The internal transcribed spacer (ITS) sequence was demonstrated as the optimal barcode for identifying Radix Astragali and its adulterants. This new analysis method is helpful in identifying Radix Astragali and expedites the utilization and data mining of DNA barcoding.Radix Astragali is a popular herb used in traditional Chinese medicine for its proimmune and antidiabetic properties. However, methods are needed to help distinguish Radix Astragali from its varied adulterants. DNA barcoding is a widely applicable molecular method used to identify medicinal plants. Yet, its use has been hampered by genetic distance, base variation, and limitations of the bio-NJ tree. Herein, we report the validation of an integrated analysis method for plant species identification using DNA barcoding that focuses on genetic distance, identification efficiency, inter- and intraspecific variation, and barcoding gap. We collected 478 sequences from six candidate DNA barcodes (ITS2, ITS,  Astragalus membranaceus and Astragalus mongholicus according to Chinese Pharmacopoeia (2010 edition). Radix Astragali is widely used for its antiperspirant, antidiuretic, and antidiabetic properties and as a tonic drug [Radix Astragali (Huang Qi), a commonly used Chinese medicinal material, is mainly sourced from the plants ofopoeia 200 editionnic drug . It possnic drug –6. Astragalus, Hedysarum, and Malva are often used in its stead [ matK (maturase K) and rbcL  of chloroplast genome, and coxI (cytochrome c oxidase 1) of the mitochondrial genome [ Astragalus) and 478 sequences from six barcodes were used to validate the new method for identifying Radix Astragali and adulterants and to accelerate the data utilization of DNA barcoding.Due to the high market demand for Radix Astragali, a diverse group of adulterants with similar-morphological characteristics from genuses, such asts stead . The trats stead , thin-lats stead , Fourierts stead , and higts stead , all, rets stead  or amplits stead , are unsts stead , 15. It ts stead , 17. In ts stead , 3′ untrts stead , ITS . Supernatant was removed and placed into a new tube before adding 700 μL isopropanol and blending for 15–20 minutes. The mixture was centrifuged in CB3 spin columns for 40 s at 12000 rpm. The filtrate was discarded and 500 μL GD (adding quantitative anhydrous ethanol before use) was added before centrifuging at 12000 rpm for 40 s. The filtrate was discarded and 700 μL PW (adding quantitative anhydrous ethanol before use) was used to wash the membrane before centrifuging for 40 s at 12000 rpm. This step was repeated with 500 μL PW, followed by a final centrifuge for 2 minutes at 12000 rpm to remove residual wash buffer. The spin column was dried at room temperature for 3–5 minutes and then centrifuged for 2 minutes at 12000 rpm to obtain the total DNA.The material specimens were naturally dried and 30 mg of dried plant material was used for the DNA extraction. Samples were rubbed for two minutes at a frequency of 30 r/s in a FastPrep bead mill , and total genomic DNA was isolated from the crushed material according to the manufacturer's instructions . We made the following modifications to the protocol: chloroform was diluted with isoamyl alcohol (24 : 1 in the same volume) and buffer solution GP2 with isopropanol (same volume). The powder, 700  psbA-trnH barcodes, as presented in μL reaction mixtures containing 2 μL DNA template (20–100 ng), 8.5 μL ddH2O, 12.5 μL 2× Taq PCR Master Mix , and 1/1-μL forward/reverse (F/R) primers (2.5 μM). The reaction mixtures were amplified in a 9700 GeneAmp PCR system . Amplicons were visualized by electrophoresis on 1% agarose gels. Purified PCR products were sequenced in both directions using the ABI 3730XL sequencer .General PCR reaction conditions and universal DNA barcode primers were used for the ITS, ITS2, and psbA-trnH, matK, rbcL, and COI), which were downloaded from the GenBank database . Complete ITS2 sequences were obtained using the HMMer annotation method, based on the Hidden Markov model (HMM) . All of database . Sequenc psbA-trnH and matK regions provided a higher rate of success than the other three barcodes using the BLAST1 method. However, matK, ITS, and psbA-trnH performed better than the other three barcodes, based on the nearest distance method. ITS and psbA-trnH obtained higher genetic distances, so the matK, ITS, and psbA-trnH barcodes were the preferable methods for identifying Radix Astragali and its adulterants based on superior sequencing efficiency and identification efficiency.A total of 478 sequences for six barcodes were analyzed, from which 161 sequences were obtained from Astragalus Radix and its adulterants. Sequence information and identification success rates are listed in  matK, and rbcL regions outperformed the ITS and psbA-trnH regions for gap rates. However, when we compared all of the average inter- and intraspecific distances, the ITS2, rbcL, matK, and psbA-trnH regions performed better than the ITS and COI regions. Therefore, in terms of intra- and interspecific variation, ITS2, matK, and rbcL are the preferable options for identifying Radix Astragali and its adulterants.Six parameters to analyze intraspecific variation and interspecific divergence were employed to assess the utility of six DNA barcodes . We expe rbcL, COI, ITS, and matK regions possessed less relative distribution of inter- and intraspecific variation than psbA-trnH and ITS2, although there were no nonoverlapping regions for the six barcodes. Hence, the rbcL, COI, ITS, and matK regions are more successful at identifying Radix Astragali and its adulterants, from the standpoint of barcoding gap analysis.Analysis of the DNA barcoding gap presents the divergence of inter- and intraspecies and indicates separate, nonoverlapping distribution between specimens in an ideal situation . In our  psbA-trnH successfully differentiated Radix Astragali and its adulterants. Furthermore, it produced areas of obvious separation for Radix Astragali. The remaining five barcodes also differentiated Radix Astragali and its adulterants. Each species clustered together, separate from other species. Considering the difficult amplification and sequencing and fast and accurate identification purpose of DNA barcoding, we did not add all the sequence data of ITS2 and psbA-trnH to build ML tree and subsequent analysis.Maximum likelihood (ML) is a general statistical criterion in widespread use for the inference of molecular phylogenies . An ML tRadix Astragali is reported to possess 47 bioactive compounds and has many bioactive properties –37. Vari psbA-trnH, rbcL, and matK from chloroplast genome; COI from mitochondrial genome), genetic distance and ML Tree were calculated by MEGA 6.0 software, and identification efficiency, intra- and interspecific variation, and barcoding gap were calculated using the Perl language algorithm. Results of the six indicators assessed are shown in  psbA-trnH outperformed other barcodes in terms of identification efficiency. ITS2 performed better in terms of genetic distance, gap rate, and inter- and intraspecific variation. RbcL performed better in terms of barcoding gap and inter- and intraspecific variation. Although ITS2 was part of the ITS sequence, it performed poorly in identification efficiency. Therefore, we suggest that the ITS sequence is the optimal barcode, and that the psbA-trnH region is a complementary barcode for identifying Radix Astragali and its adulterants.In this study, we validated a new analytical method for identifying Radix Astragali using DNA barcoding. Seventy-seven specimens of Radix Astragali and its adulterants were collected, and the sequences of 29 species reported in the literature were downloaded from the GenBank database. Based on the 478 sequences for six barcodes (ITS2, ITS from nuclear genome;In conclusion, we describe a new analytical method for the use of DNA barcoding in the identification of Radix Astragali. Six indicators, including average genetic distance, BLAST1 and the nearest distance method for identification efficiency, inter- and intraspecific variation, and gap rate were tested to evaluate six DNA barcodes using bioinformatics software and the Perl language algorithm. The ITS sequence was the optimal barcode for identifying Radix Astragali and its adulterants. This method provides a novel means for accurate identification of Radix Astragali and its adulterants and improves the utilization of DNA barcoding in identifying medicinal plant species.


AbstractCitharoceps Chamberlin was diagnosed by the presence of a very distinctive stridulatory apparatus composed of two patches of ridges on the sides of the cephalic region, and a stridulatory thorn on the prolateral region of the femur I. Currently, this genus is a junior synonym of Ariadna Audouin, with the assumption that the stridulatory apparatus could constitute an exclusive feature of its unique known species, Citharocepsfidicina Chamberlin, currently senior synonym of Citharocepscalifornica Chamberlin & Ivie. In the present study, Citharoceps is revalidated and redescribed based on the occurrence of the stridulatory apparatus in Citharocepsfidicina and Segestriacruzana Chamberlin & Ivie, and also on the presence of distinguishable characters, such as the length of the labium-sternum junction, ventral median spine on male metatarsi I, and strong sclerotized interpulmonary fold in females, forming a conspicuous median flap. Segestriacruzana is transfered to Citharoceps, with Citharocepscalifornica removed from the synonym of Citharocepsfidicina, and proposed as a junior synonym of Citharocepscruzana, due to the similarity between the additional material examined and the original description. Males of Citharocepsfidicina and Citharocepscruzana are described for the first time. Segestriidae currently comprises 120 species distributed in three genera: Segestria Latreille, 1804, Ariadna Audouin, 1826 and Gippsicola Hogg, 1900 , and Queensland Museum, Brisbane . The morphological examinations and descriptions follow The specimens examined are deposited in the American Museum of Natural History, New York , California Academy of Sciences, San Francisco , Collection of the Cabrillo National Monument Park, San Diego , Darrel Ubick collection, San Francisco (CDU), Segestriasenoculata : DENMARK: Zealand Island: Tisvilde, Tisvildeleje  (DMS), 1♂ 1♀ 3imm., 19-20.V.1991, C. Griswold & N. Scharff leg. (CAS 9032847). Ariadnamaxima : CHILE: Concepcion Province: Estación Escuadrón  (DMS), 2♂ 5♀ 3imm., 20.IX.1980, N. Cekalovic leg. (AMNH); Santiago Province: Santiago  (DMS), 1♀, 20.X.2009, T. H. Kawamoto leg. (IBSP 166664). Gippsicola sp.: AUSTRALIA: Queensland: Massey Range, 4km W of Centre Bellender Ker, 1250m  (DMS), 2♂, 9–11.X.1991, Monteith, Janetzi & Cook leg. (QM S91041); Bellender ker Range, Summit TV Station, 1560m  (DMS), 1♀, 1–7.XI.1981, Earthwatch/Queensland Museum leg. (QM S30617).Vouchers for comparative studies: PageBreakTaxon classificationAnimaliaAraneaeSegestriidaeChamberlin, 1924gen. reval.Citharoceps Chamberlin, 1924: 607.Citharocepsfidicina Chamberlin, 1924Citharoceps is distinguished from other segestriid genera by the presence in males, females and immatures of a conspicuous stridulatory apparatus composed of two patches of grooves on both sides of cephalic region , Baja California, Mexico, 4.VII.1921, Soiver leg., deposited in CAS 1392, examined). Chamberlin, 1924: 608 . – World Spider Catalog, 2015.California:UNITED STATES OF AMERICA. San Diego County: San Diego, La Jolla  (DMS), Eucalyptus grove, 1♀ 1imm., XII.1965, F. Ennik leg. (CAS 9039517); San Diego, Point Loma Peninsula  (DMS), 2♂, 8-12.IX.2003, D. Chan & T. Duffield leg. (CNMP); Baja California del Norte:MEXICO.  2.5mi S. of the Halfway House on Mex. Highway #1 (RB-3)  (DMS), 2♀ 1imm., 21.XI.1962, P. R. Craig & D. Dailey leg. .Citharocepsfidicina differ from those of Citharocepscruzana by the slightly shorter and thicker embolus . Total length 5.2; carapace 2.64 long, 1.76 wide. Palps light brownish orange . Total length 8.0; carapace 3.72 long, 2.36 wide. Palps orange, gradually darkening distally. Legs: robust, with long setae, and with ventral tibial and metatarsal spines of similar size : Total length 5.0–5.2; carapace 2.64–2.72 long; femur I 2.36–2.44. Female (n=3): Total length 6.08–8.0; carapace 3.0–3.72 long, 1.84–2.36 wide; femur I 1.92–2.56.South of California, USA and Baja California, Mexico Fig. .Citharocepsfidicina. In addition, that immature was collected with two females that match perfectly with the females collected in San Diego.We feel that species attribution is unproblematic. The immature specimen collected in Baja California del Norte by Craig and Dailey in 1962, matches perfectly with the type material of Taxon classificationAnimaliaAraneaeSegestriidaecomb. n.Segestriacruzana34°00'N; 119°45'W) (DMS), Santa Barbara County, California, USA, III–IV.1913, R. V. Chamberlin leg., deposited in AMNH, examined). –  Chamberlin & Ivie, 1935: 7  (DMS), California, USA, following  Chamberlin & Ivie, 1935: 8, pl. 5, figs 32–33  (DMS), 1♀, 22.II.2002, D. & S. Ubick leg. (CDU); Greenfield, Arroyo Seco Camping, Indians Road, el. 1126’  (DMS), 1♂ 2imm., 18.VI – 24.X.2004, D. & S. Ubick leg. (CDU); Coast Ridge Trail  (DMS), 0.8 mi SE Nacimiento Rd., el. 3000’, 1♀ 2imm., 1.VI.1991, D. Ubick leg. (CDU); San Luis Obispo County: Cayucos  (DMS), 300’, 1♀, 15.XI.1937, O. Bryant leg. (CAS 9039527); Santa Barbara County: gully SW of U. C. Field Station, 80-90m, Santa Cruz Island  (DMS), 1♂ 1imm., 22.IV.1994, D. H. Kavanaugh leg. .Citharocepscruzana differ from those of Citharocepsfidicina by the presence of a relatively longer and slender embolus . Total length 8.4; carapace 4.0 long, 2.76 wide. Palps light orange . Total length: 11.92; carapace 4.44 long, 2.6 wide. Palps reddish orange, gradually darkening distally. Legs reddish orange, with pairs I-II darker. Femur, patella and tibia I-II darkly marbled mainly on the ventral, prolateral and retrolateral regions : Total length 8.0–8.4; carapace 3.92–4.0 long, 2.4–2.76 wide; femur I 3.08–3.2. Female (n=3): Total length 9.32–11.92; carapace 4.24–4.44 long, 2.6–2.76 wide; femur I 2.72–2.92.Monterey County to Santa Barbara County, California, USA Fig. .PageBreakCitharoceps was found from the type locality of Segestriacruzana. After the examination of the immature holotype of Segestriacruzana, it was detected that it possessed the stridulatory apparatus exclusive for Citharoceps, not detected by Citharoceps were found from the middle coast of California that resemble those described by Citharocepscalifornica, together with a male, from the same region, that is similar to the one from Santa Cruz Island. Thus, Citharocepscalifornica is removed from its synonym with Citharocepsfidicina, due to its greater size and darker coloration, and placed as junior synonym of Citharocepscruzana.One male of Citharoceps Chamberlin, 1924 comprises only coastal regions of the state of California, USA, and Baja California, Mexico  in the carapace; and thorns (plectron) in the femur I. This type was also described by The occurrence of a stridulatory apparatus in spiders has been reported in at least 25 spider families , and alsplogynae  and Enteelegynae . LegendrPageBreakCitharoceps is unknown, but considering that it occurs in males, females and immatures, it seems unlikely that it has a courtship function. Citharocepsfidicina making a loud buzzing sound while rubbing the carapace ridges. Considering this information, the function of the stridulatory apparatus in Citharoceps could involve defensive buzzing sounds, as described by Micrathenagracilis , but future ethological studies are needed to corroborate this hypothesis.The function of the stridulatory apparatus in


The fully human monoclonal antibodies nivolumab (Nivo) and ipilimumab (Ipi) block the interaction between the immune checkpoint receptors programmed death-1 (PD-1) and cytotoxic T lymphocyte antigen-4 (CTLA-4), respectively, and their cognate ligands, restoring antitumor immune response. Phase 1 studies of Nivo monotherapy  or Nivo+Ipi combination therapy  demonstrated durable clinical activity and objective response rates (ORRs) of 31% to 40%, respectively, in patients with advanced melanoma (MEL). Evaluation of PD-L1 expression using immunohistochemistry (IHC) suggested a correlation between pretreatment tumor PD-L1 expression and clinical response to Nivo monotherapy [MEL patients received Nivo monotherapy (n=107) or Nivo+Ipi combination therapy . Tumor and tumor infiltrating lymphocyte (TIL) surface programmed death ligand 1 (PD-L1) expression in formalin-fixed, paraffin-embedded (FFPE) tumor tissue was evaluated by IHC with an automated assay (Dako) using the 28-8 Ab. Tumor PD-L1 positivity (PD-L1+) was defined as ≥5% cell membrane staining of any intensity; any expression on TILs was considered positive. Absolute lymphocyte counts (ALC) were measured in serial peripheral blood samples and lymphocyte subsets were evaluated using flow cytometry.P< 0.05).Tumor PD-L1-positive expression was observed in 45% and 37% of samples from the 003 and 004 studies, respectively. In 003, inclusion of any immune cell staining increased PD-L1 positivity to 92%. A numerically higher ORR was observed in MEL patients with PD-L1+ tumors with Nivo monotherapy or with sequential but not concurrent combination therapy. Neither study demonstrated an obvious change in ALC; however, phenotypic changes in T-cell subsets, including increases in the percentage of CD4 and CD8 expressing HLA-DR, ICOS and/or Ki67, were seen with combination therapy. In both studies, responses were observed irrespective of tumor PD-L1 or ALC status. In an exploratory analysis low pretreatment myeloid derived suppressor cells (MDSC) correlated with higher ORR with combination therapy (PD-L1 positivity is associated with tumor response with Nivo monotherapy; however, some responses were observed independent of PD-L1 or ALC status. No correlation between response and PD-L1 or ALC status was seen with combination therapy. MDSC levels may correlate with response to combination therapy. Future phase 3 randomized studies will explore these markers and other phenotypic changes in immune cell populations that might predict activity of Nivo in patients with MEL and other advanced cancers.


In this perspective article, we question how well model organisms, the ones that are easy to cultivate in the laboratory and that show robust growth and biomass accumulation, reflect the dynamics and interactions of microbial communities observed in nature. Today’s -omics toolbox allows assessing the genomic potential of microbes in natural environments in a high-throughput fashion and at a strain-level resolution. However, understanding of the details of microbial activities and of the mechanistic bases of community function still requires experimental validation in simplified and fully controlled systems such as synthetic communities. We have studied methane utilization in Lake Washington sediment for a few decades and have identified a number of species genetically equipped for this activity. We have also identified co-occurring satellite species that appear to form functional communities together with the methanotrophs. Here, we compare experimental findings from manipulation of natural communities involved in metabolism of methane in this niche with findings from manipulation of synthetic communities assembled in the laboratory of species originating from the same study site, from very simple (two-species) to rather complex (50-species) synthetic communities. We observe some common trends in community dynamics between the two types of communities, toward representation of specific functional guilds. However, we also identify strong discrepancies between the dominant methane oxidizers in synthetic communities compared to natural communities, under similar incubation conditions. These findings highlight the challenges that exist in using the synthetic community approach to modeling dynamics and species interactions in natural communities. For centuries, Biology has relied on model organisms for understanding the details of metabolism, regulation, evolution trends, etc. For example, rats, mice, nematode worms and yeast are all used as models for understanding complex metabolism and regulation in humans . MicrobiMethane oxidation is an important environmental process that mitigates release of large quantities of methane, mostly generated biogenically, via degradation of complex organic matter . This prMethylomonas (gammaproteobacteria) and Methylosinus  of major players, as observed at pure culture level, compared to activities/conditions approximating environmental settings. The existence of such discrepancies would have major implications for the selection of laboratory models and the experimental conditions. It would affect not only how we interpret the data on their performance, but also how we scale up these findings to the level of predicting and modeling global elemental cycles. Ultimately, we question whether our model choices and their manipulation in simplified laboratory settings allow for realistic interpretation of microbial activities in natural environmental settings.Figure 1), to address the discrepancies between the performance of natural communities in semi-in situ conditions and laboratory performance of pure culture isolates. Even though all 50 organisms originated from Lake Washington sediment, we did not follow any specific design for community structure, assuming that species that thrive in the specific incubation conditions that we apply would increase in their abundance and species that are not fit for these conditions would decrease in their abundance. Live cultures were mixed together in similar cell counts and were incubated for several weeks, with periodic transfers and dilutions, as previously described (2), low methane/high oxygen , and high methane/high oxygen . While the first two conditions mimicked gas concentrations in the opposing parts of methane and oxygen gradients in the natural lake sediment, the third condition represented a typical laboratory setting. We addressed two main questions: (i) whether Methylomonas and Methylosinus would outperform Methylobacter and Methylosarcina, to reflect their robust performance in the laboratory, and (ii) whether species other than Methylophilaceae, Flavobacteriales or Burkholderiales would persist in methane-fed communities, also based on laboratory performance of the cultivated species. In each of the communities, rapid loss of species complexity was observed after a few days of incubation, as previously observed for natural communities of high complexity  while methanotrophs of the family Methylocystaceae were undetectable. However, significant differences compared to natural community dynamics were uncovered in terms of the dominant methanotrophs. While Methylobacter species were found as dominant under the HL condition in natural samples . The dynamics of the Methylosarcina population also differed from the ones previously observed in natural communities. While Methylosarcina was reported to outperform Methylobacter under the HH condition .We assembled synthetic communities of 10 methanotrophs, 36 non-methanotrophic methylotrophs, and 4 non-methylotrophic heterotrophs . The results from these incubations clearly demonstrate that the performance/fitness of the three model organisms, as pure cultures, did not correlate well with their performance/fitness as parts of synthetic communities, even when simple two- to three-species communities were considered . In turn, their performance/fitness as parts of synthetic communities did not correlate well with the performance/fitness of their closely related counterparts as parts of natural communities . However, performance/fitness of the simple (3-species) and the complex (50 species) synthetic communities showed some agreement . Thus, we conclude that synthetic communities, at least as applied to methane-oxidizing communities and in the conditions used, appear to behave differently from the natural communities. These observations question, more generally, our reliance on data from laboratory manipulations for global predictions as related to microbe-mediated environmental processes.We next measured growth curves for pure cultures of three methanotroph species, Methylomonas spp., hardly seen in natural communities, showing robust performance, in agreement with their documented growth characteristics  The strains that we successfully isolated in pure cultures poorly reflect strains that are active in the natural habitat. However, we do have evidence of their close relatedness at the genomic level (Microbial communities involved in methane metabolism were used here as one example of a functional microbial assemblage involved into a specific biogeochemical process. In microbial ecology, and in ecology in general, fitness and performance of individual species are often considered as predictors of success in natural communities. However, the necessity of measuring performance in natural settings has been advocated . We hereeristics . In factsediment , and we ic level , understLC and ZY designed the experiments, ZY carried out experiments, SK carried out flow cytometric counting, DB carried out iTag data clustering. ZY and DB prepared figures. LC, ZY, SK, and DB wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


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).


Acute exacerbation of idiopathic pulmonary fibrosis (AE-IPF) has an extremely poor prognosis and there is currently no effective treatment for this condition. Direct hemoperfusion with a polymyxin B-immobilized fiber column (PMX-DHP) improves oxygenation, but it is unclear whether treatment of AE-IPF with PMX-DHP affects survival. This study elucidated the effectiveness and safety of PMX-DHP for the treatment of AE-IPF.This study included 31 patients with 41 episodes of AE-IPF. All patients received steroids. Of 31, 14 patients (20 episodes) were treated with PMX-DHP. The laboratory and physiological test results after the start of therapy and survival were retrospectively compared between patients treated with and without PMX-DHP.2/FiO2 ratio  and a smaller change in white blood cell count  after 2 days of treatment than patients treated without PMX-DHP. The 12-month survival rate was significantly higher in patients treated with PMX-DHP . PMX-DHP was effective in patients with more severe underlying disease . Treatment with PMX-DHP was an independent predictor of better prognosis . Mild pulmonary thromboembolism occurred in one patient treated with PMX-DHP.Patients treated with PMX-DHP had a significantly greater change in PaOTreatment of AE-IPF with PMX-DHP is tolerable and improves 12-month survival.The online version of this article (doi:10.1186/s12890-015-0004-4) contains supplementary material, which is available to authorized users. Idiopathic pulmonary fibrosis (IPF) has a devastating prognosis . Kondoh Direct hemoperfusion with a polymyxin B-immobilized fiber column (PMX-DHP) removes plasma endotoxins from gram-negative bacteria, and is an effective treatment for sepsis -11. PMX-This study investigated the effectiveness and safety of treatment of AE-IPF with PMX-DHP, and analyzed survival after treatment with and without PMX-DHP. This study also sought to identify factors affecting survival in patients with AE-IPF.Thirty-one patients who were treated for AE-IPF at our hospital between 1997 and 2013 were retrospectively reviewed. Eight patients had two episodes of AE-IPF and one patient had three episodes of AE-IPF . Eighteen patients underwent surgical lung biopsy (SLB) before developing AE-IPF, and met the 2011 consensus criteria for IPF of the American Thoracic Society (ATS), European Respiratory Society (ERS), Japanese Respiratory Society (JRS), and Latin American Thoracic Association (ALAT) . PatientAE-IPF was diagnosed according to the modified diagnostic criteria described by Collard et al. in 2007 . BrieflyClinical data, including sex, age, smoking-history, symptoms, treatments, and survival were obtained from the medical records. Laboratory and pulmonary function test results were also recorded. The severity of IPF before AE, oxygenation and HRCT findings during AE, treatments for AE, and survival were retrospectively reviewed. Disease severity of IPF within 12 months before AE was assessed using the GAP-staging system, which considers gender, age, and two lung physiology variables: forced vital capacity (FVC) and diffusion lung capacity for carbon monoxide (DLCO) .Immediately after the diagnosis of AE-IPF, all patients were started on high-dose corticosteroid pulse therapy  followed by a tapering dose of prednisolone with or without immunosuppresants (cyclophosphamide or cyclosporine). Some patients also received intravenous sivelestat sodium hydrate.Some patients were treated with PMX-DHP  starting at the same time as the corticosteroid and immunosuppressant therapy. Starting in 2006, patients were treated with PMX-DHP whenever practicable. Inclusion criteria of PMX-DHP for the treatment of AE-IPF were as follows: 1) current diagnosis of AE-IPF, 2) able and willing to provide informed consent. Exclusion criteria of PMX-DHP for the treatment of AE-IPF were as follows: 1) ≥ 85 year-old, 2) history of hypersensitivity for blood purification or extracorporeal circulation therapy, 3) hemodynamic instability, 4) severe cardiovascular disease, 5) severe hemorrhagic disease, 6) terminal cancer, 7) pregnant or lactating, 8) considered ineligible for PMX-DHP by an attending doctor. A flow chart of treatment with PMX-DHP according to these inclusion and exclusion criteria is shown in Figure Blood samples were collected before and after PMX-DHP. The serum level of angiopoietin-2 was measured using enzyme-linked immunosorbent assay .The extent of lung opacities was measured on three HRCT slices: at the bifurcation of the trachea, at the bases of the lower lobes, and at the midpoint between the other two slices. The extent of fibrosis in each lobe was scored using following system: 0, none; 1, 1–10%; 2, 11–25%; 3, 26–50%; 4, 51–75%; and 5, 76–100%. The sum of the scores from five lobes (0–25) was used to express the extent of reticular opacity, honeycombing, consolidation opacity, and ground-glass opacity (GGO) in each patient. The pattern of AE-IPF on HRCT was classified as 1) peripheral, 2) multifocal, or 3) diffuse as reported by Akira . The HRCt-test. Overall survival of patient groups was estimated using Kaplan-Meier curves, and was compared between groups using the log-rank test. The relationships between variables, including treatment with PMX-DHP and mortality, were evaluated using Cox’s proportional hazards regression analysis. All tests were two-sided and statistical significance was set at p < 0.05.Statistical analyses were performed using StatView J-4.5 . Categorical data were compared using the Chi-square test or Fisher’s exact probability test for independence, and continuous data were compared using the paired 2 and the distance walked in the 6-minute walk test (6MWT) were also impaired . The severity of IPF within 12 months before AE-IPF was staged using the GAP system [The clinical characteristics of the patients, including the laboratory and physiological test results at the time of the first AE-IPF, are shown in Table P system . The mos2 at rest, 6MWT results, serum levels of Krebs von den Lungen-6 (KL-6) and surfactant protein D (SP-D), or extent score based on HRCT between the two groups. There were also no significant differences in PaO2/FiO2 (P/F) ratio at the time of AE-IPF, time from admission to the start of treatment for AE-IPF, HRCT-patterns at the time of AE-IPF, administration of corticosteroids, immunosuppresants and other agents, or intubation rate between the two groups. Serum endotoxin levels were below the level of detection in all patients treated with PMX-DHP.The demographic data, preceding IPF treatment, laboratory and physiological tests results, HRCT findings, and treatment for AE-IPF in patients treated with and without PMX-DHP are shown in Table The details of PMX-DHP are shown in Table 4/μL vs. -0.9 ± 1.5 × 104/μL, p = 0.010; Figure p = 0.022; Figure Changes after 2 days of treatment were compared between patients treated with and without PMX-DHP  died within 12 months of the first episode of AE-IPF. Eighteen patients died of respiratory failure, four died of infection, and one died of an unknown cause. The Kaplan-Meier survival curves from the time of AE-IPF are shown in Figure .2% died Survival curves according to the severity of disease before AE-IPF showed that patients with less severe IPF (GAP-stage I) did not have a significant benefit from treatment with PMX-DHP , platelet-derived growth factor (PDGF), and transforming growth factor-β (TGF-β) in patients with AE-IPF . TherefoThe severity of IPF before AE-IPF was evaluated using the GAP staging system. The GAP system is an easy and reliable method of staging the severity of IPF , that coIn the analysis of prognostic factors, PMX-DHP, ΔP/F ratio, and ΔLDH were significantly associated with a more favorable prognosis. However, there was no difference in ΔLDH between PMX and non-PMX groups. We suspect that slight hemolysis may have occurred in patients treated with PMX-DHP, and this hemolysis may be why ΔLDH was equivalent between PMX and non-PMX groups.This study has a number of limitations. First, only a small number of patients with AE-IPF were included. Second, the data were collected retrospectively. Third, the treatment for AE-IPF other than PMX-DHP and steroids was not uniform. Finally, AE-IPF in a Japanese population may not be the same as that in other ethnic groups . AE-IPF The present study found that treatment of AE-IPF with PMX-DHP was safe, was not associated with severe adverse events, and improved oxygenation. The 12-month survival rate was better in patients treated with PMX-DHP than in patients treated without PMX-DHP. The effects of treatment with PMX-DHP were evident in patients with a more severe stage of IPF. Treatment with PMX-DHP was also a favorable prognostic factor in patients with AE-IPF. Recent randomized controlled trials reported promising results using novel medications for chronic stage of IPF, such as nintedanib and pirfenidone ,35. Howe


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.