governed by an organizational principle whereby unaddressed participants shift their gaze at a point that appears interactionally most optimal: It provides unaddressed participants with access to much of the visual, bodily behavior that accompanies both the current speaker’s  The contrast formed by the white sclera surrounding a darker iris and pupil is unique to the human eye . This coIn social interaction, a system of turn-taking organizes opportunities to speak. According to Recently, a new experimental paradigm has been developed for the study of the cognitive processes that underpin turn-taking from a third-person perspective. The general procedure involves participants being presented with a pre-recorded dialog or conversation between two people on a computer screen while their eye movements are tracked and timed with respect to the turns at talk they hear. Experimental studies using this novel paradigm have shed light on the precise timing of eye movements and turns at talk by measuring where observers of dialogs look and when they do so.before he or she begins to speak  dialogs have not found evidence for anticipatory looks to the next speaker. While there is some discrepancy in findings, studies using the novel third-person perspective eye-tracking paradigm described above have provided us with valuable first insights into how adults may process turns at talk and transitions between them, as well as how children acquire this skill during development. However, two issues emerge from this work.The first issue has already been alluded to in the preceding section and concerns the third-person perspective as such. Recently, less cognitive resources available for projection in live conversation, meaning gaze shifts may be primarily responsive to the next speaker beginning to speak rather than anticipatory. However, it could also be that eye movements in face-to-face interaction do not reflect the projection of current or next turns at all, but that the social norms and dynamics of conversation determine where participants look and when. Thus, while third-person perspective eye-tracking paradigms allow for a high degree of experimental control and manipulation to investigate eye movements during turn-taking, and the role semantics and prosody play in this context  and ten dyadic (two of the three participants) conversations Figure 1 provides an overview of the laboratory set-up and equipment.The recordings took place in a sound proof room equipped with professional lighting suitable for high quality audio and video recording. Participants sat in standard height chairs with armrests, arranged in a triangle with the chairs equidistantly placed from one another. A ceiling microphone recorded the entire conversation. Each participant wore a head-mounted lightweight uni-directional microphone (Shure SM10A), which recorded only the respective participant’s voice, and a pair of eye-tracking glasses . In addition, three HD video cameras  recorded frontal views of each person . Due to the spatial arrangement of the chairs with respect to the cameras, each person was also visible from the right and left side in the recordings made by the respective other two video cameras. Figure 2). This resulted in a time resolution of approximately 41 ms, the duration of a single frame. The synchronization was based on audible and visible information from a clapperboard used at the beginning and end of each session.For each session, the recorded material resulted in three individual videos from the cameras, three individual videos from the eye-trackers , three individual audio files, and the audio file from the ceiling microphone. The audio tracks were recorded in sync using a four-channel audio recorder (Edirol/Roland R-44). The six video recordings and three individual audio recordings were combined and synchronized in Adobe Premier Pro CS4 and then exported as a single audio–video file for analysis (MP4) at 24 frames per second  and were handed study-packs, including information about the study and procedure of the session, forms asking about their language background, screening questionnaires ruling out motor and speech impairments, as well as consent forms and questionnaires about handedness and a variety of social dimensions. Once the study-packs had been completed by all participants  and any queries had been answered, participants were seated in their chairs in the recording room. All equipment was prepared beforehand, allowing immediate fitting of the microphones and the eye-trackers .Each recording session lasted approximately 40 min. in total, with the first 20 min. constituting a trialogue phase and the second 20 min. a dialog phase. Upon completion of the initial fitting procedure, the two investigators left the room and waited in an adjacent area until the first 20 min. had elapsed. At this point, they compared performance of the three eye-trackers and asked the person wearing the eye-tracker with the poorest calibration to leave the room. Once the remaining two participants had talked for another 20 min., all three were reunited in the recording room and asked to complete the social questionnaires contained in the study-packs. This was to ensure that questions about human communication and behavior  would not influence participants’ behavior during the conversations.  Participants were then asked one more time for their written informed consent relating to how their data should be handled, thanked, and financially compensated for their participation (26 euro per person). The entire test session lasted around 120 min. The study was approved by the Social Sciences Faculty Ethics Committee, Radboud University Nijmegen.N = 35). In a small number of cases (N = 2) the response was exclusively non-verbal ; in those cases the beginning of the response was timed to the first frame of visible movement. These annotations were then imported into ELAN 4.61  sequences in which the question was addressed to a single participant who then produced a response. All QR sequences were identified by an experienced conversation analyst (Kobin H. Kendrick), resulting in a total of 281 questions and their responses . Criteria for identifying QR sequences in our dataset were based on the coding scheme proposed by LAN 4.61 .All questions in the dataset were analyzed for the presence and location of points of possible completions before the end of the turn, drawing on conversation-analytic research on turn construction . For a pThe analysis of points of possible completion revealed a number of recurrent types. If a turn contained two complete questions, a point of possible completion – represented here by a vertical bar – was identified after the completion of the first, whether the two questions were produced one after the other  or with a short silence between the two  or is it just (0.2) any money is good?”). If a turn contained a possibly complete question together with an increment, a contingent addition to a turn that continues its grammatical structure , a pointK = 0.72  who independently identified the presence and precise location of points of possible completion in the dataset revealed strong reliability, The ELAN files containing the QR annotations were linked to the synchronized videos in order to annotate the unaddressed participants’ eye movements during the QR sequences. These annotations were done manually, on a frame-by-frame basis. At each frame during the QR sequence, the gaze fixation point generated by the SMI software for the unaddressed participant (henceforth referred to as the gaze cursor) was categorized as being (1) on speaker A, (2) on speaker B, (3) on self , (4) on the surroundings , or (5) as not identifiable from the eye-tracker data . Based on this coding scheme, 45 of the originally 281 QR sequences (16.0%) were discarded from further analysis of the unaddressed participants’ eye movements due to insufficient data. .Out of the remaining 235 QR sequences, unaddressed participants moved their gaze from speaker A to speaker B in 105 (45.5%) QR sequences. In order to be considered a valid gaze shift for our analysis, the trajectory had to be one that clearly moved from A to B, without the gaze pausing elsewhere in between (such as on self or background objects). In the remaining 131 sequences, unaddressed participants either did not shift their gaze at all and instead fixated speaker A, speaker B, themselves, or the surroundings throughout, or they did move their eyes but in the opposite direction, that is, from speaker B to speaker A. While these cases are interesting in themselves, they tap into a different phenomenon than the one under investigation here and require analysis and discussion elsewhere.Regarding those 105 QR sequences that did reveal a gaze shift from speaker A to speaker B , the average question duration was 2018 ms , and the average response duration was 1899 ms . Due to the highly dynamic nature of conversation brought about by, amongst other things, differences in personality, age, gender, closeness of friendship, and topic of discussion, the seven triads of course differed in the number of QR sequences they contributed to our analysis . Likewise, participants within the triads differed in the extent to which they contributed to the conversation by asking questions, but none of the conversations excluded participants . Basing analyses of QR sequences in conversation on samples that are determined by the participants’ spontaneous behavior, thus resulting in varying numbers of QR sequences across separate interactions, is the standard procedure for corpus studies and in line with existing research e.g., .when exactly this gaze shift occurred. The time window we took into consideration for identifying gaze shifts relevant for this analysis stretched from the beginning of A’s turn to the end of B’s turn. In all cases of gaze shifting from speaker A to speaker B within this time window, unaddressed participants looked at the face of speaker A and then moved their gaze from there to the face of speaker B. Using the frame-by-frame gaze annotations, we identified the first frame at which the gaze cursor left speaker A, defined as the frame at which the gaze cursor was no longer on, overlapping with, or directly adjacent to speaker A’s head or technical head-gear . At what time point before or during B’s turn the unaddressed participant’s gaze arrived at speaker B was not of relevance for the present analysis. Annotations were made in ELAN to measure the duration from the first gaze shift away from speaker A by the unaddressed participant to two points within the question turn: (1) the end of the turn and (2) the first point of possible completion of the question, for those questions that had a possible completion before the end of the turn. In addition to measuring the duration of these intervals (in ms), the values were set as either positive or negative. This was done to identify the temporal order of the respective events, with negative values indicating an anticipatory gaze shift before a point of possible completion or the end of a turn and positive values indicating the inverse.For these 105 QR sequences that did reveal a shift of the unaddressed participant’s gaze from speaker A to speaker B, we identified However, we need to consider that it takes time to plan and launch these eye movements before they are observable. This process is estimated to take on average around 200 ms . We therGaze coding was performed by two independent coders (LD and MvdG) blind to the study’s predictions and assumptions. In addition, their coding was checked by one of the two senior analysts (JH and KK), and any errors in coding  were discussed and corrected. Due to the considerably more objective coding criteria applied for our gaze analysis in comparison to the identification of points of possible completion, formal reliabilities were calculated for the latter only.estimate of the mode, all mode values given should be considered close approximations of the true value and decimal places are not stated for those values. Note also that these distributions are based on binned data brought about by our video frame rate providing a measure every 41.7 ms (24 fps).All statistical analyses were conducted in R 3.1.1 . The denN = 57) of the questions had at least one possible completion before the end of the turn. Here, we focus our analyses first on the end of the turn and then on its first possible completion (which corresponded to the end of the turn for 45.7% of questions).Out of the 105 QR sequences analyzed here, 54.3% . Because, as we have already noted, experimental research indicates that overt eye movements are planned about 200 ms in advance of them being observed, the covert initiation of unaddressed participants’ eye movements from speaker A to speaker B occurred most frequently around 250 ms prior to the end of questions .First, we measured the time point of each first observed gaze shift away from speaker A (and toward speaker B) with respect to the end of speaker A’s turn. This showed that the estimate of the mode of these data is located very close to the end of the question, namely just 50 ms prior to turn end (see N = 63) of QR sequences were associated with observable gaze shifts that anticipated the end of the question turn. When taking into account the time it takes to prepare these gaze shifts, the percentage of anticipatory gaze shifts increases to 73.3% (N = 77).On the whole, 60.0% . Taking into account the 200 ms required to plan and launch observed eye movements, the distribution yields a mode of 40 ms just prior to the first possible completion .Because many of the questions in our data had a point of possible completion prior to turn end (as seen above), we carried out a second analysis in which we timed unaddressed participants’ first gaze shift away from speaker A with respect to the first possible completion of each question; this corresponded to the end of the turn for those questions with only one possible completion. When plotting our data with respect to this reference point, the distribution yields a mode of about 160 ms just after the first possible completion (see N = 36) of cases, unaddressed participants shifted their gaze from current to next speaker before the first possible completion, and in 55.2% (N = 58) of cases unaddressed participants’ gaze shifts had at least been planned prior to this point.When timing the gaze shifts with respect to the first possible completion of the question, we still see that a considerable number of gaze shifts from current to next speaker happen prior to the first possible completion, but much less so than when timing these gaze shifts with respect to the end of the turn: in 34.3%  = 0.234, p < 0.05). This means that for responses that coincide with first possible completions of questions, gaze shifts could either be due to unaddressed participants recognizing the possible completion or reacting to the onset of the response. In order to tease these two factors apart, we carried out two further analyses by looking at two subsets of our data.Above we have shown that unaddressed participants are sensitive to first possible completions, as can be seen from the timing of their gaze shifts. However, considering that first possible completions mark points at which transition to the next speaker becomes relevant, addressed participants, too, are likely to be sensitive to these points and time their responses to them. This means that the first possible completion of speaker A’s question and the onset of speaker B’s response may often coincide. We therefore also measured the timing of the response 3 after the first possible completion (N = 54). If the timing of the unaddressed participants’ gaze shifts we observed based on the sample as a whole is explained by response onset rather than by first possible completions, then the mode for this subset of data should be at least 200 ms later than the mode for the distribution based on the entire sample. However, as can be gleaned from Figure 6, the mode for this subset is 105 ms (Range = –2337–2419), which differs only slightly from the mode of 160 ms for the entire sample. If anything, unaddressed participants’ observed gaze shifts occur slightly earlier when B’s response occurs 200 ms after the first possible completion, and certainly no later than when we consider the entire sample. Thus, unaddressed participants’ eye movements in our data do indeed appear to reflect sensitivity to the first possible completion of the question, rather than being a mere reaction to the onset of the response.For the first analysis, we considered only those QR sequences where speaker A’s first possible completion and speaker B’s response onset did not coincide but where the response comes after the possible completion. For this comparison, we selected those sequences where the response occurred more than 200 ms N = 15). If response onset alone also attracts unaddressed participants’ attention and, as a consequence, their gaze, then we should see that the mode of the distribution of gaze shifts for this subset is earlier than that for the distribution based on the sample as a whole. As can be seen from Figure 7, this was indeed the case, with the mode of observed gaze shifts for the subset of early responses being -35 ms, compared to an overall mode of 160 ms. This means that the eye movements within this subset must have been planned -235 ms before the first possible completion, which corresponds closely to the onset of these early responses at -200 ms or less.However, we of course do acknowledge that response onset may also play a role in the timing of unaddressed participants’ eye movements. In order to explore this further, we looked at another subset of our data, namely those cases in which speaker B’s response began at least 200 ms before speaker A’s first possible completion , they move their gaze from one speaker to the next in about half of all question–response sequences. This confirms that, even when participating in actual ‘on-line’ interaction, unaddressed participants show a tendency to track current speakers cf., , at leasWhen we examined the timing of gaze shifts with regard to turn ends, we found that the most frequent gaze shifts from current to next speaker were planned around 250 ms and observable around 50 ms prior to turn end. Thus, the results suggest that unaddressed participants’ gaze shifts are predominantly anticipatory in nature when timed with respect to the end of question turns. As such, it replicates the findings from third-person perspective eye-tracking studies that have found evidence for adults shifting their gaze to the next speaker prior to turn end e.g., . OverallHowever, despite the comparability, gaze shifts prior to turn end appear to be more common in actual conversation. In the present study, the majority of gaze shifts from current to next speaker occurred prior to turn end (60% of cases) or were planned and launched prior to this point (73% of cases). In contrast, in studies using the third-person perspective paradigm, either only a relatively small proportion of gaze shifts occurred prior to turn end e.g.,  or none Figure 5) would occur after the possible completion, not before.The present study went further than just measuring eye movements with respect to turn ends. Here, we have taken into account the intricate structure of questions, and, more specifically, the first possible completion of each question, which for half of our questions was not the actual end of the turn. These points of possible completion create opportunities for a next speaker to take the turn, and it has been argued that participants in conversation are sensitive to these transition-relevance places . Indeed,well before it reaches possible completion  At the same time, however, we have been able to show that early responses which precede the first possible completion also attract unaddressed participants’ gaze, and thus may certainly be a contributing factor in those instances where first possible completion and onset of the response coincide. Hence, taking overlap between current and next speakers into account appears crucial if we aim to understand unaddressed participants’ eye movements in natural conversation. Importantly, the effect of both factors – first possible completions and response onset – is based on a process of To date, all studies of the phenomenon under investigation here – the redirection of gaze by unaddressed participants from current to next speakers at turn transitions – have used it to gain insight into processes involved in turn-taking. But the conclusion that the eye movements of unaddressed participants do not anticipate the first possible completion of the current turn, and thus do not necessarily reflect a projection of it, leads us to reconsider the nature of the phenomenon and to look elsewhere for principles that can account for the fine temporal coordination that we observe.It has long been argued that among the many functions of gaze behavior in social interaction the use of gaze to display attention, engagement in the interaction, and recipiency to the current speaker is paramount . Gazing 4. Our findings that unaddressed participants’ gaze behavior during question–response sequences appears to be organized according to a principle that optimizes recipiency also fits well with the notion of an ‘intrinsic motivation for participation,’ as it were (Although the gaze behavior of unaddressed participants does not necessarily reflect projection of the current turn, optimizing recipiency between current and next speakers does make use of the turn-taking system in other ways. Our results provide new and quantitative evidence that the recognition of points of possible completions are indeed core to the turn-taking system in conversation, as argued in  it were . Both ScIn addition, it appears from our results that response onset can trump first possible completions, at least when these responses come prior to the first possible completion. In such cases, the timing of the response appears marked and may signal a marked social action see . That unThe present study looked at eye movements with respect to one particular type of turn, that is, questions. The present study has provided us with a first glimpse of the intricate connections between turns at talk and unaddressed participants’ eye movements in spontaneous, multi-person interaction. On the one hand, we have here reproduced the basic findings from studies using the third-person perspective eye-tracking paradigm in spontaneous, live conversation. On the other, our data have provided us with stronger evidence that gaze shifts by unaddressed participants toward next speakers precede the end of the current turn than previous studies have. As such, our findings corroborate the notion that interactive paradigms do, at least in part, provide different insights than paradigms involving passive observation . FurtherThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.