Elevated expression of specific transposable elements (TEs) has been observed in several neurodegenerative disorders. TEs also can be active during normal neurogenesis. By mining a series of deep sequencing datasets of protein-RNA interactions and of gene expression profiles, we uncovered extensive binding of TE transcripts to TDP-43, an RNA-binding protein central to amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Second, we find that association between TDP-43 and many of its TE targets is reduced in FTLD patients. Third, we discovered that a large fraction of the TEs to which TDP-43 binds become de-repressed in mouse TDP-43 disease models. We propose the hypothesis that TE mis-regulation contributes to TDP-43 related neurodegenerative diseases. Accumulation of TAR DNA-binding protein 43 (TDP-43) containing cytoplasmic inclusions is a shared pathological hallmark in a broad spectrum of neurodegenerative disorders, including ALS, FTLD and Alzheimer's disease Transposable elements (TEs) are highly abundant mobile genetic elements that constitute a large fraction of most eukaryotic genomes. Retrotransposons, which copy themselves through an RNA intermediate, represent approximately 40% of the human genome Several recent studies used deep sequencing to profile the RNA targets that co-purify with immunoprecipitated mouse, rat or human TDP-43 and also to profile gene expression changes in mouse after knockdown or over-expression of TDP-43 in vivo crosslinking-immunoprecipitation sequencing (CLIP-seq) datasets per se. To address these issues, and to test the robustness of our observations, we also tested two additional mapping methods for the rat and human datasets  dataset datasets ; MethodsWith all three mapping strategies we find a dramatic enrichment of sequences that derive from each major class of TE . With thOverall, we detect the most extensive binding to TEs with the MULTI method, and these findings are not an artifact of the way we assigned weights with the MULTI method because even with the more restricted UNIQ analysis, we identify ∼80% of the rat elements that are differentially enriched when all mappable reads are included , S1F. Mop-value<2.2e−16). Thus our findings show specificity for TDP-43 and are not a byproduct of inherent biases in library construction or analysis.As a test of the biological specificity of our finding that TDP-43 selectively binds to TE derived transcripts, we applied the UNIQ mapping method to a CLIP-seq dataset for an unrelated RNA binding protein. For this purpose we chose fused in sarcoma (FUS), which like TDP-43, is an hnRNP RNA binding protein that plays diverse roles in RNA biology, including splicing Because TDP-43 has a known binding motif among its mRNA targets, we used MEME  to identBecause the human dataset p-value< = 0.01, p-values were assigned with 100 iterations on randomly chosen sets containing 50% of original data; see This reduced binding in FTLD patients of TDP-43 to TE-derived transcripts also is apparent when we examine over-all enrichment for the UGUGU pentamer motif  and S1 rThe reduced binding of TE transcripts in FTLD patients suggested that TDP-43 pathology might include a loss of TE regulation. We investigated this possibility in two ways. First, we analyzed the repetitive sequence reads from two different mRNA-seq datasets from mouse models of TDP-43 pathology.The first mRNA-seq study that we analyzed TDP-43 aggregation and neuropathology plays a fundamental role in a broad spectrum of neurodegenerative disorders A large fraction of the genetic material of multicellular organisms is made up of mobile elements as well as inactivated TEs. A fraction of these TEs retain the capacity to copy themselves and insert at new genomic locations. During the co-evolution of TEs with their host genomes, organisms have evolved elaborate and efficient mechanisms to prevent or at least regulate such transposition events. As a result, even the potentially active TE copies rarely mobilize within the germline and are also largely constrained in somatic tissue. Several recent studies demonstrate, however, that LINE-1 elements are normally active and mobile during neurogenesis in both rodent and human tissue Our findings support three conclusions. First, that TDP-43 broadly targets TE-derived transcripts, including many SINE, LINE and LTR classes as well as some DNA elements. This conclusion is replicated in three independent datasets from rat, mouse and human. Second, the association between TDP-43 and TE-derived RNA targets is reduced in FTLD patients relative to healthy subjects, consistent with the idea that loss of TE control might be part of the disease pathology. Third, we observe broad over-expression of TE derived transcripts in each of two different mouse models with TDP-43 dysfunction. And there is a striking overlap between the TE targets identified in the CLIP study and those that are over-expressed with TDP-43 misexpression. Taken together, our findings raise the hypothesis that TDP-43 normally functions to silence or regulate TE expression. When TDP-43 protein function is compromised, TEs become over-expressed. Unregulated TE expression can have a number of detrimental impacts including genome instability, activation of DNA-damage stress response or toxic effects from accumulation of TE-derived RNAs or proteins. Such toxicity from activation of mobile genetic elements may contribute to TDP-43-mediated neurodegenerative disorders.The CLIP-seq data of human healthy and FTLD brain tissues was obtained from EMBL-EBL Array Express Archive EMTAB-530 We used Bowtie i≈ei * LrL where rL is the reference library, iL is one other library, and ie is the correlation coefficient of library iL to rL. We chose a bin correlation approach as described in PeakSeq p-value of the difference of the read counts was calculated as described in DESeq iw with reads iAk and iBk from libraries A and B, and iAk+iBk = iSk, the p-value of  is the probabilities of all pairs with probabilities less than or equal to p among all combinations, i.e.,p is p(a)*p(b), by assuming the two libraries are independent, and p(x) was computed using either the Poisson distribution or a negative binomial distribution. The null hypothesis we are testing against with a negative binomial model states that it is statistically unlikely for a combination of (1) random selection of transcripts sequenced and (2) biological variation between replicates to create a differential enrichment of reads within the given window that is larger than what we see in the TDP-43 IP data as compared to the control. For the rat samples, which did not include replicates, we can only test against a Poisson model null hypothesis that random selection of sequenced transcripts would be statistically unlikely to result in differential enrichment greater than what is seen in the data. These p-values were corrected for multiple hypotheses testing using the Benjamini-Hochberg correction. We set a significance threshold, adjusted p-value<0.00001, for identifying differentially enriched regions. We next advanced the sliding window by 100 bp and repeated the previous step. Enriched regions with a gap of less than 500 bp and with the same direction for differential enrichment  were merged.To identify potential differential binding sites of TDP-43, a sliding window with size of 500 bp and moving step size of 100 bp was used to scan the genome and compute the number of reads falling in the window in both samples. The reason for partially overlapping windows is to increase the resolution at which optimal peaks can be discovered. As described above, the counts in each bin are weighted by the number of loci to which they were mapped. For the rat data, the read counts were modeled with a Poisson distribution, similarly to two popular ChIP-seq analysis approaches, MACS The above differential binding analysis method was used to analyze the datasets in three different ways. For the UNIQ method (see text), only uniquely mapped reads were included. For the UNIQ+SameEle method (see text), unique reads and those that mapped to multiple locations were included, so long as they mapped to the same element. For MULTI (see text), we included all mappable reads.A predicted region was annotated as ‘RefGene’, if it overlaps with exons of a gene, or as ‘repeat’ if it overlaps with a repetitive element. If a differential binding site overlaps with a repeat region, but this repeat region is inside an exon, then the region will be annotated as the corresponding gene. Simple repeats that overlap with other repeat classes are not considered. The annotations were obtained from the UCSC genome website, as described above, which provides 4 levels of classification for most repeat elements: Class, Family, Element, and Instance. This nomenclature approximates that used by the RepBase group, from which these annotations were derived MEME p-value for a cross-link site with cDNA counts x was computed as adjp(x) = (xμ+xσ)/xp, where xμ and xσ are the mean and standard deviation of frequency of cDNA counts x in the randomized background across 100 iterations, and xp is the observed probability. This method is not as robust as that used for the rat and human peak identification due to the non-random rates of transcription in the genome. The p-values shown in The approach described above is not suitable to the mouse dataset, because of a lack of control samples. Therefore, a similar method RNA short sequences were aligned to the whole genome in order to assess the RNA profiles of repetitive elements. The alignment software and most of the parameter settings were the same as that used for aligning the CLIP-seq datasets . The same weighting scheme was applied to each alignment as described above. Read abundances of a repeat element were computed by summing up the alignment weight of all reads mapped to the correct strand, within the TE annotation boundaries, and normalized by the length of that element. DESeq Figure S1Additional bioinformatics analyses. (A) Total candidate differentially enriched peaks annotated as transposable elements (TEs) found using two normalization methods for the Rat TDP-43-IP samples. The left panel shows overlap in the Rat TDP-43 total number of enriched repetitive element TE peaks identified using the two normalization methods, the right panel shows overlap in the number of candidate depleted repetitive element TE peaks. In both orange circles represent  the number of differential TEs identified when a “bin correlation” approach is used to normalize the reads in each sample, while the blue circles represent the differential TEs using a “library size” normalization approach. The library size normalization approach, which is commonly used, simply normalizes all samples by the total mapped mass of reads in each sample ; the underlying assumption would be that the background is approximately the same for both samples genome-wide. We noticed that the backgrounds of the control and TDP-43-IP samples were highly non-random, and that some regions had much higher or lower reads than other genomic loci, even outside of the identified binding peaks. Therefore, we modeled the background using a sliding window of non-overlapping 10 kb bins, computing the correlation coefficient between the control and IP samples in each bin, e.g., a “bin correlation” approach to normalization. As is evident from the Venn diagrams in this figure, this approach is more conservative than a simple RPM or “library size” normalization method (please see B) The whole genome was separated into non-overlapping adjacent 10 Kbp bins. Each dot (black) represents read counts of a bin. Those bins selected to compute the normalization factors were colored in red. (left) Read counts of TDP-43-IP sample and control-IP sample from Rat RIP-seq (right) Read counts of two human healthy brain samples from CLIP-seq data. (C) Motif logos for the most enriched motifs as identified by MEME in the TDP-43 binding peaks overlapping repetitive regions. (top) Rat RIP-seq data (bottom) Human CLIP-seq healthy brain tissue samples. (D) Enrichment for the UGUGU pentamer motif across a 51 nt window surrounding the binding site  relative to the genome is shown among RefGene and repeat sequences. Mouse (top panel) and rat (bottom panel). (E) For each read having multiple alignments (multi-read), the fraction of the most frequently appearing TE among all those alignments is computed. And the distribution of all multi-reads with different common TE alignment fractions is computed. For about 80% of the multi-reads, all alignments corresponded to the same TE element in Rat (top panel) and about 50% in Human (bottom panel). (F) Overlaps of detected TEs with each of three mapping methods for Rat (left panel) and Human (right panel) are shown. The three mapping methods are: UNIQ (uniquely mapped reads), UNIQ+SameEle (uniquely mapped reads and multi-reads mapped to the same elements), and MULTI (unique reads and multi-reads). (G) Extensive overlap is observed between TE transcripts that were de-repressed with over-expression H) For TDP-43 and FUS, distributions of peaks (UNIQ+SameEle) over RefGene targets are not significantly different from each other. Mean peak heights of TDP-43 and FUS are 68 and 32 respectively. The distance between TDP-43 and FUS is less than 15 with a p-value of 0.98. (I) For RefGene and repeat sequences that bind to TDP-43 in tissue from healthy human subjects, distributions of peak heights are not significantly different from each other. (J) For Refgene and repeat sequences from the mouse TDP-43 overexpression dataset, distributions of expression levels are not significantly different from each other.(DOCX)Click here for additional data file.Table S1Number of aligned reads for each TDP-43 dataset. Human dataset is from EMBL-EBL ArrayExpress Archive EMTAB-530 and rat dataset is from NCBI GEO DATASET Accession Number: GSE25032. The mouse datasets are from NCBI GEO DATASET Accession Numbers: GSE22351 and GSE27394.(DOCX)Click here for additional data file.Table S2Number of aligned reads for FUS datasets. FUS dataset is from DDBJ Sequence Read Archive (DRA) Accession Number: SRA025082.(DOCX)Click here for additional data file.Table S3Enriched and depleted repetitive elements rat TDP-43-IP/control-IP RIP-seq. Enriched and depleted repetitive elements in Human TDP-43 Healthy/FTLD CLIP-seq. Enriched repetitive elements in mouse TDP-43 CLIP-seq. Induced repetitive elements expression after depletion of TDP-43 with ASO (mouse mRNA-seq). (overlap with mouse CLIP binding sites shown). Induced repetitive elements expression in transgenic mice overexpressing human TDP-43 (mouse mRNA-seq). (overlap with mouse CLIP binding sites shown).(XLS)Click here for additional data file.


Clostridium perfringens. However, the genes responsible for the resistance have not yet been characterized. Ninety-nine C. perfringens isolates recovered from broilers and turkeys were tested for phenotypic bacitracin resistance. Bacitracin MIC90 (>256 µg/ml) was identical for both turkey and chicken isolates; whereas MIC50 was higher in turkey isolates (6 µg/ml) than in chicken isolates (3 µg/ml). Twenty-four of the 99 isolates showed high-level bacitracin resistance (MIC breakpoint >256 µg/ml) and the genes encoding for this resistance were characterized in C. perfringens c1261_A strain using primer walking. Sequence analysis and percentages of amino acid identity revealed putative genes encoding for both an ABC transporter and an overproduced undecaprenol kinase in C. perfringens c1261_A strain. These two mechanisms were shown to be both encoded by the putative bcrABD operon under the control of a regulatory gene, bcrR. Efflux pump inhibitor thioridazine was shown to increase significantly the susceptibility of strain c1261_A to bacitracin. Upstream and downstream from the bcr cluster was an IS1216-like element, which may play a role in the dissemination of this resistance determinant. Pulsed-field gel electrophoresis with prior double digestion with I-CeuI/MluI enzymes followed by hybridization analyses revealed that the bacitracin resistance genes bcrABDR were located on the chromosome. Semi-quantitative RT-PCR demonstrated that this gene cluster is expressed under bacitracin stress. Microarray analysis revealed the presence of these genes in all bacitracin resistant strains. This study reports the discovery of genes encoding for a putative ABC transporter and an overproduced undecaprenol kinase associated with high-level bacitracin resistance in C. perfringens isolates from turkeys and broiler chickens.Phenotypic bacitracin resistance has been reported in  Clostridium perfringens is a Gram-positive, anaerobic spore-forming bacterium that causes a wide variety of diseases in humans and animals. A classification based on the production of four major toxins  divides the C. perfringens into five toxigenic biotypes (A to E) C. perfringens is responsible for gas gangrene, enteritis necroticans, food poisoning, and non-foodborne gastrointestinal infections in humans C. perfringens has particular significance in poultry where it may cause necrotic enteritis Bacillus licheniformis and some strains of Bacillus subtilis, in feed. It inhibits cell wall synthesis by binding to undecaprenyl pyrophosphate (UPP) and preventing its dephosphorylation, thereby blocking the recycling of UPP to undecaprenol monophosphate , a lipid carrier implicated in the transport of peptidoglycan monomer units through the cytoplasmic membrane Necrotic enteritis is usually controlled by the addition of bacitracin antimicrobial, a polypeptide antibiotic synthesized by bacA gene encodes for an undecaprenyl pyrophosphate phosphatase (UppP) and was discovered in 1992 in Escherichia coliuppP in 2004 Streptococcus pneumonia, Staphylococcus aureus and Enterococcus faecalis V583 bcrABC genes were identified in 1995 in Bacillus licheniformisB. subtilis, Streptococcus mutans and E. faecalisbcrD gene of E. faecalis identified by Manson et al. Clostridium thermocellum. The last mechanism involves exopolysaccharide molecules. In Xanthomonas campestris, Sphingomonas strains S-88 and NW11, and E. coli K-12 S. mutans, it is speculated that rhamnose-glucose polysaccharide may act as a barrier that prevents some antibiotics from reaching their targets To date, four major bacitracin resistance mechanisms have been described. The E. faecalis, an ABC transporter and an overproduced undecaprenol kinase were both reported to be encoded by the bcrABD operon under the control of a regulator, bcrRbcrABD expression E. faecalis, these genes have been associated with high-level bacitracin resistance with a minimum inhibitory concentration (MIC) >256 mg/L E. faecalisIn C. perfringens in North America which show high levels of resistance C. perfringens isolates from turkeys and broiler chickens. We also demonstrate that bcrABDR genes are located on the chromosome.Despite studies on bacitracin resistance in The handling of animals of this study was performed in accordance with current national Canadian legislation  under the enforcement of the Canadian Food Inspection Agency (CFIA) inspectors. All fecal samples of this study were taken from already slaughtered animals in federally registered slaughter facilities where federal measures are in place to protect food animals during handling and slaughter.C. perfringens from conventionally raised birds were recovered from the normal intestinal microbiota of chicken and turkey taken at five (four chicken and one turkey) processing plants  located in the province of Quebec, Canada. Only three isolates were associated with necrotic enteritis. Antimicrobial regimens were not available. The contents of the caeca were cultured in cooked meat broth  for 48 h in anaerobic conditions at 35°C. Ten µl were then plated on anaerobic sheep blood agar (PML) and incubated for 24 h under anaerobic conditions. Three greyish colonies with a typical double zone of hemolysis were subcultured for purity on blood agar. Gram staining was performed on every isolate and cultures with Gram-positive square-ended rods were selected for PCR identification and genotyping. One to two C. perfringens isolates per bird were kept for this study.Isolates of cpa , cpb (beta), etx (epsilon), iA (iota), cpb2 (β2- toxin) and cpe (enterotoxin) of C. perfringens. Primers and conditions used were as previously described cpa, cpb, etx, iA, cpe, and cpb2. DNA amplification reactions were carried out using a Whatman Biometra thermocycler  programmed as follows: 35 cycles of 20 sec at 94°C, 20 sec at 55°C and 40 sec at 72°C with a hot start of 5 min at 94°C and a final elongation time of 5 min at 72°C. For visualization, 5 µl of the PCR reaction were subjected to electrophoresis in 1% agarose gel stained with ethidium bromide. A 100 bp ladder  was used as a marker. C. perfringens type E  and C. perfringens type B  A multiplex PCR was used for the detection of the toxin genes Multiple-locus variable number of tandem repeats analysis (MLVA) was performed as previously described by Chalmers et al. 5 CFU/ml allowed the growth of a lawn of well-developed colonies and an easily permitted discernment of growth inhibition by bacitracin. Consequently, an inoculum of 105 CFU/ml was used for evaluation of bacitracin susceptibilities. Stock cultures were thawed then plated on blood agar plates and incubated for 24 h in anaerobic conditions. Colonies were randomly selected, combined and then diluted in Brucella broth to yield concentrations of 105 CFU/ml. C. perfringens suspensions were spread with a cotton swab on a Brucella blood agar plate. After the surface of the inoculated plate had completely dried, a bacitracin Etest strip was placed on the surface according to the manufacturer’s instructions. Agar plates were then incubated at 35°C in anaerobic conditions for 48 h with a first reading at 24 h and a second reading at 48 h. At both times, colonies could be detected and MICs were read by determining where the zone of growth inhibition intersected the MIC scale on the strip. S. aureus ATCC 29213 and C. perfringens ATCC 3110 were used as quality controls. The concentrations that inhibited growth of at least 50% and 90% of the isolates tested were calculated as MIC50 and MIC90, respectively. Low level bacitracin resistance breakpoint was set at >16 µg/ml while high-level bacitracin resistance was set at >256 ug/ml according to previous studies Bacitracin minimum inhibitory concentrations (MICs) were determined using the Etest  according to the manufacturer’s instructions. Preliminary tests indicated that an inoculum size of 10bcr operon was performed on a bacitracin resistant C. perfringens strain c1261_A isolated from the normal flora of a turkey caecum using the primer walking technique. Primers for the bcrR and bcrB genes , sequenced on an ABI PRISM 310 (Applied Biosystems), and results were used for primer walking. Briefly, the targeted sequences were further divided in different section for which primer pairs were designed for PCR and sequencing  efflux pump inhibitors on bacitracin susceptibility was evaluated by a checkerboard microdilution technique as previously described by Hendricks et al. C. perfringens strain c1261_A was purified by an alkaline lysis method using a plasmid midi-kit (Qiagen) with the addition of 5 µg/ml of lysozyme in the resuspension buffer before incubation at 35°C for 30 min. The plasmid DNA was visualized on a 1% agarose gel after an electrophoresis of 60 min at 100 V. The plasmid bands sizes were determined using the software 1Dscan EX 3.01 (BD Biosciences BioImaging) of the Gel Doc 2000 (Bio Rad) by reference to plasmids of known band sizes . Plasmid curing experiments were also performed as previously described Plasmid DNA of the bacitracin resistant rrn genes), which are localized only on the chromosome. Thus, chromosomal fragments can be distinguished from plasmid bands with the rrn probe. DNA fragments were then separated by contour-clamped homogenous electric field (CHEF) electrophoresis with the following conditions: migration of 16 h at 15°C in a 0.8% agarose gel, with a voltage of 6 V/cm and switch times of 0.5 to 40 s. A low range PFG marker (New England Biolabs) was used as a DNA ladder. Gels were stained with ethidium bromide and bands were visualized with the software 1Dscan EX 3.01 (BD Biosciences BioImaging) of the Gel Doc 2000 (Bio Rad).All genomic DNAs were prepared as described previously bcrB and rrn genes were used as hybridization probes. The PCR products were labelled with digoxigenin using the PCR DIG probe synthesis kit (Roche Diagnostics) and were detected with the DIG nucleic acid detection kit (Roche Diagnostics), both according to the manufacturer’s instructions.The PFGE genomic DNAs and plasmid extractions were transferred to positively charged nylon membranes  using a Vacuum Blotter Model 785 (Bio-Rad). Amplicons derived from the C. perfringens c1261_A RNA was extracted with the RNeasy mini kit (Qiagen) from 1 ml of an overnight BHI culture supplemented with 0, 10, 50, 100 or 256 µg/mL of bacitracin. RNA was quantified and stored −80°C. Two µg of RNA was converted to cDNA using the Quantitect reverse transcriptase kit (Qiagen) according to the manufacturer’s instructions. PCR for the genes bcrA, bcrB, bcrD and bcrR were done on the same amount of cDNA from each condition. 16 s rRNA gene (rrn) was used as a control. PCR products were separated in 1% agarose gel and stained with ethidium bromide. Also, RT-PCR was used to amplify the intergenic spaces between bcr genes to determine whether these genes are cotranscribed.Total bcrD, bcrB, bcrA and bcrR genes from C. perfringens were deposited. in GenBank database under accession numbers GU810179, GU810180 GU810181 and GU810182, respectively.Sequences of the C. perfringens were recovered from the caeca of 50 turkeys (81 isolates) and 13 chickens (18 isolates). Isolation rates of C. perfringens from caeca samples were 66% for turkeys and 30% for broilers. Of those 99 field isolates, genotyping results revealed that 96 were of type A  and 3 were of type E . The three isolates associated with necrotic enteritis were of type A.Ninety-nine field isolates of C. perfringens, 24 of those showed bacitracin resistance . The remaining 75 isolates were susceptible to bacitracin with MICs ranging from 0.75 µg/ml to 16 µg/ml  were analyzed for their genetic relatedness using MLVA. The BioNumerics dendrogram yielded a total of 15 MLVA types for the 24 isolates. These types were separated in three clonal clusters, the first one containing the majority of the isolates (n = 16) (Bacitracin resistant strains ((n = 16) . The firC. perfringens strain c1261_A with a MIC of >256 ug/ml and isolated from the caecum of a turkey was selected for sequencing analysis. Putative bacitracin resistance genes of this strain were sequenced using PCR amplification with walking primers that were designed based on the sequence of the E. faecalis bcrABDR operon. Analysis of the sequence data showed that the putative bacitracin resistance genes were contiguous on a DNA fragment of 4486 bp with four open reading frames (ORFs) that were oriented in the same direction and were designated bcrR (621 bp), bcrA (702 bp), bcrB (528 bp), and bcrD (654 bp) based on sequence identity with bacitracin resistance genes identified in E. faecalisbcrABDR genes of C. perfringens strain c1261_A showed 88% sequence identity to the bcrABDR genes previously described in E. faecalis plasmid pJM01 1216-like transposase gene of E. faecalis  to a number of permeases associated with ABC transporters. BcrA is a 234 amino acid putative protein with an estimated molecular weight of 26.4 kDa. It has 62% identity and 72% similarity to the E. faecalis BcrA protein with an ATP-binding domain and also showed identity (between 40% and 45%) to other ATP binding proteins of ABC transporters. The complete amino acid sequences of BcrA and BcrB are predicted to be an homodimeric ABC transporter similar to the one recently described by Manson et al. (2004). The complete 207 amino acid sequence of BcrR contained a xenobiotic response element (XRE) conserved domain of the transcriptional regulators family from residue 2 to residue 55. BcrR shared 69% identity and 80% similarity to E. faecalis BcrR which is suspected to act as a sensor and a transducer of bacitracin availability The bcr genes (bcrABDR) on both PCRs and microarrays. They were all also positive for tetA(P), which mediates active tetracycline efflux tetB(P), which is related to the ribosomal protection family of tetracycline-resistance determinants erm(B) gene that encodes a ribosomal methylase that mediates MLSB (macrolide-lincosamide-streptogramin B) resistance C. perfringens ATCC 13124 was negative for all the DNA probes. Plasmid curing experiments revealed the lost of the tet genes by PCR, indicating plasmid localization of the tet genes. However, in no cases, the bacitracin resistance genes proved to be curable. Also, to positively correlate the absence of a complete bcr locus with phenotypic susceptibility to bacitracin, 70 susceptible C. perfringens strains of this study were analyzed for the presence of bcrABDR genes and all strains were negative for all bcr genes.All 24 bacitracin resistant isolates were positive for the four Of all efflux pump inhibitors tested, only thioridazine increased significantly the susceptibility of strain c1261_A to bacitracin . The MICbcrABDR gene cluster in presence of bacitracin, mRNA levels were analysed by semi-quantitative RT–PCR. Data showed negligible or no bcrABD mRNA in absence of bacitracin . None of these bands hybridized with the bcrB probe. I-CeuI and MluI double-digested genomic DNA of the bacitracin resistant C. perfringens strain c1261_A hybridization experiments with bcrB and rrn probes showed cohybridization of these probes on one band corresponding to a ∼97 kb chromosomal fragment  and chickens (18). The C. perfringens isolation rates from caecal samples were higher for turkeys (66%) than for broiler chickens (30%). This is in agreement with a previous study C. perfringens isolation was more difficult after the chickens reached 22 days of age due to modifications of their intestinal flora. Some birds might have been fed with a bacitracin supplemented diet that could have contributed to a lower recovery rate, as it has been found that bacitracin reduces the count of C. perfringens in the caeca of the treated birds While phenotypic bacitracin resistance has been reported in the past in C. perfringens, the high-level bacitracin resistant (MIC >256 µg/ml) C. perfringens strain c1261_A was selected for further studies using a primer walking strategy based on an enterococcal bcrABD operon. Sequence analyses of the amplicons generated by the bcr primers showed four contiguous ORFs. Two of these genes had high levels of amino acid sequence identity to the bcrA and bcrB genes of E. faecalis suggesting that the genes identified in this study encode for an ABC transporter. This is further supported by the high-level bacitracin resistance observed in the bcrABDR positive strains of C. perfringens. Reports in the literature suggest that the level of bacitracin resistance given by the ABC transporter is higher than those produced by any other mechanism bcrA and bcrB genes of E. faecalis were sufficient for high-level bacitracin resistance To elucidate the genetic basis of bacitracin resistance in C. perfringens strain c1261_A was exposed to efflux pump inhibitors to investigate their influence on susceptibility to bacitracin. Drug efflux pumps belonging to the ABC, MATE , MFS and SMR  families are found in Gram-positive bacteria, with MFS-class pumps being predominant bcrABDR genes can confer bacitracin resistance in these strains awaits construction and complementation of deletion mutants  which is presently underway in our laboratory. Semi-quantitative RT-PCR demonstrated that the bcrABDR gene cluster expresses when C. perfringens are subjected to bacitracin stress. It was also found that the bcrABD genes are transcribed as a polycistronic message and thus are part of an operon. These results are in agreement with the results obtained by Manson et albcrABD operon under the control of a regulator bcrR, which was described as a membrane bound sensor and a transducer of bacitracin availability to regulate bcrABD expression.In addition, bcr genes of the C. perfringens c1261_A strain was not on plasmid fragments. To further explore the bcr genes location, PFGE was performed with chromosomal DNAs. The I-CeuI restriction enzyme that was used cuts only in the 23S rRNA genes (rrn genes), which are localized only on the chromosome. Thus, chromosomal fragments could be differentiated from plasmid bands with the rrn probe. In this study, both the bcrB and rrn probes colocalized on a ∼97 kb fragment indicating that the bcr are chromosomal genes. This is in agreement with previous reports where all other bacterial genera, except for E. faecalisE. faecalisB. licheniformis, B. subtilis and S. mutansE. faecalis regulator, BcrR B. licheniformis and B. subtilis, regulation is performed by a two-component system of a sensor kinase and a response regulator localized on a chromosomal operon. There are also some differences between the bcrABDR cluster of this study and the one described in E. faecalis. The C. perfringens bcrD, bcrB and bcrA are smaller than their E. faecalis counterparts . The distance between the bcrA and the bcrB genes is larger in C. perfringens . We also report, for the first time to our knowledge, on the insertion of IS1216-like transposase gene flanking the chromosomal bacitracin genes of C. perfringens c1261_A. These results suggest that this IS1216-like element originated from E. faecalis and that we may anticipate the emergence of novel, IS1216-based composite mobile elements in C. perfringens.Plasmid and hybridization analyses revealed that the location of the tetA(P) and tetB(P) genes, which mediate active tetracycline efflux and ribosomal protection, respectively B resistant gene, erm(B). This is in agreement with previous studies C. perfringens. However, no other antimicrobial resistant genes were detected in our isolates indicating a low degree of resistance to most other antimicrobials tested as previously described Microarray results revealed that the bacitracin resistant strains of this study all contained both C. perfringens strain c1261_A of poultry origin. Zinc bacitracin is used in poultry production in the United-States and Canada. This practice has likely selected for C. perfringens strains encoding for bacitracin resistance genes. Bacitracin resistance genes have been described in E. faecalis and these genes have high sequence identities with the genes described in this study; thus, a common origin for the bacitracin resistance genes between the two genera is possible. Further investigations are needed to determine the role of each gene and how widespread these genes are in Clostridium strains and species from different origins.In conclusion, this study reports for the first time the characterization of putative bacitracin efflux pump and an overproduced undercaprenol kinase genes associated with acquired bacitracin resistance in a high-level bacitracin resistant Figure S1Dendrogram of the MLVA types of C. perfringens resistant isolates. Cluster analysis was performed with UPGMA using Pearson coefficient. P: chicken strains; the remaining isolates are of turkey origin.(TIF)Click here for additional data file.Table S1Primers for sequencing novel bacitracin resistant genes of C. perfringens strain c1261_A using the primer walking method and new primer designs for PCR screening of bcrA, bcrB, bcrD and bcrR.(DOC)Click here for additional data file.Table S2a.New probe sequences added to the antimicrobial resistance microarraya The bcrR probe sequence was already on the array. bermQ, a ribosomal methylase gene that mediates MLSB resistance bcrABD genes, ABC transporter genes (our study); tetB(P), tetracycline ribosomal protection protein gene (DOCX)Click here for additional data file.


Based on our data, we discuss the involvement of blood Vpr in the clinical symptoms of acquired immunodeficiency syndrome (AIDS).Viral protein R (Vpr), a protein of human immunodeficiency virus type-1 (HIV-1) with various biological functions, was shown to be present in the blood of HIV-1-positive patients. However, it remained unclear whether circulating Vpr in patients’ blood is biologically active. Here, we examined the activity of blood Vpr using an assay system by which retrotransposition of long interspersed element-1 (L1-RTP) was detected. We also investigated the We first discovered that rVpr was active in induction of L1-RTP. Biochemical analyses revealed that rVpr-induced L1-RTP depended on the aryl hydrocarbon receptor, mitogen-activated protein kinases, and CCAAT/enhancer-binding protein β. By using a sensitive L1-RTP assay system, we showed that 6 of the 15 blood samples from HIV-1 patients examined were positive for induction of L1-RTP. Of note, the L1-RTP-inducing activity was blocked by a monoclonal antibody specific for Vpr. Moreover, L1-RTP was reproducibly induced in various organs, including the kidney, when rVpr was administered to hL1-Tg mice.Blood Vpr is biologically active, suggesting that its monitoring is worthwhile for clarification of the roles of Vpr in the pathogenesis of AIDS. This is the first report to demonstrate a soluble factor in patients’ blood active for L1-RTP activity, and implies the involvement of L1-RTP in the development of human diseases. Viral protein R (Vpr), an accessory gene of human immunodeficiency virus type-1 (HIV-1), encodes a virion-associated nuclear protein of ~15 kDa [2/M phase and apoptosis of T cells and neuronal cells  [de novo L1 insertions were detected in the vicinity of tumor suppressor genes, suggesting that L1-RTP is actively involved in carcinogenesis [Long interspersed element-1  and Alu are major endogenous retroelements, accounting for ~17 and ~10% of the human genome, respectively ,9. As an(L1-RTP) ,12. Of n(L1-RTP) -16, rece(L1-RTP) . In tumoogenesis ,22. Becaogenesis ,24, DNA ogenesis , apoptosogenesis  and immuogenesis , deregulHere we present evidence that Vpr is active for induction of L1-RTP, and further demonstrate that 6 of 15 blood samples from HIV-1 patients were positive for Vpr-induced L1-RTP. Interestingly, rVpr reproducibly induced L1-RTP in various organs, including the kidney, when administered to mice that harbored human L1 as a transgene (hL1-Tg mice) ,29. ClinmneoI/ColE1 (pL1-NeoR) . rVpr caused no apparent cytotoxicity . Notably, rVpr-induced L1-RTP was completely blocked by 8D1 and C217, monoclonal antibodies (mAbs) against Vpr  [To determine the effects of rVpr e Figure B. To deme (RTIs) ,40. As se (RTIs) . As show4 cells after six administrations of 10 ng of rVpr . Co-administration of 4′-Ed4T significantly blocked L1-RTP induced by repetitive injection of 250 ng of rVpr . By contrast, down-regulation of ARNT1 by siRNA , a tryptophan photoproduct , which has been shown to associate with other cellular molecules via an LxxLL motif in the counterpart molecule (amino acids denoted by single letters) . InteresA Figure C did notllutants . Both AhTo determine the importance of the LxxLL motif of Vpr for the induction of L1-RTP, we investigated the activity of a Vpr mutant containing AQQAA instead of LQQLL . To further show that ORF1 and AhR form a complex, we transfected a plasmid DNA encoding a chimeric protein of ORF1 and EGFP (pORF1-EGFP) into HuH-7 cells, and then performed IP-WB analysis. IP using α-AhR followed by WB analysis using α-EGFP revealed that ORF1 and AhR were associated  and ORF2, which are ~40 and ~150 kDa, respectively, and are present in cytoplasmic ribonucleoprotein complexes and cytoplasmic stress granules . MoreovePreviously, we reported that FICZ is a potent activator of L1-RTP, and that its activity was dependent on ARNT1, but not on AhR . To deteAhR and C/EBP-β mRNAs, and MAPK inhibitors revealed that rVpr-induced L1-RTP depends on an AhR-p38-C/EBP-β cellular cascade . A PCR-based assay revealed that the ΔC12 mutant was not active for the induction of L1-RTP , a food-borne carcinogen, induced L1-RTP in the mouse mammary gland, a target organ of carcinogenesis, when it was administered orally to hL1-Tg mice [In addition to their clinical relevance for HIV-1 pathogenesis, our findings should have a general impact on the involvement of L1-RTP in human diseases. By analysis of tumors using second-generation sequencing technology, or genes ,22, suggor genes . Althoug-Tg mice . Given t-Tg mice  and has -Tg mice , it is pin vivo, shedding light on the possible involvement of L1-RTP in human diseases. In further research, it will be important to detect L1-RTP-inducing activity under pathological conditions.Six of the 15 blood samples from HIV-1-positive patients examined were positive for Vpr-induced L1-RTP. L1-RTP-inducing activity was detected in blood samples with low viral titres. Monitoring circulating Vpr in relation to clinical outcomes is important to clarify the roles of Vpr in AIDS symptoms. The present study is the first to show that L1-RTP-inducing activity is present HuH-7 cells (RCB1366) and HEK293T cells (RCB2202) were obtained from the Riken BioResource Centre Cell Bank. They were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum . The transfection efficiencies were ~70 and ~30%, respectively, as determined by fluorescence-activated cell sorting (FACS) on day 2 after transfection of plasmid DNA encoding enhanced green fluorescent protein (EGFP) (data not shown). MNF was kindly provided by Dr. Gabriele Vielhaber . SB20358, SP60012, PD98059 and lipopolysaccharide (L8274) were from Sigma-Aldrich. FICZ was obtained from Enzo Life Sciences . Protease inhibitors  were also purchased.Antibodies against AhR, , ARNT1 (Santa Cruz Biotechnology), β-tubulin , H2AX , C/EBP-β , FLAG (Sigma-Aldrich), EGFP , aquaporin 1  and glyceraldehyde 3-phosphate dehydrogenase  were used as the primary antibodies. A rabbit polyclonal antibody against human ORF1 was generated using the peptide MGKKQNRKTGNSKTQSAC (amino acids denoted by single letters) as an immunogen . An Alexa Fluor 546-conjugated antibody to phalloidin  was purchased. As secondary antibodies, α-mouse IgG , α-rabbit IgG , and α-goat IgG (Santa Cruz Biotechnology), all of which were conjugated with horseradish peroxidase, were used. For immunohistochemical analysis, Alexa Fluor 555-conjugated goat α-rabbit IgG (Invitrogen) and Alexa Fluor 488 goat α-mouse IgG (Invitrogen) were used as the secondary antibodies. Hoechst 33258 was purchased from Invitrogen.Based on recent reports that RTIs efficiently blocked L1-RTP ,40, we uEscherichia coli BL21 and purified as described previously  [R, which were inserted in reverse orientation. When L1-RTP occurs, the intron within each reporter gene is spliced out, and then pEF06R expresses functional EGFP, whereas pL1-NeoR expresses a functional neomycin-resistance gene (NeoR). Cells were transfected with pEF06R or pL1-NeoR using Lipofectamine 2000 (Invitrogen) or Xfect . Cells were selected for 2 days with puromycin  for pEF06R, or with hygromycin  for pL1-NeoR. Next, cells were treated for additional 2 or 3 days with the indicated amounts of rVpr.rVpr was expressed using pGEX-6P-1 in igure R) ,35-37, fFor the PCR assay, genomic DNA was prepared from harvested cells using a DNA extraction system . For semi-quantitative PCR, primers that were designed for each exon would amplify a product of ~1040 bp, whereas they would generate a product of ~140 bp upon L1-RTP. Thus, occurrence of L1-RTP was determined by evaluating the size of the amplified product ,29,37. AEGFP gene, were used as forward and reverse primers, respectively. A TaqMan-probe (5′-FAM- TGC AG * C TGG CCG AC -MGB-3′) (Invitrogen) was used to detect an amplicon of 87 bp in length (* denotes the exon junction). Template DNA was amplified with Eagle Taq Master Mix (Roche Diagnostics) and a CFX Connect Real-Time System (Bio-Rad) using the following amplification conditions: 95°C for 10 min, followed by 45 cycles of 95°C for 15 sec and 64°C for 15 sec. To obtain a standard curve for EGFP-qPCR, EGFP DNA generated after the induction of L1-RTP was amplified using the above primers and cloned into the pGEM-T Easy vector . After confirmation of the nucleotide sequence, standard samples were prepared by mixing human or mouse genomic DNA with the EGFP-containing plasmid to give 1.0, 10-1, 10-2, 10-3 and 10-4 copies/cell. To normalize the amounts of input DNA, human β-globin or mouse β-actin was quantified by qPCR with SYBR Premix Ex Taq (TaKaRa) and the CFX Connect Real-Time System (Bio-Rad). For human β-globin, the forward primer was 5′-TTG GAC CCA GAG GTT CTT TG-3′ and the reverse primer was 5′-GAG CCA GGC CAT CAC TAA AG-3′; for mouse β-actin, the forward primer was 5′-TGA CGT TGA CAT CCG TAA AGA CC-3′ and the reverse primer was 5′-AAG GGT GTA AAA CGC AGC TCA-3′.For qPCR analysis, 5′-GAA CGG CAT CAA GGT GAA CT-3′ and 5′-GGG GTG TTC TGC TGG TAG TG-3′, which were designed for each exon of the 6 cells were transfected with pL1-NeoR and selected with 25 μg/mL Hygro, and 1.0 × 105 cells were re-plated to new plates (Split). Next, cells were treated for 2 days with rVpr and further cultured in the presence of neomycin (800 μg/mL) [In the colony formation assay, ~2.0 × 100 μg/mL) -37. In t0 μg/mL) . To deteThe effects of mAbs against Vpr on the induction of L1-RTP were investigated by applying 5 ng of rVpr with 500 ng of 8D1 and C217 , giving R or pEF06R. Two days later, the cells were re-plated, incubated for 2 days with rVpr, and subjected to analysis. Silencer Negative Control siRNAs  were used as controls.For each gene, two small interfering RNAs (siRNAs) were prepared , and their functions were evaluated by transfection into cells followed by WB analysis. The nucleotide sequences of each siRNA are shown in Additional file EGFP genes derived from L1-RTP, ~250 ng of DNA were used as the qPCR template. To amplify β-globin gene as an internal control, ~50 ng of DNA were used as the qPCR template.HuH-7 cells were transfected with pEF06R and selected for 2 days with 0.5 μg/mL Puro. On day 3 after transfection, cells were re-plated and subjected to an L1-RTP assay. To examine the effects of MAPK inhibitors, SB202190, SP600125 and PD98059 at concentrations of 1, 100 and 20 μM, respectively, were added 1 h before the addition of rVpr. The cells were exposed to 10 ng/mL rVpr for 3 days and subjected to qPCR analysis. Genomic DNA was isolated using the QuickGene DNA Tissue Kit S and QuickGene-800 (Fujifilm). To selectively detect in vivo experiments, we used two transgenic mouse lines, #4 and #67, in which the L1-DNA fragment of pEF06R had been introduced as a transgene  Vpr were expressed as FLAG-tagged proteins using the pFLAG-CMV2 expression vector (Sigma-Aldrich). To obtain comparable levels of expression of each protein, the molar ratio of 1:4 of plasmid DNA for the wild-type Vpr and the LA mutant were transfected respectively. On the next day of transfection, cells were subjected to the PCR-based assay.We used the pORF1-TAP (tandem affinity purification) construct , which eHuH-7 or HEK293T cells were transfected with the plasmid constructs pFLAG-Vpr-Wt or pFLAG-Vpr-LA mutant, pORF1-EGFP and pFLAG-EGFP, which encode FLAG-tagged Vpr, a chimeric protein of ORF1 and EGFP, and FLAG-tagged EGFP, respectively. On day 2 after transfection, cells were treated with 10 ng/mL rVpr for 1 day to evaluate the dependence of the protein-protein interaction on Vpr. Then, cells were subjected to IP-WB analysis. To analyze the ORF1-AhR association, cells were suspended in a buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 1% NP40, 1 mM EDTA and a protease inhibitor cocktail and subjected to brief sonication. For analysis of the Vpr-AhR association, cells were suspended in a buffer containing 25 mM HEPES (pH 7.5), 200 mM NaCl, 0.1% NP40, 10% glycerol and a protease inhibitor cocktail, and were completely lysed by passage through 22 G and 27 G needles (in that order) ten times. Cell extracts (500 to 2000 μg) were pre-cleared with protein G Sepharose beads , reacted with 4 μg of α-AhR, α-EGFP, α-FLAG or α-SARS, and then recovered with protein G beads. As an “input” sample, about 5 or 10% of each extract subjected to immunoprecipitation, was assessed simultaneously.After perfusion fixation, organs were immersed in 0.1 M phosphate buffer (PB) (pH 7.4) supplemented with 4% paraformaldehyde at 4°C. On the following day, samples were serially immersed at 4°C in PB supplemented with 10% saccharose for 1 h, 20% saccharose until immersed completely, and then 30% saccharose overnight. Next, samples were embedded in Optimal Cutting Temperature compound  for cryosectioning. Using a cryostat , three slices (5 μm thick) were prepared from different sections of the fixed kidney: a first slice from the middle part of the kidney, a second section is from the part that contained mainly cortex with little amount of medulla, and the third section that is composed mainly of cortex. Samples were washed three times with 0.1 M phosphate-buffered saline (PBS) (10 min per wash), and incubated for 30 min at room temperature in Image-iT Fx signal enhancer (Invitrogen). After rinsing three times with 25 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.05% Tween 20 (TBST) (10 min each), sections were then reacted with rabbit α-EGFP antibody  in TBST supplemented with 1% bovine serum albumin (BSA) at 4°C. On the following day, specimens were rinsed three times with TBST, and then incubated with Alexa Fluor 555-conjugated goat α-rabbit IgG antibody  for 2 h at room temperature. Nuclear DNA was stained with Hoechst 33258 at a final concentration of 0.36 μM. Fluorolabeled sections were examined under a fluorescence microscope . Using the cellSens system (Olympus), total cell numbers in each section were first counted automatically. Next, numbers of EGFP-positive cells were counted manually. The frequency of EGFP-positive cells was calculated using the numbers of total and EGFP-positive cells. Three independent sections were prepared from a single specimen and subjected to analysis. The significance of the frequency of EGFP-positive cells was then evaluated statistically.To identify RTECs positive for L1-RTP, immunohistochemistry was performed as described previously  using thTaq I , which is specific for the methylated sequence, after sodium bisulfite treatment. Digested products were resolved by 3% agarose gel electrophoresis and stained with ethidium bromide.We performed sodium bisulfite treatment of genomic DNA using the EZ DNA Methylation Kit , according to the manufacturer’s instructions. One microlitre of the aliquot was used as the template for combined bisulfite restriction analysis (COBRA) . PrimersP value < 0.05 was deemed to indicate statistical significance.Statistical significance was evaluated using the Mann-Whitney U-test. A All authors declare that they have no competing interest for the current work.NO, MT, YS, KI, MS, AD and SH carried out biochemical analyzes using cell lines. KY, TO and TD NO, MT, YS, MG, AD and TO performed experiments using hL1-Tg mice. YK, TO, KI and YS established qPCR of L1-RTP. AM and NO analyzed methylation status of CpG in the L1-5′UTR. YS, NO, MT, TI and MY carried out immunohistochemistry of cells positive for rVpr-induced L1-RTP. NO, SH, JT, HG and SO analyzed correlation of the activity of Vpr-induced L1-RTP in blood of HIV-positive patients and clinical manifestations. NO, MB and MT examined the effects of RTIs on rVpr-induced L1-RTP. SK and YI designed experiments. NO, KI, MT, AD, YS and YI were involved in preparation of the manuscript. All authors read and approved the final manuscript.No cytotoxicity of rVpr.Click here for fileStandard curve of qPCR assay with a TaqMan probe.Click here for fileL1-RTP induced by low dose of rVpr.Click here for fileSummary viral titres and L1-RTP activity.Click here for fileDetection of Vpr in blood samples of HIV-1 positive patients.Click here for filein vivo.Summary of the PCR-based assay Click here for fileEffects of d4T on rVpr-induced L1-RTP.Click here for fileNo changes of methylation status of CpG in the L1-5′UTR.Click here for fileInhibitory effects of MNF on rVpr-induced L1-RTP.Click here for fileAhR, ARNT1, CREB and c-Jun on expression of endogenous proteins.Effects of siRNAs of Click here for fileCYP1A1 expression under down-regulation of AhR or ARNT1.Click here for fileEffects of MAPK inhibitors on rVpr-induced L1-RTP.Click here for fileEffects of AhR siRNA on chromatin recruitment of ORF1.Click here for fileConstitutive association of ORF1 and AhR under the conditions competent for the induction of L1-RTP.Click here for fileNo apparent changes of expression of L1 mRNA after the addition of rVpr.Click here for fileL1-RTP by Vpr required a carboxy-terminal region.Click here for fileEffects of benzonase on the interaction of AhR and ORF1 or Vpr.Click here for fileNucleotide sequence of siRNA used in the current study.Click here for file


We report on the synthesis and magnetic characterization of ultralong (1 cm) arrays of highly ordered coaxial nanowires with nickel cores and graphene stacking shells . Carbon-containing nickel nanowires are first grown on a nanograted surface by magnetron sputtering. Then, a post-annealing treatment favors the metal-catalyzed crystallization of carbon into stacked graphene layers rolled around the nickel cores. The observed uniaxial magnetic anisotropy field oriented along the nanowire axis is an indication that the shape anisotropy dominates the dipolar coupling between the wires. We further show that the thermal treatment induces a decrease in the coercivity of the nanowire arrays. This reflects an enhancement of the quality of the nickel nanowires after annealing attributed to a decrease of the roughness of the nickel surface and to a reduction of the defect density. This new type of graphene–ferromagnetic-metal nanowire appears to be an interesting building block for spintronic applications. Magnetic nanowires have been widely investigated during the last two decades for fundamental physics –7, and nThe past few years have witnessed the rise of graphene as an extraordinary functional material with unique properties –16. ThisIn a previous study, we demonstrated the possibility to synthesize an array of aligned Ni nanowires on a patterned silicon surface . In thisThe SEM micrographs of the post-annealed carbon-containing nickel nanowires  show theIn order to prove that these nanowires have a core–shell structure with a nickel core and graphene stacking shell, they were placed on a carbon-coated copper grid and their surfaces were examined by TEM . A typichk bands  are probably absent due to the low number of analyzed stacked graphene layers. The other rings observed on the diffraction pattern are attributed to face-centered cubic (fcc) nickel. The formation of the stacked graphene layers results from the phase separation and the nickel-catalyzed crystallization of carbon by thermal annealing. During the post-annealing stage of the carbon-containing nickel nanowires, the carbon atoms diffuse and homogenously dissolve in the nickel phase [Indeed, in both cases only the 002 reflections corresponding to crystalline hexagonal graphite were present –30. Theyel phase –32. As tel phase ,33–34. = 1500 Oe) and the larger squareness (Mr/Ms = 0.4) when the external magnetic field is applied parallel to the nanowire axis, compared to the perpendicular configuration (roughly  = 3100 Oe and Mr/Ms = 0.14), indicate that the nanowire array exhibits a preferential magnetic orientation along the wire axis .The magnetic behavior of the nanowire arrays after post-annealing has been investigated at 300 K by using a Quantum Design SQUID magnetometer. The in-plane magnetization hysteresis loops were measured for an applied field parallel (black curve) and perpendicular (red curve) to the wire axis . The sat = 32 Oe) when the external magnetic field is applied parallel to the nanowire array, than the one measured for the perpendicular configuration (= 21 Oe). It can be seen that the measured values are very low compared to the ones reported in literature for nickel nanowires with the same diameter (roughly 350 Oe) [In the case of polycrystalline ferromagnetic nanowires, such uniaxial magnetic anisotropy originates from the shape anisotropy resulting from the very high aspect ratio of these nanostructures ,35–43. Cex and the domain wall width λw whose values for nickel are about 20 and 90 nm, respectively [Here, for nickel nanowires with a 150 nm diameter, a multidomain configuration is expected. Indeed, this diameter is significantly larger than the exchange length λectively . Moreoveectively ,42. For ectively ,38,41. Aectively .The presence of carbon impurities within the as-grown C–Ni nanowires may also be another factor resulting in higher coercive fields before annealing, since these impurities may generate some defects in the crystalline structure of the nickel phase. In addition, the small coercive fields recorded after annealing suggest that there is no oxide layer surrounding the nickel core, which would produce a bias exchange between the magnetizations of nickel and nickel oxide. This non-oxidized nickel nanowire is expected due to the presence of the stacked graphene layers as a barrier.In summary, an efficient method for the synthesis of an array of ultralong and organized coaxial nanowires, with nickel cores and graphene stacking shells, has been demonstrated. The TEM analysis revealed that the stacked graphene layers forming the shell have a turbostratic structure and a nanotextural order. We have further demonstrated the presence of a preferential magnetic orientation along the wire axis, which has been attributed to the shape anisotropy. The low coercive fields reflect the low roughness and low structural defects as well as dipolar coupling between the nanowires. This new type of graphene ferromagnetic metal nanowire appears to be an interesting building block for spintronics, for example, for the injection of a spin-polarized current from the metal to the high-carrier-mobility graphene structure. Its integration in a planar configuration opens the way to further device characterization. Moreover, the metal-catalyzed crystallization of carbon by thermal annealing, which is the mechanism employed in this work, allows the synthesis of graphene sheets of a few layers with low defects –33. Ther2, and the periodicity of the nanograting patterns was 240 nm. The width of each nanograte was about 140 nm. The details of the plasma process employed for the synthesis of the (C–Ni) nanowires are presented elsewhere [−4 Pa, whereas the deposition argon pressure was fixed to 0.67 Pa. The deposition was performed for 2 min at a floating potential and at a low temperature (T < 150 °C). After the growth, the postannealing treatment of the C–Ni nanowires at 400 °C for 60 min was performed in an oven at atmospheric pressure and under argon flow. After annealing, the samples were cooled down at a rate of 12 °C/min. Scanning electron microscopy (SEM) imaging was performed at 5 kV on a JEOL JSM 7600 F microscope. Transmission electron microscopy (TEM) imaging and selected-area electron diffraction (SAED) were performed on a Hitachi H-9000 NAR microscope . After the postannealing procedure of the carbon-containing Ni nanowires, the TEM specimens were prepared by a simple scratching of the sample surface with a pair of tweezers over a carbon-coated copper grid. Then, a drop of ethanol was placed on to the copper grid for the purpose of dispersion of the collected nanostructures.As described elsewhere , the nanlsewhere . BrieflyThe optimization of the thermal-annealing procedure and the magnetic characterization of the as-grown nanowires are available in the Supporting Information.File 1Annealing procedure and hysteresis loops of the as-grown C–Ni nanowires.


Fibrosis is defined as an excessive accumulation of extracellular matrix (ECM) components with an associated loss of normal tissue architecture and function. Fibrosis occurs in many different auto-inflammatory (auto-immune) diseases. Current therapies targeting fibrosis are limited and we are in great need for novel therapeutic regimens.In fibrosis, abnormal fibroblast proliferation and ECM production by fibroblasts is stimulated by factors released by different cell types, amongst which macrophages. Macrophages produce many pro-fibrotic factors, such as IL-6, CCL2, PDGF and TGF-beta.In rodent models of rheumatoid arthritis and inflammatory bowel disease, somatostatin analogues exerted strong anti-inflammatory effects, by inhibition of pro-inflammatory cytokine secretion. Somatostatin analogues bind to somatostatin receptors (sst) and we demonstrated sst subtype 2 expression on human macrophages.In the present study we investigated the effects of somatostatin, octreotide, a clinically used somatostatin analogue, and cortistatin, an endogenously expressed somatostatin-like peptide, on macrophage-induced fibroblast proliferation.A human fetal lung fibroblast cell line (HFL1) was used in this study. Monocytes were isolated from healthy donors and allowed to differentiate into macrophages. Fibroblasts were co-cultured with macrophages or incubated with conditioned macrophage media in the presence or absence of lipopolysaccharide, somatostatin, octreotide or cortistatin. Thymidine incorporation was used to measure fibroblast proliferation.Culture of fibroblasts with media of unstimulated and LPS stimulated macrophages resulted in an approximately 2-fold and 3-fold increase in proliferation, respectively.1 expressed on fibroblasts, at least the effects of octreotide on fibroblast proliferation should be mediated via macrophages.Conditioned media from octreotide-treated LPS-activated macrophages showed a statistically lower activation of fibroblast proliferation. In co-culture experiments somatostatin, octreotide and cortistatin significantly inhibited unstimulated macrophage-induced fibroblast proliferation by approximately 30%, whereas these compounds significantly inhibited LPS-stimulated macrophage-induced fibroblast proliferation by 20%. As octreotide does not bind to sstin vitro. Treatment of activated macrophages with somatostatin analogues significantly decreased proliferation of fibroblasts. Ongoing studies will reveal the cytokines and growth factors involved in these actions, and whether their release by macrophages is affected by somatostatin analogues. Based on these preliminary findings somatostatin analogues may be promising agents in treatment of fibrotic diseases.In conclusion, macrophages significantly stimulate human fetal lung fibroblast proliferation


A molecular dynamics study on nanoindentation experiments is carried out for some single-layer rectangular graphene films with four edges clamped. Typical load–displacement curves are obtained, and the effects of various factors including indenter radii, loading speeds, and aspect ratios of the graphene film on the simulation results are discussed. A formula describing the relationship between the load and indentation depth is obtained according to the molecular dynamics simulation results. Young’s modulus and the strength of the single-layer graphene film are measured as about 1.0 TPa and 200 GPa, respectively. It is found that the graphene film ruptured in the central point at a critical indentation depth. The deformation mechanisms and dislocation activities are discussed in detail during the loading-unloading-reloading process. It is observed from the simulation results that once the loading speed is larger than the critical loading speed, the maximum force exerted on the graphene film increases and the critical indentation depth decreases with the increase of the loading speed. The perfect graphene is a kind of carbonaceous material which consists of two-dimensional honeycomb lattice structures of single layer of carbon atoms. It is the basic unit to build other dimensional carbonaceous materials, such as zero-dimensional fullerenes, one-dimensional carbon nanotubes, and three-dimensional graphite,2. GraphIt is still debatable whether a graphene sheet is truly a two-dimensional structure or if it should be regarded as a three-dimensional structure since it exhibits a natural tendency to ripple, as observed in recent experiments,14-16. Cab initio total energy calculation[Although the above research has been carried out for mechanical properties of graphene, the variation of the values of elastic modulus has not yet reached a consensus. Especially for rectangular graphene films, the relationship between the load and the indentation depth is not clear. Furthermore, there are few papers available which describe the deformation mechanisms and dislocation activities of graphene film during the nanoindentation processes in detail. These investigations are concentrated on tension deformation-28 and sculation, and quaculation. Researcculation also repIn this article, some MD simulations of nanoindentation experiments are performed on a set of single-layer rectangular graphene films with four clamped edges. The dislocation activities and the deformation mechanism are discussed, and a formula is introduced in order to describe the relationship of load and indentation depth and to measure the mechanical properties of graphene.In order to carry out the nanoindentation experiments, one diamond sphere was introduced to simulate the indenter. Figure When performing MD simulations, we use the canonical  ensemble and control the temperatures at an ideal temperature of 0.01 K. In order to avoid the complex effects of the atomic thermal fluctuations, the temperature is regulated with the Nosé-Hoover method and the time step was set to 1 fs. During the simulation, one key step, named energy minimization and relaxation, should be carried out to make the system remain in the equilibrium state with lowest energy. Then, the indentation experiment was executed and the simulation results were output for further research.F) and indentation depth (d) calculated in MD simulations. The moment the load–displacement curve drops suddenly is considered to be a critical moment. In our simulations, the load suddenly decreased once the indentation depth exceeded 5.595 nm, defined as the critical indentation depth (dc), and the corresponding maximum load (Fmax) is 655.08 nN.We take the case of the graphene film with an aspect ratio of 1.2 and the diamond indenter with a radius of 2 nm as an example to describe the indentation experiment in the following. The indenter was placed over the geometric center of the graphene film and forced to move in the direction perpendicular to the original graphene surface. Figure Figure Generally speaking, elastic deformation which is reversible and plastic deformation which is irreversible are two typical kinds of deformation of an object or material in the view of engineering. In order to determine whether the deformation of the graphene film is elastic or plastic, a set of experiments of loading-unloading-reloading processes are conducted. As shown in Figure dc. The graphene sheet almost can make a complete recovery, i.e., restore its initial structures, and the curves of reloading processes almost perfectly match the initial loading curve while the unloading curve shows very small deviations from the initial one, as shown in the inset of Figure • Stage I. The unloading process is done before the indentation depth reaches the critical depth, dc that the unloading process begins. The graphene sheet cannot make a complete recovery, and there exited broken covalent bonds after the unloading process. In the reloading process, the maximum force exerting on graphene is much smaller than that in Figure • Stage II, i.e., the yellow region in Figure According to the available correlation for the indentation experiments of a circular single-layer graphene film in,22,37, od is the indentation depth and F denotes the concentrated force gotten by the graphene film. In Equation 1, the load F consists of two parts: the first part, Fσ(d), represents the term due to the axial tension of the two-dimensional (2-D) film,where σ02D is the pre-tension of the single-layer graphene film, r is the indenter radius, β denotes the aspect ratio and is equal to L/b, and Requ represents the equivalent radius of the rectangular graphene sheet, (Lb/π)1/2. The second one, FE(d), represents the large deformation term,where E2D is the 2-D elastic modulus, i.e., Young’s modulus, of the single layer graphene film. The strain energy density of graphene, as a standard 2-D material, can be represented by the energy of per unit area. Then, the corresponding pre-tension and elastic modulus can be expressed as σ02D and E2D, respectively, with the unit N/m. The common pre-tension and elastic modulus of a 3-D bulk material can be obtained through these 2-D values divided respectively by the effective thickness which is always treated as the layer spacing of the graphite crystal, i.e., 3.35 Å. q is an nondimensional value, q = 1/(1.05 - 0.15ν - 0.16ν2) = 0.9795, where ν denotes Poisson’s ratio, ν = 0.165[r/R > 0.1, the indenter radius has a significant influence on the load–displacement properties[r/R > 0.1; thus, Equations 2 and 3 are corrected by a factor of (r/R)3/4 and (r/R)1/4, respectively.where  = 0.165,21. It ioperties,39. In oAccording to the expression of maximum stress in the plane of a circular elastic film in, the maxFigure ab initio methods[Table  methods. Lee et  methods. Neek-Am methods. In addi methods,22,43,44Fmax and dc are not that obvious. When the loading speed decreases from 1.00 to 0.50 Å/ps, dropping by 50%, the value of dc increases by 33.35%, and the value of Fmax decreases by 8.43% correspondingly. Nevertheless, when the loading speed decreases from 0.20 to 0.10 Å/ps, dropping by 50%, the changes of Fmax and dc are only 1.68% and 0.21%, respectively. The results may be attributed to the fact that the higher the loading speed of the indenter, the less time it takes to go through the graphene sheet, resulting in a higher load and lower indentation depth than those at a lower loading speed, in which situation the load process is much slower. Secondarily, the spherical indenter’s influences on results are observed by changing the indenter radius. The simulations of various indenter radii  are carried out at the speed of 0.20 Å/ps. The results of the load–displacement curve are shown in Figure Fmax increases obviously and the critical indentation depth also becomes greater with the increase of the indenter radius. Finally, after changing the aspect ratio of the graphene film to 1.1 and 1.5, Young’s modulus and the maximum stress of the graphene are obtained using the methods mentioned above. It can be deduced from Figure For further study of nanoindentation properties, a series of simulations have been carried out with different loading speeds, indenter radii, and aspect ratios of graphene film. It is indicated that the speed of 0.20 Å/ps can be regarded as the critical loading speed, as shown in Figure Some MD simulations of nanoindentation experiments on single-layer rectangular graphene sheets have been carried out in order to obtain the mechanical properties of graphene. A correlation between the load and the indentation depth is constructed, and Young’s modulus and the strength of graphene are obtained in the end. The simulation results show that the unloaded graphene film could make a complete recovery if the maximum indentation depth is less than the critical indentation depth, and the graphene film undergoes elastic deformation during the whole loading-unloading-reloading process. However, if the maximum indentation depth is larger than the critical indentation depth, the graphene sheet could not restore its original structures after unloading and the graphene deforms plastically. Based on the simulations of nanoindentation at different loading speeds and indenter radii, it can be observed that the maximum load increased and the critical indentation depth decreased with the increase of loading speed. In addition, the indenter radius has a remarkable influence on the force-displacement curve. As the indenter radius increases, the critical load and the critical indentation depth also increase.2-D: two-dimensional; AFM: atomic force microscope; AIREBO: adaptive intermolecular reactive empirical bond order; GPa: gigapascals; MD: molecular dynamics; NVT: constant number of particles, volume, and temperature; SW: Stone-Wales 5-7-7-5; TPa: terapascals.The authors declare that they have no competing interests.The analysis of the simulation results was mainly carried out by WDW. The simulation processes were mainly conducted by SL, JJM, and CLY. Some fairly helpful proposals about the construction of models were made by YJZ and MLL. All authors read and approved the final manuscript.


Group II introns are self-splicing RNAs that act as mobile retroelements in the organelles of plants, fungi and protists. They are also widely distributed in bacteria, and are generally assumed to be the ancestors of nuclear spliceosomal introns. Most bacterial group II introns have a multifunctional intron-encoded protein (IEP) ORF within the ribozyme domain IV (DIV). This ORF encodes an N-terminal reverse transcriptase (RT) domain, followed by a putative RNA-binding domain with RNA splicing or maturase activity and, in some cases, a C-terminal DNA-binding (D) region followed by a DNA endonuclease (En) domain. In this study, we focused on bacterial group II intron ORF phylogenetic classes containing only reverse transcriptase/maturase open reading frames, with no recognizable D/En region . On the basis of phylogenetic analyses of the maturase domain and its C-terminal extension, which appears to be a signature characteristic of ORF phylogenetic class, with support from the phylogeny inferred from the RT domain, we have revised the proposed new class F, defining new intron ORF varieties. Our results increase knowledge of the lineage of group II introns encoding proteins lacking the En-domain. Group II introns are self-splicing RNAs that act as mobile retroelements and are thought to be the ancestors of nuclear spliceosomal introns et al. http://webapps2.ucalgary.ca/~groupii/index.html#), class F contained only seven introns, whereas 10 unclassified introns also lacked a recognizable D/En region.Class F is the most recently identified bacterial ORF phylogenetic class. It currently includes a small group of bacterial group II introns with a typical class IIB RNA secondary structure, similar in overall organization to the introns of class E We previously showed that the C-terminal portion of class D RT/maturase proteins constitutes a distinctive, characteristic signature of the ORF class, which has been conserved throughout evolution. It constitutes a group-specific functionally important protein region contributing to both maturase function and intron mobility We searched non redundant  databases for class D group II IEPs, using BlastP or TBlastN, with the consensus amino-acid sequence obtained from a multiple sequence alignment (MSA) of domain X and the C-terminal extension of 35 bacterial class D ORFs We focused on group II intron ORF phylogenetic classes containing only reverse transcriptase (RT)/maturase open reading frames (ORFs), with no recognizable D/En region , using all available sequences from the database until 07-03-2012, and the new sequences identified in this work. The phylogenetic relationships between unclassified and class F intron ORFs available from the database were first investigated by comparing phylogenetic trees estimated by maximum likelihood methods from alignments of sequences for the RT and for the X-domain plus its C-terminal extension. These alignments were generated with Geneious Pro software (Biomatters Ltd.) and Clustal W.http://guidance.tau.ac.il. The species delimitation plugin for Genious The alignment used for the phylogenetic analysis of intron ORFs lacking a recognizable D/En region was initially based on the X-domain and C-terminal sequences. It covered a mean of 130 amino acids corresponding to gap-free lengths of 174 unique sequences that were ≤75% identical . The only exceptions were the class A ORF sequences E.c.I4 and S.f.I1 (99% identity), and the Ni.haI1 (class F) and Ni.haI2 (unclassified) ORFs (99% identity). The larger RT-domain, containing a mean of 257 amino acids, was also used for the phylogenetic analysis of a subset of 123 unique sequences with recognizable RT amino-acid sequences . We included representatives Other types of analysis were performed to investigate the influence of the various phylogenetic estimation methods on our results. We carried out Bayesian analysis with the parallel version of MrBayes 3.1 Nucleotide sequences (4–5 kb) containing putative intron-encoded ORFs were analyzed with Mfold for the detection of potential group II intron RNA structures et al. As mentioned above, Simon Bacterial group II intron ORF phylogenetic classes containing only reverse transcriptase/maturase open reading frames, with no recognizable D/En region, belong to class A, which had only three almost identical (99% identity) members, class C, class E and class D. Moreover, class E was split into two clades, E1 and E2 Ricinus communis).We also updated the compilation of class D intron-encoded ORFs and identified 41 not reported in the intron database, 18 of which had a complete RT-X sequence (Table S1). The 67 class D ORFs included in the final dataset originated from 49 different bacterial species belonging to seven subdivisions of Eubacteria ; four originated from nonculturable environmental bacteria, one from an archaea (Euryarchaeota), one from a nonculturable environmental archaea sample and, surprisingly, one was found in a draft plant genome sequence , revealed an associated group II intron RNA structure. Moreover, 13 of the 27 newly identified ORFs related to former class F or unclassified introns were full-length, as were 14 of the 41 new class D ORFs.Phylogenetic analyses were performed on an amino-acid sequence alignment  for domain X and its C-terminal extension for 174 bacterial group II introns, by the maximum likelihood estimation method. The initial set of intron ORFs included all class A, C, D, E, F and unclassified introns reported in the database and the new introns identified in this work. The final dataset used for the analyses contained only selected sequences that were ≤75% identical .et al. The estimated phylogenetic tree supports the existence of the previously described classes C, D and E , but notg1), supported by a bootstrap value of 93%, contains eight different ORFs, including those of the Ca.ac.I1 and Rh.js.I1 introns previously described as “unclassified”. A second group (hereafter referred to as g2) with strong support (99% boostrap) contained 10 different ORFs, including those of the Mx.xa.I1 intron, which had previously been assigned to class F, and the unclassified introns W.e.I5, Ma.sp.I2, Sg.ce.I1 and B.th.I3. A third group (hereafter referred to as g3), with a high posterior probability (1) and high bootstrap values (77%), contained eight introns, including the class F introns D.a.I1 and Ni.ha.I1 and the previously unclassified intron Ni.ha.I2. Finally, a fourth group (hereafter referred to as g4) with strong statistical support (100% boostrap) had eight members, including the class F intron Pe.ca.I3. The relationships between these groups and the major classes remain unclear, because the deeper branches of the tree have yet to be resolved. Notably, maximum likelihood estimation on the basis of a reduced multiple sequence alignment , with the removal of unreliable columns by the GUIDANCE g4 (96%) and g2 (94%). Likewise, high levels of statistical support were obtained for class D (73%) and class E (82%), whereas the short alignment decreased the bootstrap value for classes C and g1 to 57% and 65%, respectively, and provided no significant support for g3 (not shown). In the short MSA tree, phylogenetic resolution was lower, but nonetheless consistent with the monophyly of g2 and g4.Class D, C and other ORFs branched from a common node with a high bootstrap value (79%), but a low posterior probability (0.51). The phylogenetic tree for the domain X and C-terminal amino-acid sequences also suggested the existence of additional varieties of introns, with strong statistical support  and a high posterior probability (≥0.99). One of these new groups . Nevertheless, this phylogeny identifies several specific features. Some of the class C intron ORFs  lie outside the main node, suggesting that they might be divergent ORFs within this class. Intron Sr.me.I2, which remained unclassified in the maturase tree , clustered within g1 in the RT tree. The intron ORFs clustered in g2 contained a characteristic FADD consensus motif, rather than YADD, in the RT5 domain. There was strong support for sister clade status for classes D and E . This phylogeny was not altered by the addition to the MSA of intron ORF RT-sequences representative of classes B, CL1, CL2 and ML, all of which have an En-domain in their IEPs. This intron lineage branched from a strongly supported (95% bootstrap) node, suggesting that the En domain was acquired only once, in a common ancestor , by the maximum likelihood estimation method and Bayesian inference. In general, the inferred phylogeny was congruent with that obtained with the maturase and C-terminal amino-acid sequences. The estimated phylogenetic tree  supporteancestor .−5). The interspecies distance of the monophyletic g1-4 ranged from 1.325 (g3:g4) to 2.740% (g1:g2) divergence, but only g1 and g2 have significant P(AB) values, with g1 identified as the most divergent clade.The interspecies distance between the currently recognized classes C, D and E  ranged fg1-4). Group g1, which contains no introns previously classified as class F, displayed a significant interspecies distance and may constitute a separate ORF-based phylogenetic subclass. By contrast, g2-4 each included group F members and shared a common node with a basal group containing the rest of the former class F introns , and the unclassified intron c-Ku.st.I1. The grouping together of g2-4 and the small basal F group does not seem to be supported by high bootstrap values in the tree. However, we nevertheless propose that class F should be represented by the four former clades until their relationships can be resolved.We have increased the number of known introns in bacteria and added to knowledge concerning the lineage of group II introns encoding RT/maturase proteins lacking a D/En region. We have identified several new intron ORF varieties and revised the definition of the previously proposed class F. We found that the introns used to established class F The relationships between the intron ORF classes of the intron lineage lacking the En-domain remain unclear, because the deeper branches of the tree have yet to be resolved. Nevertheless, the phylogeny based on the RT-domain suggests that classes E and D may be sister clades. We therefore obtained no evidence to support the hypothesis that class E introns form part of the lineage of introns with an En domain in their IEPs The large number of bacterial genome sequences already available and the continual production of additional sequence data provide the basis for successful analyses of group II intron diversity. We expect even greater intron diversity in bacteria to be detected through future surveys of sequence databases, providing insight into the importance of these catalytic RNAs and retroelements in bacterial evolution.Figure S1MSA based on the maturase and C-terminal extension sequences.(TIF)Click here for additional data file.Figure S2MSA based on the RT-domain (RT0-7).(TIF)Click here for additional data file.Table S1Class D group II introns.(XLS)Click here for additional data file.Table S2New varieties of group II introns lacking the En domain.(XLS)Click here for additional data file.